Evaluation of Cytotoxic Effects of Dichloromethane Extract of Guduchi (Tinospora cordifolia Miers ex Hook F & THOMS) on Cultured HeLa Cells

Extracts of Tinospora cordifolia (TCE) have been shown to possess anti-tumor properties, but the mechanism of the anti-tumor function of TCE is poorly understood. This investigation elucidates the possible mechanism underlying the cytotoxic effects of dichlormethane extracts of TCE, after selecting optimal duration and concentration for treatment. HeLa cells were exposed to various concentrations of TCE, which has resulted in a concentration-dependent decline in the clonogenicity, glutathione-S-transferase (GST) activity and a concentration-dependent increase in lipid peroxidation (TBARS) with a peak at 4 h and lactate dehydrogenase (LDH) release with a peak at 2 h. Our results suggest that the cytotoxic effect of TCE may be due to lipid peroxidation and release of LDH and decline in GST

The grape fruit flavonone naringin protects mice against doxorubicin-induced cardiotoxicity

Doxorubicin (DOX), an anthracycline drug widely used for the treatment of various cancers, causes a cumulative dose-dependent cardiac toxicity that is characterized by an irreversible cardiomyopathy and congestive heart failure. The cardioprotective effect of 2.5, 5, 7.5 and 10 mg/kg naringin (NIN) was studied in mice treated with 15 mg/kg DOX. The animals were killed 30 h after DOX treatment. The latter induced acute cardiotoxicity indicated by a significant elevation in glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT), creatine kinase (CK-MB) and lactate dehydrogenase (LDH) in mice serum. Treatment of mice with NIN before DOX administration significantly reduced serum levels of GPT, GOT, CK-MB and LDH indicating that NIN protected against the DOX-induced cardiotoxicity. DOX induced a significant increase in the 8-OHdG DNA adducts and the PARP activity in the heart and liver of mice, whereas NIN treatment of mice before DOX administration significantly reduced 8-OHdG DNA adducts and PARP activity in the heart and liver. Similarly, NIN inhibited the DOX-induced decline in the glutathione concentration, catalase and superoxide dismutase activities and abated DOX-induced lipid peroxidation in the heart and liver of mice. Intraperitoneal administration of 1.25 mg/kg DOX significantly elevated survival of Ehrlich ascites carcinoma (EAC) bearing mice, whereas the combination of 10 mg/kg NIN with DOX did not alter the tumor cell growth, median survival time or average survival time of tumor bearing mice when compared to DOX treatment alone, indicating that NIN does not interfere with the antineoplastic activity of DOX. Our study demonstrates that naringin reduced the doxorubicin-induced cardiotoxicity, without affecting its antineoplastic activity, which may be due to reduction in the DOX-induced 8-OHdG DNA adducts and PARP activity, increase in the antioxidant enzymes and alleviation of lipid peroxidation by naringin

The Role of the Focal Adhesion Protein PINCH1 for the Radiosensitivity of Adhesion and Suspension Cell Cultures

Focal adhesion (FA) signaling mediated by adhesion to extracellular matrix and growth factor receptors contributes to the regulation of the cellular stress response to external stimuli. Critical to focal adhesion assembly and signaling is the adapter protein PINCH1. To evaluate whether the prosurvival function of PINCH1 in radiation cell survival depends on cell adhesion, we examined PINCH1fl/fl and PINCH1−/− mouse embryonic fibroblasts and human cancer cell lines. Here, we found that the enhanced cellular radiosensitivity mediated by PINCH1 depletion observed under adhesion conditions is conserved when cells are irradiated under suspension conditions. This unsuspected finding could not be explained by the observed modification of adhesion and growth factor associated signaling involving FAK, Paxillin, p130CAS, Src, AKT, GSK3β and ERK1/2 under suspension and serum withdrawal relative to adhesion conditions with serum. Our data suggest that the adapter protein PINCH1 critically participates in the regulation of the cellular radiosensitivity of normal and malignant cells similarly under adhesion and suspension conditions

Evaluation of Cytotoxic Effects of Dichloromethane Extract of Guduchi (Tinospora cordifolia Miers ex Hook F & THOMS) on Cultured HeLa Cells

Extracts of Tinospora cordifolia (TCE) have been shown to possess anti-tumor properties, but the mechanism of the anti-tumor function of TCE is poorly understood. This investigation elucidates the possible mechanism underlying the cytotoxic effects of dichlormethane extracts of TCE, after selecting optimal duration and concentration for treatment. HeLa cells were exposed to various concentrations of TCE, which has resulted in a concentration-dependent decline in the clonogenicity, glutathione-S-transferase (GST) activity and a concentration-dependent increase in lipid peroxidation (TBARS) with a peak at 4 h and lactate dehydrogenase (LDH) release with a peak at 2 h. Our results suggest that the cytotoxic effect of TCE may be due to lipid peroxidation and release of LDH and decline in GST. Keywords: cytotoxicity -glutathione-S-transferase -Guduchi -HeLa -lactate dehydrogenaselipid peroxidation Introduction Tinospora cordifolia Miers, commonly known as 'Guduchi' in India, contains tinosporine, tinosporide, tinosporaside, cordifolide, cordifol, heptacosanol, clerodane furano diterpene, diterpenoid furanolactone tinosporidine, columbin and b-sitosterol. The aqueous extract of guduchi stem has shown the presence of arabinogalactan that showed immunological activity (1). The methanolic extract of the plant contains phenylpropanoids, norditerpene furan glycosides, diterpene furon glycosides and plytoecdysones (2). The stem is used in dyspepsia, fevers and urinary diseases. The bitter principle present shows antiperiodic, antispasmodic, antiinflammatory and antipyretic properties Guduchi has been reported to treat throat cancer in humans (9). The preliminary studies on the stem extracts of T. cordifolia have shown promising responses in cultured HeLa cells, where various extracts of guduchi at concentrations of 0, 5, 10, 25, 50 and 100 mg ml À1 reduced the cell survival in a dose-dependent manner. Dichloromethane extract is the most promising one as far as cytotoxic effect is concerned (5). These preliminary studies were done to establish the cytotoxic effect of guduchi at higher doses. A polysaccharide present in T. cordifolia inhibit metastases in the lungs of syngeneic C57BL/6 mice, when the drug was administered simultaneously with tumour challenge (10). Singh et al. (11) have reported that an alcoholic extract of T. cordifolia enhanced the differentiation of TAM to dendritic cells, in response to granulocyte/macrophage-colonystimulating factor, interleukin-4 and tumor necrosis factor. Our own earlier studies on mice transplanted with Ehrlich ascites carcinoma have shown that 50 mg kg À1 body weight of dichloromethane extract of guduchi inhibited the proliferation of tumor cells (12). In earlier studies higher concentrations of dichloroethane extracts were evaluated after exposing the cells arbitrarily for 2 h, where no attempt was made to study the effect of treatment time. A maximum cytotoxic effect was reported for dichloromethane extract of guduchi in comparison with the aqueous and methanol extracts. Therefore, Materials and Methods Collection and Extraction of Plant Material The stems of Tinospora cordifolia (Willd.) Miers ex Hook. F. & Thoms. (Family: Menispermaceae) were collected from Manipal, India during the month of March, 2002, shade dried and coarsely powdered with the help of a ball mill. The plant material (20 kg) was exhaustively extracted with 5 l each of petroleum ether (60-80 C), chloroform and dichloromethane, respectively, using a Soxhlet continuous extraction apparatus for 1 week. The final dichloromethane extracts (henceforth TCE) were concentrated in vacuo and dried under reduced pressure. An approximate yield of 1.2% w/w was obtained. TCE and doxorubicin hydrochloride (DOX) were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 5 mg ml À1 and diluted in sterile MEM in such a way so as to obtain required concentrations. All drug solutions were prepared afresh immediately before use. Cell Line and Culture Methods HeLa S3 cells procured from National Centre for Cell Science, Pune, India, were used throughout the study. The HeLa S3 cells have a doubling time of 20 ± 2 h. The cells were routinely grown in the 25 cm 2 culture flasks (Techno Plastic Products, Trasadingën, Switzerland) with loosened caps containing Eagle's minimum essential medium (MEM) supplemented with 10 percent fetal calf serum, 1 percent l-glutamine and 50 mg ml À1 gentamicin sulfate at 37 C in an atmosphere of 5 percent CO 2 in humidified air in a CO 2 incubator (NuAir, Plymouth, USA). The whole cell growth experiments were done on already established monolayers of HeLa cells. A fixed number (5 · 10 5 ) of exponentially growing cells were seeded into several culture flasks (Techno Plastic Products, Trasadingën, Switzerland) and were allowed to reach plateau phase. Selection of Optimum Duration The optimum duration for drug exposure was evaluated by Pratt and Willis test (13) in HeLa cells. Briefly, 1 · 10 5 cells were seeded in 25 cm 2 culture dishes (Cellstar, Greiner, Germany). They were allowed to grow for 24 h. An aliquot of 8 mg ml À1 of TCE was added and after 0, 1, 2, 4, 6, 8, 12, 18 or 24 h, the TCE-containing medium was replaced with a fresh drug-free medium. After 72 h of initiating of cultures, the cells were harvested and counted using a hemocytometer (American Optics, USA) under an inverted microscope (Labovert microscope, Ernst, Leitz GmbH, Wetzlar, Germany). The viability of cells was determined using Trypan blue dye-exclusion test. The results obtained from Pratt and Willis assay (13) were confirmed by clonogenic assay Effect of Various Concentrations of TCE The plateau phase cell cultures were divided into the following groups according to the treatment: DMSO group. The cells of this group were treated with 2 ml ml À1 of DMSO (negative control). TCE group. The cell cultures of this group were exposed to 0, 1, 2, 4, 5, 6 or 8 mg ml À1 of TCE. The TCE-containing medium was removed from each culture flask of each group after 4 h and the cells were washed twice with sterile PBS. The cells from each group of flasks were dislodged by trypsin EDTA treatment and divided into two parts and the following assays were carried out. Cytotoxicity of various treatments was measured by Pratt and Wills test (13) as described earlier, except that HeLa cells were treated with 0, 1, 2, 4, 5, 6 or 8 mg ml À1 TCE for 4 h. Results obtained from Pratt and Willis assay were confirmed by clonogenic assay (14) as described above, except that HeLa cells were treated with 0, 1, 2, 4, 5, 6 or 8 mg ml À1 TCE for 4 h. Assay of Enzyme Activity A separate experiment was carried out to examine the effect of 0, 1, 2, 4, 6 or 8 mg ml À1 TCE on enzyme activities in cell homogenates [lipid peroxidation and glutathione-S-transferase (GST)] or medium (lactate dehydrogenase, LDH) at different post-TCE treatment times. The grouping and other conditions were essentially similar to those as described above, except that 10 mg ml À1 DOX was used as a positive control. The enzyme contents released in the medium (LDH) or cell homogenates of all groups were determined at 0, 0.5, 1, 2, 4, 8 or 12 h post-treatment. The absorbance was recorded for all assays using a UV-visible double beam spectrophotometer (UV-260, Shimadzu Corp., Tokyo, Japan). Lipid peroxidation (TBARS) was measured by the method of Buege and Aust (15). Briefly, the cell homogenate was mixed with TCA-TBA-HCl and heated for 15 min in a boiling water bath. After centrifugation the absorbance was recorded at 535 nm. Lipid peroxidation in the samples was determined against the standard curve of MDA (Malondialdehyde) and expressed as TBARS, U mg À1 protein. The activity of LDH 268 Evaluation of cytotoxic effects of dichloromethane extract of Guduchi was estimated at 0, 0.5, 1, 2, 4, 8 or 12 h post-drug treatment in the culture medium of all three groups simultaneously. The estimation of LDH release in the culture medium was carried out by the method described by Decker and Lohman-Matthes (16) with minor modifications. The whole medium from each cell culture of each group was removed and collected separately immediately after TCE treatment and was considered 0 h after treatment. The cells were fed with a fresh 5 ml medium and the above procedure (removal of media) subsequently repeated at 0.5, 1, 2, 4, 8 and 12 h. Briefly, the tubes containing media were centrifuged and 50 ml of the medium was transferred to the individual tubes containing Tris-EDTA-NADH buffer followed by 10 min incubation at 37 C and the addition of pyruvate solution. The absorbance was read at 339 nm and the data expressed as units per litre (U l À1 ). The cytosolic GST activity was determined spectrophotometrically at 37 C according to the procedure of Habig et al. (17). Briefly, the reaction mixture (2.7 ml of 100 mM phosphate buffer (pH 6.5) and 0.1 ml of 30 mM CDNB) was preincubated at 37 C for consecutive 5 min, and the reaction initiated by adding 0.1 ml of supernatant and the absorbance recorded for 5 min at 340 nm. Reaction mixture without the enzyme was used as a blank. The GST activity is expressed as U mg À1 protein. Statistical Analysis The statistical analyses were performed using GraphPad Prism 2.01 statistical software (GraphPad Software, San Diego, CA, USA). The significance among all groups was determined by one-way ANOVA and Bonferroni's post hoc test applied for multiple comparisons. All the investigations were carried out from the same stock of cells concurrently. The whole cell growth experiments were done on already established monolayers of HeLa cells. The experiments were repeated for confirmation of results that are the average of five individual experiments. The test of homogeneity was applied to determine variation among each experiment. The data of each experiment did not differ significantly from one another and hence, all the values have been combined and means calculated. A P-value < 0.05 was considered statistically significant. Results The results are expressed as percent viability for Pratt and Willis assay and surviving fraction (SF) for clonogenic assay in Selection of Optimum Duration DMSO (negative control) treatment did not alter the spontaneous viability of HeLa cells significantly with time Various Concentrations of TCE Cause a Decline in Viability The spontaneous viability of HeLa cells remained unaltered after treatment of DMSO, the negative control TCE (Dichloromethane Extracts) Affect Enzyme Activity DMSO treatment (negative control) did not alter the spontaneous levels of TBARS, LDH and GST significantly, with assay time. Treatment of HeLa cells with 0, 1, 2, 4, 6 or 8 mg ml À1 TCE, showed a time-dependent elevation in the lipid peroxidation for all post-treatment times. The TBARS reached a peak at 4 h post-treatment and plateaued thereafter. The concentration of TBARS was 1.2-fold greater than that of DOX treatment (positive control), whereas it was 2.8-fold higher when compared with DMSO treatment, negative control Discussion The cytotoxic effect of TCE increased in a dose-dependent manner in HeLa cells, and an 80 percent decline in SF was observed for 4 h treatment duration. Therefore, further studies Evaluation of cytotoxic effects of dichloromethane extract of Guduchi were carried out using this time of TCE exposure. The reports on the use of various treatment time of TCE are unavailable. Earlier, different extracts of guduchi were found to be cytotoxic and cytoxicity increased in a concentration-dependent manner. The greatest cytotoxic effect was observed for dichloromethane extract followed by the methanol extract of Tinospora, whereas aqueous extract was least toxic among all the extracts of Tinospora evaluated. Further, in earlier studies the concentrations used were higher than that used in the present study and no attempts were made to examine the effect of treatment duration on the cytotoxicity after treatment (5). Reports on the cytotoxic effects of Tinospora, except our own are unavailable, however, alcoholic extract of Tinospora has been reported to be cytotoxic in a transplantable mouse tumor (11). The exact mechanism of cytotoxic effect of guduchi is not known. Increased lipid peroxidation, LDH release accompanied by a decline in GST concentration by guduchi are some of the important events leading to cell death. Lipid peroxidation is an important event related to cell death and has been reported to cause severe impairment of membrane function through increased membrane permeability and membrane protein oxidation and eventually cell death by damaging the cellular DNA The cytotoxic action of guduchi could be attributed to the presence of alkaloids, diterpenoid lactones, glycosides, steroids, sesquiterpenoid, phenolics, aliphatic compounds or polysaccharides. Alkaloids like berberine, palmatine, tembetarine, choline, tinosporin, isocolumbin, palmatine, tetrahydropalmatine and magnoflorine have been isolated from the non-polar fraction of extracts of stem and roots of T. cordifolia The present study demonstrates that TCE exerted cytotoxic effect in a concentration-dependent manner and the cytotoxic effect could be due to DNA damage, inhibition of topoisomerase II, increased TBARS and LDH accompanied by a decrease in GST

Voreloxin Is an Anticancer Quinolone Derivative that Intercalates DNA and Poisons Topoisomerase II

Topoisomerase II is critical for DNA replication, transcription and chromosome segregation and is a well validated target of anti-neoplastic drugs including the anthracyclines and epipodophyllotoxins. However, these drugs are limited by common tumor resistance mechanisms and side-effect profiles. Novel topoisomerase II-targeting agents may benefit patients who prove resistant to currently available topoisomerase II-targeting drugs or encounter unacceptable toxicities. Voreloxin is an anticancer quinolone derivative, a chemical scaffold not used previously for cancer treatment. Voreloxin is completing Phase 2 clinical trials in acute myeloid leukemia and platinum-resistant ovarian cancer. This study defined voreloxin's anticancer mechanism of action as a critical component of rational clinical development informed by translational research.Biochemical and cell-based studies established that voreloxin intercalates DNA and poisons topoisomerase II, causing DNA double-strand breaks, G2 arrest, and apoptosis. Voreloxin is differentiated both structurally and mechanistically from other topoisomerase II poisons currently in use as chemotherapeutics. In cell-based studies, voreloxin poisoned topoisomerase II and caused dose-dependent, site-selective DNA fragmentation analogous to that of quinolone antibacterials in prokaryotes; in contrast etoposide, the nonintercalating epipodophyllotoxin topoisomerase II poison, caused extensive DNA fragmentation. Etoposide's activity was highly dependent on topoisomerase II while voreloxin and the intercalating anthracycline topoisomerase II poison, doxorubicin, had comparable dependence on this enzyme for inducing G2 arrest. Mechanistic interrogation with voreloxin analogs revealed that intercalation is required for voreloxin's activity; a nonintercalating analog did not inhibit proliferation or induce G2 arrest, while an analog with enhanced intercalation was 9.5-fold more potent.As a first-in-class anticancer quinolone derivative, voreloxin is a toposiomerase II-targeting agent with a unique mechanistic signature. A detailed understanding of voreloxin's molecular mechanism, in combination with its evolving clinical profile, may advance our understanding of structure-activity relationships to develop safer and more effective topoisomerase II-targeted therapies for the treatment of cancer

Efficacy of EGFR Inhibition Is Modulated by Model, Sex, Genetic Background and Diet: Implications for Preclinical Cancer Prevention and Therapy Trials

Molecule-targeted therapies are being widely developed and deployed, but they are frequently less effective in clinical trials than predicted based upon preclinical studies. Frequently, only a single model or genetic background is utilized using diets that are not relevant to that consumed by most cancer patients, which may contribute to the lack of predictability of many preclinical therapeutic studies. Inhibition of epidermal growth factor receptor (EGFR) in colorectal cancer was used to investigate potential causes for low predictive values of many preclinical studies. The efficacy of the small molecule EGFR inhibitor AG1478 was evaluated using two mouse models, ApcMin/+ and azoxymethane (AOM), both sexes on three genetic backgrounds, C57BL/6J (B6) and A/J (A) inbred strains and AB6F1 hybrids, and two diets, standard chow (STD) or Western-style diet (WD). AG1478 has significant anti-tumor activity in the B6-ApcMin/+ model with STD but only moderately on the WD and in the AOM model on an A background with a WD but not STD. On the F1 hybrid background AG1478 is effective in the ApcMin/+ model with either STD or WD, but has only moderate efficacy in the AOM model with either diet. Sex differences were also observed. Unexpectedly, the level of liver EGFR phosphorylation inhibition by AG1478 was not positively correlated with inhibition of tumor growth in the AOM model. Model-dependent interactions between genetic background and diet can dramatically impact preclinical results, and indicate that low predictive values of preclinical studies can be attributed to study designs that do not account for the heterogeneous patient population or the diets they consume. Better-designed preclinical studies should lead to more accurate predictions of therapeutic response in the clinic

Modulation of Hydrogen Peroxide Production in Cellular Systems by Low Level Magnetic Fields

Increased generation of reactive oxygen species (ROS) and an altered redox status have long been observed in cancer cells, suggesting that ROS might be involved in the development of these cells. However, recent studies suggest that inducing an excess of ROS in cancer cells can be exploited for therapeutic benefits. Cancer cells in advanced stage tumors frequently exhibit multiple genetic alterations and high oxidative stress, suggesting that it might be possible to preferentially modulate the development of these cells by controlling their ROS production. Low levels of ROS are also important for the development and survival of normal cells. In this manuscript, we present data on the influence of the suppression of the Earth's magnetic field (low level magnetic fields or LLF) which magnitudes range from 0.2 µT to 2 µT on the modulation of hydrogen peroxide (H2O2) in human fibrosarcoma cancer cell line HT1080, pancreatic AsPC-1 cancer cell line, and bovine pulmonary artery endothelial cells (PAEC) exposed to geomagnetic field (control; 45 µT–60 µT). Reduction of the Earth's magnetic field suppressed H2O2 production in cancer cells and PAEC. The addition of catalase and superoxide dismutase (SOD) mimetic MnTBAP inhibited the magnetic field effect. Modulating ROS production by magnetic fields may open new venues of biomedical research and therapeutic strategies

Overexpression of Inosine 5′-Monophosphate Dehydrogenase Type II Mediates Chemoresistance to Human Osteosarcoma Cells

overexpression in osteosarcoma patients with poor response to chemotherapy. The aim of this study was to provide evidence for direct involvement of IMPDH2 in the development of chemoresistance..IMPDH2 is directly involved in the development of chemoresistance in osteosarcoma cells, suggesting that targeting of IMPDH2 by RNAi or more effective pharmacological inhibitors in combination with chemotherapy might be a promising means of overcoming chemoresistance in osteosarcomas with high IMPDH2 expression

In situ Biological Dose Mapping Estimates the Radiation Burden Delivered to ‘Spared’ Tissue between Synchrotron X-Ray Microbeam Radiotherapy Tracks

Microbeam radiation therapy (MRT) using high doses of synchrotron X-rays can destroy tumours in animal models whilst causing little damage to normal tissues. Determining the spatial distribution of radiation doses delivered during MRT at a microscopic scale is a major challenge. Film and semiconductor dosimetry as well as Monte Carlo methods struggle to provide accurate estimates of dose profiles and peak-to-valley dose ratios at the position of the targeted and traversed tissues whose biological responses determine treatment outcome. The purpose of this study was to utilise γ-H2AX immunostaining as a biodosimetric tool that enables in situ biological dose mapping within an irradiated tissue to provide direct biological evidence for the scale of the radiation burden to ‘spared’ tissue regions between MRT tracks. Γ-H2AX analysis allowed microbeams to be traced and DNA damage foci to be quantified in valleys between beams following MRT treatment of fibroblast cultures and murine skin where foci yields per unit dose were approximately five-fold lower than in fibroblast cultures. Foci levels in cells located in valleys were compared with calibration curves using known broadbeam synchrotron X-ray doses to generate spatial dose profiles and calculate peak-to-valley dose ratios of 30–40 for cell cultures and approximately 60 for murine skin, consistent with the range obtained with conventional dosimetry methods. This biological dose mapping approach could find several applications both in optimising MRT or other radiotherapeutic treatments and in estimating localised doses following accidental radiation exposure using skin punch biopsies

Vitamin A Enhances Antitumor Effect of a Green Tea Polyphenol on Melanoma by Upregulating the Polyphenol Sensing Molecule 67-kDa Laminin Receptor

BACKGROUND: Green tea consumption has been shown to have cancer preventive qualities. Among the constituents of green tea, (-)-Epigallocatechin-3-O-gallate (EGCG) is the most effective at inhibiting carcinogenesis. However, the concentrations of EGCG that are required to elicit the anticancer effects in a variety of cancer cell types are much higher than the peak plasma concentration that occurs after drinking an equivalent of 2-3 cups of green tea. To obtain the anticancer effects of EGCG when consumed at a reasonable concentration in daily life, we investigated the combination effect of EGCG and food ingredient that may enhance the anticancer activity of EGCG on subcutaneous tumor growth in C57BL/6N mice challenged with B16 melanoma cells. METHODOLOGY/PRINCIPAL FINDINGS: All-trans-retinoic acid (ATRA) enhanced the expression of the 67-kDa laminin receptor (67LR) and increased EGCG-induced cell growth inhibition in B16 melanoma cells. The cell growth inhibition seen with the combined EGCG and ATRA treatment was abolished by treatment with an anti-67LR antibody. In addition, the combined EGCG and ATRA treatment significantly suppressed the melanoma tumor growth in mice. Expression of 67LR in the tumor increased upon oral administration of ATRA or a combined treatment of EGCG and ATRA treatment. Furthermore, RNAi-mediated silencing of the retinoic acid receptor (RAR) alpha attenuated the ATRA-induced enhancement of 67LR expression in the melanoma cells. An RAR agonist enhanced the expression levels of 67LR and increased EGCG-induced cell growth inhibition. CONCLUSIONS/SIGNIFICANCE: Our findings provide a molecular basis for the combination effect seen with dietary components, and indicate that ATRA may be a beneficial food component for cancer prevention when combined with EGCG