Abstract
Objective
The purposes of this study were to determine whether autophagy was involved in cisplatin (CDDP) resistance and to investigate the role of the autophagy in the regulation of chemosensitivity to CDDP in laryngeal cancer Hep-2 cells.
Methods
A WST-1 assay was performed to determine cell viability and cell proliferation. Autophagy activation and proapoptotic effects were characterized using monodansylcadaverine labeling and Hoechest staining, respectively. Western blot analysis was used to detect the expression of apoptotic and autophagy-related genes. Flow cytometry was used to assess cell apoptosis ratio.
Results
Exposure to CDDP induced the aggregation of autophagosomes in the cytoplasms of Hep-2 cells and up-regulated the expression of Beclin 1 and LC3II. However, CDDP treatment could not lead to obvious inhibition of cell proliferation, which implies that the autophagy may protect CDDP-treated cells from undergoing cell death. Meanwhile, the WST-1 assay indicated that knockdown of the autophagic gene Beclin 1 sensitized Hep-2 cells to CDDP. Furthermore, CDDP-mediated apoptotic cell death was further potentiated by pretreatment with autophagy inhibitor 3-methyladenine or small interfering RNA against Beclin 1. For the definite mechanism of Beclin 1–enhancing chemosensitivity to CDDP, we found that Beclin1 augmented CDDP-induced apoptotic signaling via enhancing caspase-9 and caspase-3 activity but not caspase-8.
Conclusion
Our results suggest that functional autophagy in response to CDDP may lead to cell survival in Hep-2 cells, whereas defective autophagy may contribute to CDDP-induced apoptosis in Hep-2 cells. Thus, modulators of autophagy may be used beneficially as adjunctive therapeutic agents during the treatment of laryngeal cancer with CDDP therapy.
1
Introduction
Larynx squamous cell carcinoma constitutes almost 2% to 3% of all malignant tumors, representing the second most common malignant neoplasm of the respiratory tract . Early laryngeal cancer can usually be managed successfully with either radiotherapy or surgery. Advanced-stage cancer often requires a combination of treatment modalities. Chemotherapy is an important option in curing or controlling various cancers, but laryngeal cancer is insensitive to cisplatin (CDDP)-based chemotherapy in a clinical setting. The underlying mechanism has not been illuminated. Evidence suggests that autophagy plays a role in CDDP-induced cell death or CDDP resistance. Kim et al found that autophagy increases the cytotoxicity of irradiation in apoptosis-efficient cells, which suggests the ability of autophagy to overcome multidrug resistance in cancer cells. Notably, autophagy can serve either to promote cell/tumor survival at certain stages or to promote cell death at other stages . Despite this complication, it has been suggested by Mishima et al that the blocking of autophagy could be a new strategy in the treatment of chronic myelogenous leukemia.
Prior studies have led to conflicting views of the role of autophagy in cancer chemotherapy. It has been established that autophagy mediates cell death of acute lymphoblastic leukemia cells by dexamethasone , promotes growth inhibition of PC3 cells by phenethyl isothiocyanate , promotes cell death by histone deacetylase inhibitors in chondrosarcoma cell lines , and may constitute a key mechanism by which transforming growth factor β promotes the generation of antitumor responses . On the other hand, autophagy represents a protective mechanism against apoptotic cell death under starvation as well as contributes to resistance against therapy-induced apoptosis in cancer cells. It has been shown that autophagy is activated as a protective mechanism against 5-fluorouracil–induced apoptosis ( , autophagy blockade sensitizes prostate cancer cells toward sulforaphane , autophagy serves a protective role in imatinib-mediated cell killing , and autophagy inhibition augments the anticancer activity of the histone deacetylase inhibitor suberoylanilide hydroxamic acid .
Markers for autophagy induction include Beclin 1 and LC3 conversion. We detected a high level of Beclin 1 expression in intact laryngeal cancer Hep2 cells. Hence, we speculated that autophagy may play a role in CDDP resistance in laryngeal cancer. Then, we examined whether increased cellular autophagy contributes to CDDP resistance in laryngeal cancer Hep2 cells and whether autophagy alteration affects the molecular events associated with cell death. Autophagy inhibitor 3-methyladenine (3-MA) or small interfering RNA (siRNA) against the autophagic gene Beclin 1 was used to study the effect of autophagy on drug resistance.
2
Materials and methods
2.1
Cell culture and transfection
The human laryngeal cancer Hep-2 cell line was purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Hep-2 cells were cultured in Dulbecco modified Eagle medium containing 10% fetal bovine serum (Gibco [New York, NY, USA], catalog number: 16000-044), penicillin (10 U/mL; Sigma [Los Angeles, CA, USA], catalog number: P0781) and streptomycin (0.1 mg/mL; catalog number: P0781) and were kept in a humidified atmosphere of 5% CO 2 at 37°C. Cells were seeded into 6-cm Petri dishes (5 × 10 5 cells/well) and were transfected at 80% confluence with pSUPER-Bec transfectants ( Beclin 1 gene partially silenced) using lipofectamine 2000 reagent (Invitrogen Life Technologies [CA, USA], catalog number: 1668027) as described by the manufacturer. The similar fashion with pSUPER-non (scramble RNA) was transfected, and the parental Hep-2 cells were used as the control.
2.2
Western blot analysis
The cultured cells were washed twice with phosphate-buffered saline and lysed with cold radio immunoprecipitation assay lysis buffer containing protease inhibitors (phenylmethylsulfonyl fluoride 1 mmol/L and leupeptin 0.1 g/L). Cell lysates were collected from culture plates using a rubber policeman, and protein was collected by centrifugation. Protein concentrations were determined using the bicinchoninic acid protein assay (Pierce, catalog number: 23225). Aliquots containing 40 μ g of proteins were boiled in 2 × loading buffer (0.1 M Tris-Cl, pH 6.8, 4% sodium dodecyl sulfate, 0.2% bromophenyl blue, and 20% glycerol) for 10 minutes, loaded into 10% Tris-HCl polyacrylamide gels, and transferred electrophoretically to Immobilon-P membrane (Millipore Corporation, catalog number: IPVH07850). Membranes were incubated with primary antibodies and appropriate horseradish peroxidase secondary antibodies. Membranes were additionally probed with an antibody against actin (Santa Cruz Biotechnology [Santa Cruz, CA], catalog number: sc-1616) to ensure equal loading of protein between samples. Detection was performed with chemiluminescent agents (Pierce, catalog number: 26651).
2.3
Polyclonal antibodies
Anti-Beclin 1 antibody (Santa Cruz, catalog number: sc-10086) recognizes a single 60-kd band on Western blot. Anti-LC3 antibody (Santa Cruz, catalog number: sc-16756) recognizes 16- and 18-kd bands. Anti–caspase-9 antibody (Cell Signaling, catalog number: 9502), anti–caspase-3 antibody (Cell Signaling, catalog number: 9662), anti–caspase-8 antibody (Cell Signaling, catalog number: 4790), anti–cleaved caspase-9 antibody (Cell Signaling, catalog number: 9501), anti–cleaved caspase-3 antibody (Cell Signaling, catalog number: 9661), and anti–cleaved caspase-8 antibody (Cell Signaling, catalog number: 9749) recognize single 35-, 57-, 47-, 17-, 37-, and 18-kd band on Western blot, respectively.
2.4
WST-1 assay for cell viability and cell proliferation
Hep-2 cells were plated in 96-well plates at 5000 or 6000 cells/well. The next day, cells were treated with or without 0.25, 0.5, and 1.0 μ g/mL CDDP with 5 to 6 replicates. According to the experiment, the cytotoxity or cell viability was assessed by incubating cells with WST-1 reagent (Beyotime [SuZhou, China], catalog number: C0035) for 2 hours and measuring the absorbance at 450 and 630 nm (as reference) with a microplate reader (Bio-Rad). The cell survival rate was calculated as follows: (OD experimental group − OD control group )/OD control group × 100%.
2.5
Hoechest 33258 staining
Replicate cultures of 1 × 10 6 cells/well were plated on a 24-well plate. The cells were transfected with plasmid and/or treated with CDDP. After a change of fresh medium 24 hours later, the cells were incubated with 5 μ L of Hoechst 33258 (Beyotime, catalog number: C0003) solution per well at 37°C for 10 minutes, followed by observation under a fluorescence microscope. Strong fluorescence can be observed in the nuclei of apoptotic cells, whereas weak fluorescence was observed in nonapoptotic cells. Quantification of apoptotic cells was performed by taking the images in random fields and counting at least 200 cells in 4 random fields in each well.
2.6
Quantification of monodansylcadaverine cell labeling
Monodansylcadaverine (MDC; Sigma, catalog number: 30432) is a specific autophagolysosome marker for analyzing the autophagic process. Hep-2 cells were treated with CDDP (0.5 μ g/mL) for 24 hours. Autophagic vacuoles were labeled with MDC by incubating cells with 0.05 mM MDC in Dulbecco modified Eagle medium at 37°C for 10 minutes, at various times after CDDP treatment. After incubation, cells were washed 3 times with phosphate-buffered saline and immediately analyzed with a fluorescence microscope (Nikon Eclipse TE 300, Beijing, China) equipped with a filter system (V-2A excitation filter: 380/420 nm; barrier filter: 450 nm). Images were captured with a CCD camera and imported into Adobe Photoshop.
2.7
Statistical analysis
Statistical analysis of results was performed using analysis of variance and Tukey multiple range tests using Prism version 2.0 software (GraphPad Software, San Diego, CA); statistical significance was defined as P ≤ .05.
2
Materials and methods
2.1
Cell culture and transfection
The human laryngeal cancer Hep-2 cell line was purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Hep-2 cells were cultured in Dulbecco modified Eagle medium containing 10% fetal bovine serum (Gibco [New York, NY, USA], catalog number: 16000-044), penicillin (10 U/mL; Sigma [Los Angeles, CA, USA], catalog number: P0781) and streptomycin (0.1 mg/mL; catalog number: P0781) and were kept in a humidified atmosphere of 5% CO 2 at 37°C. Cells were seeded into 6-cm Petri dishes (5 × 10 5 cells/well) and were transfected at 80% confluence with pSUPER-Bec transfectants ( Beclin 1 gene partially silenced) using lipofectamine 2000 reagent (Invitrogen Life Technologies [CA, USA], catalog number: 1668027) as described by the manufacturer. The similar fashion with pSUPER-non (scramble RNA) was transfected, and the parental Hep-2 cells were used as the control.
2.2
Western blot analysis
The cultured cells were washed twice with phosphate-buffered saline and lysed with cold radio immunoprecipitation assay lysis buffer containing protease inhibitors (phenylmethylsulfonyl fluoride 1 mmol/L and leupeptin 0.1 g/L). Cell lysates were collected from culture plates using a rubber policeman, and protein was collected by centrifugation. Protein concentrations were determined using the bicinchoninic acid protein assay (Pierce, catalog number: 23225). Aliquots containing 40 μ g of proteins were boiled in 2 × loading buffer (0.1 M Tris-Cl, pH 6.8, 4% sodium dodecyl sulfate, 0.2% bromophenyl blue, and 20% glycerol) for 10 minutes, loaded into 10% Tris-HCl polyacrylamide gels, and transferred electrophoretically to Immobilon-P membrane (Millipore Corporation, catalog number: IPVH07850). Membranes were incubated with primary antibodies and appropriate horseradish peroxidase secondary antibodies. Membranes were additionally probed with an antibody against actin (Santa Cruz Biotechnology [Santa Cruz, CA], catalog number: sc-1616) to ensure equal loading of protein between samples. Detection was performed with chemiluminescent agents (Pierce, catalog number: 26651).
2.3
Polyclonal antibodies
Anti-Beclin 1 antibody (Santa Cruz, catalog number: sc-10086) recognizes a single 60-kd band on Western blot. Anti-LC3 antibody (Santa Cruz, catalog number: sc-16756) recognizes 16- and 18-kd bands. Anti–caspase-9 antibody (Cell Signaling, catalog number: 9502), anti–caspase-3 antibody (Cell Signaling, catalog number: 9662), anti–caspase-8 antibody (Cell Signaling, catalog number: 4790), anti–cleaved caspase-9 antibody (Cell Signaling, catalog number: 9501), anti–cleaved caspase-3 antibody (Cell Signaling, catalog number: 9661), and anti–cleaved caspase-8 antibody (Cell Signaling, catalog number: 9749) recognize single 35-, 57-, 47-, 17-, 37-, and 18-kd band on Western blot, respectively.
2.4
WST-1 assay for cell viability and cell proliferation
Hep-2 cells were plated in 96-well plates at 5000 or 6000 cells/well. The next day, cells were treated with or without 0.25, 0.5, and 1.0 μ g/mL CDDP with 5 to 6 replicates. According to the experiment, the cytotoxity or cell viability was assessed by incubating cells with WST-1 reagent (Beyotime [SuZhou, China], catalog number: C0035) for 2 hours and measuring the absorbance at 450 and 630 nm (as reference) with a microplate reader (Bio-Rad). The cell survival rate was calculated as follows: (OD experimental group − OD control group )/OD control group × 100%.
2.5
Hoechest 33258 staining
Replicate cultures of 1 × 10 6 cells/well were plated on a 24-well plate. The cells were transfected with plasmid and/or treated with CDDP. After a change of fresh medium 24 hours later, the cells were incubated with 5 μ L of Hoechst 33258 (Beyotime, catalog number: C0003) solution per well at 37°C for 10 minutes, followed by observation under a fluorescence microscope. Strong fluorescence can be observed in the nuclei of apoptotic cells, whereas weak fluorescence was observed in nonapoptotic cells. Quantification of apoptotic cells was performed by taking the images in random fields and counting at least 200 cells in 4 random fields in each well.
2.6
Quantification of monodansylcadaverine cell labeling
Monodansylcadaverine (MDC; Sigma, catalog number: 30432) is a specific autophagolysosome marker for analyzing the autophagic process. Hep-2 cells were treated with CDDP (0.5 μ g/mL) for 24 hours. Autophagic vacuoles were labeled with MDC by incubating cells with 0.05 mM MDC in Dulbecco modified Eagle medium at 37°C for 10 minutes, at various times after CDDP treatment. After incubation, cells were washed 3 times with phosphate-buffered saline and immediately analyzed with a fluorescence microscope (Nikon Eclipse TE 300, Beijing, China) equipped with a filter system (V-2A excitation filter: 380/420 nm; barrier filter: 450 nm). Images were captured with a CCD camera and imported into Adobe Photoshop.
2.7
Statistical analysis
Statistical analysis of results was performed using analysis of variance and Tukey multiple range tests using Prism version 2.0 software (GraphPad Software, San Diego, CA); statistical significance was defined as P ≤ .05.
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