Pharbitis Nil (PN) induces apoptosis and autophagy in lung cancer cells and autophagy inhibition enhances PN-induced apoptosis

Abstract

Ethnopharmacological relevance: Pharbitis Nil (PN) is used as a main component of the existing drug, DA-9701, which was developed to treat functional dyspepsia (FD) in Korea. PN extracts isolated from its seeds have been reported to have anticancer effects. Aim of the study: The purpose of this study was to investigate the underlying mechanism of the chemotherapeutic effects of PN in lung cancer cells. Materials and methods: We performed MTT assays, colony formation assays, flow cytometry assays, Western blot analysis, reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence analysis, and cell counting assays to study the molecular mechanism of chemotherapeutic effects of PN in lung cancer cells. Results: Our results indicate that PN induced autophagy as well as apoptosis. PN inhibited cell proliferation and survival by inducing apoptosis in several lung cancer cell lines. PN-treated cells also exhibited induction of autophagy, as evidenced by increased protein expression levels and punctuate patterns of LC3 II. Moreover, activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), which plays an important role in autophagy activation, was shown to be related with PN-induced autophagy. Interestingly, pharmacological blockade of autophagy activation with wortmannin and inhibition of ERK1/2 phosphorylation by U0126 markedly enhanced PN-induced apoptosis and reduced cell viability, suggesting that autophagy induced by PN may have a cytoprotective effect by suppressing apoptosis. PN- induced apoptosis was regulated by signal transducer and activator of transcription 3 (STAT3) deactivation. Moreover, decrease of STAT3 activation in PN-treated cells was associated with reduced survivin expression, further demonstrating that PN-induced apoptosis was regulated by STAT3 deactivation. Conclusion: We believe that PN, which is already proven to treat human patients with FD, might be a potential anticancer drug for human lung cancer. In addition, our data suggest that the combination of PN treatment with an autophagy inhibitor or traditional anticancer agents may be an effective anticancer therapy.