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-3β 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-3βTyr216, the active form of GSK-3β 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-3β 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.

No abstract

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.

Glycogen synthase kinase-3β (GSK-3β), 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-3β 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-3β by phosphorylation at serine 9 and activation of PI3K/Akt, MAPK/ERK and β-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-3β thereby inducing trafficking of β-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-3β. Furthermore, nimbolide attenuated cell proliferation and induced apoptosis as evidenced by increased p-cyclin D1Thr286 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.

No abstract