Abstracts - Invited Speakers
|Date of Web Publication||17-Jun-2011|
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. Abstracts - Invited Speakers. J Carcinog 2011;10:16
IS 1: Life-threatening, yet life-saving: Cell-targeted therapy with plant toxins
Sudarshan Gadadhar, Anjali A Karande
Department of Biochemistry, Indian Institute of Science, Bangalore, India
Immunotherapy is fast emerging as one of the leading modes of treatment of cancer, in combination with chemotherapy and radiation. Use of immunotoxins, proteins bearing a cell-surface receptor-specific antibody conjugated to a toxin, is expected to enhance the efficacy of cancer treatment. Of the numerous known molecules that inhibit translation, abrin, isolated from the Abrus precatorius plant, is one of the most potent ones but has not been exploited in designing immunotoxins. Abrin, a type II ribosome inactivating protein, has a catalytic efficiency higher than any other toxin belonging to this class of proteins. To overcome the drawbacks of the holotoxin that enters any and every cell, and also increase the specificity of the conjugate, we used the recombinant A chain of abrin to conjugate to antibodies raised against the human Gonadotropin releasing hormone receptor. The conjugate inhibited translation and also induced apoptosis in cells expressing the receptor but not in cells lacking the receptor.
IS 2: Generation of tumorigenic breast cells with cancer stem cell properties
Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, India
Several in vitro transformation strategies have been established to understand the mechanisms of breast carcinogenesis. Some of these models led to the generation of squamous cell or medullary carcinomas which represent a small minority of breast cancers. Others required the expression of supra-physiological levels of oncogenic Ras for transformation, while mutations in the Ras oncogene are very rare in naturally arising breast cancers. Furthermore, while recent studies suggest that cancers may originate in adult stem/progenitor cells, most previous studies were undertaken in adherent HMECs that are mostly differentiated. Thus, the cell type of origin of breast cancer, and a suitable model system that closely resembles the vast majority of human breast cancers remains elusive. Using the 'mammosphere' system that selectively facilitates growth of breast stem/progenitor cells in suspension, but not that of differentiated cells, we have generated breast cancer cells that generate invasive adenocarcinomas, the most commonly encountered breast cancer in the oncology clinic. In addition these cells showed stem-like properties of self-renewal and differentiation, and harboured a subpopulation of CD44+/CD24- cells that closely resembled the identified breast cancer stem cell signature. Thus, our study strongly supports the possible stem/progenitor origin of a subtype of breast cancer.
IS 3: Cancer related anemia: Novel insights
Professor of Medicine, Hematology-Oncology, and Head of the Department of Medical Oncology St. Johns Medical College & Hospital, Bangalore, India
Anemia is a common finding in cancer patients with approximately 30% of patients with solid tumors reported to suffer from anemia. Anemia can result from the disease process itself or to its treatment, whether it is chemotherapy or radiotherapy. The factors associated with anemia and thought to contribute to its development encompass disorders of iron metabolism, reduced numbers of erythroid progenitor cells in the bone marrow, increased levels of inflammatory cytokines, extracorpuscular hemolysis, and a relative deficiency of erythropoietin. The presence of preexisting micronutrient deficiencies (iron, B12, and folic acid) may also act in concert to worsen cancer related anemia especially in a country where the prevalence of (nutritional) anemia is already high. The prevalence and etiology of cancer related anemia, the role of anemia as an independent predictor of survival in cancer, the mechanisms of cancer related anemia, and the recent advances in the management of cancer related anemia will be discussed in this presentation.
IS 4: p53/TA-p63/p73 suppresses cancer progression, reprogramming and generation of cancer stem cells through activation-induced cytidine deaminase and miRNAs
Genome Discovery, 49, Nattar Street (Main), Murungapakkam, Modiliarpet, Pondicherry-605 004.
The tumor suppressor JunB has been shown to be a transcriptional target of p63/p73 (Boominathan L, 2007; Invention disclosure 2002/2003). It has been shown that loss of JunB expression in myeloid cells results in the development of chronic myeloid leukemia like disease, suggesting that it functions as a tumor suppressor gene. Interestingly, JunB has also been shown to increase the transcription of miRNA-155, which in turn appears to target the expression of AID (activation-induced cytidine deaminase). Over expression of AID could result in increased number of translocation of chromosomes, genomic instability, and cancer. Therefore, fine-tuning its expression is essential to inhibit cancer. These data together suggest that p63/p73 can increase the transcription of miR-155 through its ability to induce JunB expression. In turn, increased miRNA-155 expression could suppress the expression of AID and thereby it could function as a tumor suppressor in a cell context dependent manner. Interestingly, miR-181b/miR-34b*/34c-3p*/30c*/29*/26*/192*/215*/103*/107*/143*(target scan; *-predicted), a transcriptional target of the tumor suppressor p53/p73/p63, has also been shown to inhibit the expression of AID. Together, the p53/p73/p63-JunB-miR155/miR-181b-AID-dependent tumor suppressor pathway may play a vital role in the control of lymphoma/leukemia. Further, p53 has been shown to inhibit reprogramming of differentiated cells into induced pluripotent stem cells. However, the mechanism by which it inhibits reprogramming remains abstruse. Remarkably, it has recently been shown that AID is required for promoter demethylation and increased expression of reprogramming factors Oct-4 and Nanog. This results in initiation of reprogramming towards pluropotency. This data suggests that p53/p73/p63, by suppressing the expression of AID through its target miR-181b/miR-155/miR-34b*/34c-3p*/30c*/29*/26*/192*/215*/103*/107*/143*, it could suppress reprogramming of differentiated cells. This data further suggests that the loss of p53/p63/p73 during cancer progression may aid reprogramming of malignant cells into cancer stem cells. Therefore, the mechanism identified in this study may be of significance in cancer therapy.
IS 5: Combinational chemopreventive approaches for colorectal cancer
Chinthalapally V Rao
Center for Chemoprevention and Cancer Drug Development, Oklahoma Cancer Center, Department of Medicine, Medical Oncology, University of Oklahoma Health Sciences Center, Oklahoma City, OK; USA
Clinical and preclinical studies suggest that molecular targeted small molecule drug development approaches very promising at the preclinical and clinical level for the prevention and treatment of several epithelial cancers. NSAIDs such as aspirin, sulindac, and celecoxib, a cyclooxygenase (COX)-2 inhibitor; DFMO, an ODC inhibitor, atorvastatin, a HMG-R inhibitor, Targretin, a RXR-modulator and Gefitinib, a EGFR inhibitors have been shown to reduce the risk of colon and other epithelial cancers. However, their prolonged administration at higher doses ad significant efficacy but cause for a concern for unwanted toxicity. Thus, developing different molecular targeted chemopreventive combinations may provide additive and synergistic efficacy at low-doses, without any unwanted side-effects.
Using combinational molecular targeting in preclinical models we have generated impressive data using both single agents and multiple agents. These targets include 5-Lipoxygenase (5-LOX)/COX-2; COX-2 and HMG-R; ODC and COX; and p53 and COX-2; and RXR and ER-beta. Thus, development of agents with 5-LOX/COX inhibition or combinations of agents that represent different modes of action provide a practical approach for improving chemopreventive and therapeutic efficacy without unwanted side effects. For example, experiments were designed to test the chemopreventive/therapeutic efficacy of licofelone, a novel 5-LOX/COX- inhibitor. To test the efficacy of licofelone (50-300 ppm in diet) in the colon, we utilized azoxymethane (AOM)-induced rat colon carcinogenesis and Apc-min intestinal tumor model and AOM-induced rat colon adenocarcinoma model. In the rat colon model, licofelone suppressed AOM-induced colonic aberrant crypt foci (ACF) in a dose-dependent manner (Total ACF, P<0.0.05-0.0001; multicrypt foci, P<0.01-0.0001); similarly, Apc -min intestinal tumor formation was also significantly suppressed (P<0.01-0.0001). We will discuss on going progress on the combinational targets and identification select chemopreventive agents and promise for the human clinical trials. (Supported by NCI CN-N01-53300 and NCI R01CA094962 and CA102947)
IS 6: Resveratrol-induced mitochondrial dysfunction in cancer
Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, New York, USA
Although resveratrol triggers cell death in multiple types of cancer, the underlying molecular mechanism is unclear. We show that resveratrol induces p53- independent Bax translocation and oligomerization on mitochondria to execute apoptotic cell death. Resveratrol causes loss of mitochondrial-membrane potential, and induces caspase-dependent apoptosis. Bax recruitment and oligomerization on mitochondria following resveratrol treatment is associated with cytochrome c release from mitochondria. Interestingly, we observed that resveratrol induces Bax oligomerization in the cytosol. Translocation of Bax to mitochondria and its oligomerization on this organelle precludes p53 involvement, but requires Bim up-regulation and Bid cleavage. Immunoprecipitation and cross linking analyses demonstrate that both Bax/Bax primarily undergo homo-oligomerization, but Bax seems to play a major role in cytochrome c release as Bax knockout cancer cells inhibited cytochrome c release. Additionally, p53 knockout cells did not prevent resveratrol-induced cytochrome c release further supporting that p53 does not significantly regulate Bax/Bak-mediated cytochrome c release upon resveratrol treatment. Caspase activation and apoptosis is initiated by caspase-9 activation as silencing of caspase-9 inhibited caspase activation and apoptosis, whereas knocking down caspase-8 did not modulate caspase activation. Altogether, we characterize the molecular mechanisms of resveratrol-induced caspase activation, and apoptosis in cancer cells. Since our findings demonstrated that resveratrol induces Bax activation in the cytosol, further analysis on how to enhance Bax oligomerization on mitochondria in a p53-independent manner may provide a novel approach to enhance apoptotic cell death in cancer.
IS 7: "Breast cancer beyond": Impact of psycho neuro immunal logical parameters in breast cancer management
Editor -in-chief,The Indian Journal of Surgical Oncology, President ASI 2006, Director, Bangalore Institiute of Oncology, No.20, Srinivasa, BSK III Stage, Bangalore
Breast cancer is the second commonest malignancy seen in women in Indian sub continent. Annually 93 thousand new cancer cases are detected in India as per national cancer registry programme. An old disease with new therapeutic strategies from radical mastectomy of Hallstead to present day Breast conservative surgery with or without reconstructive procedure. We are able to prognosticate the disease using biological, biochemical parameters and plan adjuvant chemo, radiation, hormone and target therapy.
Breast Cancer beyond what do we mean?
Breast cancer is a profoundly stressful disease posing both physical and psychological threats to the patient. Patients have to endure distressing side effects of multimodal treatments over a long period of time leading to anxiety, depression and other psychological reactions that can affect treatment compliance. The lessons learnt from clinical interaction with the patients resulting in various life time events in the women who suffer from breast disease in the form of events in the family resulting in patient presenting with breast cancer. This made us to think what is the Impact? Of stress and breast cancer and can we look at stress as a carcinogenic potential and also to plan for preventive programmes in future for breast cancer. Hence the study under took was to evaluate impact of psycho neuro immunological parameters in breast cancer. These stress perceptions coupled with cancer-related intrusive thoughts, age, sociodemographic characteristics and financial concerns along with a tendency towards negativity (neuroticism) may conspire to heighten a women's risk for psychological distress, reduce their quality of life and also inhibit innate and anti tumor immune responses against cancer. We are presenting our experiences in impact of psycho, neuro immunological parameters in management of Breast cancer. The out come of this study let us to establish psycho oncology as part and parcel of treatment modality including yoga. Now we have establish "Cooper" an educational initiative and an institute to train medical and paramedical health professionals to give good quality care to the breast cancer patients.
IS 8: Estrogens, T regulatory cells and vascular endothelial growth factor in high risk premenopausal breast cancer: Their role in carcinogenesis and disease progression
Francesco Recchia 1,2 , Giampiero Candeloro 1 , Stefano Necozione 3 , Giovambattista Desideri 4 , Maurizio S D'Andrea 4 , Silvio Rea 2,5
1 Oncologia, Ospedale Civile, Avezzano; 2 Fondazione Carlo Ferri, Monterotondo; 3 Epidemiologia Clinica, Università degli Studi, L'Aquila; 4 Geriatria, Università degli Studi, L'Aquila; 5 Oncologia Chirurgica, Università degli Studi, L'Aquila, Italy
Estrogen is important in reproductrion and breast cancer carcinogenesis and there is evidence in vitro, that it modulates T-regs proliferation and VEGF expression in breast cancer cells through transcriptional activation. However few clinical studies have investigated the association between VEGF and estrogens in humans. Objective of this study was to evaluate weather estrogen suppression with a lutenizing hormone releasing hormone (LH-RH) analogue was able to downregulate VEGF expression, and to decrease the number of circulating T-regs, in premenopausal patients with high-risk early breast cancer. From April 2003 to October 2008 we conducted this study on 100 premenopausal early breast cancer patients. After surgery, before any type of therapy, plasma VEGF, T-regs and sex hormones (estradiol, progesterone, FSH, LH) were evaluated as baseline, and yearly thereafter. Patients received an LH-RH analogue for 5 years, a chemotherapy that was tailored to the biological characteristics of each patient, radiation therapy, and hormonal therapy in estrogen receptor positive (ER+) tumors. Primary end popint was the evaluation of VEGFand T-regs. Secondary end points were progression-free survival (PFS) and overall survival (OS). Mean age was 43 years (range 26-45), mean number of positive axillary nodes was 3.28 ± 0.5, fifty-three patients were ER+ and progesterone receptors (PGR) +, 30 were ER+ and PGR negative (-), 17 were ER- and PGR-, mean KI-67 was 33.9 % ± 2.5%, 20 patients were Herb-2 positive by FISH. After one year, estradiol was suppressed to values < 40 pg/ml, and a statistically significant decrease of VEGF and T-regs were observed. Plasma VEGF continued to decrease (P<0.001), during the study period, in 94% of patients that were disease-free, while in 6% of patients VEGF increased with respect to baseline values, at disease relapse. No unexpected toxicity of chemotherapy was observed, while hot flashes and G1 osteopenia occurred after LH-RH analogues administration. After a median follow-up of 50 months (range 24-90), 5-year progression free survival and overall survival rate were 94.6% and 100%, respectively. Estrogen deprivation with an LH-RH analogue is able to decrease estradiol, plasma VEGF levels and T-regs in premenopausal high risk breast cancer patients. These data show how estrogens mediated increase of T-regs and induction of free extracellular VEGF may be one mechanism involved in sex steroid dependent breast carcinogenesis and in the worst prognosis observed in premenopausal breast cancer patients.
IS 9: Cell based cancer therapy: Cell encapsulation and imaging cell function
Assistant Professor of Radiology and of Medicine, Director, Biomedical NMR Research Center, Dartmouth Medical School,
Cells can be engineered to secrete therapeutic molecules and can be grafted as a permanent and regenerative source of therapy. The encapsulation of such cells in semi-permeable, nanoporous microcapsules is required to protect the cells from host immune attack and to prevent their undesirable proliferation and migration in the host. The presentation will highlight strategies to engineer cell encapsulating microcapsules with nanoporous surfaces that permit the transport of small molecules including nutrients, gases, ions and biotherapeutic molecules secreted by the cellular grafts, but that prevent the passage of large molecules of the immune system. The talk will also outline approaches to image encapsulated cell function in vivo, using MRI as well as optical methods.
IS 10: RLIP76 is a R-Ras signaling effector in vascular cells regulating endosomal trafficking, cell spreading and migration, and tumor vascularization
Jeremy GT Wurtzel, Sharad S Singhal 1 , Sanjay Awasthi 1 , Lawrence E Goldfinger
Department of Anatomy & Cell Biology and The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA, USA, 1 Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, TX, USA
RLIP76/RalBP1 is a glutathione-conjugate transporter which facilitates export of chemotherapeutic drugs from cancer cells. As such, RLIP76 promotes cancer cell survival, and ablation or blockade of RLIP76 causes regression of various types of solid tumors, while RLIP76-null mice are strongly resistant to chemically-induced neoplasia. We have found that RLIP76 also participates in cancer progression by additional mechanisms: regulating cell motility which can affect tumor progression at multiple stages, and by regulating angiogenic neo-vascularization of nascent tumors. We originally found RLIP76 in a proteomic screen for signaling effectors of the Ras family small G protein, R-Ras, and we found that through its interaction with R-Ras, RLIP76 is required for cells' ability to spread on ECM substrates, and for cell migration. We have previously shown that RLIP76 mediates these cellular functions by driving a signaling cascade comprised of the GTPases Arf6 and Rac1. R-Ras has weak oncogenic activity (compared to other Ras paralogues which are potent proto-oncogenes) but it is an important regulator of cell migration and tumor angiogenesis. We now find that the R-Ras effector, RLIP76, modulates tumor progression in part by regulating the growth of new blood vessels from surrounding tissue into the tumor mass (tumor angiogenesis). Solid tumors derived from B16 melanoma cells implanted in RLIP76-null mice were significantly smaller compared to those in wild type mice, consistent with the literature. Additionally, neo-vascularization of the tumors in RLIP76-null mice was also significantly reduced. Thus, RLIP76 is a positive regulator of tumor angiogenesis in mice. We have further characterized the signaling mechanism and found that RLIP76 and activated R-Ras co-localize at the membranes of recycling endosomes in cells, and RLIP76 recruits the Arf6 activator protein, ARNO, to these sub-cellular sites. ARNO anchored to endosomal membranes via an R-Ras/RLIP76 complex enhances localized activation of Arf6, which drives microtubule-dependent anterograde traffic of the recycling endosomes to the leading edge of the cell, stimulating cell migration. We propose that RLIP76 promotes tumor angiogenesis in part due its roles in migration of vascular endothelial cells. Thus, the RLIP76 signaling nexus is an attractive cancer therapy target as RLIP76 enhances tumor progression at multiple stages.
IS 11: Global cancer prevention efforts: Imperatives and Impediments in the developing world
Department of Genetics, 145 Bevier Road Rutgers University, Piscataway, NJ 08854 USA.
Email: Kovvali@Biology.Rutgers.Edu, firstname.lastname@example.org
In 2000, 5.3 million men and 4.7 million women developed a malignant tumor and 6.2 million of them died of the disease. It is estimated in the World Cancer Report that Cancer incidence rates could increase by 50 percent to 15 million new cases in the year 2020. The predicted sharp increase in new cases will mainly be due to steadily aging populations in both developed and developing countries and also due to current trends in smoking prevalence and the growing adaption of unhealthy lifestyles. Do cancer prevention strategies used in the west, especially the USA, work in the developing world? Are molecular etiologies of known and emerging cancers the same in the western and developing world? It is commonly cited that tobacco, alcohol, low fruit and vegetable intake, physical inactivity, over weight and obesity are key risk factors for cancer incidence. While tobacco use is declining in USA, its use in the developing countries is yet to show perceptible decline as there are no disincentives like steep health insurance premiums for smokers in the USA. Lack of proper nutrition may be responsible for many cancers in the developing world while excessive caloric intake from fat is attributed to cancers in the developed world. It appears that modifiable and avoidable lifestyles account for a significant proportion of cancers in the developing world. As the developing countries are aggressively aspiring to reach the status of developed nations through industrialization and occupational adjustments, the developing societies are under the evolutionary pressure witnessed by the developed world. It will be interesting to investigate the innate carcino-resistant and carcino-preventive abilities of the conservative clans of the developing countries as opposed to those who embrace the modern lifestyle.
IS 12: C17orf37 mediated cancer cell migration and metastasis
Jamboor K Vishwanatha, Subhamoy Dasgupta
University of North Texas Health Science Center, Institute for Cancer Research and Department of Molecular Biology and Immunology, Fort Worth, TX, 76107, USA
Post-translational modification by covalent attachment of isoprenoid lipids regulates the functions and biological activities of several proteins implicated in the oncogenic transformation and metastatic progression of cancer. The largest group of prenylated proteins contains a 'CAAX' motif (C denotes cysteine, A represents aliphatic amino acids, and X any amino acid) at the carboxyl-terminal which serves as a substrate for a series of post-translational modifications that convert these otherwise hydrophilic proteins to lipidated proteins, facilitating membrane localization. A novel gene named Chromosome 17 open reading frame 37 (C17orf37) located in the 17q12 amplicon is overexpressed in different forms of human cancer and contains a putative prenylation domain at the C-terminal tail. The present study is designed to explore the importance of prenyl-modification on the function role of C17orf37 in cancer. C17orf37 promotes migration and invasion of cancer cells. This function of C17orf37 is due to its ability to act as a membrane-bound signaling molecule modulating the PI-3K/Akt pathway, there by transcriptionally upregulating NF-κB downstream target genes MMP-9, uPA and VEGF. Via this process, C17orf37 overexpression in tumors contributes to the migratory and invasive phenotype, facilitating the malignant progression of the disease. Here we show that C17orf37 contains a functional CAAX motif and is post-translationally modified by protein geranylgeranyltransferase-I. Geranylgeranylation of C17orf37 at the CAAX-motif results in translocation of the protein to the inner-leaflet of plasma membrane, and enhances migratory phenotype of cells by inducing increased filopodia formation and potentiation of directional migration. A prenylation-deficient mutant of C17orf37 is functionally inactive and fails to trigger dissemination of injected cells in a mouse model of metastasis. These findings demonstrate that prenylation is required for the function of the C17orf37 protein in cancer cells and imply that the modification may functionally regulate metastatic progression of disease.
IS 13: Early detection of lung cancer
Chief of Thoracic Surgery, Vice Chair, Surgical Quality, North Shore University Health System, 2650 Ridge Avenue, 3507 Walgreen Bldg., Evanston, IL 60201
Lung cancer is the leading cause of cancer-related mortality in the U.S. and worldwide. Screening has been controversial because prior studies with chest x-ray have found no mortality benefit, while others that saw an improvement in detection with low-dose helical CT (also called spiral CT) had methodological limitations and raised concerns about false positive screens. So in August 2002, the National Cancer Institute in the United States launched the National Lung Screening Trial to clarify the issue. The researchers enrolled 53,454 current and former heavy smokers ages 55 to 74. These high-risk patients were assigned to receive three annual screens with either low-dose helical CT or standard chest x-ray. As of Oct. 20, 2010, there were a total of 354 deaths from lung cancer in the CT group, compared with 442 in the chest x-ray group. That amounts to a 20.3% reduction in lung cancer mortality -- a finding that the study's independent data and safety monitoring board decided was statistically significant enough to halt the trial and declare a benefit. A concern remains about the high rate of false positive exams particularly in areas with endemic granulomatous disease. The goal of our study at the University of Cincinnati was to determine whether lung cancer screening could be done while minimizing the number of benign biopsy specimens taken in an area endemic for histoplasmosis. The subjects were recruited by letters mailed to area physicians and local advertisement. The inclusion criteria were age older than 50 years and at least a 20 pack-year smoking history. The exclusion criteria were symptoms suggestive of lung cancer or a history of malignancy in the previous 5 years. The participants completed a questionnaire and underwent a chest CT scan at baseline and annually for 5 years. The management of positive screening results was determined using a defined algorithm: annual follow-up CT scan for nodules less than 5 mm; 6-month follow-up CT scan for nodules 5 to 7 mm; review by our multidisciplinary tumor board for nodules 8 to 12 mm; and biopsy for nodules greater than 12 mm. A total of 132 patients were recruited. Of the 132 patients, 61% had positive baseline CT findings and 22% had positive findings on the annual CT scans. Six cancers were detected. Of these 6 patients, 5 had stage I disease and underwent lobectomy, and 1 had stage IIIA disease and underwent induction chemotherapy and radiotherapy followed by lobectomy. All patients were alive and disease free at a mean follow-up of 41.7 ± 18.6 months. No biopsies were performed for benign lesions. Also, no cancers were missed when the protocol was followed. We concluded that screening with CT can be done effectively in an area endemic for histoplasmosis while minimizing benign biopsies.
IS 14: An investigation on the cytotoxic and apoptotic activities of carvacrol in Hep-G2 cells
Hulya Sivas, Ozlem Tomsuk
Department of Biology, Anadolu University, Faculty of Sciences, Molecular Biology Section, 26470 Eskisehir, Turkey.
Carvacrol is a predominant aromatic compound in essential oils of the family Labiatae with a wide range of biological activities such as antibacterial, antiviral, antifungal and antiparasital effects. Here, cytotoxicity and apoptotic activities of carvacrol were investigated in a human liver hepatocellular carcinoma cell line Hep-G2. Effects of carvacrol on the viability of the cells were investigated by WST-1, neutral red and trypan blue staining assays. Inhibition of DNA synthesis was investigated using BrdU incorporation method. Further, an apoptotic activity of carvacrol was determined by acridine orange/ethidium bromide staining. According to data, carvacrol exhibited a dose-dependent effect on the viability of Hep-G2 cells, indicating an IC50 value around 500 mM. DNA synthesis was also inhibited after carvacrol treatment parallel to the inhibition of viability of the cells. Apoptotic morphology such as chromatin condensation and cytoplasmic blebbing was observed after incubation of the cells with carvacrol for 48 hours. In conclusion, carvacrol exhibited cytotoxic and apoptotic activities in Hep-G2 cells, indicating its potential for use as an anticancer agent.
IS 15: Polyamine inhibition as tertiary colorectal cancer prevention
Jason A Zell
Department of Medicine (Division of Hematology/Oncology), & Department of Epidemiology, School of Medicine University of California, Irvine, 101 The City Drive South, Orange, CA, 92629; USA
Polyamines are naturally-occurring substances found in the diet, and produced in epithelial tissues via the conversion of arginine-derived ornithine to polyamines. This conversion occurs by the rate-limiting enzyme ornithine decarboxylase (ODC) to form the major polyamines: putrescine, spermidine, spermine. Polyamines are involved in cellular processes, such as tissue growth and repair, cell proliferation, and regulation of transcription. However, in excess they cause cancer formation within epithelial tissues, and are implicated in colorectal carcinogenesis. Eflornithine (difluoromethylornithine, DFMO) is a suicide inhibitor of ODC, and through this action inhibits polyamine synthesis. Sulindac, like other NSAIDs, has polyamine-inhibitory properties, via induction of spermidine spermine acetyltransferase (SSAT) and resultant cellular polyamine export. Together, eflornithine and sulindac decrease cellular polyamine pools. In a phase III clinical trial of eflornithine and sulindac compared with placebo, eflornithine and sulindac produced a 70% reduction in recurrent adenomas, and a 91.5% reduction in advanced adenomas. Together with the U.S. National Cancer Institute (NCI)-Division of Cancer Prevention, investigators at SWOG have developed a phase III clinical trial among colorectal cancer patients to study eflornithine, sulindac, and placebo as tertiary prevention: SWOG 0820 "A Double Blind Placebo-Controlled Trial of Eflornithine and Sulindac to Prevent Recurrence of High Risk Adenomas and Second Primary Colorectal Cancers in Patients with Stage 0-III Colon Cancer, Phase III" (Study Coordinators: J. Zell, P. Brown). Projected accrual will be 1340 patients, and the primary endpoint is a reduction in high risk adenomas and second primary colorectal cancers. Translational pharmacogenetic analyses are proposed as secondary endpoints, and will be discussed in light of recent translational research. This large tertiary colorectal cancer (CRC) prevention trial will be placed in the context of other relevant phase III CRC clinical trials underway within the NCI-supported cooperative oncology group system.
IS 16: Molecular biology of de-regulation of gene expression in Ni(II) compound transformed C3H/10T1/2 mouse fibroblasts
Joseph R Landolph Jr, Aruni T Pehl-DeSilva, Duy Mai, Jim KH Lin, Preethi Samala, Sara Keliipaakaua
Departments of Molecular Microbiology/Immunology and Pathology, USC Cancer Center, Keck School of Medicine, Univ. Southern California, Los Angeles, Calif., 90033, USA.
Nickel (Ni) refinery workers who inhaled Ni-containing sulfidic ore dusts and also smoked cigarettes in Ni refineries contracted lung and nasal cancers. Inhalation of Ni 3 S 2 /green NiO also induces respiratory cancer in rats. We showed Ni 3 S 2 and green and black nickel oxides were phagocytosed into and induced chromosomal aberrations, cytotoxicity, and morphological, A. I., and neoplastic transformation in C3H/10T1/2 Cl 8 (10T1/2) mouse embryo cells. We found that 130 genes were differentially expressed between non-transformed and two 3-methylcholanthrene (MCA)-/four Ni-transformed 10T1/2 cell lines by mRNA differential display. Ni/MCA-transformed cell lines displayed a) ect-2 gene amplification/higher levels of ect-2 gene mRNA/protein; higher steady-state levels of b) calnexin mRNA/protein and c) Wdr1 gene mRNA; and d) no detectable levels of DRIP-80 and β-2-centaurin mRNAs. We hypothesized Ni +2 -induced 1) amplification of ect-2 gene/higher levels of rhoA-GTP led to higher levels of microtubules (MTs), and 2) silencing of β-2-centaurin gene, caused higher levels/aggregation of microfilaments (MFs), and 3) silencing of the DRIP-80 gene caused aberrations in Ca +2 ion gradients, in transformed 10T1/2 cells. To test these hypotheses, we stained cells with fluorescent phalloidin to decorate MFs, separately with fluorescent antibody to α-tubulin/β-tubulin to decorate MTs, then separately with Fluo 3AM to stain Ca +2 ions, then examined cells by confocal microscopy. In non-transformed 10T1/2 cells, MFs/MTs were arranged homogeneously in long thin fibers. In NiS/green NiO transformed cell lines, MFs and MTs were over-expressed and aggregated in some areas, absent in other areas, changing shapes of transformed cells, rounding them/altering their contact with extra-cellular matrix. In non-transformed cells, Ca +2 ions were found in two arrangements in non-transformed 10T1/2 cells: State I, in low density cells, with a heavy concentration of Ca +2 ions in the nucleus, and lesser amounts in the cytoplasm; and State II, in high density cells near confluence, where there were few Ca +2 ions in the nucleus, most in the cytoplasm. Non-transformed 10T1/2 cells cycled between States I and II. In Ni/MCA transformed cell lines, Ca +2 ions were predominantly cytoplasmic (State II). Our model suggests activating mutations or silencing methylations in 15 genes led to differential expression of 130 total genes. We conclude Ni ions caused amplification of the ect-2 gene, and silenced expression of the β-centaurin-2 gene, leading to expression of higher steady-state levels of MTs and MFs, respectively, causing changes in cell shape, hence changes in global gene expression. Ni ions also silenced the DRIP-80 gene, which likely led to alterations in Ca +2 ion gradients in transformed cells, further altering activities of Ca +2 ion-dependent enzymes and cellular physiology. These Ni ion-induced events cumulatively led to differential expression of 130 genes in transformed cell lines, altered cell shapes, and altered Ca +2 ion gradients, contributing to induction and maintenance of transformed phenotypes of transformed cell lines. Supported by grants R01 ES03341/NIEHS (PI, JRL) and 5 T32 AI07078/NIAID, funding from M. S. Program, Department of Molecular Microbiology and Immunology at USC, and USC Discretionary Funding at USC to JRL.
IS 17: Principle of peptide-based cancer imaging & therapy
Head Molecular Imaging HCG, Bangalore, India
Receptors for regulatory peptides are over expressed in a variety of human cancers. They represent the molecular basis for in-vivo imaging with radiolabeled peptide probes. Somatostatin-derived tracers, designed to image the sst2-over expressing neuroendocrine tumors, have enjoyed almost 2 decades of successful development and extensive clinical applications. More recent developments include second- and third-generation somatostatin analogs, with a broader receptor subtype profile or with antagonistic properties. Emerging tracers for other peptide receptors, including cholecystokinin/gastrin and GLP-1 analogs for neuroendocrine tumors, bombesin and neuropeptide-Y analogs for prostate or breast cancers, or Arg-Gly-Asp peptides for neoangiogenesis labeling, are also in current development. Application fields include both SPECT/CT and PET/CT. In somatostatin-based cancer imaging a stable somatostatin analog linked to a chelator that can bind radioactive metals such as 111In, 99mTc, or 68Ga is injected intravenously into the circulation of a patient with a potential neuroendocrine tumor. The radiotracer will distribute in the body. If the patient has a tumor expressing somatostatin receptors in large amounts, the tracer will selectively bind to these somatostatin receptors and will actively be taken up by the cells through a process called receptor-ligand internalization. The internalization will ultimately lead to an accumulation of radioactivity in the tumor, compared with the rest of the organs; it will be amenable to detection through gamma-camera scanning and PET. Normally, rapid and specific uptake is observed in the tumor, and concomitant labeling of kidney and bladder is also found, as a consequence of the predominant urinary excretion pathway of most radioligands. The sensitivity of this type of cancer imaging is excellent, with tumors of a few millimeters in diameter often being identified; several large studies have proposed somatostatin receptor scintigraphy as the method of choice for the detection of some of the gastroenteropancreatic tumors. Which Isotope to Choose? The widely used somatostatin-based peptides were labeled with a variety of radionuclides for SPECT (111In, 99mTc, and 67Ga) and for PET (18F, 68Ga, and 64Cu). Gamma Emitters appear to have undergone a kind of renaissance due to the availability of sophisticated SPECT/CT. 111In is an attractive radionuclide because it often can be used as a surrogate of the b-emitters 90Y and 177Lu. 99mTc, still one of the workhorses, has its obvious advantages; powerful kit formulations are in use for the HYNICTOC and N4 TATE conjugates. The application of 68Ga-labeled peptides has attracted much attention because of the availability of a cost-effective generator and the available chemistry (22). Mainly, 68Ga-DOTA-TOC, 68Ga-DOTA-TATE, and 68Ga-DOTA-NOC are being studied in clinical trials. In addition, 68Ga-based bombesin derivatives were studied in gastrointestinal stromal tumors (23). The 68-min half-life of this radionuclide is suitable for the pharmacokinetics of most peptides. Another widely used, attractive metallic positron emitter is 64Cu. It can be produced in large scale at high specific activity with a medical cyclotron. Although its half-life of 12.7 h may be superior to that of 68Ga for many applications, its decay characteristics (19% b1; 39% b2) may not be ideal for patient dosimetric reasons. Nevertheless, somatostatin-based 64Cu-labeled conjugates show promising preclinical properties. This also applies for RGD- and bombesin-based radiopeptides; as already indicated, 18F-labeled peptides were developed and those based on somatostatin and RGD were successfully studied in patients. Considering the success of somatostatin receptor targeting as a niche indication in neuroendocrine tumors, we can extrapolate to the large number of novel candidate peptides and expect a boom in molecular imaging when peptide receptors over expressed in key tumors such as breast, prostate, or pancreas carcinomas can finally be targeted. This will largely depend on the development of adequate probes, which continues to be a considerable challenge. I will share my experiences and results of the peptide receptor labeling and PRRNT
IS 18: Molecular MRI techniques for in vivo targeted drug screening and biomarker detection
School of Biological and Health Systems Engineering, Arizona State University, 501 E Tyler, ECG Bldg, Room 334, Tempe, AZ. 85287
There is a clear, recognized need for drugs and imaging contrast agents that are specific to the molecular makeup of individual patients. Concurrently, there are a multitude of techniques to generate and rapidly screen libraries of drug candidates for their chemical structures and efficacy in vitro. There is a clear need for an intermediate in vivo diagnostic step between drug development and the testing required to move the drugs quickly into the clinic. The field of molecular imaging has revolutionized in vivo diagnostics, and has the potential to provide highly sensitive detection of targets within the body. Of existing imaging modalities, magnetic resonance imaging (MRI) is uniquely three-dimensional, noninvasive, and rich in contrast. However, traditional MRI has suffered from a lack of molecular sensitivity. This talk will summarize the state-of-the art for molecular MRI, and will identify current novel techniques to create contrast and increase sensitivity, with the aim of developing molecular MRI as a high-throughput technique for whole-body screening of drug toxicity, target molecule detection, and identifying targeted drug candidates.
IS 19: Potential role of Insulin-like growth factor binding proteins isoforms in cancer progression
Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore-560012, India.
The insulin-like growth factor binding proteins (IGFBP) isoforms are a set of molecules that are structurally similar and 11 isoforms (IGFBPs1-10 and -L1) have been described. IGFBPs1-6 are known to bind to IGFs with relatively higher affinity than others. The principal role attributed to IGFBPs is to modulate the actions of IGFs mainly by controlling the amount of free IGF that can bind to the receptors. However, they are also known to regulate cell survival in an IGF independent pathway wherein they can affect cell growth, adhesion, migration and apoptosis. For example, IGFBP-1 and -2 contain an RGD motif that facilitates invasion of tumor cells by interaction with integrins. IGFBP-3 has been reported to induce apoptosis whilst IGFBP-5 may inhibit apoptosis. By microarray experiments, we identified IGFBPs 2-5 and 7 as over expressed genes in gliomas. In breast cancers IGFBP isoforms 2, 3 and 5 have been reported as tumor promoting genes. Immunohistochemical staining revealed expression of IGFBP2 in high grade gliomas and invasive breast cancers. Also, IGFBP2 expression was associated with poor prognosis of glioblastoma patients. IGFBPs 3 and 5 were also found to be prognostic indicators in GBMs. Inhibition of IGFBP-2 by shRNA in glioma and breast cancer cells revealed IGFBP-2 as a survival/growth promoting factor. Administration of IGFBP-2 neutralizing antibodies to breast cancer cells resulted in apoptosis of the cells supporting a growth/survival role for IGFBP2 in breast cancer cells. Expression profiling of genes by microarray analysis of IGFBP-2 positive and negative breast tumors revealed differential gene expression pattern. Microarray analysis of control and IGFBP-2 knock down breast cancer cells revealed down regulation of several interesting genes that are reported to be involved in EMT/invasion/metastasis. Taken together, our data and published literature suggest a tumor promoting role for some IGFBP isoforms.
IS 20: Imaging assessment of therapeutic response in cancer: New ideas in radiology for personalized medicine
Matthew A Lewis
UT Southwestern Medical Center at Dallas, Texas, USA
There is a need for rapid assessment of cancer therapeutics, both experimentally in development and in the clinic. Physicians would like to know within days or hours (rather than weeks) if a given therapy is working in a patient. Optical imaging and ultrasound are low-cost imaging modalities that may be well-suited for this specific task. In this talk, an established ultrasound technique (tissue characterization) and a new optical method (Cerenkov luminescence) will be presented in the context of cancer theragnostic imaging.
IS 21: Systematic approaches in cancer chemo-prevention
Nagi B Kumar
Division of Population Sciences, H. Lee Moffitt Cancer Center & Research Institute, University of South Florida, College of Medicine, USA
Prevention is the ultimate approach to controlling cancer. Prevention efforts require a thorough understanding of the mechanism of carcinogenesis. Knowledge of the molecular basis of carcinogenesis is critical, elucidating signaling, metabolic pathways and defining genetic progression models. New technologies in genomics and proteomics have spurred this field of research. The use of this knowledge to develop pharmacologic agents (including nutrient-derived agents) to reverse or halt the process of carcinogenesis is called Chemoprevention. Cancer Chemoprevention is the prevention of cancer or treatment of identifiable precancers (histopathological or molecular intraepithelial neoplasia). During the past two decades epidemiological and laboratory studies have demonstrated that several nutrient-derived agents to have multiple molecular targets and influence multiple biochemical and molecular cascades that inhibit mutagenesis, proliferation, induce apoptosis and suppress the formation and growth of human cancers. Funded by the National Cancer Institute, National Institute of Health, USA., Dr. Kumar has led a inter-disciplinary collaborative effort and initiated several epidemiological, preclinical, mechanistic and early phase I-II clinical trials, laying the foundation to develop a program to systematically accelerate anticancer agent development and validation at the Moffitt Cancer Center. The presentation will specifically include this experience and describe the approaches used by Dr. Kumar and her team to identifying and prioritizing agents for chemoprevention, target, research design, selection of intermediate and surrogate endpoint biomarkers and molecular targets.
IS 22: A novel natural product for the treatment of prostate cancer
Partha P Banerjee
Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, Washington DC
The etiology of human prostatic carcinoma remains largely undefined. However, it is becoming clear that epigenetic inactivation of various tumor suppressor genes could play pivotal role in the development of various cancers including prostate cancer. One of such tumor suppressor is Ras-association domain family 1 (RASSF1) gene. Epigenetic inactivation of RASSF1A is probably the most frequently methylated gene described thus far in human cancer. Ectopic expression of RASSF1A in cancer cells that lack endogenous RASSF1A transcripts resulted in reduced growth of cancer cells in vitro and in nude mice supporting a role for RASSF1A as a tumor suppressor gene. Since, the restoration of RASSF1A expression in tumor cells impairs their tumorigenicity, factors that restore RASSF1A expression have immense importance in preventing tumor growth. Plant derived bioactive compounds might have enormous benefit in preventing various cancers in human. The incidence of latent (microscopic) prostate cancer is remarkably similar throughout the world, but the rates of clinically significant, aggressive prostate tumors are much lower in Asian countries than in the United States and other Western countries, suggesting that environmental factors, such as diet may underlie the East-West differences in prostate cancer progression. Traditional Asian diets are not only high in starch and fiber, but also rich in many bioactive compounds that have anti-cancer properties. Therefore, plant derived bioactive compounds might have enormous benefit in preventing various cancers in human. In this investigation, we demonstrated that, mahanine, a plant derived carbazole alkaloid, restores epigenitically silenced tumor suppressor gene, RASSF1A expression in both androgen-responsive (LNCaP) and androgen-negative (PC3) prostate cancer cells by inhibiting DNA methyltransferase (DNMT) activity. Mahanine-induced expression of RASSF1A in turn significantly reduces cyclin D1 but not other cyclins. As a result of down-regulation of cyclin D1, cancer cells arrest at G0/G1 phase. To understand the inverse relationship between RASSF1A and cyclin D1, we observed that mahanine treatment down-regulates cyclin D1 and addition of RASSF1A siRNA prevents this inhibition. Inactivation of both Akt and CREB by mahanine and RASSF1A is associated with the down-regulation and translocation of cyclin D1. This study shows that mahanine can reverse an epigenetically silenced gene, RASSF1A in prostate cancer cells by inhibiting DNMT activity that in turn down-regulates a key cell cycle regulator, cyclin D1. Mahanine, therefore, promises an encouraging therapeutic choice for advanced prostatic cancer. (Supported by NIH grant 5R01CA131123).
IS 23: Arsenic in drinking water and cancer risk: A global public health issue
Dean of the School of Public Health, UIC, Chicago, IL
In the past decade, high concentrations of arsenic in drinking water supplies have posed major public health problems in many parts of the world, including India and Bangladesh, with tens of millions of people are at risk. Among other health effects, arsenic exposure has been linked to cancers of the skin, liver, lung and bladder. This problem has been extensively studied in Bangladesh, providing many valuable insights into the extent of the problem and potential interventions to mitigate its impact.
IS 24: CYP3A4 is an epoxygenase that mediates growth of ER+ breast cancer cells, in part, through biosynthesis of (±)-14,15-EET and activation of stat3§
Ranjana Mitra¶, ψ *, Zhijun Guo¶, ψ* , Monica Milani¶,ψ, Clementina MesarosΦ, Mariangellys Rodriguez¶,ψ, Julia Nguyen¶,ψ, Xianghua Luoχψ, Duncan Clarkeͳ, Jatinder Lambaθ, Erin Schuetz∋, David B. Donnerͳ, Narender PuliΩ, John R. FalckΩ, Jorge Capdevilaͳ, Kalpna Gupta¶,ψ, Ian A. BlairΦ and David A. Potter¶,ψ,1
¶ Division of Hematology, Oncology and Transplantation, University of Minnesota and theψ Masonic Cancer Center, University of Minnesota, ΦCenter of Excellence in Environmental Toxicology and Department of Pharmacology, University of Pennsylvania, χDivision of Biostatistics, University of Minnesota School of Public Health, ͳDepartment of Genetics, Cell Biology and Development, θDepartment of Experimental and Clinical Pharmacology, University of Minnesota, ∋Pharmaceutical Sciences, St. Jude Children's Research Hospital, ͳDepartment of Surgery, University of California San Francisco, Ω Department of Biochemistry and Pharmacology, University of Texas Southwestern Medical Center, ͳDepartment of Medicine, Vanderbilt University and Vanderbilt/Ingram Cancer Center
CYP3A4 expression correlates with decreased survival in breast cancer, but its mechanisms in cancer biology are unknown. CYP gene profiling by RNAi silencing demonstrates that CYP3A gene expression is specifically required for growth of breast cancer lines. CYP3A4 silencing blocks the cell cycle at the G2/M checkpoint and induces apoptosis in the MCF7 line, thereby affecting mitotic activity, survival and anchorage-dependent growth. Stat3 (Tyr 705) phosphorylation, recently linked to CYP epoxygenase metabolism of arachidonic acid (AA), is inhibited by CYP3A4 silencing, suggesting that CYP3A4 may synthesize epoxyeicosatrienoic acids (EETs) that drive Stat3 phosphorylation. In vitro, CYP3A4 metabolizes AA to the epoxygenase products (±)-8,9-, (±)-11,12- and (±)-14,15-EET (together ~2 pmol/pmol CYP3A4/minute), in an NADPH-dependent fashion. Furthermore, the (±)-14,15-EET content of growing MCF7 cells is CYP3A4-dependent. In a dose-dependent fashion, the (±)-14,15-EET regioisomer selectively increases breast cancer cell proliferation, in part, by increasing mitotic rates, rescuing cells from CYP3A4 silencing and promoting anchorage-dependent cloning. Silencing of Stat3 blocks breast cancer cell growth and abrogates (±)-14,15-EET-induced proliferation, indicating that (±)-14,15-EET requires Stat3 to induce breast cancer cell growth. Silencing of CYP3A4 reduces nuclear pY705-Stat3, while (±)-14,15-EET restores this signaling process. Breast cancer cell growth is therefore mediated, in part, by CYP3A4-mediated biosynthesis of (±)-14,15-EET that induces nuclear pY705-Stat3. These studies indicate that CYP3A4 is an AA epoxygenase that promotes Stat3-mediated breast cancer cell growth, in part, through (±)-14,15-EET biosynthesis. These results have implications for paracrine signaling in the tumor microenvironment and cancer progression.
IS 25: Cancer chemopreventive activity of a naturally occurring novel coumarin compound
Rana P Singh
School of Life Sciences, Central University of Gujarat, Gandhinagar, Gujarat, India; Cancer Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India
There is a large wealth of plant resources unexplored for its potential medicinal values. Recent studies suggest that chemoprevention could be an alternative strategy for the control and management of cancer. Prostate cancer (PCa) is a very common malignancy in men, especially in aging men, and thus chemopreventive strategy could be a rational approach to control PCa. In this regard, screening as well as study of many naturally occurring agents have shown very good efficacy in laboratory studies. Recently, we found that a coumarin compound, decursin, inhibits the growth of different human PCa cells in culture without any considerable effect on non-neoplastic prostate epithelial cells. Whereas decursinol showed relatively reduced activity against human PCa cells. The cell growth inhibitory effect of decursin was accompanied by the inhibition of cell cycle progression in G1 phase in PCa cells. Decursin modulated the protein expression of cyclin-dependent kinases, cyclins and cyclin-dependent kinase inhibitors. It also affected the interaction of these G1 phase cell cycle proteins. At higher doses, it also induced cell death in cancer cells, which was mostly apoptotic in nature. Our in vivo antitumor efficacy study in DU145 xenograft in nude mice showed that oral treatment with decursin causes strong tumor growth inhibition that became statistically significant only after one week of the treatment and accounted for strong decrease in tumor volume. Decursin treatment did not show any considerable effects on body weight gain and diet consumption. IHC analysis of the tumor showed decreased cell proliferation, increased apoptosis, and decreases in cytokine levels. In DU145 cell culture, decursin inhibited EGF-induced EGFR signaling. In other studies, decursin inhibited HUVEC cell proliferation without any considerable effect on cell death. It also reduced the invasion, migration and tube formation on matrigel, and MMP activity in HUVEC. These studies suggest that decursin has strong anticancer efficacy against advanced human prostate cancer growth both in vitro and in vivo, and, therefore, could be a novel coumarin chemopreventive agent in PCa control and management,
IS 26: Tumor microenvironment: Promise and potential in chemoprevention research
Department of Pharmaceutical Sciences, University of Colorado Denver School of Pharmacy, Aurora, CO 80045,
Tumor microenvironment refers to the dynamic cellular and extra-cellular components surrounding tumor cells at each stage of carcinogenesis. It is now considered an integral part of carcinogenesis that plays critical role in tumor growth, angiogenesis, epithelial to mesenchymal transition (EMT), invasion, migration and metastasis. Beside its vital role in carcinogenesis, tumor microenvironment is also a better drug target because of its relative genetic stability with lesser probability for drug-resistance development. Therefore, we believe that cancer chemopreventive strategies targeting both tumor and tumor microenvironment would be better and effective towards preventing, retarding or reversing the process of carcinogenesis. Our completed and ongoing studies clearly show that cancer chemopreventive agent 'silibinin' targets tumor growth, angiogenesis, EMT, invasion, migration and metastasis. In my presentation, I will elaborate these studies with major focus on the effect of silibinin on tumor microenvironment. Results from these studies are highly encouraging as silibinin targets tumor microenvironment effectively and more importantly, at much lower concentrations compared to its inhibitory effect on cancer cells. Silibinin is already in clinical trials, and based upon our results we recommend that its chemopreventive effectiveness should be verified through its effect on biological end points in both tumor and tumor microenvironment.
IS 27: Issues on digital image processing techniques for oncology
DR Ramesh Babu
Department of Computer Sciences, Dayananda Sagar College of Engineering, Kumaraswamy Layout, Bangalore, India
Cancer has been an immense threat to human life for many years and is expected to become the leading cause of death in the next few decades. In the fight against cancer, the emerging field of Computer Aided Detection and Diagnosis of cancer has attracted significant attention. Digital Image processing plays a key role in the development of CAD techniques for Detection, recognition and analysis of cancer, evaluation of the effectiveness of cancer treatment and prediction of the risk of cancer development. Image processing for oncology has attracted much attention from both the image processing and cancer communities.
IS 28: DNA damage control
Department of Physiology, National University of Singapore, Singapore
Regulation of cell death is critical for cellular homeostasis, and defective apoptosis is a hallmark of cancers. The cellular response to various genotoxic stimuli that cause DNA-damage, as well as to oncogenes, is cell cycle arrest that allows for repair of damaged DNA, or apoptosis that eliminates cells with damaged genomes. A failure of this surveillance response can result in genetic alterations that lead to genome instability and development of cancers. The identification and characterization of genes that play a role in regulating apoptosis is thus critical to understanding the molecular basis of tumorigenesis. Overexpression of the transcription factor Stra13 causes growth arrest and mice lacking the Stra13 develop an autoimmune disorder. Stra13 expression is rapidly induced by many genotoxic stimuli, suggesting that it may be a critical regulator of cellular responses to stress signals. Preliminary data from Stra13-/- mice support this hypothesis. Thymocytes from Stra13-/- mice exhibit defective radiation-induced apoptosis, which is dependent on the tumor suppressor p53. Consistently Stra13-/- cells express lower levels of p53 and its transcriptional targets. Together, these results suggest that Stra13 regulates DNA-damage induced apoptosis in both p53-dependent and independent ways. The molecular circuitry leading to the anti-proliferative responses mediated by Stra13 will be discussed.
IS 29: MicroRNA biomarkers for lung cancer
Roswell Park Cancer Institute
MicroRNAs have emerged as an important class of genes involved in cancer. While their study helps clarify tumor biology, their unique characteristics makes them good potential biomarkers. MicroRNAs are stable in body fluids and in formalin fixed paraffin embedded tissue, simplifying their study as potential biomarkers. The most common methods of high-throughput quantification of microRNAs are RT-PCR arrays, microarrays and deep sequencing. While the replicability of these methods for samples with high RNA content is excellent, this replicability decreases dramatically at low RNA input amounts. In addition, the cross-platform correlation for microRNA quantification is modest at best. Therefore, the selection of an assay platform is application-specific. MicroRNAs have been used to prognosticate early lung cancer. A number of investigators have demonstrated the potential of microRNA profiling to predict patients at a higher risk of recurrence after resection. However, good validation studies are pending. Similarly, early studies have examined the potential of microRNA quantification in serum to diagnose lung cancer. However, this area has yet to overcome methodologic limitations. The most common approach is to use RT-PCR based assay of serum. However, the selection of normalizing microRNAs is a problem in these studies. MicroRNA expression profiling has also been used to distinguish histological sub-types of lung cancer - a clinically important area. Good validation data exists for such an application. Chemosensitivity prediction using microRNA profiling is in its infancy and further developmental work is required to make this a reality. In conclusion, microRNA expression profiling of tissue and body fluids have emerged as an interesting area of study, but has its limitations. Further well designed validation studies are required to judge the utility of microRNA biomarkers in lung cancer.
IS 30: Cancer prevention and therapy through targeting RLIP76
Molecular Biology & Immunology, Director of Cancer Prevention, University of North Texas Health Science Center, Fort Worth, Texas, USA
Targeted depletion of the RALBP1 encoded 76 kDa splice variant, RLIP76, causes marked and sustained regression of human xenografts of lung, colon, prostate, and kidney cancer without toxicity in nude mouse models. We proposed that the remarkable efficacy and broad-spectrum of RLIP76-targeted therapy is because of its glutathione-conjugate (GS-E) transport-activity which is required for clathrin-dependent endocytosis (CDE). We studied RLIP76 mutant proteins, and the functional consequences of their expression into RLIP76 -/- MEFs, and the correlation of CDE, RLIP76 protein expression, and consequences of RLIP76 depletion in a panel of 24 cell lines representing 10 histologies of cancer, and 4 non-malignant cell lines. Results of these studies identify key residues for GS-E binding in RLIP76, establish the requirement of RLIP76-mediated GS-E transport for CDE, and demonstrate a direct correlation between GS-E transport activities with CDE. Depletion of RLIP76 nearly completely blocked down-stream signaling of EGF in a CDE-dependent manner, and Wnt5a signaling in a CDE-independent manner. RLIP76-targeted therapy caused regression of breast and pancreatic xenografts. RLIP76-targeted cancer therapeutics have high specificity and broad-spectrum of activity because of disruption of key cancer-critical signaling mechanisms that are regulated by CDE, suggesting the intriguing possibility that RLIP76 is an anti-apoptotic mechanism required not only for the survival of cancer cells, but also for their very existence. In support of this assertion, well known carcinogens, benzo[a]pyrene (B[a]P), dimethylbenzanthracene (DMBA) and phorbol esters (PMA or TPA) were ineffective in causing neoplasia in RLIP76 -/- mice. PMA-induced skin carcinogenesis in RLIP76 +/+ mouse was suppressed completely by depletion of either PKCα or RLIP76 by siRNA or antisense, that could be restored by topical application of RLIP76 protein in RLIP76 -/- mouse skin. Likewise, B[a]P-induced lung and stomach adenocarcinoma were absent in female and nearly absent in male RLIP76 -/- mice. In RLIP76 -/- mice, p53, P38, and JNK activation did not occur in response to either carcinogen. Our findings demonstrated for the first time that GS-E efflux by RLIP76, the rate limiting step or mercapturic acid metabolism, is also the rate-determining step of clathrin-dependent endocytosis, and a key mechanism necessary for cancer initiation, progression, and metastasis.
IS 31: Prostate cancer prevention by dietary agents
Shivendra V Singh
Department of Pharmacology & Chemical Biology and Urology, and University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA
Epidemiological studies suggest that increased consumption of Allium vegetables (e.g., garlic) may be protective against risk of different cancers, including cancer of the prostate. Anticancer effect of Allium vegetables is attributable to organosulfur compounds, including diallyl trisulfide (DATS). In this presentation, I have summarized the results of our published and unpublished studies documenting chemopreventive and anti-aging effects of DATS in vivo. DATS is highly effective in inhibiting growth of cultured and xenografted human prostate cancer cells. DATS treatment prologs life span of C. elegans. Gavage of DATS delays progression of prostate cancer development in TRAMP mice. DATS causes apoptotic cell death in human prostate cancer cells irrespective of their p53 status or androgen responsiveness and the cell death correlates with c-Jun N-terminal kinase mediated phosphorylation of Bcl-2. These results merit future clinical investigation of DATS for prevention of prostate. These studies were supported by the NCI grant CA113363.
IS 32: 15-lipoxygenase-1 has tumor suppressive properties in colorectal carcinogenesis
Ismail Çimen, Seda Tuncay, Erhan Astarci, Sreeparna Banerjee
Department of Biological Sciences, Middle East Technical University, Ankara, Turkey.
15-lipoxygenase-1(15-LO-1), a member of the inflammatory eicosanoid pathway, oxidatively metabolizes linoleic acid and its expression is repressed in colorectal cancer (CRC). In this study, we investigated the hypothesis that the lack of 15-LO-1 expression in CRC cells may contribute to tumorigenesis. Therefore we have introduced 15-LO-1 in HCT-116 and HT-29 cells that do not have detectable levels of 15-LO-1. Our data indicate that expression of 15-LO-1 significantly decreased cell proliferation and increased apoptosis. In addition, we observed a reduction in adhesion to fibronectin, anchorage independent growth on soft agar, cellular motility and ability to heal a scratch wound and migratory and invasive capacity across Matrigel. 15-LO-1 expression also reduced the expression of MTA-1 protein, a part of the NuRD silencing complex. Mechanistically, the anti-tumorigenic property of 15-LO-1 was by inhibition of the anti-apoptotic inflammatory transcription factor nuclear factor kappa B (NF-κB). Ectopic expression of 15-LO-1 gene in CRC cell lines inhibited the degradation of inhibitor of kappa B (IκBα), decreased nuclear translocation of NF-κB subunits p65 and p50, decreased DNA binding in the nucleus and decreased transcriptional activity of NF-κB. The 15-LO-1 enzymatic product 13(S)-HODE is known to be a PPARgamma (PPARγ) agonist, and NF-κB can be inhibited by PPARγ. We have observed that the inhibition of both early and late stages of NF-κB activation could rescued by the PPARγ antagonist GW9662 indicating that the inhibition was most likely mediated via PPARγ. We therefore propose that 15-LO-1 has tumor suppressive properties and should be exploited for therapeutic benefits.
IS 33: Notch signaling in human cancers: mechanisms and therapeutic challenges
National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore, India
Our laboratory has been interested in the progression of human cervical cancers. We have focussed on identifying the Notch pathway as a key second signal that complements the function of human papillomavirus oncogenes (for review see Maliekal et al., Oncogene, 2008). Our key collaborators have been Apurva Sarin at NCBS and several investigators from the Kidwai Institute of Oncology. More recently, we have been looking at metatatic progression in the context of Notch signaling and the cancer stem cell paradigm and I will review our recent published (Srivatsava et al., British Journal of Cancer, 2010) and upublished observations (Bajaj et al., in preparation). In addition, primarily with Cecil Ross, we have started a education-cum research initiative on hematological malignancies at St. John's Medical College. I will discuss some of the directions we are taking in terms of studying aspects of the Notch pathway in the context of human Chronic Myeloid leukemias.
IS 34: Preliminary study of proteomic analyses of serum from breast cancer patients for possible diagnostic protein markers
Nonglak Yoonim, Sittiruk Roytrakul 1 , Sukkid Yasothornsrikul
Department of Biochemistry, Faculty of Medical Science, Naresuan University, Phitsanulok Thailand, 1 Genome Institute, National Center for Genetic Engineering and Biotechnology, Thailand Science Park, Pathumthani, Thailand.
Breast cancer is the most common form of cancer among women and also is a major public health problem. It is one of the leading causes of cancer-related death in women throughout the world. Early diagnosis is the key step to improve the prognosis. Because of the mechanism of breast cancer in human is very complicated and there are many factors such as biological, physiological and genetics that involved in such complex mechanism. In addition, genetic alterations and modifications in gene expression are found during different steps of tumor progression. These changes are translated at the protein level where quantitative and qualitative modifications are found in tumor compared to normal. This study aimed to identify type and quantities of protein molecules involved in breast cancer using proteomics technology and then apply these results for using as the biomarkers and index that specific for breast cancer. 151 breast cancer sera were separated to 4 groups depend on their stage of breast cancer and 149 normal sera were also separated to 4 groups as a control for each stage of breast cancer group. Serum protein concentration was determined according to Lowry protein assay. An equal amount of serum proteins from each sample within each group was pooled and analyzed by SDS-PAGE. The protein bands were identified by in-gel digestion followed by mass spectrometry. Tryptic peptides were recovered for a subsequent analysis with LC-MS/MS. For proteins quantitation, DeCyderMS Differential Analysis software was used. The DeCyderMS analyzed data were submitted to database search using the Mascot. The results of SDS-PAGE-LC/MS/MS showed two candidate proteins that may be possible for being used as biomarkers for early diagnosis because the quantity of them decrease more than 15% in stage I and II in breast cancer samples compare to normal controls. They are unnamed protein product (gi/34532201) that has the most similar protein sequences to Nucleoporin 210kDa-like (NUP210L) and alpha 1 type XIII collagen isoform 8 (gi/22027587). In addition, we also confirmed the results of pooled sera by using individual sera of 14 breast cancer patients stage I. The results showed that the quantity of unnamed protein product (gi/34532201) of 12 breast cancer sera (12/14 = 85.7%) decrease or disappear compare to normal controls; therefore, this protein may be using as a biomarker for early detection of breast cancer. Our data showed that proteomics can be applied in identification of serum proteins or biomarkers useful for the early detection of breast cancer. The unnamed protein product (gi/34532201) candidate proteins represented in these breast cancer sera may be using as new biomarkers for early detection of breast cancer. However, further study for validation of the potential of these candidate proteins for diagnosis and early detection of breast cancer is needed.
IS 35: Multivalent bifunctional chelator scaffold design for PET/SPECT imaging of cancer
Department of Radiology, UT Southwestern Medical Center, Dallas, Texas, USA
Multimerization of a targeting molecule on one scaffold has been recognized as an efficient way to improve cell-specific binding affinity. As such, various approaches have been reported to exploit multivalent scaffolds for the construction of molecular imaging probes. However, the chemistries are often complicated and become even more so when a bifunctional chelator (BFC) must be conjugated to introduce a metal radionuclide for Positron Emission Tomography (PET) or Single Photon Emission Computed Tomography (SPECT). In this talk, a unique approach will be presented and discussed for the construction of simplified but potentially versatile multivalent scaffolds for multimeric presentation of targeting molecules. This type of multivalent scaffolds features a chelator that forms a stable and neutral complex with a radiometal and multiple functional groups for the anchoring of targeting molecules.
IS 36: Virtual prototype for screening heat shock protein 90 directed cancer chemotherapeutics
Shireen Vali, Rani Pallavi 1 , Utpal Tatu
Cellworks Group, ITPL, Bangalore India, 1 Department of Biochemistry, Indian Insitute of Science, Bangalore, India
With the advent of shift towards molecularly targeted therapies for cancer,the understanding of pathways is imperative for successful translation of research to clinical deployment. The inherent complexity of the maze of pathways due to cross-talk, positive and negative feed-forward and feed-back loops, compounds the challenges. The added dimension of differing tumor signatures across patient groups causes impact on responder/non-responder groups. Through constructing the virtual map for all the circuitries of one of the key target, Hsp90, in cancer, we have developed a model which can be used to examine the efficacy of its inhibitors in context dependent manner. The dynamicity of this model allows us to incorporate the clinical differences between the patients with respect to Hsp90 activity and expression and activities of its client proteins into the map and can be used to understand the efficacy of chemotherapeutic drugs. Cancer being a disease of multiple phenotypes mandates that the therapy analysis per patient group be across a wide set of bio-markers across all the relevant phenotypes. The approach based on creating a functional proteomics derived virtual co-culture of cell systems with comprehensive coverage of pathways across all phenotypes, allows screening different experiments across different environments. This approach addresses the key translational research gaps which is linking diagnostic determination of tumor signature to appropriate therapy.
IS 37: Clinical aspects and advances in the treatment of advanced head and neck squamous cell carcinoma
Victoria M Villaflor
University of Chicago medical center, 5841 S Maryland Ave. MC 2115, Chicago Illinois, USA 60637
There are approximately 500,000 cases of head and neck carcinoma annually worldwide and 47,000 cases in the United States of which 95% are squamous cell carcinomas. Worldwide men are affected more often than women and mortality is greater in men. Most often head and neck cancers are associated with tobacco and alcohol use. In India, betel nut and quid use play an important role in the development of these tumors, especially of the oral cavity. Additionally, viral infection (particularly HPV), occupational exposure, dietary factors, genetic factors and previous radiation may play a role. Clinical presentation varies depending on the primary site affected. Patients may present with oral ulcers/mass, pain, dental changes, epistaxis, nasal obstruction, headache, otalgia, laryngitis, dysphagia, chronic cough, hemoptysis, neck mass and/or stridor. Once a head and neck malignancy is suspected, physical examination should include oral cavity visual assessment, palpation of oral cavity and neck, an oral mirror examination with subsequent fiberoptic endoscopy. Once a mass is identified a biopsy should be performed. Squamous cell carcinoma should prompt a panendoscopy and CT scan of the head (orbits), neck, chest and abdomen to adequately stage the patient. The majority of patients present with late stage disease. Treatment for the patient with early stage (I/II) disease is typically single modality using surgery or radiotherapy. Patients who have locally advanced disease are treated with multimodality treatment based on the site and extent of disease. Surgical resection is not always an option for patients with locally advanced tumors due to anatomical location, inability to achieve wide margins, comorbidities or due to loss of function. Resectability is subjective, variable and surgeon dependant. In the past, treatment was limited to surgical resection and/or radiotherapy. Chemotherapy has demonstrated improved locoregional control and overall survival when added to radiotherapy. Additionally, organ preservation may occur in patients who undergo concurrent chemoradiotherapy. Toxicities are increased due to enhanced radiation delivery but also due to the effects of the chemotherapy used. The benefits of chemoradiotherapy have been established by several randomized trials and subsequent meta-analysis. Few studies have directly compared regimens and the meta-analysis depicts variable chemotherapy regimens and radiation schedules however, the locoregional control and survival improvements with chemoradiotherapy are consistent. Currently, in the United States, chemoradiotherapy is standard of care. The regimens amongst institutions vary greatly with most institutions using cisplatin 100 mg/m2 given on days 1, 22, and 43 with radiotherapy. The recent RTOG 0129 demonstrates that by using an accelerated concomitant boost with 2 cycles of cisplatin days 1 and 22 are equivalent to the current once daily radiation with cisplatin every three weeks. Induction chemotherapy has recently resurged and may have a role in the treatment of advanced head and neck cancer. Recently, taxanes added to platinum and 5-fluorouracil have shown improved response rate and survival compared to platinum and 5-fluorouracil alone. Ongoing trials are aimed at evaluating the role of induction chemotherapy in the setting of highly active concurrent chemoradiotherapy regimens in patients with hi-risk squamous cell cancers of the head and neck. (Decide, Paradigm and Italian trials). The epidermal growth factor receptor (EGFR) is overexpressed and abnormally activated in head and neck squamous cell cancers. The use of a monoclonal antibody directed against EGFR evaluated in head and neck squamous cell cancers appears to have activity both as a radiosensitizing agent and as an antineoplastic drug. Cetuximab has improved locoregional control and overall survival when compared to radiation alone. Currently, this is not considered standard of care as it has not been compared to more conventional chemoradiotherapy. However, with the low rate of toxicities observed with this regimen it should be considered in medically unfit and poor performance status patients where the risks for addition of chemotherapy outweigh the benefits. Studies evaluating cetuximab added to chemoradiotherapy are ongoing. Research directed at targeted therapies is of interest in this population.
IS 38: Screening for oral cancers: The role of auto-fluorescence visualization
Departments of Dentistry and Head & Neck Surgery, Roswell Park Cancer Institute, Buffalo, NY
Oral cancer accounts for more than 3% of all cancers identified globally every year, making it the 6th most common cancer worldwide. In India, oral cancers account for 30% of all cancers. It ranks number one among men and number three among women among all diagnosed cancers in India. Globally, over two-thirds of the oral cancer patients are diagnosed with an advanced disease resulting in a poor overall 5-year survival of 50%. Even specialized health care professionals often find it difficult to clinically differentiate between benign and early cancerous changes in the oral cavity. Therefore, it is essential to develop efficient visual-aids that can detect early cancerous changes. Recently, there has been considerable interest in using autofluorescence visualization (AFV) as a potential screening tool for oral cancers. At Roswell Park Cancer Institute (RPCI), we undertook a prospective study to evaluate AFV as a surveillance and detection tool for oral premalignant lesions and cancers. We utilized autofluorescence blue excitation centered at a wave length of 405 nm to visualize oral lesions. A total of 124 high-risk patients were screened and 441 biopsies were obtained. On baseline histopathological evaluation, 40% were low grade lesions - LGL (hyperplasia, parakeratosis with atypia and mild dysplasia), 18% were high grade lesions - HGL (moderate dysplasia, severe dysplasia and CIS) and 9% were oral cancers (OC). Of these, 35% of LGL, 32% of HGL and 15% of OC were identified only on AFV and were not visible on conventional visual examination. We also evaluated 115 leukoplakias in 52 high risk patients. The relative sensitivity of detection of dysplastic changes in leukoplakia was 20% greater with AFV compared to conventional oral exam. Addition of AFV to clinical evaluation can improve the ability to identify the subset of leukoplakias at a higher risk of malignant transformation. Overall, AFV is a promising adjunct tool for detecting pre-malignant and malignant changes in the oral cavity of high risk patients. Cost-effectiveness studies and efficacy studies in low-risk population are needed to confirm its place in diagnostic armamentarium.
IS 39: Imaging hypoxia in tumors: new probes and techniques
Vikram D Kodibagkar
Department of Radiology, UT Southwestern Medical Center, Dallas, Texas, USA.
Measuring absolute tissue oxygen tension (pO 2 ) non-invasively may provide significant information and insight into the functional mechanisms of tissues and in clinical prognosis of diseases. Tumor oxygenation plays a vital role in determining the efficacy of radiotherapy, photodynamic therapy and some chemotherapies, and imaging hypoxic regions could be clinically important. Although it has been hoped that modulation of tumor oxygenation could be applied to enhance therapeutic efficacy, up to now effective oximetry methods have been lacking in the clinic. This talk focuses on imaging techniques to asses hypoxia in tumors. Two novel magnetic resonance (MR) approaches developed in our lab for assessment of hypoxia are also described: 1) a quantitative 1 H MR oximetry method PISTOL and related developments and 2) a novel Gd-based agent for targeted imaging of hypoxic regions in tumors.
IS 40: When natural products meet cellular target: Targeting Skp2 by flavokawains in chemoprevention of prostate carcinogenesis
Departments of Urology and Pharmaceutical Sciences and Chao Family Comprehensive Cancer Center, University of California, Irvine, Orange, CA 92868, USA
Natural products or secondary metabolites have co-evolved with biological macromolecules, such as nucleic acids, proteins and others for over three billion years. Consequently, natural products or secondary metabolites may interact with specific nucleic acid or protein targets within cells and thus offer a privileged starting point in the search for highly specific and potent modulators of cellular function. During recent years, my laboratory has focused on identification of novel natural products that can target cancer-specific signaling pathways and/or molecular targets, leading to selective modulation of cancer cell functions. In doing this, we can provide novel cancer preventive or therapeutic candidate agents for future individualized prevention and /or therapy. S-phase kinase-associated protein 2 (Skp2), a putative E2F1/ the retinoblastoma protein (pRb) down-stream target gene, has been found to be overexpressed in the majority of examined primary prostate cancer (PCa) specimens (over 89%). In addition, recent studies reported that blockade of Skp2 function abolished tumorigenesis in Pten, ARF and pRb knockout mice. In this study, we found that flavokawains, novel chalcones from the kava plant, selectively inhibited the growth of pRb deficient mouse fibroblast and epithelial cells. We demonstrated that flavokawains treatment resulted in Skp2 degradation and accumulation of Skp2 substrates p27 and FOX3a/Bim, leading to induction of apoptosis and cell growth inhibition in PCa cell lines. Moreover, we showed that transfection of a dominant-negative Cullin 1 to interfere with Cullin1 neddylation rescued the flavokawains induced Skp2 degradation. In an in vitro assay, we further showed that flavokawains directly inhibited Cullin neddylation. These results suggested that flavokawains target neddylation for proteasome and ubiquitin mediated SKP2 degradation. In in vivo experiments, TRAMP mice were fed with flavokawain A supplemented food. The number of mouse prostate intraepithelial neoplasia was significantly decreased and the average genitourinary weights of flavokawain A treated mice were significantly reduced compared with control food fed mice (1.45±0.88 gram vs.2.22±0.77 gram, p<0.05, Mann-Whitney U and Kolmogorov-Smirnov test). No toxicities of flavokawain A treatments were observed. Taken together, these studies suggested that flavokawains or their analogues deserve further studies as novel Skp2 targeting agents for prevention and treatment of PCa patients with loss of Rb function or Skp2 overexpression.
IS 41: Palm gamma-t3 as an effective agent in targeting prostate cancer stem cell-like population
Yee Leng Yap, Wei Ney Yap, Ming-Tat Ling 1 , Yong-Chuan Wong
Davos Life Science Pte. Ltd., Cancer Research Laboratory, 16 Tuas South Street 5, Singapore 637795, 1 Australian Prostate Cancer Research Centre-Queensland & Institute of Health and Biomedical Innovation, Queensland University of Technology, Qld 4120, Australia.
Emerging evidences support that prostate cancer is originated from a rare sub-population of cells, namely prostate cancer stem cells (CSCs). Conventional therapies for prostate cancer are believed to target mainly the majority of differentiated tumor cells but spare CSCs, which may account for the subsequent disease relapse after the treatment. Therefore, successful elimination of CSCs may be an effective strategy to archive complete remission from this disease. Tocotrienols (T3) are vitamin-E constituents that naturally found in palm oil and have been shown to have anticancer effect against a wide-range of human cancers. Recently, we have reported that gamma-T3, one of the four T3 isomers, was the most potent form of T3 against prostate cancer. Meanwhile, we found that gamma-T3 treatment not only inhibits prostate cancer cell invasion, but also sensitizes the cells to Docetaxel-induced apoptosis, suggesting that gamma-T3 may be an effective therapeutic agent against advanced stage prostate cancer. Here, we demonstrate for the first time that gamma-T3 can down-regulate the expression of prostate cancer stem cell markers (CD133/CD44) in androgen independent (AI) prostate cancer cell lines (PC-3 & DU145), as evident from Western blotting and flow cytometry analysis. Meanwhile, spheroid formation ability of the prostate cancer cells was significantly hampered by gamma-T3 treatment. More importantly, pre-treatment of PC-3 cells with gamma-T3 was found to interfere with the tumor initiation ability of the cells. Our data suggest that gamma-T3 may be an effective agent in targeting prostate CSCs, which may account for its anti-cancer and chemosensitizing effects reported in previous studies.
IS 42: Role of RLIP76 in mechanisms of carcinogenesis
Yogesh C Awasthi
University of North Texas Health Science Center, Fort Worth, Texas, USA
Studies by our group over the past decade have established that RLIP76 (syn. RALBP1), a previously characterized GTPase activating protein also involved in clathrin pit coated endocytosis is the major transporter of the glutathione-conjugates of endogenous and exogenous electrophilic compounds. It has been established by us and other investigators that the electrophilic end-product of lipid peroxidation, 4hydroxynonenal (HNE), is a major second messenger involved in the regulation of cellular homeostasis. Our studies suggest that HNE levels above its basal constitutive intracellular concentrations promote apoptosis while those below the basal levels promote proliferation. RLIP76 by transporting the GHS-conjugate of HNE (GS-HNE) regulates the intracellular concentration of HNE. Most of the cancer cells studied by our group so far over express RLIP76, suggesting that these cells acquire resistance to apoptosis by lowering the intracellular concentrations of HNE through RLIP76-mediated ATP-dependent transport of GS-HNE. Inhibition of RLIP76 transport function or ablation of RLIP76 expression results in apoptosis of most of the cancer cells studied so far. Our results showing that the xenografts of lung, kidney, pancreas, and colon cancer cell lines in nude mice are completely regressed by anti-RLIP76 antibodies or RLIP76 siRNA, also suggest a central role of RLIP76 in progression of cancer. Furthermore, our studies suggest that the existence of RLIP76 in cells seems to be essential for initiation and progression of chemical carcinogenesis. This is indicated by the results of our studies showing that phorbol ester and DMB-induced chemical carcinogenesis is absent in RLIP76 knockout mice. These studies suggesting a central role of RLIP76 in the initiation and progression of cancer will be discussed. (NIH grants ES012127 and EY04396).