Nimbolide, a neem limonoid inhibits cytoprotective autophagy to activate apoptosis via modulation of the PI3K/Akt/GSK-3\u03b2 signalling pathway in oral cancer

Josephraj Sophia,Sujit K Bhutia,Siddavaram Nagini,Rajakishore Mishra,Jaganathan Kowshik,Bramanandam Manavathi,Anju Dwivedi

doi:10.1038/s41419-018-1126-4

Abstract

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.

Highlights

Oral squamous cell carcinoma (OSCC), the sixth most common malignancy worldwide with an annual incidence of over 300,000 newly diagnosed cases is one of the major public health problems due to the rising incidence of the disease especially among the younger population[1,2,3]
We investigated the effect of nimbolide on autophagy and the time point at which the phosphorylation status of glycogen synthase kinase-3β (GSK-3β) and PI3K dictates the choice between autophagy and apoptosis in nimbolide treated SCC131 and SCC4 oral cancer cell lines
To assess whether nimbolide-induced cytotoxicity is caused by apoptosis, we examined the nuclear morphology of SCC131 and SCC4 cells using the fluorescent DNA-binding dye, DAPI

Summary

Introduction

Oral squamous cell carcinoma (OSCC), the sixth most common malignancy worldwide with an annual incidence of over 300,000 newly diagnosed cases is one of the major public health problems due to the rising incidence of the disease especially among the younger population[1,2,3]. Novel molecularly targeted intervention strategies are required to inhibit the development and progression of OSCC. Nimbolide was shown to inhibit the development and progression of 7,12-dimethylbenz[a]anthracene (DMBA)induced hamster buccal pouch (HBP) carcinomas that closely emulate human OSCCs in histology, precancerous lesions, propensity for invasion and metastasis and gene expression signatures[14,15,16,17,18]

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