Activation of PI3K/Akt/NF-kB Signaling Mediates Swedish Snus Induced Proliferation and Apoptosis Evasion in the Rat Forestomach: Modulation by Blueberry.

BACKGROUND AND OBJECTIVES: The present study was undertaken to ascertain whether the modulatory effects of blueberries on cell proliferation induced by Swedish snus in the rat forestomach epithelium is mediated via abrogation of the PI3K/Akt/NFκB signaling axis that regulates cell fate decision. METHODS: The transcript and protein expression of genes involved in cell cycle progression and apoptosis, as well as canonical PI3K/Akt/NF-κB signaling pathways, were analyzed by qRT-PCR, immunoblotting and ELISA. Expression profiling of noncoding RNAs (ncRNAs) that influence PI3K/Akt/NF-κB signaling was undertaken. TUNEL assay was performed using flow cytometry. RESULTS: Administration of snus induced basal cell hyperplasia in the rat forestomach with increased cell proliferation and inhibition of apoptosis. This was associated with the activation of PI3K/Akt/NFκB signaling. Coadministration of blueberries significantly suppressed snus-induced hyperplasia. Analysis of the molecular mechanisms revealed that blueberries suppress the phosphorylation of Akt, NF-κB and IKKß, prevent nuclear translocation of NF-κB and modulate the expression of microRNAs that influence PI3K/Akt/NF-κB signaling. CONCLUSION: Taken together, the results of the current study provide compelling evidence that blueberries exert significant protective effects against snus-induced soft tissue changes in the rat forestomach epithelium mediated by inhibiting key molecular players in the PI3K/Akt/NF-κB signaling axis. Long-term studies on the impact of snus exposure on various cellular processes, signaling pathways, and the interplay between genetic and epigenetic mechanisms are however warranted. The results of this investigation may contribute to the development of protection against soft tissue changes induced by smokeless tobacco in the human oral cavity.

Astaxanthin inhibits hallmarks of cancer by targeting the PI3K/NF-κΒ/STAT3 signalling axis in oral squamous cell carcinoma models.

Aberrant activation of the PI3K/Akt signalling pathway, a major driving force of diverse cellular processes has been implicated in tumour development and progression. Here, we report that astaxanthin (AXT), a potent antioxidant ketocarotenoid prevents cancer hallmarks by inhibiting PI3K/Akt and the associated downstream NF-κB and STAT-3 signalling pathways in SCC131 and SCC4 oral cancer cells as well as in the hamster buccal pouch carcinogenesis model. Using small molecule inhibitors of NF-κB, STAT-3 and PI3K and by overexpression of PI3K, we provide evidence to show that AXT inhibits NF-κB and STAT-3 signalling and cancer hallmarks by restraining the kinase activity of PI3K/Akt. Additionally, AXT downregulated the noncoding RNAs (ncRNAs), miR-21 and HOTAIR that influence PI3K/Akt signalling emphasising its modulatory effects on epigenetic regulation. Ethyl cellulose-based AXT nanoparticles showed greater chemotherapeutic efficacy in the hamster oral carcinogenesis model compared to native AXT. We suggest that AXT prevents cell proliferation, apoptosis evasion, invasion and angiogenesis by intercepting the crosstalk between the PI3K/Akt, NF-κB and STAT-3 signalling circuits both in vitro and in vivo. Astaxanthin that abrogates the PI3K/Akt signalling axis, a central hub that orchestrates acquisition of cancer hallmarks is a promising candidate for anticancer drug development.

Nimbolide, a neem limonoid inhibits cytoprotective autophagy to activate apoptosis via modulation of the PI3K/Akt/GSK-3ß signalling pathway in oral cancer.

Of late, nimbolide, a limonoid from the neem tree (Azadirachta indica) has gained increasing research attention owing to its potent antiproliferative and apoptosis-inducing effects. The present study was designed to investigate the effect of nimbolide on autophagy and the time point at which the phosphorylation status of GSK-3ß and PI3K dictate the choice between autophagy and apoptosis in SCC131 and SCC4 oral cancer cells. Additionally, we analysed changes in the expression of proteins involved in autophagy and apoptosis after therapeutic intervention with nimbolide in a hamster model of oral oncogenesis. Furthermore, we also demonstrate changes in the expression of key genes involved in apoptosis and autophagy during the stepwise evolution of hamster and human OSCCs. Nimbolide-induced stereotypical changes in oral cancer cells characteristic of both apoptosis and autophagy. Time-course experiments revealed that nimbolide induces autophagy as an early event and then switches over to apoptosis. Nimbolide negatively regulates PI3K/Akt signalling with consequent increase in p-GSK-3ßTyr216, the active form of GSK-3ß that inhibits autophagy. Downregulation of HOTAIR, a competing endogenous RNA that sponges miR-126 may be a major contributor to the inactivation of PI3K/Akt/GSK3 signalling by nimbolide. Analysis of key markers of apoptosis and autophagy as well as p-AktSer473 during sequential progression of hamster and human OSCC revealed a gradual evolution to a pro-autophagic and antiapoptotic phenotype that could confer a survival advantage to tumors. In summary, the results of the present study provide insights into the molecular mechanisms by which nimbolide augments apoptosis by overcoming the shielding effects of cytoprotective autophagy through modulation of the phosphorylation status of Akt and GSK-3ß as well as the ncRNAs miR-126 and HOTAIR. Development of phytochemicals such as nimbolide that target the complex interaction between proteins and ncRNAs that regulate the autophagy/apoptosis flux is of paramount importance in cancer prevention and therapeutics.

Gedunin, A Neem Limonoid in Combination with Epalrestat Inhibits Cancer Hallmarks by Attenuating Aldose Reductase-Driven Oncogenic Signaling in SCC131 Oral Cancer Cells.

BACKGROUND AND OBJECTIVES: Aldose Reductase (AR), a polyol pathway enzyme that mediates diabetic complications is implicated in tumour development and progression. This study was undertaken to determine whether gedunin, a neem limonoid prevents the hallmarks of cancer by inhibiting AR and the associated downstream PI3K/Akt/mTOR/ERK/NF-κB signalling axis in the SCC131 oral cancer cell line. METHODS: The expression of AR and key molecules involved in cell proliferation, apoptosis, autophagy, invasion and angiogenesis was analysed by qRT-PCR, and immunoblotting. ROS generation and cell cycle were analysed by FACS. Alamar blue assay and scratch assay were used to evaluate cell proliferation and migration in the endothelial cell line Eahy926. RESULTS: Gedunin and the AR inhibitor epalrestat inhibited AR expression and ROS generation. Cell cycle arrest at G1/S was associated with cell death by autophagy with subsequent switch over to apoptosis. Furthermore, hypoxia-induced cell migration was inhibited in Eahy926 cells with downregulation of pro-invasive and proangiogenic proteins in SCC131 as well as Eahy926 cells. Co-inactivation of Akt and ERK was coupled with abrogation of IKK/NF-κB signaling. However, the combination of gedunin and epalrestat was more effective than single agents. CONCLUSION: Inhibition of AR-mediated ROS signalling may be a key mechanism by which gedunin and epalrestat exert their anticancer effects. Our results provide compelling evidence that the combination of gedunin and epalrestat modulates expression of key oncogenic signalling kinases and transcription factors primarily by influencing phosphorylation and subcellular localisation. AR inhibitors such as gedunin and epalrestat are novel candidate agents for cancer prevention and therapy.

Nimbolide upregulates RECK by targeting miR-21 and HIF-1α in cell lines and in a hamster oral carcinogenesis model.

Reversion-inducing cysteine-rich protein with Kazal motifs (RECK), a potent inhibitor of matrix metalloproteinases (MMPs) is a common negative target of oncogenic signals and a potential therapeutic target for novel drug development. Here, we show that sequential RECKlessness stimulates angiogenesis and Notch signalling in the 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis model, a paradigm for oral oncogenesis and chemointervention. We also report the chemotherapeutic effect of nimbolide, a limonoid from the neem tree (Azadirachta indica) based on the upregulation of RECK as well as modulation of the expression of key molecules involved in invasion and angiogenesis. We demonstrate that nimbolide upregulates RECK by targeting miR-21, and HIF-1α resulting in reduced MMP activity and blockade of VEGF and Notch signalling. Nimbolide reduced microvascular density, confirming its anti-angiogenic potential. Molecular docking analysis revealed interaction of nimbolide with HIF-1α. Additionally, we demonstrate that nimbolide upregulates RECK expression via downregulation of HIF-1α and miR-21 by overexpression and knockdown experiments in SCC4 and EAhy926 cell lines. Taken together, these findings provide compelling evidence that targeting RECK, a keystone protein that regulates mediators of invasion and angiogenesis with phytochemicals such as nimbolide may be a robust therapeutic approach to prevent oral cancer progression.

Exposure to welding fumes activates DNA damage response and redox-sensitive transcription factor signalling in Sprague-Dawley rats.

BACKGROUND: Occupational exposure to welding fumes containing a complex mixture of genotoxic heavy metals, radiation, gases and nanoparticles poses a serious health hazard to welders. Since their categorization as possible carcinogens, welding fumes have gained increasing attention as high priority agents for risk assessment. OBJECTIVE: The present study was undertaken to investigate the effects of welding fume inhalation on oxidative stress, DNA damage response (DDR), and nuclear factor erythroid 2-related factor-2 (Nrf2) and nuclear factor kappa B (NFκB) signalling in the lung tissues of male Sprague-Dawley rats. METHODS: Animals were divided into five groups. Group 1 animals served as control. Rats in groups 2-5 were exposed to 50mg/m3 stainless steel (SS) welding fumes for 1h for 1day, 1 week, 2 weeks, and 4 weeks respectively. Reactive oxygen species (ROS) generation, 8-oxo-2'-deoxyguanosine (8-oxodG), xenobiotic-metabolizing enzymes (XMEs) and antioxidants were analysed. DNA damage sensors, DNA repair enzymes, inflammatory mediators, cell cycle progression, apoptosis and key players in Nrf2 and NFκB signalling were assessed by flow cytometry, quantitative real-time reverse transcriptase PCR, immunoblotting, immunohistochemistry and immunofluorescence. RESULTS: Rats exposed to welding fumes showed increased levels of chromium and ROS in lung tissues associated with accumulation of 8-oxodG and enhanced expression of XMEs and antioxidants. This was accompanied by upregulation of DNA damage sensors, cell cycle arrest in G1/S phase, overexpression of a multitude of DNA repair enzymes and caspase-mediated apoptosis. In addition, exposure to welding fumes induced activation of Nrf2 and NFκB signalling with enhanced expression of inflammatory mediators. CONCLUSION: The results of the present study unequivocally demonstrate that exposure of rats to SS welding fumes alters the expression of 37 genes involved in oxidative stress, detoxification, inflammation, DNA repair, cell cycle progression, and apoptosis. Activation of DDR and the ROS-sensitive Nrf2 and NFκB signalling pathways may be key molecular events that mediate adaptive cellular response to welding fume exposure.

Blueberry inhibits invasion and angiogenesis in 7,12-dimethylbenz[a]anthracene (DMBA)-induced oral squamous cell carcinogenesis in hamsters via suppression of TGF-ß and NF-κB signaling pathways.

Aberrant activation of oncogenic signaling pathways plays a pivotal role in tumor initiation and progression. The purpose of the present study was to investigate the chemopreventive and therapeutic efficacy of blueberry in the hamster buccal pouch (HBP) carcinogenesis model based on its ability to target TGF-ß, PI3K/Akt, MAPK and NF-κB signaling and its impact on invasion and angiogenesis. Squamous cell carcinomas were induced in the HBP by 7,12-dimethylbenz[a]anthracene (DMBA). The effect of blueberry on the oncogenic signaling pathways and downstream events was analyzed by quantitative real-time PCR and immunoblotting. Experiments with the ECV304 cell line were performed to explore the mechanism by which blueberry regulates angiogenesis. Blueberry supplementation inhibited the development and progression of HBP carcinomas by abrogating TGF-ß and PI3K/Akt pathways. Although blueberry failed to influence MAPK, it suppressed NF-κB activation by preventing nuclear translocation of NF-κB p65. Blueberry also modulated the expression of the oncomiR miR-21 and the tumor suppressor let-7. Collectively, these changes induced a shift to an anti-invasive and anti-angiogenic phenotype as evidenced by downregulating matrix metalloproteinases and vascular endothelial growth factor. Blueberry also inhibited angiogenesis in ECV304 cells by suppressing migration and tube formation. The results of the present study suggest that targeting oncogenic signaling pathways that influence acquisition of cancer hallmarks is an effective strategy for chemointervention. Identification of modulatory effects on phosphorylation, intracellular localization of oncogenic transcription factors and microRNAs unraveled by the present study as key mechanisms of action of blueberry is critical from a therapeutic perspective.

The Hamster Buccal Pouch Model of Oral Carcinogenesis.

The hamster buccal pouch (HBP) carcinogenesis model is one of the most well-characterized animal tumor models used as a prelude to investigate multistage oral carcinogenesis and to assess the efficacy of chemointervention. Hamster buccal pouch carcinomas induced by 7,12-dimethylbenz[a]anthracene (DMBA) show extensive similarities to human oral squamous cell carcinomas. The HBP model offers a number of advantages including a simple and predictable tumor induction procedure, easy accessibility for examination and follow-up of lesions, and reproducibility. This model can be used to test both chemopreventive and chemotherapeutic agents.

Nimbolide, a neem limonoid inhibits Phosphatidyl Inositol-3 Kinase to activate Glycogen Synthase Kinase-3ß in a hamster model of oral oncogenesis.

Glycogen synthase kinase-3ß (GSK-3ß), a serine/threonine kinase is frequently inactivated by the oncogenic signalling kinases PI3K/Akt and MAPK/ERK in diverse malignancies. The present study was designed to investigate GSK-3ß signalling circuits in the 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis model and the therapeutic potential of the neem limonoid nimbolide. Inactivation of GSK-3ß by phosphorylation at serine 9 and activation of PI3K/Akt, MAPK/ERK and ß-catenin was associated with increased cell proliferation and apoptosis evasion during stepwise evolution of HBP carcinomas. Administration of nimbolide inhibited PI3K/Akt signalling with consequent activation of GSK-3ß thereby inducing trafficking of ß-catenin away from the nucleus and enhancing the expression of miR-126 and let-7. Molecular docking studies confirmed interaction of nimbolide with PI3K, Akt, ERK and GSK-3ß. Furthermore, nimbolide attenuated cell proliferation and induced apoptosis as evidenced by increased p-cyclin D1(Thr286) and pro-apoptotic proteins. The present study has unravelled aberrant phosphorylation as a key determinant for oncogenic signalling and acquisition of cancer hallmarks in the HBP model. The study has also provided mechanistic insights into the chemotherapeutic potential of nimbolide that may be a useful addition to the armamentarium of natural compounds targeting PI3K for oral cancer treatment.

Ellagic acid inhibits VEGF/VEGFR2, PI3K/Akt and MAPK signaling cascades in the hamster cheek pouch carcinogenesis model.

BACKGROUND: Blocking vascular endothelial growth factor (VEGF) mediated tumor angiogenesis by phytochemicals has emerged as an attractive strategy for cancer prevention and therapy. METHODS: We investigated the anti-angiogenic effects of ellagic acid in a hamster model of oral oncogenesis by examining the transcript and protein expression of hypoxia-inducible factor-1alpha (HIF-1α), VEGF, VEGFR2, and the members of the PI3K/Akt and MAPK signaling cascades. Molecular docking studies and cell culture experiments with the endothelial cell line ECV304 were performed to delineate the mechanism by which ellagic acid regulates VEGF signaling. RESULTS: We found that ellagic acid significantly inhibits HIF-1α-induced VEGF/VEGFR2 signalling in the hamster buccal pouch by abrogating PI3K/Akt and MAPK signaling via downregulation of PI3K, PDK-1, p-Akt(ser473), mTOR, p-ERK, and p-JNK. Ellagic acid was also found to reduce the expression of histone deacetylases that could inhibit neovascularization. Analysis of the mechanism revealed that ellagic acid inhibits hypoxia-induced angiogenesis via suppression of HDAC-6 in ECV304 cells. Furthermore, knockdown of endogenous HDAC6 via small interfering RNA abrogated hypoxia-induced expression of HIF-1α and VEGF and blocked Akt activation. Molecular docking studies confirmed interaction of ellagic acid with upstream kinases that regulate angiogenic signaling. CONCLUSIONS: Taken together, these findings demonstrate that the anti-angiogenic activity of ellagic acid may be mediated by abrogation of hypoxia driven PI3K/Akt/mTOR, MAPK and VEGF/VEGFR2 signaling pathways involving suppression of HDAC6 and HIF-1α responses. GENERAL SIGNIFICANCE: Ellagic acid offers promise as a lead compound for anticancer therapeutics by virtue of its ability to inhibit key oncogenic signaling cascades and HDACs.