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The antibiotic heliomycin (resistomycin), which is generated from Streptomyces resistomycificus, has multiple activities, including anticancer effects. Heliomycin was first described in the 1960s, but its clinical applications have been hindered by extremely low solubility. A series of 4-aminomethyl derivatives of heliomycin were synthesized to increase water solubility; studies showed that they had anti-proliferative effects, but the drug targets remained unknown. In this study, we conducted cellular thermal shift assays (CETSA) and molecular docking simulations to identify and validate that heliomycin and its water-soluble derivative, 4-(dimethylaminomethyl)heliomycin (designated compound 4-dmH) engaged and targeted with sirtuin-1 (SIRT1) in p53-functional SAS and p53-mutated HSC-3 oral cancer cells. We further addressed the cellular outcome of SIRT1 inhibition by these compounds and found that, in addition to SIRT1, the water-soluble 4-dmH preferentially targeted a tumor-associated NADH oxidase (tNOX, ENOX2). The direct binding of 4-dmH to tNOX decreased the oxidation of NADH to NAD+ which diminished NAD+-dependent SIRT1 deacetylase activity, ultimately inducing apoptosis and significant cytotoxicity in both cell types, as opposed to the parental heliomycin-induced autophagy. We also observed that tNOX and SIRT1 were both upregulated in tumor tissues of oral cancer patients compared to adjacent normal tissues, suggesting their clinical relevance. Finally, the better therapeutic efficacy of 4-dmH was confirmed in tumor-bearing mice, which showed greater tNOX and SIRT1 downregulation and tumor volume reduction when treated with 4-dmH compared to heliomycin. Taken together, our in vitro and in vivo findings suggest that the multifaceted properties of water-soluble 4-dmH enable it to offer superior antitumor value compared to parental heliomycin, and indicated that it functions through targeting the tNOX-NAD+-SIRT1 axis to induce apoptosis in oral cancer cells.

Colorectal cancer is the most common cancer that affects both sexes and has a poor prognosis due to aggressiveness and chemoresistance. Essential oils isolated from Calocedrus formosana (CF-EOs) have been shown to demonstrate anti-termite, antifungal, anti-mosquito, and anti-microbial activities. However, the anticancer effects of CF-EOs are not yet fully understood. Therefore, the present study aimed to explore the molecular mechanism underlying CF-EOs-mediated anti-proliferative activity in colon cancer cells. Here, cell impedance measurements showed that CF-EOs inhibit proliferation in colon cancer cells with wild-type or mutant p53. Flow cytometry revealed that CF-EOs at 20, 50 µg/mL significantly induced ROS generation and autophagy in both HCT116 p53-wt and HCT116 p53-null cell lines, whereas pretreatment with the ROS scavenger N-acetyl cysteine (NAC) markedly attenuated these changes. CF-EOs also induced apoptosis at 50 µg/mL in both lines, as determined by flow cytometry. Protein analysis showed that CF-EOs markedly induced apoptosis markers, including Trail, cleaved caspase-3, cleaved caspase-9, and cleaved PARP, as well as autophagy markers, such as the levels of ULK1, Atg5, Atg6, Atg7, and the conversion of LC3-I to LC3-II. CF-EOs were further found to inhibit the activity and expression of the NAD+-dependent deacetylase SIRT1 to increase the levels of acetylated p53 (Ac-p53) in p53-wt cells and acetylated c-Myc (Ac-c-Myc) in p53-null cells, ultimately inducing apoptosis in both lines. Interestingly, suppression of SIRT1 by CF-EOs enhanced the acetylation of ULK1, which in turn prompted ROS-dependent autophagy in colon cancer cells. The induction of apoptosis and autophagy by CF-EOs suggests that they may have potential as a promising new approach for treating cancer. Collectively, our results suggest that essential oils isolated from Calocedrus formosana act as a promising anticancer agent against colon cancer cells by targeting SIRT1 to induce ROS-mediated autophagy and apoptosis.

Immunotherapy has emerged as a promising new treatment modality for head and neck cancer, offering the potential for targeted and effective cancer management. Squamous cell carcinomas pose significant challenges due to their aggressive nature and limited treatment options. Conventional therapies such as surgery, radiation, and chemotherapy often have limited success rates and can have significant side effects. Immunotherapy harnesses the power of the immune system to recognize and eliminate cancer cells, and thus represents a novel approach with the potential to improve patient outcomes. In the management of head and neck squamous cell carcinoma (HNSCC), important contributions are made by immunotherapies, including adaptive cell therapy (ACT) and immune checkpoint inhibitor therapy. In this review, we are focusing on the latter. Immune checkpoint inhibitors target proteins such as programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) to enhance the immune response against cancer cells. The CTLA-4 inhibitors, such as ipilimumab and tremelimumab, have been approved for early-stage clinical trials and have shown promising outcomes in terms of tumor regression and durable responses in patients with advanced HNSCC. Thus, immune checkpoint inhibitor therapy holds promise in overcoming the limitations of conventional therapies. However, further research is needed to optimize treatment regimens, identify predictive biomarkers, and overcome potential resistance mechanisms. With ongoing advancements in immunotherapy, the future holds great potential for transforming the landscape of oral tumor treatment and providing new hope for patients.

Background/purpose: Oral submucous fibrosis (OSF) is a precancerous condition of oral cancer with a complex etiology. Our previous work has demonstrated that non-coding RNA miR-1246 contributes to the cancer stemness of oral cancer. In the current study, we sought to investigate the effect of the inhibition of miR-1246 on the oral fibrogenesis. Methods: The expression levels of miR-1246 in OSF tissues and fibrotic buccal mucosal fibroblasts (fBMFs) were examined by qRT-PCR. Collagen gel contraction and migration assays were conducted to evaluate the myofibroblast activities. The relationship between miR-1246 and type I collagen was assessed and the protein expression of type I collagen was determined by Western blot. Results: MiR-1246 expression was upregulated in both OSF specimen and fBMFs compared to the normal counterparts. Inhibition of miR-1246 successfully suppressed the myofibroblast activities, including collagen gel contractility and migration capacity. Moreover, the expression of miR-1246 was positively correlated with type I collagen and the expression of type I collagen was abrogated by repression of miR-1246. Conclusion: MiR-1246 is not only critical to the maintenance of oral stemness but also important to the activation of myofibroblasts. Our results showed that miR-1246 is positively associated with the type I collagen, which may be a downstream effector of miR-1246 and responsible for the fibrosis effect on fBMFs. Keywords: Oral submucous fibrosis, MicroRNA-1246, Type I collagen, Buccal mucosal fibroblasts

Abstract 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.

Capsaicin (8-methyl-N-vanillyl-6-noneamide), which is an active component in red chili peppers, is used as a chemopreventive agent that shows favorable cytotoxicity against cancer cells. Accumulating evidence indicates that capsaicin preferentially inhibits a tumor-associated NADH oxidase (tNOX, ENOX2) that is ubiquitously expressed in cancer but not in non-transformed cells. This attenuates cancer cell growth by inducing apoptosis. The capsaicin-mediated inhibition of tNOX was recently shown to prolong the cell cycle. However, the molecular events underlying this regulation have not yet been investigated. In the present study, we used a cellular thermal shift assay (CETSA) to detect “target engagement” of capsaicin and its consequent impact on cell cycle progression. Our results indicated that capsaicin engaged with tNOX and triggered the proteasomal degradation of tNOX, which leads to the inhibition of NAD+-dependent SIRT1 deacetylase. Ultimately, the acetylation levels of c-Myc and p53 were enhanced, which suppressed the activation of G1 cyclin/Cyclin-dependent kinase complexes and triggered cell cycle arrest in cancer cells. The results obtained when tNOX was overexpressed in non-cancer cells validated its importance in cell cycle progression. These findings provide the first molecular insights into the regulatory role of tNOX and the anti-proliferative property of capsaicin in regulating the cell cycle of bladder cancer cells.

Bladder cancer is one of the most frequent cancers among males, and its poor survival rate reflects problems with aggressiveness and chemo-resistance. Recent interest has focused on the use of chemopreventatives (nontoxic natural agents that may suppress cancer progression) to induce targeted apoptosis for cancer therapy. Capsaicin, which has anti-cancer properties, is one such agent. It is known to preferentially inhibit a tumor-associated NADH oxidase (tNOX) that is preferentially expressed in cancer/transformed cells. Here, we set out to elucidate the correlation between tNOX expression and the inhibitory effects of capsaicin in human bladder cancer cells. We showed that capsaicin downregulates tNOX expression and decreases bladder cancer cell growth by enhancing apoptosis. Moreover, capsaicin was found to reduce the expression levels of several proteins involved in cell cycle progression, in association with increases in the cell doubling time and enhanced cell cycle arrest. Capsaicin was also shown to inhibit the activation of ERK, thereby reducing the phosphorylation of paxillin and FAK, which leads to decreased cell migration. Finally, our results indicate that RNA interference-mediated tNOX depletion enhances spontaneous apoptosis, prolongs cell cycle progression, and reduces cell migration and the epithelial-mesenchymal transition. We also observed a downregulation of sirtuin 1 (SIRT1) in these tNOX-knockdown cells, a deacetylase that is important in multiple cellular functions. Taken together, our results indicate that capsaicin inhibits the growth of bladder cancer cells by inhibiting tNOX and SIRT1 and thereby reducing proliferation, attenuating migration, and prolonging cell cycle progression.

Capsaicin has been reported to preferentially inhibit the activity of tumor-associated NADH oxidase (tNOX), which belongs to a family of growth-related plasma membrane hydroquinone oxidases in cancer/transformed cells. The inhibitory effect of capsaicin on tNOX is associated with cell growth attenuation and apoptosis. However, no previous study has examined the transcriptional regulation of tNOX protein expression. Bioinformatic analysis has indicated that the tNOX promoter sequence harbors a binding motif for POU3F2, which is thought to play important roles in neuronal differentiation, melanocytes growth/differentiation and tumorigenesis. In this study, we found that capsaicin-mediated tNOX downregulation and cell migration inhibition were through POU3F2. The protein expression levels of POU3F2 and tNOX are positively correlated, and that overexpression of POU3F2 (and the corresponding upregulation of tNOX) enhanced the proliferation, migration and invasion in AGS (human gastric carcinoma) cells. In contrast, knockdown of POU3F2 downregulates tNOX, and the cancer phenotypes are affected. These findings not only shed light on the molecular mechanism of the anticancer properties of capsaicin, but also the transcription regulation of tNOX expression that may potentially explain how POU3F2 is associated with tumorigenesis.

Surface-modified nano-SiO2 is a common additive in many products. However, the safety of nano-SiO2 products under various modifications is still unclear. In this study, we investigated the genotoxicity and acute pulmonary toxicity of nano-SiO2 with or without modification. The samples used in this study included: sample A (SA, 55.16 nm, 411.3 mg/mL), modified sample A (mSA, 82.29 nm, 37.7 mg/mL), sample B (SB, 22 nm, 358.0 mg/mL), and modified sample B (mSB, 86.64 nm, 37.7 mg/mL). In the genotoxicity study, we conducted an Ames test, chromosomal aberration test (CA), and a micronucleus (MN) test. The SA, mSA, and mSB groups showed negative results in all these genotoxicity tests. Only SB showed a weakly positive reaction in these assays, but the genotoxicity could be reversed after S9 metabolism or modification. In the acute pulmonary toxicity test, the rats were given an intratracheal instillation (IT) (0.5 mL/kg) of diluted samples and sacrificed after 1 or 14 days. The mortality rate, number of leukocytes and cytokines of TNF-α in the bronchoalveolar lavage fluid (BALF), and the pathology in the lungs were determined. The results revealed that mSA posed acute toxicity in rats. After modification, the pulmonary toxicity was increased in mSA but decreased in mSB on Day 1, and no significant difference was observed on Day 14. In conclusion, there was no observed genotoxicity in either SA or SB, while mSA posed acute inhalation toxicity to rats that decreased in mSB after modification. This indicates that the decrease in pH level in SA and decrease in the solid content in SB are considered after the trifluorosilane surface-modified amorphous nano-silica.

Epithelial-to-mesenchymal transition (EMT) is a crucial event involved metastasis of certain tumors. Thus, identifying chemical agents that can block EMT is highly warranted for the development of anti-cancer chemoprevention/chemotherapies. In this study, we found that Antrodin C (ADC), a maleimide derivative isolated from Antrodia cinnamomea health food product inhibits TGF-β1-induced EMT and breast cancer cell metastasis in vitro. Pretreatment of MCF-7 cells with ADC significantly blocked TGF-β1-induced phenotypic changes and actin cytoskeleton remodeling. In addition, ADC was able to up-regulate epithelial markers such as E-cadherin and occludin, whereas mesenchymal markers including N-cadherin and vimentin were significantly inhibited, possibly through the modulation of transcriptional regulators Smad/Smad3. ADC blocked TGF-β1-induced migration and invasion of MCF-7 cells through the down-regulation of matrix-metalloproteinases (MMP-2, -9) and urokinase plasminogen activator (uPA). The inhibition of MMPs and uPA activity by ADC was reasoned by suppression of its corresponding transcription factor β-catenin. Taken together, our data suggested that ADC attenuates the TGF-β1-induced EMT, migration and invasion of human breast carcinoma through the suppression of Smad2/3 and β-catenin signaling pathways.

Ursolic acid (UA), a naturally occurring pentacyclic triterpene acid found in many medicinal herbs and edible plants, triggers apoptosis in several tumor cell lines but not in human bone cancer cells. Most recently, we have demonstrated that UA exposure reduces the viability of human osteosarcoma MG-63 cells through enhanced oxidative stress and apoptosis. Interestingly, an inhibitor of osteoclast-mediated bone resorption, zoledronic acid (ZOL), also a third-generation nitrogen-containing bisphosphonate, is effective in the treatment of bone metastases in patients with various solid tumors. In this present study, we found that UA combined with ZOL to significantly suppress cell viability, colony formation, and induce apoptosis in two lines of human osteosarcoma cells. The pre-treatment of the antioxidant had reversed the oxidative stress and cell viability inhibition in the combined treatment, indicating that oxidative stress is important in the combined anti-tumor effects. Moreover, we demonstrated that ZOL combined with UA significantly induced autophagy and co-administration of autophagy inhibitor reduces the growth inhibitory effect of combined treatment. Collectively, these data shed light on the pathways involved in the combined effects of ZOL and UA that might serve as a potential therapy against osteosarcoma.