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Background Oxaliplatin belongs to the platinum-based drug family and has shown promise in treating cancer by binding to DNA to induce cytotoxicity. However, individual patients show diverse therapeutic responses toward oxaliplatin due to yet-unknown underlying mechanisms. We recently established that oxaliplatin also exert its anti-cancer activity in gastric cancer cell lines by targeting tumor-associated NADH oxidase (tNOX), attenuate NAD+ generation and reduce NAD+-dependent sirtuin 1 (SIRT1) deacetylase activity, which in turn enhances p53 acetylation and apoptosis. Methods In this study, differential cellular outcomes in response to oxaliplatin exposure of p53-wild-type versus p53-null HCT116 human colon cancer cells were examined. Cell growth profile was determined by cell impedance measurements and apoptosis was analyzed by flow cytometry. The engagement between oxaliplatin and tNOX protein was studied by cellular thermal shift assay. Furthermore, western blot analysis revealed that p53 was important in regulating tNOX expression in these cell lines. Results In p53-wild-type cells, we found that oxaliplatin inhibited cell growth by inducing apoptosis and concurrently down-regulating tNOX at both the transcriptional and translational levels. In p53-null cells, in contrast, oxaliplatin moderately up-regulated tNOX expression and yielded no apoptosis and much less cytotoxicity. Further experiments revealed that in p53-wild-type cells, oxaliplatin enhanced ROS generation and p53 transcriptional activation, leading to down-regulation of the transcriptional factor, POU3F2, which enhances the expression of tNOX. Moreover, the addition of a ROS scavenger reversed the p53 activation, POU3F2 down-regulation, and apoptosis induced by oxaliplatin in p53-wild-type cells. In the p53-null line, on the other hand, oxaliplatin treatment triggered less ROS generation and no p53 protein, such that POU3F2 and tNOX were not down-regulated and oxaliplatin-mediated cytotoxicity was attenuated. Conclusion Our results show that oxaliplatin mediates differential cellular responses in colon cancer cells depending on their p53 status, and demonstrate that the ROS-p53 axis is important for regulating POU3F2 and its downstream target, tNOX. Notably, the depletion of tNOX sensitizes p53-null cells to both spontaneous and oxaliplatin-induced apoptosis. Our work thus clearly shows a scenario in which targeting of tNOX may be a potential strategy for cancer therapy in a p53-inactivated system.

Gastric cancer is a common human malignancy and a major contributor to cancer-related deaths worldwide. Unfortunately, the prognosis of most gastric cancer patients is poor because they are generally diagnosed at a late stage after the cancer has already metastasized. Most current research, therefore, emphasizes selective targeting of cancer cells by apoptosis-inducing agents. One such therapeutic agent is capsaicin, a component of chili peppers that has been shown to possess anti-growth activity against various cancer cell lines. Here, we examined the effect of capsaicin on SNU-1 and TMC-1 gastric cancer cells and found differing outcomes between the two cell lines. Our results show that capsaicin induced significant cytotoxicity with increases in oxidative stress, PARP cleavage, and apoptosis in sensitive SNU-1 cells. In contrast, TMC-1 cells were much less sensitive to capsaicin, exhibiting low cytotoxicity and very little apoptosis in response to capsaicin treatment. Capsaicin-induced apoptosis in SNU-1 cells was associated with down-regulation of tumor-associated NADH oxidase (tNOX) mRNA and protein. On the contrary, tNOX expression was scarcely affected by capsaicin in TMC-1 cells. We further showed that tNOX-knockdown sensitized TMC-1 cells to capsaicin-induced apoptosis and G1 phase accumulation, and led to decreased cell growth, demonstrating that tNOX is essential for cancer cell growth. Collectively, these results indicate that capsaicin induces divergent effects of the growth of gastric cancer cells that parallel its effects on tNOX expression, and demonstrate that forced tNOX down-regulation restored capsaicin-induced growth inhibition in TMC-1 cells.

Monoclonal antibodies were generated in mice to a 34-kDa circulating form of a drug-responsive hydroquinone (NADH) oxidase with a protein disulfide-thiol interchange activity specific to the surface of cancer cells and the sera of cancer patients. Screening used Western blots with purified 34-kDa tNOX from HeLa cells and the sera of cancer patients. Epitopes were sought that inhibited the drug-responsive oxidation of NADH with the sera of cancer patients, but which had no effect on NADH oxidation with the sera of healthy volunteers. Two such antisera were generated. One, designated monoclonal antibody (mAb) 12.1, was characterized extensively. The NADH oxidase activity inhibited by mAb 12.1 also was inhibited by the quinone site inhibitor capsaicin (8-methyl- N-vanillyl-6-noneamide). The inhibition was competitive for the drug-responsive protein disulfide-thiol interchange activity assayed either by restoration of activity to scrambled RNase or by cleavage of a dithiodipyridine substrate, and was uncompetitive for NADH oxidation. Both the mAb 12.1 and the postimmune antisera immunoprecipitated drug-responsive NOX activity and identified the same 34-kDa tNOX protein in the sera of cancer patients that was absent from sera of healthy volunteers, and was utilized as immunogen. Preimmune sera from the same mouse as the postimmune antisera was without effect. Both mouse ascites containing mAb 12.1 and postimmune sera (but not preimmune sera) slowed the growth of human cancer cell lines in culture, but did not affect the growth of non-cancerous cell lines. Immunocytochemical and histochemical findings showed that mAb 12.1 reacted with the surface membranes of human carcinoma cells and tissues.

Retinol stimulates the formation of transition vesicles in situ and in all free systems based on rat liver. The stimulation is on vesicle formation from transitional endoplasmic reticulum and not on vesicle fusion with donor membranes. Vesicle budding in the cell free system requires a nucleoside triphosphate and is sensitive to inhibition by thiol reagents. In this report we develop and test a model whereby a retinol-modulated NADH:protein disulfide reductase (NADH oxidase) with protein disulfide-thiol interchange activity is implicated in the vesicle budding mechanism. The protein has the ability to restore activity to scrambled, inactive RNase A and is stimulated or inhibited by retinol depending on the redox environment. Under reducing conditions and in the presence of a chemical reductant such as GSH, the partial reaction stimulated by retinol appears to be the oxidation of membrane thiols. This is the first report of an enzymatic mechanism to explain specific retinol effects both in vivo and in vitro on membrane trafficking not given by retinoic acid.

Plasma membrane vesicles of HeLa cells are characterized by a drug-responsive oxidation of NADH. The NADH oxidation takes place in an argon or nitrogen atmosphere and in samples purged of oxygen. Direct assay of protein thiols by reaction with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB; Ellman's reagent), suggests that protein disulfides may be the natural electron acceptors for NADH oxidation by the plasma membrane vesicles. In the presence of NADH, protein disulfides of the membranes were reduced with a concomitant stoichiometric increase in protein thiols. The increase in protein thiols was inhibited in parallel to the inhibition of NADH oxidation by the antitumor sulfonylurea LY181984 with an EC50 of ca. 30 nM. LY181984, with an EC50 of 30 nM, also inhibited a protein disulfide–thiol interchange activity based on the restoration of activity to inactive (scrambled) RNase and thiol oxidation. The findings suggest that thiol oxidation, NADH-dependent disulfide reduction (NADH oxidation), and protein disulfide–thiol interchange in the absence of NADH all may be manifestations of the same sulfonylurea binding protein of the HeLa plasma membrane. A surface location of the thiols involved was demonstrated using detergents and the impermeant thiol reagent p-chloromercuriphenylsulfonic acid (PCMPS). The surface location precludes a physiological role of the protein in NADH oxidation. Rather, it may carry out some other role more closely related to a function in growth, such as protein disulfide–thiol interchange coupled to cell enlargement.