Investigating impacts of the mycothiazole chemotype as a chemical probe for the study of mitochondrial function and aging.

Small molecule inhibitors of the mitochondrial electron transport chain (ETC) hold significant promise to provide valuable insights to the field of mitochondrial research and aging biology. In this study, we investigated two molecules: mycothiazole (MTZ) - from the marine sponge C. mycofijiensis and its more stable semisynthetic analog 8-O-acetylmycothiazole (8-OAc) as potent and selective chemical probes based on their high efficiency to inhibit ETC complex I function. Similar to rotenone (Rote), MTZ, a newly employed ETC complex I inhibitor, exhibited higher cytotoxicity against cancer cell lines compared to certain non-cancer cell lines. Interestingly, 8-OAc demonstrated greater selectivity for cancer cells when compared to both MTZ and Rote, which has promising potential for anticancer therapeutic development. Furthermore, in vivo experiments with these small molecules utilizing a C. elegans model demonstrate their unexplored potential to investigate aging studies. We observed that both molecules have the ability to induce a mitochondria-specific unfolded protein response (UPRMT) pathway, that extends lifespan of worms when applied in their adult stage. We also found that these two molecules employ different pathways to extend lifespan in worms. Whereas MTZ utilizes the transcription factors ATFS-1 and HSF1, which are involved in the UPRMT and heat shock response (HSR) pathways respectively, 8-OAc only required HSF1 and not ATFS-1 to mediate its effects. This observation underscores the value of applying stable, potent, and selective next generation chemical probes to elucidate an important insight into the functional roles of various protein subunits of ETC complexes and their regulatory mechanisms associated with aging.

Investigating impacts of marine sponge derived mycothiazole and its acetylated derivative on mitochondrial function and aging.

Small molecule inhibitors of the mitochondrial electron transport chain (ETC) hold significant promise to provide valuable insights to the field of mitochondrial research and aging biology. In this study, we investigated two molecules: mycothiazole (MTZ) - from the marine sponge C. mycofijiensis and its more stable semisynthetic analog 8-O-acetylmycothiazole (8-OAc) as potent and selective chemical probes based on their high efficiency to inhibit ETC complex I function. Similar to rotenone (Rote), a widely used ETC complex I inhibitor, these two molecules showed cytotoxicity to cancer cells but strikingly demonstrate a lack of toxicity to non-cancer cells, a highly beneficial feature in the development of anti-cancer therapeutics. Furthermore, in vivo experiments with these small molecules utilizing C.elegans model demonstrate their unexplored potential to investigate aging studies. We observed that both molecules have the ability to induce a mitochondria-specific unfolded protein response (UPRMT) pathway, that extends lifespan of worms when applied in their adult stage. Interestingly, we also found that these two molecules employ different pathways to extend lifespan in worms. Whereas MTZ utilize the transcription factors ATFS-1 and HSF-1, which are involved in the UPRMT and heat shock response (HSR) pathways respectively, 8-OAc only required HSF-1 and not ATFS-1 to mediate its effects. This observation underscores the value of applying stable, potent, and selective next generation chemical probes to elucidate an important insight into the functional roles of various protein subunits of ETC complexes and their regulatory mechanisms associated with aging.

IL-13 and the hydroperoxy fatty acid 13(S)HpODE play crucial role in inducing an apoptotic pathway in cancer cells involving MAO-A/ROS/p53/p21 signaling axis.

This study depicted the effect of IL-13 and 13(S)HpODE (the endogenous product during IL-13 activation) in the process of cancer cell apoptosis. We examined the role of both IL-13 and 13(S)HpODE in mediating apoptotic pathway in three different in vitro cellular models namely A549 lung cancer, HCT116 colorectal cancer and CCF52 GBM cells. Our data showed that IL-13 promotes apoptosis of A549 lung carcinoma cells through the involvement of 15-LO, PPARγ and MAO-A. Our observations demonstrated that IL-13/13(S)HpODE stimulate MAO-A-mediated intracellular ROS production and p53 as well as p21 induction which play a crucial role in IL-13-stimulated A549 cell apoptosis. We further showed that 13(S)HpODE promotes apoptosis of HCT116 and CCF52 cells through the up-regulation of p53 and p21 expression. Our data delineated that IL-13 stimulates p53 and p21 induction which is mediated through 15-LO and MAO-A in A549 cells. In addition, we observed that PPARγ plays a vital role in apoptosis as well as in p53 and p21 expression in A549 cells in the presence of IL-13. We validated our observations in case of an in vivo colon cancer tumorigenic study using syngeneic mice model and demonstrated that 13(S)HpODE significantly reduces solid tumor growth through the activation of apoptosis. These data thus confirmed that IL-13 > 15-LO>13(S)HpODE > PPARγ>MAO-A > ROS > p53>p21 axis has a major contribution in regulating cancer cell apoptosis and further identified 13(S)HpODE as a potential chemo-preventive agent which can improve the efficacy of cancer treatment as a combination compound.

Alkaloid-rich fraction of Ervatamia coronaria sensitizes colorectal cancer through modulating AMPK and mTOR signalling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Ervatamia coronaria, a popular garden plant in India and some other parts of the world is known traditionally for its anti-inflammatory and anti-cancer properties. The molecular bases of these functions remain poorly understood. AIM OF THE STUDY: Efficacies of the existing therapies for colorectal cancer (CRC) are limited by their life-threatening side effects and unaffordability. Therefore, identifying a safer, efficient, and affordable therapeutic is urgent. We studied the anti-CRC activity of an alkaloid-rich fraction of E. coronaria leaf extracts (AFE) and associated underlying mechanism. MATERIALS AND METHODS: Activity guided solvant fractionation was adopted to identify the activity in AFE. Different cell lines, and tumor grown in syngeneic mice were used to understand the anti-CRC effect. Methodologies such as LCMS, MTT, RT-qPCR, immunoblot, immunohistochemistry were employed to understand the molecular basis of its activity. RESULTS: We showed that AFE, which carries about six major compounds, is highly toxic to colorectal cancer (CRC) cells. AFE induced cell cycle arrest at G1 phase and p21 and p27 genes, while those of CDK2, CDK-4, cyclin-D, and cyclin-E genes were downregulated in HCT116 cells. It predominantly induced apoptosis in HCT116p53+/+ cells while the HCT116p53-/- cells under the same treatment condition died by autophagy. Notably, AFE induced upregulation of AMPK phosphorylation, and inhibition of both of the mTOR complexes as indicated by inhibition of phosphorylation of S6K1, 4EBP1, and AKT. Furthermore, AFE inhibited mTOR-driven conversion of cells from reversible cell cycle arrest to senescence (geroconversion) as well as ERK activity. AFE activity was independent of ROS produced, and did not primarily target the cellular DNA or cytoskeleton. AFE also efficiently regressed CT26-derived solid tumor in Balb/c mice acting alone or in synergy with 5FU through inducing autophagy as a major mechanism of action as indicated by upregulation of Beclin 1 and phospho-AMPK, and inhibition of phospho-S6K1 levels in the tumor tissue lysates. CONCLUSION: AFE induced CRC death through activation of both apoptotic and autophagy pathways without affecting the normal cells. This study provided a logical basis for consideration of AFE in future therapy regimen to overcome the limitations associated with existing anti-CRC chemotherapy.

Induction of monoamine oxidase A-mediated oxidative stress and impairment of NRF2-antioxidant defence response by polyphenol-rich fraction of Bergenia ligulata sensitizes prostate cancer cells in vitro and in vivo.

Prostate cancer (PCa) is a major cause of mortality and morbidity in men. Available therapies yield limited outcome. We explored anti-PCa activity in a polyphenol-rich fraction of Bergenia ligulata (PFBL), a plant used in Indian traditional and folk medicine for its anti-inflammatory and antineoplastic properties. PFBL constituted of about fifteen different compounds as per LCMS analysis induced apoptotic death in both androgen-dependent LNCaP and androgen-refractory PC3 and DU145 cells with little effect on NKE and WI38 cells. Further investigation revealed that PFBL mediates its function through upregulating ROS production by enhanced catalytic activity of Monoamine oxidase A (MAO-A). Notably, the differential inactivation of NRF2-antioxidant response pathway by PFBL resulted in death in PC3 versus NKE cells involving GSK-3ß activity facilitated by AKT inhibition. PFBL efficiently reduced the PC3-tumor xenograft in NOD-SCID mice alone and in synergy with Paclitaxel. Tumor tissues in PFBL-treated mice showed upregulation of similar mechanism of cell death as observed in isolated PC3 cells i.e., elevation of MAO-A catalytic activity, ROS production accompanied by activation of ß-TrCP-GSK-3ß axis of NRF2 degradation. Blood counts, liver, and splenocyte sensitivity analyses justified the PFBL safety in the healthy mice. To our knowledge this is the first report of an activity that crippled NRF2 activation both in vitro and in vivo in response to MAO-A activation. Results of this study suggest the development of a novel treatment protocol utilizing PFBL to improve therapeutic outcome for patients with aggressive PCa which claims hundreds of thousands of lives each year.

Targeting cellular microtubule by phytochemical apocynin exhibits autophagy-mediated apoptosis to inhibit lung carcinoma progression and tumorigenesis.

BACKGROUND: Lung cancer is the leading cause of cancer-related deaths worldwide. Several targets have been identified for lung cancer therapy, amongst which 'Microtubule' and its dynamics are the most widely studied and used in therapy. Tubulin-microtubule polymer dynamics are highly sought after targets in the field of anti-cancer drug designing. Natural compounds are important sources for developing anticancer therapeutics owing to their efficacy and lower cytotoxicity. Evidence suggested that therapeutic targeting of microtubule by natural compounds is amongst the most widely used interventions in numerous cancer therapies including lung cancer. PURPOSE: To determine the efficacy of apocynin (a natural compound) in suppressing the progression of lung carcinoma both in vitro and in vivo, along with the identification of targets and the underlying mechanism for developing a novel therapeutic approach. METHODS: We have demonstrated themicrotubule depolymerizing role of apocynin by established protocols in cellular and cell-free system. The efficacy of apocynin to inhibit lung carcinoma progression was studied on A549 cells.The tumoricidal ability of apocynin was studied in BALB/c mice model as well.Mice were classified into 4 groups namely-group II mice as tumor control; group III-IV mice asalso tumor-induced but treated with differential apocynin doses whereas group I mice were kept as normal. RESULTS: Apocynin, showed selective cytotoxicity towards lung cancer cells rather than normal lung fibroblast cells. Apocynin inhibited oncogenic properties including growth, proliferation (p < 0.05), colony formation (p < 0.05), invasion (p < 0.05) and spheroid formation (p < 0.05) in lung cancer cells. Apart from other established properties, apocynin was found to be a novel and potent component to bind with tubulin and depolymerize cellular microtubule network. Apocynin mediated cellular microtubule depolymerization was the driving mechanism to trigger autophagy-mediated apoptotic cell death (p < 0.05) which in turn retarded lung cancer progression. Furthermore, apocynin showed tumoricidal characteristics to inhibit lung tumorigenesis in mice as well. CONCLUSION: Targeting tubulin-microtubule equilibrium with apocynin could be the key regulator to catastrophe cellular catabolic processes to mitigate lung carcinoma. Thus, apocynin could be a potential therapeutic agent for lung cancer treatment.

S-allyl cysteine inhibits TNF-α-induced inflammation in HaCaT keratinocytes by inhibition of NF- κB-dependent gene expression via sustained ERK activation.

Tumor necrosis factor-α (TNF-α)-induced keratinocyte inflammation plays a key role in the pathogenesis of multiple inflammatory skin diseases. Here we investigated the anti-inflammatory effect of S-allyl cysteine (SAC) on TNF-α-induced HaCaT keratinocyte cells and the mechanism behind its anti-inflammatory potential. SAC was found to inhibit TNF-α-stimulated cytokine expression. Further, SAC was found to inhibit TNF-α-induced activation of p38, JNK and NF-κB pathways. Interestingly, SAC was found to differentially regulate ERK MAP kinase in cells. TNF-α-induced transient ERK activation and SAC treatment resulted in sustained ERK activation both in the presence and absence of TNF-α. Additionally, SAC failed to inhibit the TNF-α-induced expression of the pro-inflammatory cytokines TNF-α and IL-1ß when cells were treated with the MEK inhibitor PD98059, suggesting that the anti-inflammatory effect of SAC is via sustained activation of the ERK pathway. Since ERK activation has been reported to negatively regulate NF-κB-driven gene expression and we find that SAC activates ERK and negatively regulates NF-κB, we investigated whether there existed any crosstalk between the ERK and the NF-κB pathways. NF-κB-dependent reporter assay, visualization of the nuclear translocation of NF-κB-p65 subunit and determination of the cellular levels of I-κB, the inhibitor of NF-κB, revealed that SAC inhibited TNF-α-induced NF-κB activation, and PD98059 treatment reversed this effect. These results collectively suggest that SAC inhibits TNF-α-induced inflammation in HaCaT cells via a combined effect entailing the inhibition of the p38 and the JNK pathways and NF-κB pathway via the sustained activation of ERK.

Azadiradione ameliorates polyglutamine expansion disease in Drosophila by potentiating DNA binding activity of heat shock factor 1.

Aggregation of proteins with the expansion of polyglutamine tracts in the brain underlies progressive genetic neurodegenerative diseases (NDs) like Huntington's disease and spinocerebellar ataxias (SCA). An insensitive cellular proteotoxic stress response to non-native protein oligomers is common in such conditions. Indeed, upregulation of heat shock factor 1 (HSF1) function and its target protein chaperone expression has shown promising results in animal models of NDs. Using an HSF1 sensitive cell based reporter screening, we have isolated azadiradione (AZD) from the methanolic extract of seeds of Azadirachta indica, a plant known for its multifarious medicinal properties. We show that AZD ameliorates toxicity due to protein aggregation in cell and fly models of polyglutamine expansion diseases to a great extent. All these effects are correlated with activation of HSF1 function and expression of its target protein chaperone genes. Notably, HSF1 activation by AZD is independent of cellular HSP90 or proteasome function. Furthermore, we show that AZD directly interacts with purified human HSF1 with high specificity, and facilitates binding of HSF1 to its recognition sequence with higher affinity. These unique findings qualify AZD as an ideal lead molecule for consideration for drug development against NDs that affect millions worldwide.