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Bladder cancer is one of the most frequent cancers among males, and a poor survival rate reflects problems with aggressiveness and chemo-resistance. Accumulating evidence indicates that SIRT1 is involved in bladder cancer tumorigenesis and is positively associated with chemo-resistance and poor prognosis. We recently synthesized water-soluble chemical derivatives of heliomycin, an antibiotic from Streptomyces resistomycificus , and demonstrated that they possess anticancer properties. In this present study, we used the cellular thermal shift assay (CETSA) in T24 bladder cancer cells to show that heliomycin (designated compound (H1)) and its 4-( tert -butylamino)methyl derivative (HD2) directly engaged with SIRT1 in the native cellular environment, whereas another derivative (HD3) did not. Upon binding, heliomycin downregulated SIRT1 protein expression without altering its transcript level, and subsequently induced autophagy. Interestingly, the derivative (HD2) triggered apoptosis. The interaction between SIRT1 protein and heliomycin or its derivatives was also speculated by a molecular docking simulation, suggesting heliomycin (H1) and derivative (HD2) acting with the different binding modes to SIRT1. Given the increased water-solubility, hydrogen bonds were found on Ala262 and Ile347 residues in the docked complex of derivative (HD2) to produce more steady interaction and initiate signaling pathways that were not observed in the case of heliomycin. Meanwhile, it is evident that derivative (HD3) did not engage with SIRT1 by CETSA or molecular docking studies, nor did it downregulate SIRT1 expression. Taken together, these findings clearly show that SIRT1 is targeted and downregulated by heliomycin and its water-soluble 4-aminomethylated derivative (HD2) possibly through autophagic and/or proteasomal degradation, leading to cell death and growth suppression of T24 bladder cancer cells., Competing Interests: None., (AJCR Copyright © 2022.)

Although considered a sporadic type of skin cancer, malignant melanoma has regularly increased internationally and is a major cause of cancer-associated death worldwide. The treatment options for malignant melanoma are very limited. Accumulating data suggest that the natural compound, capsaicin, exhibits preferential anticancer properties to act as a nutraceutical agent. Here, we explored the underlying molecular events involved in the inhibitory effect of capsaicin on melanoma growth. The cellular thermal shift assay (CETSA), isothermal dose-response fingerprint curves (ITDRF CETSA ), and CETSA-pulse proteolysis were utilized to confirm the direct binding of capsaicin with the tumor-associated NADH oxidase, tNOX (ENOX2) in melanoma cells. We also assessed the cellular impact of capsaicin-targeting of tNOX on A375 cells by flow cytometry and protein analysis. The essential role of tNOX in tumor- and melanoma-growth limiting abilities of capsaicin was evaluated in C57BL/6 mice. Our data show that capsaicin directly engaged with cellular tNOX to inhibit its enzymatic activity and enhance protein degradation capacity. The inhibition of tNOX by capsaicin was accompanied by the attenuation of SIRT1, a NAD + -dependent deacetylase. The suppression of tNOX and SIRT1 then enhanced ULK1 acetylation and induced ROS-dependent autophagy in melanoma cells. Capsaicin treatment of mice implanted with melanoma cancer cells suppressed tumor growth by down-regulating tNOX and SIRT1, which was also seen in an in vivo xenograft study with tNOX-depleted melanoma cells. Taken together, our findings suggest that tNOX expression is important for the growth of melanoma cancer cells both in vitro and in vivo , and that inhibition of the tNOX-SIRT1 axis contributes to inducting ROS-dependent autophagy in melanoma cells., Competing Interests: None., (AJCR Copyright © 2021.)

Despite the progress that has been made in diagnosing and treating oral cancers, they continue to have a poor prognosis, with a 5-year overall survival rate of approximately 50%. We have intensively studied the anticancer properties of capsaicin (a burning constituent of chili pepper), mainly focusing on its apoptotic properties. Here, we investigated the interplay between apoptosis and autophagy in capsaicin-treated oral cancer cells with either functional or mutant p53. Cytotoxicity was determined by cell impedance measurements and WST-1 assays, and cell death was analyzed by flow cytometry. The interaction between capsaicin and tumor-associated NADH oxidase (tNOX, ENOX2) was studied by cellular thermal shift assay (CETSA) and isothermal dose-response fingerprint curves (ITDRF CETSA ). Our CETSA data suggested that capsaicin directly engaged with tNOX, resulting in its degradation through the ubiquitin-proteasome and the autophagy-lysosome systems. In p53-functional SAS cells, capsaicin induced significant cytotoxicity via autophagy but not apoptosis. Given that tNOX catalyzes the oxidation of NADH, the direct binding of capsaicin to tNOX also inhibited the NAD + -dependent activity of sirtuin 1 (SIRT1) deacetylase, we found that capsaicin-induced autophagy involved enhanced acetylation of ULK1, which is a key player in autophagy activation, possibly through SIRT1 inhibition. In p53-mutated HSC-3 cells, capsaicin triggered both autophagy and apoptosis. In this case, autophagy occurred before apoptosis: during this early stage, autophagy seemed to inhibit apoptosis; at a later stage, in contrast, autophagy appeared to be essential for the induction of apoptosis. Western blot analysis revealed that the reduction in tNOX and SIRT1 associated with enhanced ULK1 acetylation and c-Myc acetylation, which in turn, reactivated the TRAIL pathway, ultimately leading to apoptosis. Taken together, our data highlight the potential value of leveraging capsaicin and tNOX in therapeutic strategies against oral cancer., Competing Interests: None., (AJCR Copyright © 2020.)

We have studied the chemopreventive property of capsaicin, a major active component in chili pepper, and found that it exhibited apoptotic activity against various lines of cancer cells. Interestingly, accumulating data has revealed that, in addition to cytotoxicity, capsaicin also plays regulatory role on cell migration and invasion. However, its effect on cell migration is paradoxical and not completely understood. Here, we set out to elucidate the molecular events underlying capsaicin-inhibited cell migration in bladder cancer cells. Our results show that the capsaicin-reduced cell migration was associated with down-regulation of sirtuin 1 (SIRT1) deacetylase, possibly through proteasome-mediated protein degradation. More importantly, we employed a cellular thermal shift assay (CETSA) to demonstrate that there was a direct binding between capsaicin and SIRT1. The engagement with capsaicin and protein degradation diminished the deacetylase of SIRT1, which in turn, enhanced acetylation of cortactin and β-catenin to decrease MMP-2 and MMP-9 activation, resulting in cell migration impairment in bladder cancer cells., Competing Interests: None.

Tumor-associated NADH oxidase (tNOX, ENOX2), which belongs to a family of growth-related NADH oxidases, was originally identified as a plasma membrane protein of rat hepatoma and is inhibited or downregulated by several anti-cancer drugs. The objective of this study was to evaluate the anti-tumor effects of tNOX used as an immunogen against Lewis lung cancer. Human tNOX was expressed in Escherichia coli , purified by His-Tag affinity chromatography, and emulsified with the adjuvant, ISA 201 VG. Immunological analyses of the generated tNOX vaccine were performed in mice. The results of ELISA and ELISpot were significantly higher in tNOX vaccine group compared to the control group. In vivo , we examined the anti-tumor effects of mice that received the tNOX vaccine via the intraperitoneal or subcutaneous routes. Mice were vaccinated three times at 2-week intervals, challenged at 2 weeks after the final vaccination, and terminated at 34 days post-challenge. Antibody titers, tumor volume and histopathological scores were used to evaluate the anti-tumor effects of the tNOX vaccine. Our results revealed that tNOX-vaccinated mice had significantly higher antibody titers than negative control (NC) and challenge control (CC) mice. When compared to the corresponding CC groups, the intraperitoneal and subcutaneous vaccination with tNOX showed a significantly smaller tumor mass volume ( P < 0.05) and a significantly lower histological lesion score ( P < 0.05), respectively. Our results demonstrate that the use of a xenogeneic tNOX as an immunogen in mice activates immune responses and anti-tumor effects against Lewis lung cancer., Competing Interests: None.