| BackgroundAccording to the2008Global Cancer Statistics, esophageal cancer ranks among the10most frequent cancers in the world, with most occurring in developing countries. Mortality rates are very similar to incidence rates (4,82,300cases per year), due to the relatively late stage of diagnosis and the poor efficacy of treatment. Esophageal cancer is one of the leading causes of cancer-related death in China. Esophageal cancer is the sixth leading cause of cancer deaths worldwide, with an estimated482,000new cases and407,000deaths in2008. The crude incidence and mortality rates of esophageal cancer were due to chemotherapy failure chemoresistance. Surgery has been the primary course of treatment for patients with esophageal cancer. However, local recurrence and distant metastases remain an issue after surgery. Nearly50%of patients with a diagnosis of esophageal cancer present with over metastatic disease, and chemotherapy is the main stay of palliation in this setting.With the increasing use of chemotherapy as an adjunct to surgical management, systemic chemotherapy will ultimately be used to treat the majority of patients with esophageal cancer. Cisplatin is the first line chemotherapeutic agent for several cancers.Resistance of esophageal cancer to cisplatin is the main reason of chemotherapy failure. Resistance to chemotheraptic agents is a significant issue in the management of patients with esophageal cancer.Although some study has provided some mechanism of cisplatin-resistance, it is still a challenge for further research to identify the related mechanisms.Autophagy, which literally means’self eating’, is a lysosome-dependent patway involved in protein and organelle degradation. Autophagy is hallmarked by the formation of double-membrane bound organelles and protein known as autophagosomes. Fushion of the autophagosomes with a lysosome provides hydrolases and lysis of the autophagosome inner membrane and breakdown of the contents occurs in the autolysosome. In cancer therapy, the role of autophagy is paradoxical, in which this cellular process may serve as a pro-survival or pro-death mechanism to counteract or mediate the cytotoxic action of anticancer agents. The pro-survival function is that autophagy acts in tumour suppression by removing damaged organelles and possibly growth factors, and reduces chromosome instability. On the contrary, autophagy acts as a cytoprotective mechanism that helps cancer cells resist anti-cancer treatments and survive in conditions of low nutrient supply. More importantly, many of the cancer therapeutic agents are capable of inducing autophagy via various pathways. Among various cancer therapeutic agents, cisplatin is one of the most important anticancer drugs used in the treatment of solid tumors, especially for ovarian, testicular, cervical, small cell lung and esophageal carcinomas. Notably, the role of autophagy in mediating cellular response to the cytotoxicity of cisplatin in esophageal cancer cells, especially in cisplatin-resistant cancer cells, remains elusive. Whether ERK activation contributes to the autophagic response in resistant cancer cells is unclear. Moreover, the role of cisplatin in the regulation of mTOR and Beclin 1pathways remains elusive.ObjectivesActivation of autophagy is a hallmark in tumor cells treated with chemotherapy. Cell populations that respond with autophagy are more resistant and will recover following withdrawal of the chemotherapeutic agents. Notably, the role of autophagy in mediating cellular response to the cytotoxicity of cisplatin in esophageal cancer cells, especially in cisplatin-resistant cancer cells, remains elusive.In this study, our aim was to investigate the role of autophagic response in mediating cellular resistance to cisplatin-induced cytotoxicity and the mechanisms involved. In addition, we propose to evaluate the antitumor activity of cisplatin in combination with autophagy inhibitor in human esophageal squmous carcinoma cells. We also propose that the antitumor activity of cisplatin in combination with autophagy inhibitor should be investigated in nude mice of cisplatin-resistant esophageal squmous carcinoma xenograft model. Findings of this study will help us better understand the role of autophagic response in mediating chemo-resistance and facilitate the identification of novel approaches for overcoming drug resistance in esophageal cancer cells.MethodsTo investigate the role and mechanism of autophagy induced by cisplatin-resistant human esophageal squamous carcinoma cells in vitro and vivo, various commonly used methods are listed and discussed below. Such as western blot assay, immunofluorescence assay, autophagy detection with acridine orange (AO) staining, transmission electron microscopy (TEM), colony formation assay, SA beta Gal staining, transfection of adherent cells with siRNA, flow cytometry to detect the extent of apoptosis, nude mice xenograft model etc.1. Western blot assay was used to determine LC3-Ⅱ and p62level (1) The conversion from endogenous LC3-I (cytosolic form) to LC3-II (membrane-bound lipidated form) can be detected by western blot with antibodies against LC3. Although the actual molecular weight (MW) of LC3-II (a PE-conjugated form) is larger than that of LC3-I, LC3-Ⅱ (apparent MW is14kD) migrates faster than LC3-I (apparent MW is16kD) in SDS-PAGE because of extreme hydrophobicity of LC3-II. LC3is useful in biochemical assays to assess autophagosome numbers.The accumulation of autophagosomes is not always indicative of autophagy induction and may represent either the increased generation of autophagosomes and/or a block in autophagosomal maturation and the completion of the autophagy pathway. As described above, if cells are treated with lysosomo-tropic reagents such aschloroquine, or bafilomycin A1, which inhibit acidification inside the lysosome or inhibit autophagosome-lysosome fusion, the degradation of LC3-Ⅱ is blocked, resulting in the accumulation of LC3-Ⅱ. Through this assay, we studied whether cisplatin is able to induce autophagy and if so, what is the function of autophagy in cisplatin-induced cell death. Moreover, we tested whether the addition of CQ could further increase LC3-Ⅱ level.(2) Several reports have described p62, which is also named se-questosome1(SQSTM1), has both LC3-binding (the mammalian homologue of the autophagy-related protein Atg8) and ubiquitin-binding domains, allowing it to mediate the recognition of protein aggregates by a protein (LC3) in the membrane of the forming autophagosome. Cells were harvested in radioimmunoprecipitation buffer containing protease and phosphotase inhibitors as described previously. Equal amounts of protein were resolved with SDS-PAGE and transferred to PVDF membranes (Roche Diagnos-tics Corp., Indianapolis, IN). The membranes were probed with primary antibodies overnight at4℃and incubated for1h with secondary peroxidase-conjugated anti-body at room temperature. Chemiluminescent signals were then developed with Lumiglo reagent (Cell Signaling Technology) and exposed to X-ray film (Fuji Photo Film, Tokyo, Japan).2. Immunofluorescence assay.Cells grown on petri-dish were fixed with4%(v/v) paraformaldehyde for15min and then made permeable with ice-cold100%methanol for10min in freezer. After being rinsed in PBS for5min, the cells were covered with10%(v/v) normal goat serum (Invitrogen) for1h at room temperature. After this procedure, the cells were incubated with primary antibody at4℃overnight. After being rinsed three times in PBS for5min each, cells were probed with Alexa Fluor488goat anti-rabbit secondary antibody (Invitrogen) and incubated for2h at room temperature. Finally, cells were incubated with4,6-diamidino-2-phenylindole (DAPI) at room temperature for10min. Fluorescent signals were detected using a confocal microscope (C1-si, Nikon, Japan). The acquired images were analyzed by the Nikon software (NIS-Elements AR, Nikon, Japan).3. Autophagy detection with acridine orange (AO) staining.The volume of the cellular acidic compartment, as a marker of autophagy, was visualized by lysosomotropic agent acridine orange. Acridine orange moves freely across biological membrane and accumulates in acidic compartment, where it is seen as fluorescence bright red. Cells were treated for4h and stained with5μg/ml acridine orange at room temperature for1min. Then cells were washed with PBS and visualized under a fluorescent microscope.4. Flow cytometry was used to determine the cellular acidic vesicleCells were stained with acridine orange for15min, removed from the plate with trypsin-EDTA, and collected in growth medium. Green (510-530nm) and red (.650nm) fluorescence emission from104cells illuminated with blue (488nm) excitation light was measured with a FACSCalibur from Becton Dickinson (BD FACSCanto TM II flow cytometry, BD Biosciences, CA). The red:green fluorescence ratio for individual cells was calculated using FlowJo7.6software.5. Transmission electron microscopy (TEM).To quantify morphologic features by TEM, cells were scored for the presence of autophagic vacuoles, multivesicular bodies and multilamellar bodies, and comparisons between conditions were made with a paired t-test.6. Colony formation assay.EC109and EC109/CDDP cells were treated as designated for48h before reseeded in six-well plates (3,000cells/well) respectively. After ten days, the survival clones were stained by0.5%hematoxylin stain for10min and photos were taken using digital camera and the colony was counted with the ImageJ software.7. SA beta Gal staining.Aspirate the growth medium from the cells, wash the cells twice with1ml of1x PBS per well/plate, carefully remove the entire wash solution by aspiration, so the cells do not detach, add1.5ml per well of1×Fixation Buffer and incubate the plate for6-7minutes at room temperature. During the fixation process, prepare the Staining Mixture as described in the Preparation Instructions and rinse the cells3times with1ml of1xPBS per well/plate.8. Transfection of adherent cells with siRNA.The day before transfection, seed1×105cells per well of a6-well plate in0.5ml of an appropriate culture medium containing serum and antibiotics.Incubate the cells under normal growth conditions (typically37℃and5%CO2). On the day of transfection, dilute150ng siRNA in100μl culture medium without serum (this will give a final siRNA concentration of5nM after adding complexes to cells in step5). Add12μl of HiPerFect Transfection Reagent to the diluted siRNA and mix by vortexing. Incubate the samples for5-10min at room temperature (15-25℃) to allow the formation of transfection complexes. Add the compleses drop-wise onto the cells. Gently swirl the plate to ensure uniform distribution of the transfection complexes.Incubate the cells with the transfection complexes under their normal growth conditions and monitor gene silencing after an appropriate time.Change the medium as required.9. Flow cytometry was used to determine the extent of apoptosis.EC109and EC109/CDDP were incubated with cisplatin (2.5βM) for48h. After drug withdrawing, cells were harvested and dye with FITC AnnexinV and PI on day1,2and3. After treatment, the ratios of apoptosis were analyzed by Flow cytometer.10. Western blot assay was used to determine activity of MAPKs and mTORCells were harvested in radioimmunoprecipitation buffer containing protease and phosphotase inhibitors as described previously. Equal amounts of protein were resolved with SDS-PAGE and transferred to PVDF membranes (Roche Diagnos-tics Corp., Indianapolis, IN). The membranes were probed with phospho-mTOR, mTOR, phospho-p85/p70S6kinase, p85/p70S6kinase, phospho-4E-BP1,4E-BP1, phospho-ERK1/2, ERK1/2, phospho-p38, p38, phospho-JNK1/2/3, JNK1/2/3and β-actin primary antibodies overnight at4℃and incubated for1h with secondary peroxidase-conjugated anti-body at room temperature. Chemiluminescent signals were then developed with Lumiglo reagent (Cell Signaling Technology) and exposed to X-ray film (Fuji Photo Film, Tokyo, Japan).11. Nude mice xenograft model.EC109/CDDP cells were trypsinized and collected by centrifugation. Cell viability was confirmed to be above95%based on trypan blue staining. The cells (2×106) were suspended in0.2ml of PBS and injected subcutaneously into the right flank or dorsal region of4-to6-week-old female BALB/c nu/nu mice (The Animal Center, Southern Medical University, Guangzhou, China). After inoculation, the mice were maintained under sterile conditions, and the size of tumor formed was measured using calipers every2days. Tumor volume was calculated by the following formula: volume=(length/2)×(width2). On day10after inoculation, all mice produced a palpable tumor. The mice were then divided randomly into four groups of eight mice each:1) control group in which vehicle alone was received;2) cisplatin-treated group in which cisplatin (diluted with physiological saline) was administered intraperitoneally once a week for4weeks;3) chloroquine-treated group in which chloroquin (suspended in PBS) was administered by lavage daily for4weeks; and4) cisplatin plus chloroquine-treated group in which cisplatin was administered once a week for4weeks, and chloroquine was administered by lavage daily for4weeks. Body weight was monitored throughout the experiments. At the end of the experiment, the mice were sacrificed and tumors were excised for further assays. Animal care and experiments were conducted in accordance with the Animal Research Committee Guidelines of Southern Medical University.12. Statistical analysisResults were expressed as mean±SD. Statistical analysis was done by using the prism statistical package (GraphPad Software, San Diego, CA). Unpaird t test was used to compare data between two groups. Both one-way ANOVA and subsequently the Turkey’s t test were used to compare data between three or more groups.Dunnett test was used to compare data with control group.P values less than0.05were considered statistically significant.Results1. Cisplatin is an antineoplastic drug in the class of alkylating agents and is used to treat various types of esophageal cancers, such as esophageal squamous cell carcinoma and adenocarcinoma. Treatment with cisplatin for48hrs led to autophagy in cisplatin-resistant esophageal cancer cell EC109/CDDP but not in parent esophageal cancer cell EC109. Ultrastructural TEM analysis was consistent with the induction of autophagy by cisplatin, with many vesicles showing muti-lamellar structure typical for autophagosomes.The presence of vesicles enqulfing cytoplasmic organelles confirmed formation of autophagolysosome. After treatment with cisplatin, a significant increase in LC3-II levels was evident in cisplatin-resistant EC109/CDDP. Redistribution of GFP-LC3from a diffuse cytosolic to a punctate autophagosome-associated pattern and formation of acidic vesicular organells were observed in EC109/CDDP.2. The EC109and EC109/CDDP cell lines are significantly more sensitive to a range of concentration of cisplatin. The EC109/CDDP and HKESC-1/cis were more resistant to cisplatin treatment compared to EC109and HKESC-1cell lines. Colonies were extremely rare in EC109and HKESC-1cells, but colonies that recover from cisplatin treatment were in excess in EC109/CDDP and HKESC-1/cis, clearly demonstrating resistance and recovery from cytotoxic cisplatin treatment-Inhibition of autophagy by CQ sensitizes EC109/CDDP and HKESC-1/cis cells to cisplatin-induced apoptosis. Knockdown of Atg5or Atg7inhibited the autophagic activity. Moreover, Knockdown of Atg5or Atg7together with cisplatin significantly reduced the colonies consistent with autophagy playing a protective role and facilitating recovery of the treated cells. Therefore, if autophagy plays a role in the cisplatin-induced resistance, it must utilize Atg5or Atg7.3. Cisplatin decreased the phosphorylation of4E-BP1, p70S6kinase and p85S6kinase. These data suggest that cisplatin-mediated autophagy was at least mediated by down-regulation of mTOR4. Phosphorylation of ERK, p38and JNK was increased in EC109and EC109/CDDP cells in response to cisplatin treatment. Inhibition of ERK by U0126decreased the sensitity of EC109and EC109/CDDP to cisplatin-indcued cell death.Taken together, these results indicate that ERK plays a role in regulation cisplatin-induced cell death of esophageal cancer cells (EC109and EC109/CDDP). Cisplatin induced apoptosis in cisplatin-sensitized cells EC109and cisplatin-resistant cellls EC109/CDDP. The induction of apoptosis in EC109was more obvious than EC109/CDDP. Pretreatment of these cells with U0126significantly reduced cisplatin-induced apoptosis. In addition, pretreatment of these cells with U0126significantly reduced cisplatin-induced senescence. These data suggest that cisplatin-induced apoptosis and senescence in esophageal cancer cells were associated with the activation of ERK.5. We evaluated the anti-tumor effects of cisplatin, chloroquine, and the combination treatment in nude mice harboring EC109/CDDP tumor xenografts. Our results showed that chloroquine alone evidenced no detectable anti-tumor activity. Cisplatin alone resulted in a relatively effective delay of tumor growth as compared with control. However, when these two agents were administered in combination, significant anti-tumor effects were observed. Collectively, our data show that chloroquine can promote the sensitization of cancer cells to cisplatin, both in vitro and in vivo.ConclusionsOur findings revealed that cisplatin induced autophagy in cisplatin-resistant esophageal cancer cells, but not in parent esophageal cancer cells. Inhibition of autophagy enhanced the cytotoxicity in resistant esophageal cancer cells, indicating autophagy as a survival mechanism promoting chemo-resistance and cellular recovery. In relation to signaling pathways involved, cisplatin induced autophagy via inhibiting mTOR activity. On the contrary, ciaplatin induced apoptosis and sencescence through ERK signaling pathway. In addition, cisplatin in combination with chloroquine elicited greater antitumor activity than cisplatin alone in vivo in the cisplatin-resistant esophageal cancer xenograft model.Collectively, these data demonstrated that cisplatin induced pro-survival autophagy in cisplatin-resistant esophageal cancer cells. The combination of CQ and cisplatin showed beneficial effect compared with cisplatin monotherapy in vivo.The combination regimen may have siganificant potential clinical application, especially in the circumvention of cisplatin-resistant esophageal cancer. |
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