OncoTargets and Therapy (Oct 2018)
16-Hydroxycleroda-3,13-dien-15,16-olide induces anoikis in human renal cell carcinoma cells: involvement of focal adhesion disassembly and signaling
Abstract
Yu-Chi Chen,1 Bu-Miin Huang,2 Wei-Chang Lee,3 Yung-Chia Chen3,4 1Department of Urology, E-Da Hospital, Kaohsiung, Taiwan; 2Department of Cell Biology and Anatomy, College of Medicine, National Cheng Kung University, Tainan, Taiwan; 3Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan; 4Department of Anatomy, School of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan Background: Clerodane diterpene, 16-hydroxycleroda-3,13-dien-15,16-olide (CD) isolated from Polyalthia longifolia Benth. & Hook. f. var. pendula was found to be a potential apoptotic inducer in human leukemia, lung cancer, and colon cancer cells. However, the molecular mechanism remains elusive in renal system. Thus, in the present study, the regulatory mechanisms of CD-induced apoptosis in clear cell renal cell carcinoma (ccRCC) cells were investigated. Materials and methods: Cell proliferation was evaluated by colony formation assay and cell cycle analyses. Protein expressions of focal adhesion (FA) related complexes were examined by immunofluorescence staining and Western blot analyses. Cell migration and invasion capabilities of renal cell carcinoma (RCC) cells were determined by wound healing and Transwell assays. Results: CD inhibited cell colony formations, induced cell arrest at G2/M phase, and increased subG1 cell population both in 786-O and A-498. During CD treatment, the “rounded-up” cells were observed. The immune-staining of phosphorylated focal adhesion kinase (pFAK), vinculin, and paxillin displayed disassembly of the FA. Moreover, disruption of actin stress fibers was noted after CD treatment. Consistent with the findings, the expressions of pSrc, pFAK, FAK, vinculin, vimentin, and paxillin were all downregulated by CD. In addition, CD attenuated cell migration and invasion activities accompanied by the reductions of pNF-κB, matrix metalloproteinase (MMP)-2, MMP-9 as well as vascular endothelial growth factor expressions. Conclusion: CD induced cell cycle arrest, FA complex disassembly, and the inactivation of migratory-related signaling pathways to induce apoptosis in ccRCC cells. Keywords: focal adhesion, signaling pathway, Polyalthia longifolia, clerodane diterpene, migration, invasion, renal cell carcinoma, RCC
