The Efrects Of Autophagic Response Mediated By Paclitaxel Or γ-irradiation On Chemosensitivity And Radiosensitivity In Folliculin-deficient Renal Cancer Cells

BackgroundRenal cancer accounts for about2%of all cancers. Most of renal cancers are sporadic cancers, and familial forms of renal cancer account for only about4%of all cases. Investigations of rare inherited forms of renal cancer have provided seminal insights into the molecular pathogenesis of both familial and sporadic renal cancer. Currently identified inherited forms of renal cancer include Von Hippel-Lindau syndrome(VHL syndrome), hereditary papillary renal cellcarcinoma, hereditary leiomyomatosis and renal cell cancer(HLRCC) and Birt-Hogg-Dube’(BHD) syndrome. BHD syndrome is an autosomal dominant genetic disease characterized by fibrofolliculomas (follicular hamartomas), lung cysts,spontaneous pneumothorax and renal cell carcinomas. BHD is caused by loss-of-function mutations in the folliculin (FLCN) gene, The FLCN gene located on chromosome17p11.2, encodes a protein of579amino acids. FLCN combined with FNIP1and FNIP2protein, interacts with AMPK in mammalian cell lines, and regulates the mTOR pathway, however, the molecular function of FLCN is still largely unknown.An effective cure for BHD disease associated renal cancer has not been established. In2006Youfeng Yang et al established UOK257cell line, the first renal cancer cell line which originated from a patient with BHD syndrome; in2011Xiaohong Lu et al established UOK257-2cell line,which was UOK257cell line restored with ectopic expression of FLCN, their study also had shown preferential toxicity of paclitaxel to UOK257cell line, although the mechanism was still unclear. In our study, we found that Paclitaxel induced more cytotoxicity in FLCN-deficient kidney cancer cells,and we also investigated the cellular and molecular mechanism of paclitaxel-induced autophagy and apoptosis in renal cancer cells with and without FLCN expression.Autophagy, which is also known as type-II programmed cell death, is an evolutionary conserved process in which cell engulfs cytoplasmic materials including organelles within a double-membrane vacuole (named autophagosome) and delivers them to the lysosome for degradation. Autophagy contributes to maintaining cellular homeostasis. When cells encounter environmental stressors such as nutrient starvation, autophagy is induced to provide nutrients and energy required for cell survival. So autophagy is recognized as a cytoprotective process against environmental stress. Meanwhile, autophagy is also an alternative route of programmed cell death. Previous study has reported that suppression of mammalian target of rapamycin (mTOR) pathway could active autophagy in lung cancer cells and liver cancer cells, and inhibit tumor cell growth through autophagic cell death. In tumor cells, the role of autophagy may depend on the type of tumor and the nature and extent of the insult. Appropriate modification of autophagy, that is, inhibition of cytoprotective autophagy or promotion of cyto-killing autophagy could augment cytotoxicity caused by anticancer therapy in tumor cells. Thus, in addition to apoptotic response, it would be very useful to determine if an antitumor agent can induce autophagy and what type of autophagies it is.In our study, we found that preferential toxicity of paclitaxel to FLCN-deficient kidney cancer cells was associated with enhanced autophagy, paclitaxel combined with an autophagy inhibitor3-MA could obviously inhibit growth of FLCN-deficient renal cancer cells.Paclitaxel (Taxol) is an effective mitotic inhibitor and apoptosis inducer, whether paclitaxel could induce autophagy in cells is still in dispute. To examine whether Paclitaxel could induce autophagy in FLCN-deficient kidney cancer cells, besides FLCN-deficient cell line UOK257, a cell line derived from a BHD patient’s kidney cancer, we also employed a RCC cell line, ACHN, with known FLCN expression and its FLCN expression could be effectively suppressed with siRNA. Our study indicated that paclitaxel could induce autophagy in FLCN-deficient kidney cancer cells but not in FLCN-expressing cells, paclitaxel combined with an autophagy inhibitor might be a potentially more effective chemotherapeutic approach for FLCN-deficient renal cancer.ObjectiveTo investigate the cellular and molecular mechanism of paclitaxel-induced autophagy in renal cancer cells with and without FLCN expression. To detect the combination with paclitaxel and an autophagy inhibitor as a potentially more effective chemotherapeutic approach for FLCN-deficient renal cancer.Materials and methods1. Cell culture Two pairs of cell lines were used: FLCN siRNA-silenced ACHN-5968cell line and scrambled ACHN line (ACHN-sc); FLCN-null UOK257cell line and UOK257-2line restored with ectopic expression of FLCN.2. Cell viability and apoptosis assay The viability of cells was measured by MTT assay. UOK257/UOK257-2and ACHN5968/ACHN-sc cells were cultured in96-well plates, and treated with100nM paclitaxel or PBS, MTT (5mg/ml) was added to each well in0、24、48、72hours, respectively. Absorbance was measured using a microplate reader (BioTek). Cell apoptosis was detected using DAPI stain and TUNEL assay. TUNEL:monolayer cells in96-well plate were treated with corresponding reagents and cultured at37℃, quantitation of apoptotic cells was measured by in situ colorimetric TUNEL assay (HT TiterTACSTMAssay kit, TREVIGEN(?)) following the manufacturer’s protocol. DAPI stain: cells with indicated reagents treatment were fixed with methanol/acetone (1:1) for5min at room temperature, and stained with DAPI (1:2000dilution, in1x PBS) for10min. The cells were subsequently rinsed with PBS and observed under a fluorescent microscope (ZEISS). Inaddition, we also examined the cleaved caspase-3protein in all cells by western blot.3. Autophagy assay (1) Transmission electron microscopy assay Transmission electron microscopy assay is the most traditional method. UOK257/UOK257-2and ACHN5968/ACHN-sc cells with paclitaxel or PBS treatment were cultured in10cm plates. Cells were handled following the transmission electron microscopy assay’s protocol, and observed with a H-800transmission electron microscope (Hitachi, Tokyo, Japan). At the ultrastructural level, an autophagosome is defined as a double-membraned structure containing undigested cytoplasmic contents.(2) GFP-LC3assay Microtubule-associated protein1light chain3(LC3) is located at autophagic membrane. LC3is proved to be associated with number of autophagosome.when autophagy is actived, cells could exhibit a high number of fluorescence punctate structures. In this study, cells were transfected with GFP-LC3(Addgene plasmid24920) with LipofectamineTM2000(Invitrogen, USA) following the manufacturer’s protocol. At24hours after transfection, the cells were treated with paclitaxel (100nM) or PBS control and cultured at37℃for24hours. The cells were subsequently examined under the fluorescence microscope (ZEISS)(3) Monodansyl cadaverine assay Monodansyl cadaverine (MDC) could mark autophagosome with blue punctate structures under fluorescence microscopy. For MDC assay, cells cultured in6-well plate were treated with0.05mM MDC and incubated at37℃for20minutes. Intracellular autophagy was detected using a fluorescence microscope (ZEISS).(4) LC3and P62western blot LC3is an important autophagy marker recruited to the autophagosome membrane. LC3has two isoforms, LC3-Ⅰ and LC3-Ⅱ. During autophagy, LC3-Ⅰ is conjugated to autophagic membrane-associated phosphatidylethanolamine and converted to LC3-Ⅱ, LC3-Ⅱ and autophagosome were deliverd to the lysosome for degradation. To exclude the possibility that the increased LC3-Ⅱ levels were resulted from the accumulation of LC3-Ⅱ due to downstream inhibition other than paclitaxel induction, generally reagent-induced LC3should be examined in presence or absence of lysosomal inhibitor bafilomycin A1.In this study, we treated the cells with paclitaxel in presence or absence of lysosomal inhibitor bafilomycin A1, and examined protein level of LC3-Ⅰ/Ⅱ. In addition, we also examined another autophagy marker P62protein in all cells.4. Examination of MAPK pathway To explore the molecular mechanism of paclitaxel induced autophagy in FLCN-deficient cells, we examined the alteration of the MAPK pathway by western blot, including the MEK, ERK and P-ERK protein level. We also examined the alteration of P-ERK, LC3-Ⅰ/Ⅱ and beclin-Ⅰ protein level in presence or absence of ERK inhibitor U0126.5. Examination of autophagic effect on cells To explore the effect of autophagy on FLCN-deficient cells, we inhibited autophagy using autophagy inhibitor or siRNA and observed the alteration of paclitaxel toxicity to FLCN-deficient cells. Cells were treated with autophagy inhibitor3-MA or transfected with beclin1siRNA, the alteration of LC3-I/II were examined by western blot, cell viability and apoptosis were measured by MTT and TUNEL assay.6. Statistical Analyses Statistical significances were analyzed by ANOVA and paired Student t test with Statistics Package for Social Science (SPSS) software (Version14). Qualitative data were expressed as means±S.D, and p<0.05was considered statistically significant difference.Results1. Paclitaxel induced cytotoxicity and apoptosis in FLCN-deficient renal cancer cells Paclitaxel could inhibit cell growth in all treated cells with or without FLCN expression. However, suppression of cell growth by paclitaxel on FLCN-deficient UOK257and ACHN-5968cells was more significant than that on matched UOK257-2and ACHN-sc cells, indicating more severe paclitaxel-induced cytotoxicity to FLCN-deficient cells. The result of DAPI stain and TUNEL assay indicated a much greater number of apoptotic cells were detected in UOK257and ACHN5968lines compared to UOK257-2and ACHN-sc lines. The levels of cleaved caspase-3were obviously higher in UOK257and ACHN5968cells upon the treatment with paclitaxel, indicating more apoptosis was induced in cells without FLCN expression. These results supported the conclusion that paclitaxel induces more apoptosis in FLCN-deficient renal cancer cells.2. Paclitaxel induced autophagy in FLCN-deficient renal cancer cells In absence of bafilomycin Al, paclitaxel increased LC3-Ⅱ level in UOK257and ACHN5968cell lines but not in UOK257-2and ACHN-sc cell lines. Although increased LC3-Ⅱ levels were detected in all of the bafilomycin A1-treated cells due to inhibition of lysosomal degradation of LC3-Ⅱ, LC3-Ⅱ levels were even higher in the paclitaxel-treated FLCN-deficient cells compared to that in the FLCN-expressing cells.MDC and GFP-LC3results showed that the FLCN-deficient cells exhibited a higher number of punctate structures compared to FLCN-expressing UOK257-2and ACHN-sc cells (p<0.05). Transmission electron microscopy assay results showed that increased autophagosome number was present in UOK257and ACHN-5968cells. These results demonstrated that autophagy was induced by paclitaxel treatment and FLCN-deficient cells presented a higher level of autophagy.3. Paclitaxel induces autophagy in FLCN-deficient cells via activation of MAPK pathway Western blot results indicated that absence of FLCN was connected with the activation of the MAPK pathway.Compared with UOK257-2and ACHN-sc cells, MAPK pathway were more actived in UOK257and ACHN-5968cells, P-MEK and P-ERK protein level were higher in FLCN-deficient cells. Previous reports indicated that MAPK pathway could interact with Beclin-1protein and regulate autophagy. In this study, paclitaxel treatment further significantly increased the expression of phospho-ERK and Beclin1in FLCN-deficient UOK257and ACHN-5968cells. Additionally, treatment with the ERK inhibitor U0126significantly reduced the expression of LC3, Beclin1, and phospho-ERK in UOK257and ACHN-5968cells, GFP-LC3results indicated that cells exhibited a lower number of punctate structures. These results suggested that paclitaxel induced autophagy in FLCN-deficient cells via the ERK pathway and Beclin1protein.4. Inhibition of autophagy enhanced paclitaxel-induced apoptosis and cytotoxicity in FLCN-deficient cells Pretreatment with autophagy inhibitor3-MA led to a significant decrease of LC3-Ⅱ levels in FLCN-deficient UOK257and ACHN-5968cells, indicating that autophagy was inhibited by3-MA in those cells.MTT and TUNEL results showed that pretreatment with3-MA effectively inhibited cell viability and enhanced paclitaxel-mediated apoptosis in UOK257and ACHN-5968cells compared to UOK257-2and ACHN-sc cells. The knockdown of Beclin1led to a significant increase of apoptosis and inhibition of cell viability in FLCN-deficient cells, which was consistent with the results obtained through3-MA treatment. These data indicated that autophagy provided protection and survival advantage to FLCN-deficient cells against cell apoptosis and cell death induced by paclitaxel. Inhibition of autophagy could increase the paclitaxel-induced cytotoxicity to these cells and might improve the efficacy of paclitaxel against these cancer cells.Conclusions1. Compared with FLCN-expressing cells, paclitaxel has preferential toxicity to FLCN-deficient kidney cancer cells.2. Paclitaxel induced autophagy in FLCN-deficient cells via the MAPK pathway.3. Inhibition of autophagy could increase the paclitaxel-induced cytotoxicity to FLCN-deficient cells, paclitaxel combined with an autophagy inhibitor might be a potentially more effective chemotherapeutic approach for FLCN-deficient renal cancer. BackgroundRenal cancer is one of the most common urological cancers, about25%-30%renal cancer patients are diagnosised with metastasis in the first visit. Radical nephrectomy is the preferred way for localized renal cancer, however,1/3patients will have metastasis after surgical treatment. Patients presenting with advanced renal cancer have a poor prognosis due to the relative chemo and radioresistance of this disease. Radiotherapy is rarely used to treat primary renal cancer although carbon ions have recently been used with some success. Previous study has shown that many renal cancer cases are associated with loss of function of the tumour suppressor gene such as VHL, FLCN and MET. Loss-of-function mutations in the folliculin (FLCN) gene always cause BHD syndrome, which can present with bilateral renal cancer. An effective cure for BHD disease associated renal cancer has not been established.In this study, we found that FLCN-deficient cells were more sensitive to y-irradiation compared to their FLCN-expressing counterparts. The enhanced effects of y-irradiation on FLCN-deficient cells were mediated by increased autophagy, but not apoptosis.Autophagy is a normal cellular process used for the generation of nutrients and energy in response to stress for example, during periods of starvation. The autophagic process is deregulated in cancer and depending on the cellular context and stress, can be pro-survival or lead to autophagic, programmed type Ⅱ death. The therapeutic effect of autophagy on tumor is a burgeoning area of research and has drawn intense interest from cancer researchers. The contribution of autophagy to radiation efficacy is unclear. The autophagy inhibitor chloroquine can increase the sensitivity to radiation in vitro and is currently being tested in clinical trials. However, the induction of autophagy has also been demonstrated to enhance the effects of radiation. Selvakumar Anbalagan et al found that the induction of pro-death autophagy by treatment with autophagy inducer increases the radiosensitivity of VHL-deficient renal cancer cells whilst having little effect on cells with normal VHL function.To explore therapeutic approaches for renal cancer in BHD syndrome, we investigated the anticancer effects of irradiation on FLCN-deficient renal cancer cells.We compared radiosensitivity between FLCN-deficient renal cancer cells and FLCN-expressing cells, as well as apoptosis and autophagy induced by y-irradiation. We also examined the alteration of radiosensitivity in FLCN-deficient renal cancer cells after autopahgy inhibition or induction, to determine the impact of autophagy on irradiation-mediated FLCN-deficient cell death.Objective1. To investigate the radiosensitivity of FLCN-deficient renal caner cells using y-irradiation.2. To investigate the apoptosis and autophagy induced by y-irradiation in FLCN-deficient renal caner cells and FLCN-expressing cells.3. To determine the impact of autophagy on irradiation-mediated FLCN-deficient cell death4. To explore the mechanism of autophagy induced by y-irradiation in FLCN-deficient renal caner cells.Materials and methods1. Cell culture To determine the impact of FLCN on radiosensitivity of renal caner cells, we used Human renal cancer cell lines ACHN,786-0,769-P, Caki-1and UOK257for Clonogenic assay. To investigate the impact of autophagy on irradiation-mediated FLCN-deficient cell death, we used two pairs of cell lines: FLCN siRNA-silenced ACHN-5968cell line and scrambled ACHN line (ACHN-sc); FLCN-null UOK257cell line and UOK257-2line restored with ectopic expression of FLCN.2. Clonogenic assay ACHN,786-0,769-P, Caki-1and UOK257cells were irradiated with OGy、1Gy、3Gy and5Gy using a Cs137irradiator. After irradiation, cells were incubated under normal culture conditions for14days. Cells were stained with0.5%crystal violet and colonies were counted. UOK257/UOK257-2, ACHN5968/ACHN-sc cells were cultured in6cm plates and irradiated with0Gy、1Gy、3Gy5Gy and7Gy,and cells were also stained and colonies were counted. Surviving fraction was calculated as (mean colony counts)/(cells inoculated) x (plating efficiency [PE]), and PE was calculated as (mean colony counts for non-irradiated controls)/(cells inoculated for non-irradiated controls).3. y-irradiation and apoptosis Cell apoptosis assay was carried out using TUNEL methods. For TUNEL assay, monolayer cells in96-well plate were treated with indicated irradiation dose and duration. After fixation in3.7%paraformaldehyde for5minutes, apoptotic cells were detected by in situ colorimetric TUNEL assay (HT TiterTACSTMAssay kit, Trevigen(?)) following the manufacturer’s protocol. The quantitation of apoptotic cells was immediately measured at450nm in a microplate reader (BioTek).The protein level of cleaved caspase-3were also examined after irradiation4. y-irradiation and autophagy irradiation-induced autophagy was measured by LC3western blot MDC and GFP-LC3assays. For MDC assay, cells cultured in6-well plates were irradiated and after7hours cells were treated with0.05mM MDC for20minutes at37℃. Following MDC, cells were fixed in4%paraformaldehyde for10minutes and intracellular autophagy was detected using a fluorescence microscope (ZEISS). For GFP-LC3assay, cells were cultured in6-well plates and transfected with GFP-LC3through LipofectamineTM2000(Invitrogen, USA) following the manufacturer’s protocol. The cells received indicated irradiation dose after transfection of24hours. The fluorescence of GFP-LC3was examined under a fluorescence microscope (ZEISS). 5. The impact of autophagy on radiosensitivity of FLCN-deficient cells We applied3-MA and Beclin1siRNA to suppress autophagy in FLCN-deficient cell lines, and observed cell surviving fraction after autophagy inhibition. To test our hypothesis that the induction of autophagic cell death in combination with irradiation increases cell killing of FLCN-deficient RCC cells, we treated UOK257and ACHN-5968cells with autophagy inducer rapamycin prior to irradiation, and observed cell surviving fraction after autophagy induction.6. The mechanism of irradiation-induced autophagy To explore the mechanism of autophagy induced by y-irradiation in FLCN-deficient renal caner cells, we examined the alteration of the MAPK pathway and Beclin1protein that are known to be associated with autophagic regulation in lung cancer cells. To further validate that the elevated Beclin1protein is a critical regulator for irradiation-induced autophagy in FLCN-deficient cells, we also knocked down Beclin1using siRNA in UOK257and ACHN-5968cells, and observed the alteration in irradiation-induced autophagy.7. Statistical Analyses Statistical significances were analyzed by ANOVA and paired Student t test with Statistics Package for Social Science (SPSS) software (Version14). Qualitative data were expressed as means±S.D, and p<0.05was considered statistically significant difference.Results1. FLCN-deficient renal cancer cells exhibited higher radiosensitivity We treated ACHN,786-0,769-P, Caki-1and UOK257cell lines with irradation from0-5Gy and clonogenic assays were performed and graphed as survival curves. The FLCN-deficient RCC cell UOK257is relatively more sensitive to IR comparing with other renal cancer cell lines.2. FLCN-deficient renal cancer cells exhibited less irradation-induced apoptosis We examined apoptosis in UOK257/UOK257-2and ACHN-sc/ACHN-5968cell lines using in situ colorimetric TUNEL assay. Irradation induced apoptosis in all treated cells with or without FLCN expression. However, a much greater number of apoptotic cells were detected in UOK257-2and ACHN-sc cell lines compared to UOK257and ACHN-5968cell lines. Furthermore, higher expression of cleaved caspase-3protein was detected in both UOK257-2and ACHN-sc cells after Irradation. Together, the above results showed that higher radiosensitivity of FLCN-deficient cells was not associated with increased apoptosis after irradation, suggesting that FLCN deficiency might lead to higher radiosensitivity through a nonapoptotic pathway in renal cancer cells.3. Irradation induced autophagy in FLCN-deficient renal cancer cells We examined the autophagosome formation using MDC and GFP-LC3assay. The results showed that punctate structures were present in all the cell lines after irradiation. However, the FLCN-deficient cells (UOK257and ACHN-5968) displayed much higher number of punctate structures compared to their corresponding FLCN-expressing counterparts. Western blot results showed that obviously enhanced LC3-II/I ratio were observed in UOK257and ACHN-5968after exposure to irradiation for7hours, while the controls, UOK257-2and ACHN-sc, showed relatively less changes. These results indicated that autophagy occurred in these FLCN-deficient RCC cells after irradiation.4. Irradiation-induced autophagy contributed to radiosensitivity of FLCN-deficient RCC cells Our results demonstrated that pretreatment with autophagy inhibitor3-MA led to a significant decrease of LC3-II/I ratio in FLCN-deficient UOK257and ACHN-5968cells compared to FLCN-expressing cells, suggesting that autophagy was inhibited by3-MA in these cells. In addition, we further observed that pretreatment of UOK257and ACHN-5968cells with3-MA effectively increased their colony survival compared to UOK257-2and ACHN-sc cells, indicating that inhibition of autophagy could enhance the radioresistance of FLCN-deficient renal cancer cells. Knockdown of Beclin1also led to a significant increase of cell survival in FLCN-deficient cells, which was consistent with the results obtained through3-MA treatment. These data demonstrated that deficiency of FLCN could enhance the radiosensitivity of RCC cells through increased autophagic cell death. We treated UOK257and ACHN-5968cells with autophagy inducer rapamycin prior to irradiation. As predicted, rapamycin treatment significantly increased LC3expression and enhanced radiation-induced killing of these FLCN-deficient cells. These results indicated that induction of autophagy could further increase radiosensitivity of FLCN-deficient renal cancer cells.6. The mechanism of irradiation-induced autophagy in FLCN-deficient renal cancer cells Elevated expressions of phospho-MEK and phospho-ERK were detected in FLCN-deficient renal cell lines, indicating that deficiency of FLCN was associated with a higher level of MAPK pathway activation. Irradiation exposure further significantly increased the expression of phospho-ERK in FLCN-deficient UOK257and ACHN-5968cells, and the Beclin1knockdown significantly inhibited LC3expression in FLCN-deficient cells after irradiation exposure, indicating that MAPK pathway and Beclin1protein are essential for irradiation-induced autophagy in FLCN-deficient cells.Conclusions1. FLCN-deficient renal cancer cells exhibited higher radiosensitivity.2. Irradation induced autophagy in FLCN-deficient renal cancer cells.3. Irradiation-induced autophagy contributed to radiosensitivity of FLCN-deficient RCC cells. Rapamycin treatment significantly enhanced radiation-induced killing of these FLCN-deficient cells.4. MAPK pathway and Beclin1protein are essential for irradiation-induced autophagy in FLCN-deficient cells.