β-elemene enhances the radiosensitivity of gastric cancer cells by inhibiting Pak1 activation

AIM: To explore the potential of beta-elemene as a radiosensitizer for gastric cancer cells and the underlying mechanisms. METHODS: SGC7901, MKN45, MKN28, N87, and AGS human gastric cancer cell lines were used to screen for radioresistant gastric cancer cell lines. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay was used to determine the effects of beta-elemene and IPA-3 on cell viability in MKN45 and SGC7901 gastric cancer cell lines. A clonogenic survival assay and annexin V-FITC/PI apoptosis detection assay were used to evaluate cellular radiosensitivity and radiation-induced cell death, respectively. A proteomic method, isobaric tags for relative and absolute quantitation (iTRAQ), was employed to screen the proteins regulated by beta-elemene pretreatment prior to ionizing radiation (IR) in SGC7901 gastric cancer cell line. IPA-3 was used as a specific small molecule inhibitor of p21-activated protein kinase 1 (Pak1) to target Pak1 signaling. Protein levels of PAK1IP1 (p21-activated protein kinase-interacting protein 1), total Pak1 (t-Pak1), phospho-Pak1 (T423), phospho-ERK1/2 (Thr202/Tyr204), and cleaved caspase-3 (17 kDa) were assessed by western blotting. RESULTS: MKN45 and SGC7901 gastric cancer cell lines were relatively more resistant to IR. beta-elemene pretreatment decreased clonogenic survival following IR in MKN45 and SGC7901 gastric cancer cell lines. Additionally, beta-elemene pretreatment prior to IR increased radiation-induced cell death compared with IR alone in MKN45 (10.4% +/- 0.9% vs 34.8%+/- 2.8%, p < 0.05) and SGC7901 (11.6% +/- 0.9% vs 46.7% +/- 5.2%, p < 0.05) human gastric cancer cell lines, respectively, consistent with the level of cleaved caspase-3 (17 kDa). Through iTRAQ analysis and western blot validation, we found that beta-elemene upregulated PAK1IP1 and downregulated phospho-Pak1 (T423) and phospho-ERK1/2 in SGC7901 gastric cancer cells. IR increased the level of phospho-Pak1 (T423). Pretreatment with beta-elemene decreased radiation-induced Pak1 and ERK1/2 phosphorylation. Inhibition of Pak1 using IPA-3 decreased clonogenic survival following IR. In addition, IPA-3 increased radiation-induced cell death in MKN45 (13.4% +/- 0.3% vs 26.6% +/- 1.0%, p < 0.05) and SGC7901 (16.0% +/- 0.6% vs 37.3% +/- 1.7%, p < 0.05) gastric cancer cell lines, respectively, consistent with the level of cleaved caspase-3 (17 kDa). Western blotting showed that IPA-3 decreased radiation-induced Pak1 and ERK1/2 phosphorylation. CONCLUSION: This is the first demonstration that beta-elemene enhances radiosensitivity of gastric cancer cells, and that the mechanism involves inhibition of Pak1 signaling.