Folliculin deficient renal cancer cells show higher radiosensitivity through autophagic cell death.

PURPOSE: Mutations in the FLCN gene are responsible for fibrofolliculoma, pulmonary and renal cysts, and renal cell carcinoma in patients with Birt-Hogg-Dubé syndrome. To explore therapeutic approaches to renal cell carcinoma in patients with Birt-Hogg-Dubé syndrome we investigated the anticancer effects of irradiation on folliculin deficient renal cancer cells. MATERIALS AND METHODS: Folliculin deficient (UOK257 and ACHN-5968) and folliculin expressing (UOK257-2 and ACHN-sc) cell lines were used in this study. Clonogenic assays were used to determine the radiosensitivity of folliculin deficient and expressing renal cell carcinoma cells. Apoptosis was detected in these cells by DAPI and TUNEL assays after irradiation. Monodansylcadaverine analysis, GFP-LC3 assay and Western blot were performed to monitor the autophagic process. RESULTS: Folliculin deficient cells were more sensitive to irradiation than their folliculin expressing counterparts. The enhanced effects of irradiation on folliculin deficient cells were mediated by increased autophagy but not by apoptosis. An increased Beclin 1 protein level and an activated mitogen-activated protein kinase pathway were identified as the key regulators of increased autophagy in these folliculin deficient cells. Inhibiting autophagy with 3-methyladenine or beclin 1 siRNA obviously increased radioresistance in folliculin deficient cells. Moreover, irradiation combined with autophagic inducer rapamycin significantly increased autophagy and radiosensitivity in folliculin deficient renal cell carcinoma cells. CONCLUSIONS: Findings suggest that folliculin deficient renal cell carcinoma cells are highly sensitive to irradiation due to increased autophagic cell death, unlike other types of renal cell carcinoma. Irradiation plus autophagy inducers, eg rapamycin, might be a potentially more effective therapeutic approach to folliculin deficient renal cell carcinoma.

Suppression of autophagy enhances preferential toxicity of paclitaxel to folliculin-deficient renal cancer cells.

BACKGROUND: Paclitaxel, a widely used chemotherapeutic drug, can induce apoptosis in variety of cancer cells. A previous study has shown preferential toxicity of paclitaxel to FLCN-deficient kidney cancer cell line, UOK257. In this report, we investigate the cellular and molecular mechanism of paclitaxel-induced autophagy and apoptosis in renal cancer cells with and without FLCN expression. METHODS: Two pairs of cell lines were used: FLCN siRNA-silenced ACHN cell line (ACHN-5968) and scrambled ACHN cell line (ACHN-sc); FLCN-null UOK257 cell line and UOK257-2 cell line restored with ectopic expression of FLCN. Autophagy was examined by western blot, GFP-LC3, transmission electron microscopy, and MDC assay. Cell viability and apoptosis were detected using MTT assay, DAPI stain and TUNEL assay. After inhibition of autophagy with 3-Methyladenine (3-MA) or Beclin 1 siRNA, cell viability and apoptosis were measured by MTT assay and TUNEL assay. RESULTS: After paclitaxel treatment, a dose-dependent decrease in cell viability and increase in apoptosis were observed in FLCN-deficient UOK257 and ACHN-5968 cells compared to their FLCN-expressing counterparts, suggesting that renal cancer cells without FLCN were more sensitive to paclitaxel. Enhanced autophagy was found to be associated with paclitaxel treatment in FLCN-deficient RCC cells. The MAPK pathway was also identified as a key pathway for the activation of autophagy in these kidney cancer cells. Inhibition of phosphorylated ERK with ERK inhibitor U0126 showed a significant decrease in autophagy. Furthermore, after inhibition of autophagy with 3-Methyladenine (3-MA) or Beclin 1 siRNA, apoptosis induced by paclitaxel was significantly increased in FLCN-deficient UOK257 and ACHN-5968 cells. CONCLUSIONS: Preferential toxicity of paclitaxel to FLCN-deficient kidney cancer cells is associated with enhanced autophagy. Suppression of autophagy further enhances paclitaxel-induced apoptosis in FLCN-deficient renal cancer cells. Our results suggest that paclitaxel combined with an autophagy inhibitor might be a potentially more effective chemotherapeutic approach for FLCN-deficient renal cancer.