The Bmi-1 polycomb protein antagonizes the (-)-epigallocatechin-3-gallate-dependent suppression of skin cancer cell survival.

The polycomb group (PcG) proteins are epigenetic regulators of gene expression that enhance cell survival. This regulation is achieved via action of two multiprotein PcG complexes--PRC2 (EED) and PRC1 [B-cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1)]. These complexes modulate gene expression by increasing histone methylation and reducing acetylation--leading to a closed chromatin conformation. Activity of these proteins is associated with increased cell proliferation and survival. We show increased expression of key PcG proteins in immortalized keratinocytes and skin cancer cell lines. We examine the role of two key PcG proteins, Bmi-1 and enhancer of zeste homolog 2 (Ezh2), and the impact of the active agent in green tea, (-)-epigallocatechin-3-gallate (EGCG), on the function of these regulators. EGCG treatment of SCC-13 cells reduces Bmi-1 and Ezh2 level and this is associated with reduced cell survival. The reduction in survival is associated with a global reduction in histone H3 lysine 27 trimethylation, a hallmark of PRC2 complex action. This change in PcG protein expression is associated with reduced expression of key proteins that enhance progression through the cell cycle [cyclin-dependent kinase (cdk)1, cdk2, cdk4, cyclin D1, cyclin E, cyclin A and cyclin B1] and increased expression of proteins that inhibit cell cycle progression (p21 and p27). Apoptosis is also enhanced, as evidenced by increased caspase 9, 8 and 3 cleavage and increased poly(adenosine diphosphate ribose) polymerase cleavage. EGCG treatment also increases Bax and suppresses Bcl-xL expression. Vector-mediated enhanced Bmi-1 expression reverses these EGCG-dependent changes. These findings suggest that green tea polyphenols reduce skin tumor cell survival by influencing PcG-mediated epigenetic regulatory mechanisms.

Selenium and selenium-antagonistic elements in nutritional cancer prevention.

Selenium is an essential trace element with antioxidative, antimutagenic, antiviral and anticarcinogenic properties. There is increasing evidence that the dietary selenium intakes are sub-optimal in the populations of many countries and that human cancer mortalities would significantly decline if additional selenium was made available either through supplementation or the fortification of certain foods. An important property of selenium is its interaction with other elements that may be present in foods, the water, the workplace and the environment, e.g. As, Cu, Ni, Co, Cr, Mn, Zn, Cd, Sn, Pb, Hg, Bi, Mo, Ag, Au, etc. The sequestration of elements by selenium represents an efficient natural detoxification mechanism for some of these elements but also results in the physiological inactivation of selenium. Animal experiments confirm that the chronic exposure to low levels of these elements abolishes the cancer-protective effect of selenium. Human cancer is likewise significantly determined by the interactions of selenium with other elements, as evidenced by epidemiological, ecological and case-control studies. Cadmium, for example, is a key risk-increasing element for prostate cancer; for breast cancer, Cd, Cr, Zn are mainly contributing; for bronchial cancer (in smelter workers), Cd, As, Cr, Sb, Co, La, all these elements are in a reciprocal relationship with Se. While selenium remains the key cancer-protective trace element, the interpretation of its mode of action necessitates consideration of the effects of selenium antagonistic elements.

The role of zinc in life: a review.

Zinc is a common element in human and natural environments and plays an important part in many biological processes. Zinc, which is defined as an essential trace element, or a micronutrient, is essential for the normal growth and the reproduction of all higher plants and animals, and of humans. In addition, it plays a key role during physiological growth and fulfills an immune function. It is vital for the functionality of more than 300 enzymes, for the stabilization of DNA, and for gene expression. This review summarizes the role and manifestations of zinc in the environment and its importance for human health and metabolism, as well as its physiological role. Toxicity, teratogenicity, carcinogenicity, and immunological functions of zinc are outlined with particular reference to the properties of zinc as an antioxidant, and its role in cancer prevention.

Interactive effects of selenium and chromium on mammary tumor development and growth in MMTV-infected female mice and their relevance to human cancer.

Evidence for interactive effects of chromium and selenium on the appearance of mammary tumors was obtained by exposing female virgin C3H mice infected with the murine mammary tumorvirus (MMTV) to subtoxic levels of Cr [as Cr(III) nitrate] and Se (as sodium selenite) in the supply water. Cr counteracted the inhibitory effect of Se on tumor development in a dose-dependent manner, shortened the tumor latency period, and accelerated tumor growth rates. Exposure to Cr also altered the levels of Se in the liver and kidneys of the mice, indicating that Cr interacts with Se and affects its organ distribution. Chromium must be added to the list of Se-antagonistic elements that weaken or abolish the antitumorigenic effects of Se. These findings are relevant to human cancer as previous studies revealed the age-corrected mortalities from breast and other major forms of cancer in different countries to be inversely correlated with the dietary Se intakes, and directly correlated with the estimated intakes of Cr and of other Se-antagonistic elements. The presence of these elements in foods must be taken into account when estimating the optimal dose of supplemental Se for cancer risk reduction.

Proteasome-mediated degradation of cell division cycle 25C and cyclin-dependent kinase 1 in phenethyl isothiocyanate-induced G2-M-phase cell cycle arrest in PC-3 human prostate cancer cells.

Phenethyl isothiocyanate (PEITC), a constituent of many cruciferous vegetables, offers significant protection against cancer in animals induced by a variety of carcinogens. The present study demonstrates that PEITC suppresses proliferation of PC-3 cells in a dose-dependent manner by causing G(2)-M-phase cell cycle arrest and apoptosis. Interestingly, phenyl isothiocyanate (PITC), which is a structural analogue of PEITC but lacks the -CH(2) spacers that link the aromatic ring to the -N=C=S group, neither inhibited PC-3 cell viability nor caused cell cycle arrest or apoptosis. These results indicated that even a subtle change in isothiocyanate (ITC) structure could have a significant impact on its biological activity. The PEITC-induced cell cycle arrest was associated with a >80% reduction in the protein levels of cyclin-dependent kinase 1 (Cdk1) and cell division cycle 25C (Cdc25C; 24 h after treatment with 10 micro M PEITC), which led to an accumulation of Tyr(15) phosphorylated (inactive) Cdk1. On the other hand, PITC treatment neither reduced protein levels of Cdk1 or Cdc25C nor affected Cdk1 phosphorylation. The PEITC-induced decline in Cdk1 and Cdc25C protein levels and cell cycle arrest were significantly blocked on pretreatment of PC-3 cells with proteasome inhibitor lactacystin. A 24 h exposure of PC-3 cells to 10 micro M PEITC, but not PITC, resulted in about 56% and 44% decrease in the levels of antiapoptotic proteins Bcl-2 and Bcl-X(L), respectively. However, ectopic expression of Bcl-2 failed to alter sensitivity of PC-3 cells to growth inhibition or apoptosis induction by PEITC. Treatment of cells with PEITC, but not PITC, also resulted in cleavage of procaspase-3, procaspase-9, and procaspase-8. Moreover, the PEITC-induced apoptosis was significantly attenuated in the presence of general caspase inhibitor and specific inhibitors of caspase-8 and caspase-9. In conclusion, our data indicate that PEITC-induced cell cycle arrest in PC-3 cells is likely due to proteasome-mediated degradation of Cdc25C and Cdk1, and ectopic expression of Bcl-2 fails to confer resistance to PEITC-induced apoptosis. Furthermore, the results of the present study point toward involvement of both caspase-8- and caspase-9-mediated pathways in apoptosis induction by PEITC.

Mitochondrial permeability transition is a central coordinating event in N-(4-hydroxyphenyl)retinamide-induced apoptosis.

The inhibitory effects of N-(4-hydroxyphenyl)retinamide (4HPR) on the process of carcinogenesis are not fully understood and may result from its ability to induce apoptosis in transformed cells. This study investigated the apoptotic properties of 4HPR in four human cutaneous squamous cell carcinoma cell lines. Apoptosis induction, detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling method, occurred in a dose- and time-dependent fashion after treatment with 4HPR. 4HPR promoted reactive oxygen species (ROS) determined by oxidation of 2',7'-dichlorofluorescin. 4HPR-induced ROS, and apoptosis could be inhibited by L-ascorbic acid. Rhodamine 123 retention revealed that 4HPR treatment promoted a gradual dissipation of mitochondrial inner transmembrane potential, and this could be inhibited by L-ascorbic acid, implying that mitochondrial permeability transition was involved in apoptosis induction. Cyclosporin A and bongkrekic acid inhibited dissipation of mitochondrial inner transmembrane potential, ROS production, and DNA fragmentation after exposure to 4HPR, demonstrating that mitochondrial permeability transition was a central coordinating feature of 4HPR-induced apoptosis.

Zinc supplementation attenuates high glucose-induced epithelial-to-mesenchymal transition of peritoneal mesothelial cells.

Zinc (Zn) plays an important role in preventing many types of epithelial-to-mesenchymal transition (EMT)-driven fibrosis in vivo. But its function in the EMT of the peritoneal mesothelial cells (PMCs) remains unknown. Here, we studied the Zn effect on the high glucose (HG)-induced EMT in the rat PMCs (RPMCs) and the underlying molecular mechanisms. We found that Zn supplementation significantly inhibited TGF-ß1 and ROS production, and attenuated the HG-induced EMT in the RPMCs, likely through inhibition of MAPK, NF-κB, and TGF-ß/Smad pathways.

Estrogenic effects of fusarielins in human breast cancer cell lines.

The fusarielins are a group of metabolites found in several Aspergillus and Fusarium species that have been reported to have with weak antifungal, antibiotic and cytotoxic effects. This study identifies fusarielin A, F, G and H isolated from Fusarium as mycoestrogens. Mycoestrogens are compounds from fungi that bind to the estrogen receptors and induce an estrogenic response in targeted cells. All four tested fusarielins stimulate MCF-7 cell proliferation with fusarielin H as the most potent, able to stimulate cell proliferation 4-fold in a resazurin metabolism assay at 25µM. MDA-MB-231 cells without the estrogen receptor-α and MCF-10a cells without estrogen receptors were not stimulated by fusarielins. Furthermore, the stimulation was prevented in MCF-7 cells when fusarielins were incubated in the presence of the estrogen receptor antagonist fulvestrant. These observations suggest that fusarielins bind to the estrogen receptor and act as weak mycoestrogens.

Resveratrol-mediated chemoprevention of diethylnitrosamine-initiated hepatocarcinogenesis: inhibition of cell proliferation and induction of apoptosis.

Hepatocellular carcinoma (HCC) is one of the most common cancers and lethal diseases. In view of the limited treatment and a grave prognosis of liver cancer, preventive control has been emphasized. Resveratrol, a polyphenol found in grape skins, peanuts, berries and red wine, has been shown to possess potent growth inhibitory effects against various human cancer cells. Although resveratrol has been found to exhibit chemopreventive actions in experimentally induced skin, breast, colon and esophagus rodent tumors, chemopreventive potential of this dietary constituent has not been explored well against experimental liver cancer. We evaluated the inhibitory effect of resveratrol using a two-stage model of rat hepatocarcinogenesis in Sprague-Dawley rats. Initiation was performed by a single intraperitoneal injection of diethylnitrosamine (DENA, 200 mg/kg), followed by promotion with phenobarbital (0.05%) in drinking water. The rats had free access to food supplemented with resveratrol equivalent to 50, 100 or 300 mg/kg body weight/day. Resveratrol treatment was started 4 weeks prior to the initiation and continued for 20 weeks. Resveratrol dose-dependently reduced the incidence, total number and multiplicity of visible hepatocyte nodules. Mean nodular volume and nodular volume as percentage of liver volume were also inhibited upon resveratrol treatment. Histopathological examination of liver tissue confirmed the protective effect of resveratrol. Immunohistochemical detection of cell proliferation and assay of apoptosis indicated a decrease in cell proliferation and increase of apoptotic cells in the livers of resveratrol-supplemented rats. Resveratrol also induced the expression of pro-apoptotic protein Bax, reduced anti-apoptotic Bcl-2 expression, with a concurrent increase in Bax/Bcl-2 ratio with respect to DENA control. The present study provides evidence, for the first time, that resveratrol exerts a significant chemopreventive effect on DENA-initiated hepatocarcinogenesis through inhibition of cell proliferation and induction of apoptosis. Resveratrol-induced apoptogenic signal during rat liver carcinogenesis may be mediated through the downregulation of Bcl-2 and upregulation of Bax expression. Due to a favorable toxicity profile, resveratrol can potentially be developed as a chemopreventive drug against human HCC.

The PERK/eIF2 alpha signaling pathway of Unfolded Protein Response is essential for N-(4-hydroxyphenyl)retinamide (4HPR)-induced cytotoxicity in cancer cells.

N-(4-hydroxyphenyl)retinamide (4HPR) is a synthetic retinoid that has been tested in clinical trials as a cancer chemopreventive drug. 4HPR is cytotoxic to cancer cells but the underlying molecular mechanisms are at present only partially understood. Here we demonstrate that in the human cervical cancer cell line HeLa and the human leukemia cell line HL-60, 4HPR caused rapid, Reactive Oxygen Species (ROS)-dependent activation of the Unfolded Protein Response (UPR). In HeLa cells, 4HPR was shown to induce cell death and activation of procaspases. These effects of 4HPR could be abolished by the over-expression of dominant negative mutants of PERK or eIF2 alpha. HeLa cells incubated with 4HPR were found to form autophagosomes that were also mediated by the PERK/eIF2 alpha pathway. While 4HPR-induced cell death could be significantly prevented by the presence of specific caspase inhibitors, 3-methyladenine (3-MA) that inhibits autophagosome formation enhanced 4HPR-induced cell death. Examination of individual 4HPR-treated HeLa cells revealed that those without the development of autophagosomes hence exhibiting an incomplete UPR were caspase-active and were not viable, while those with autophagosomes were caspase-inactive and retained cell viability. Our data suggest that the PERK/eIF2 alpha pathway is essential for the cytotoxicity of 4HPR that targets on cancer cells with malfunctional UPR.

DNA mismatch repair status may influence anti-neoplastic effects of butyrate.

HNPCC (hereditary non-polyposis colon cancer) is an autosomal-dominant disorder characterized by early-onset CRC (colorectal cancer). HNPCC is most often associated with mutations in the MMR (mismatch repair) genes hMLH1, hMSH2, hMSH6 or hPMS2. The mutator phenotype of a defective MMR system is MSI (microsatellite instability), which also occurs in approx. 15-25% of sporadic CRC cases, where it is associated with the hypermethylation of the promoter region of hMLH1. Dietary factors, including excessive alcohol consumption, ingestion of red meat and low folate intake, may increase the risk of MSI high tumour development. In contrast, aspirin may suppress MSI in MMR-deficient CRC cell lines. Butyrate, a short-chain-fatty-acid end product of carbohydrate fermentation in the colon, shares a number of anti-neoplastic properties with aspirin, including inhibiting proliferation and inducing apoptosis of CRC cells. Recent in vitro studies suggest that physiological concentrations of butyrate (0.5-2 mM) may have more potent anti-neoplastic effects in CRC cell lines deficient in MMR, but mechanisms for such a differential response remain to be established.

Effects of soy phytoestrogens genistein and daidzein on breast cancer growth.

OBJECTIVE: To determine whether genistein and daidzein, the major phytoestrogens in soy, can stimulate breast cancer growth. DATA SYNTHESIS: Systematic search through primary English-language literature on MEDLINE (1966-January 2001), EMBASE (1982-January 2001) and Current Contents (1998-January 2001). DATA SOURCES: Genistein and daidzein at low concentrations were found to stimulate breast tumor growth in in vitro and in vivo animal studies, and antagonize the antitumor effect of tamoxifen in vitro. At high concentrations, genistein inhibited tumor growth and enhanced the effect of tamoxifen in vitro. CONCLUSIONS: Genistein and daidzein may stimulate existing breast tumor growth and antagonize the effects of tamoxifen. Women with current or past breast cancer should be aware of the risks of potential tumor growth when taking soy products.

Anticarcinogenic effects of selenium.

Selenium (Se) exerts its anticarcinogenic effects by multiple mechanisms. In the physiological dosage range, Se appears to function as an antimutagenic agent, preventing the malignant transformation of normal cells and the activation of oncogenes. These protective effects of Se seem to be primarily associated with its presence in the glutathione peroxidases, which are known to protect DNA and other cellular components from damage by oxygen radicals. Selenoenzymes are also known to play roles in carcinogen metabolism, in the control of cell division, oxygen metabolism, detoxification processes, apoptosis induction and the functioning of the immune system. Other modes of action, either direct or indirect, may also be operative, such as the partial retransformation of tumor cells and the inactivation of oncogenes. However, the effects of Se in the physiological dosage range are not attributable to cytotoxicity, allowing Se to be defined as a genuine nutritional cancer-protecting agent. The anticarcinogenic effects of Se are counteracted by Se-antagonistic compounds and elements. For maximal utilization of its cancer-protective potential, Se supplementation should start early in life and be maintained over the entire lifespan. In addition, exposure to Se antagonists and carcinogenic risk factors should be minimized by appropriate dietary and lifestyle changes.

Reactive oxygen species in choline deficiency induced carcinogenesis and nitrone inhibition.

Reactive oxygen species and free radical processes have been considered important in cancer development for many years. Much research demonstrates that the choline-deficiency induced hepatocarcinogenesis model prominently involves reactive oxygen species. We present a summary of results obtained in our original studies of this model over the last 4 years. We have shown that alpha-phenyl-tert-butyl nitrone (PBN) and some of its hydroxylated derivatives (the 4- and 3-hydroxylated compounds) prevent hepatocarcinogenesis in this model. Mechanistic studies have demonstrated that isolated mitochondria from the livers of rats fed the choline-deficiency defined amino acid diet produce significantly much more H2O2 per NADH reducing equivalents oxidized. Based on these observations, we postulate that H2O2 is a primary carcinogenic factor in this model. Based on studies of the action of PBN on isolated mitochondria, we postulate that the inhibiting action of PBN involves suppression of H2O2 production of mitochondria and generally decreasing the oxidative stress within the preneoplastic lesions. The net effect of the activity of the nitrone compounds appears to be due to their ability to shift the apoptosis/neoplastic tendency balance toward apoptosis of the cells within the preneoplastic lesions. This is considered to be the primary reason the size of the preneoplastic lesions are significantly decreased and why the nitrones are potent anti-carcinogenic agents in this model.

Sodium orthovanadate potentiates EGCG-induced apoptosis that is dependent on the ERK pathway.

Epigallocatechin-3-gallate (EGCG) is a potent chemopreventive agent in many test systems and has been shown to inhibit tumor promotion and induce apoptosis. In the present study, we determined the effect of vanadate, a potent inhibitor of tyrosine phosphatase, on EGCG-induced apoptosis. Investigation of the mechanism of EGCG or vanadate-induced apoptosis revealed induction of caspase 3 activity and cleavage of phospholipase-gamma1 (PLC-gamma1). Furthermore, vanadate potentiated EGCG-induced apoptosis by mitogen-activated protein kinase (MAPK) signaling pathway. Treatment with EGCG plus vanadate for 24h produced morphological features of apoptosis and DNA fragmentation in U937 cells. This was associated with cytochrome c release, caspase 3 activation, and PLC-gamma1 degradation. EGCG plus vanadate activates multiple signal transduction pathways involved in coordinating cellular responses to stress. We demonstrate a requirement for extracellular signal-regulated protein kinase (ERK), a member of the mitogen-activated protein kinase family in EGCG plus vanadate-induced apoptosis in U937 cells. Elevated ERK activity that contributed to apoptosis by EGCG plus vanadate was supported by PD98059 and U0126, chemical inhibitor of MEK/ERK signaling pathway, prevented apoptosis. Taken together, our finding suggests that ERK activation plays an active role in mediating EGCG plus vanadate-induced apoptosis of U937 cells and functions upstream of caspase activation to initiate the apoptotic signal.

Molecular effects of genistein on estrogen receptor mediated pathways.

Genistein, a component of soy products, may play a role in the prevention of breast and prostate cancer. However, little is known about the molecular mechanisms involved. In the present study, we examined the effects of genistein on the estrogen receptor positive human breast cancer cell line MCF-7. We observed that genistein stimulated estrogen-responsive pS2 mRNA expression at concentrations as low as 10(-8) M and these effects can be inhibited by tamoxifen. We also showed that genistein competed with [3H]estradiol binding to the estrogen receptor with 50% inhibition at 5 x 10(-7) M. Thus, the estrogenic effect of genistein would appear to be a result of an interaction with the estrogen receptor. The effect of genistein on growth of MCF-7 cells was also examined. Genistein produced a concentration-dependent effect on the growth of MCF-7 cells. At lower concentrations (10(-8)-10(-6) M) genistein stimulated growth, but at higher concentrations (> 10(-5) M) genistein inhibited growth. The effects of genistein on growth at lower concentrations appeared to be via the estrogen receptor pathway, while the effects at higher concentrations were independent of the estrogen receptor. We also found that genistein, though estrogenic, can interfere with the effects of estradiol. In addition, prolonged exposure to genistein resulted in a decrease in estrogen receptor mRNA level as well as a decreased response to stimulation by estradiol.

Galectin-3 mediates genistein-induced G(2)/M arrest and inhibits apoptosis.

Many recent studies have focused on potential chemopreventive activities of dietary genistein, a natural isoflavonoid compound found in soy products. Genistein has been implicated in anticancer activities, including differentiation, apoptosis, inhibition of cell growth and inhibition of angiogenesis. In previous studies, genistein was shown to induce apoptosis and cell cycle arrest at G(2)/M in several cancer cell lines in vitro, which is associated with induction of p21(WAF1/CIP1), a universal inhibitor of cyclin-dependent kinases. At present, the molecular basis for diverse genistein-mediated cellular responses is largely unknown. In the present study, we investigated whether galectin-3, an anti-apoptotic gene product, regulates genistein-mediated cellular responses. We show that genistein effectively induces apoptosis without detectable cell cycle arrest in BT549, a human breast epithelial cell line which does not express galectin-3 at a detectable level. In galectin-3 transfected BT549 cells, genistein induced cell cycle arrest at the G(2)/M phase without apoptosis induction. Interestingly, genistein induces p21(WAF1/CIP1) expression in galectin-3-expressing BT549 cells, but not in control BT549 cells undergoing apoptosis. Collectively, the results of the present study suggest that galectin-3, at least in part, is a critical determinant for genistein-mediated cell cycle arrest and apoptosis, and genistein induction of p21(WAF1/CIP1) is associated with cell cycle arrest, but not required for apoptosis induction.

Phase I trial of alpha-tocopherol effects on 13-cis-retinoic acid toxicity.

BACKGROUND: Retinoids are under intensive study for the treatment and prevention of cancer. Substantial dose-related toxicities of retinoids are a major obstacle to this work. In a recent retrospective analysis of combined 13-cis-retinoic acid (13cRA) and alpha-tocopherol (AT) in myelodysplasia, 13cRA toxicity was reduced significantly and 13cRA activity was enhanced. These results suggested the need for prospective testing of this new combination. This trial tested the hypotheses that At can reduce toxicity of high-dose 13cRA and does not interfere with 13cRA absorption/activity as reflected by reduced 13cRA serum levels. PATIENTS AND METHODS: This was a phase I trial design in which patients received fixed-dose 13cRA (100 mg/m2/d) plus escalating-dose AT (beginning at 800 IU/d, increased 400 IU/d each month until 2000 IU/d). We collected toxicity data every four weeks from self-report forms, clinical examinations and laboratory studies. AT effects on 13cRA toxicity were determined by comparing maximum toxicity at lowest AT dose with that at highest AT dose. We also measured serum levels of both agents every four weeks. RESULTS: Of the 45 patients registered, 36 had cancer (active or prior history), 9 had premalignant lesions. Thirty-nine patients could be evaluated for initial-course toxicity; 31 for final course toxicity. Median time on treatment (all patients) was four months (range, 1-9 months); a total of 223 month-long courses of treatment were given. Eighteen percent of patients (7/39) developed grade 3 or 4 toxicity in the initial course. The rates of increase and decrease in 13cRA toxicity associated with increasing AT doses were similar: 36% decreased (11/31), 32% increased (10/31) (P = 0.84). At did not reduce 13cRA serum levels. After initial increases of mean AT plasma levels (17.9 micrograms/ ml at baseline to 45.4 micrograms/ml after first four-week course), subsequent AT plasma increases (< 2-fold) did not keep pace with increased AT doses (2-3-fold). No major activity occurred in the 21 patients with active refractory cancer. The complete response rate in patients with premalignant head-and-neck or lung lesions was 77.8% (7/9), which included two patients previously refractory to 13cRA alone. CONCLUSION: Although escalating doses of AT did not reduce 13cRA toxicity, the rate of initial-course (including 800 IU/d of AT) high-grade toxicity was substantially lower than that typical of high-dose 13cRA-alone and similar to that typical of low-dose 13cRA-alone. Indeed, a trial of 13cRA-alone followed by 13cRA plus AT may have detected a significant toxicity difference. We did not design such a trial out of ethical concern for known side effects of high-dose 13cRA. The increase in AT serum levels was not proportional with increasing doses of AT, which may explain the lack of a dose-response effect of AT on 13cRA toxicity. Previous trials have established that 13cRA has an approximate 10% complete response rate in oral premalignancy. Our small trial's 77.8% complete response rate in premalignant lesions suggests that AT may enhance 13cRA clinical activity. Future trials of 13cRA plus AT are needed to define this combinations toxicity profile, clinical activity and pharmacokinetics.