ABSTRACT
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.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Autophagy/drug effects , Estrone/deficiency , Kidney Neoplasms/drug therapy , Kidney Neoplasms/metabolism , Paclitaxel/pharmacology , Apoptosis/drug effects , Apoptosis Regulatory Proteins/deficiency , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Autophagy/genetics , Beclin-1 , Cell Line, Tumor , Dose-Response Relationship, Drug , Estrone/metabolism , Gene Knockdown Techniques , Humans , Kidney Neoplasms/enzymology , Kidney Neoplasms/pathology , MAP Kinase Signaling System/drug effects , Membrane Proteins/deficiency , Membrane Proteins/genetics , Membrane Proteins/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , TransfectionABSTRACT
To determine the effect of administration of a progestin alone on endogenous opioid peptide activity, we infused naloxone (2 mg/h for 4 h) into seven estrogen-deficient postmenopausal women before and after oral medroxyprogesterone acetate (Provera; 20 mg) daily for 30 days. Baseline serum LH levels were significantly decreased by the Provera therapy [70.3 +/- 6.6 (+/- SE) vs. 27.5 +/- 1.7 mIU/ml; P less than 0.001]. Naloxone infusion before Provera treatment had no effect on serum LH levels. In contrast, after Provera therapy, a significant (P less than 0.001) increase in LH levels toward the pre-Provera baseline occurred with naloxone infusion. These findings suggest that progestins exert their negative feedback effects on LH at least in part through an opioid peptide-mediated mechanism and that progestin treatment alone can reestablish opiatergic control of LH.
