XIAP Is Highly Expressed In Esophageal Cancer And Its Downregulation By RNAi Sensitizes Esophageal Carcinoma Cell Lines To Chemotherapeutics

ObjectiveMalignant cells commonly have defects in cell death control and apoptosis, and inhibition of apoptosis can lead to tumorigenesis and resistance to therapy. Most chemotherapeutic drugs counteract cancer by inducing cell apoptosis. Apoptosis resistance enables cancer cells to survive, although exposed to many proapoptotic factors, such as cytotoxic drugs, anoxemia, and radialization Cancer cells escape apoptosis by a number of mechanism among which overexpression of antiapoptotic genes has been shown to play a critical role, such as overexpression of some members of the IAP gene family or the Bcl-2 family. The inhibitor of apoptosis proteins (IAPs) have been identified as acting downstream of Bcl-2 by inhibiting caspases. To date, eight members of IAPs have been identified in humans, and x-linked inhibitor of apoptosis protein (XIAP) is the most potent one. XIAP can inhibit caspases-3, -7 and -9, and is the only member of IAP family able to directly inhibit both the initiation and execution phase of the caspase cascade crucial to mediate the controlled demise of malignant cells. Esophageal squamous cell carcinoma, the major histologic form of esophageal cancer, is one of the most frequent fatal malignancies in the world, especially in the northern part of China. Treatment of ESCC has primarily relied on classical modalities including surgery, radiotherapy and chemotherapy or a combination of these methods, but the outcome has not improved significantly. Most of the treatment failures are due to relapse after surgery or metastatic disease resistant to systemic therapy. Apoptosis resistance in ESCC accounts for its poor response to chemotherapy and enhanced metastasis. Therefore, it is necessary to search for new treatment strategies. Whether XIAP could be a therapeutic target in ESCC was still unknown.In this study, the expression of XIAP in ESCC cases with reference to normal mucosa, as well as the expression of XIAP in ESCC cell lines, was evaluated. Then we investigated whether the downregulation of XIAP expression in ESCC cell lines by siRNA could enhance cell apoptosis and the chemotherapeutic effects of Paclitaxel, Cisplatin, Fluorouracil and Etoposide, the chemotherapeutic agents used currently in the treatment of patients with ESCC.Materials and Methods1. Patients and Tissue Specimens: Specimens of cancer tissues and matched adjacent normal mucosa were taken from 110 consecutive patients. All patients in this study had undergone curative tumor resection in the Department of Thoracic Surgery, the First Affiliated Hospital of Anhui Medical University between Oct 2004 and Sept 2005. None of the patients had received radiotherapy or chemotherapy before surgery. Tumor tissues were dissected from the resected specimens and the normal tissue blocks were taken from the distal resection margin. The specimens for RT-PCR and Western blot were snap-frozen in liquid nitrogen, and the specimens for immunohistochemistry were fixed in 4% polyformaldehyde and embedded in paraffin.2. Cell Lines and Cell Culture: Human ESCC cell lines TE10, KYSE30, KYSE70, KYSE150, KYSE410, KYSE450, KYSE510 and EC9706 were grown in RPMI1640 medium with 10% fetal bovine serum. The human esophageal squamous cancer cell line KYSE series was a generous gift from Dr. Shimada Y and EC9706 was kindly provided by Prof. Mingrong Wang.3. Tissue Microarray Construction, Immunohistochemistry and Evaluation of the IHC Staining: Tissue microarrays (TMA) containing 110 ESCC cases were constructed with a Beecher Instruments for Tissue Array. After the construction of the array block, multiple 4μm sections were cut with a microtome using an adhesive-coated tape sectioning system. H&E-stained sections were used for histological verification of tumor and normal tissues on the arrayed samples.Then immunohistochemistry (IHC) was performed using a diaminobenzidine-base detection method. Omitting the primary antibody from the immunohistochemical procedure and replacing it with antibody diluent acted as negative controls.The percentage of XIAP positive cells was determined semiquantitatively by assessing the entire sample. Each sample was assigned to one of the following categories: 0 (0-4%), 1 (5-24%), 2 (25-49%), 3 (50-74%), or 4 (75-100%). The intensity of immunostaining was determined as 0 (negative), 1+ (weak), 2+ (moderate), or 3+ (strong). A final immunoreactive score between 0 and 12 was calculated by multiplying the percentage of positive cells with the staining intensity score. In this study, scores of 8-12 were defined as XIAP "high expression", and scores of 0-7 were defined as "negative or reduced expression". All slides were evaluated for immunostaining independently by two observers with no prior knowledge of patients' clinical data.4. siRNA Transfection and The Effect Confirmation: siRNA targeting XIAP (Hs_BIRC4₅ HP Validated siRNA 1027400, Cat. SI00299446) was designed and synthesized by Qiagen company. The control (nonsilencing) siRNA was designed by Qiagen company and synthesized by GeneChem company. Transfection was performed in 50%-60% confluent cells using Lipofectamine2000 Reagent according to the manufacture's protocol. The downregulation of XIAP by siRNA was confirmed by RT-PCR and Western blot at the indicated time points.5. RT-PCR: Total RNA was extracted from frozen tissues or cells using TRIzol reagent according to the instructions of the manufacturer. Five micrograms of total RNAs of each sample were reverse transcribed to the first strand of cDNA primed with random hexamers using Superscript? First-Strand Synthesis System for RT-PCR kit. The products were electrophoresed by 1.2% agarose-gel and semi-quantified by Gel-pro Analyzer image analysis software. 6. Western Blot Analysis: For Western blot analysis, tissues or cells were lysed with the buffer. The protein concentrations were determined using the BCA Protein Assay kit. Thirty micrograms of protein were separated on 10% SDS-PAGE gels and transferred to PVDF membrane. After blocking, the membrane was incubated with anti-XIAP antibody (1:1000) at 4℃overnight. After washing, the membrane was incubated with secondary antibody at a dilution 1:3000 at room temperature for 1 hour. Proteins were detected with the ECL kit and anti-β-actin antibody was used as loading control. Densitometry was performed by Gel-pro Analyzer software.7. Flow Cytometric Assay: Cells were seeded in 6-well plates. Forty-eight hours after transfection, both floating and attached cells (use trypsin) were collected. Cells were incubated with 5μg/ml propidium iodide and 50μg/ml RNase-A in PBS for 30 min in 37℃. Flow activated cell sorter analysis was carried out using a FACSCalibur flow cytometer with CellQuest software. A total of 10,000 cells were measured per sample. The sub-G₁-G₀ cell fraction was considered as representative of apoptotic cells.8.Treatments with Chemotherapeutic Agents and Measurement of Cell Viability: Growth inhibition of ESCC cells were determined by the colorimetric MTT cell viability/proliferation assay. In brief, cells were seeded in 96-well plates with 4000 cells per well. Twenty-four hours after transfection, the chemotherapeutics were added in varying concentrations to each well. Cells were incubated for 72 hours, then the media were replaced with 100μl MTT, dissolved in RPMI1640 at the final concentration of 0.5mg/ml. The plates were incubated for an additional 4 hours, then the medium was aspirated off leaving the dark blue formazan product in the bottom of the wells. The absorbency was detected at 570nm on Bio-Rad model 550-microplate Reader after 200μl of DMSO were added to each well to dissolve the formazan crystals. The percentage of dead (or growth inhibited) cells was normalized to untreated controls. All of the experiments were carried out at least three times in triplicate.9. Statistical Analysis: Statistical analysis was done using the SPSS statistical software. The difference between XIAP expression in tumor tissues and normal tissues was performed by unpaired Student's t test. The correlation between XIAP expression and clinicopathologic characteristics was analyzed using Chi-square test and Spearman's correlation analysis. 50% inhibiting concentration (IC50) was calculated using Probit analysis. Differences in mean values between control treatment and combination treatments were analyzed using Student-Neuman-Keuls analysis. P<0.05 was considered statistically significant.Results1. Overexpression of XIAP in Human ESCC Tissues at mRNA and Protein Level: RT-PCR analysis of XIAP mRNA expression in patient-matched normal and tumor tissues showed that XIAP mRNA was up-regulated in ESCC tissues compared with their normal counterparts (P<0.01). Western blot analysis showed that XIAP protein was overexpressed in ESCC tissues (P<0.01). And this corresponds to the result of immunohistochemistry detection of TMA samples.2. XIAP Expression with Clinical Features: To further characterize the expression of XIAP with clinical features, we performed tissue microarray analysis of 110 paired ESCC tissue specimens, among them, 102 samples could be assessed due to the loss of paraffin sections from slides. XIAP was localized to cytoplasm and rather diffuse. XIAP protein showed significantly high expression in ESCC compared with normal mucosa (P=0.000). However, the increased expression of XIAP in ESCC tissues did not show obvious correlation with any of the clinicopathologic characteristics, including patients' age, gender, and tumor histo-pathological features.3. Expression Pattern of XIAP in ESCC Cell Lines: Multiple ESCC cell lines were examined for XIAP protein expression by Western blot analysis. The highest level of XIAP expression was detected in KYSE30 KYSE150, KYSE410, KYSE450 and EC9706 cell, whereas low XIAP expression was observed in TE10, KYSE70 and KYSE510 cells. We chose both XIAP high expression cell lines KYSE150, EC9706 and XIAP low expression cell lines TE10, KYSE510 in subsequent experiments.4. Expression of XIAP was Blocked Efficiently by RNAi: The expression of XIAP was examined by RT-PCR and Western blot at 24, 48, and 72hours after siRNA transfection. We found that XIAP specific siRNA can efficiently block XIAP expression both at mRNA and protein level. And the efficiency can reach more than 95% at protein level in all the cell lines.5. Effect of RNAi on Apoptosis: The apoptosis rate was11.63%±0.058% in KYSE150 and 11.30%±0.100% in EC9706 respectively, which were significantly higher than mock and control siRNA transfection (P<0.01, Fig.5). However, no obvious apoptosis was observed in either TE10 or KYSE150 cells whose XIAP expression was low.6. XIAP Downregulation Sensitizes ESCC Cells to Chemotherapeutics: In KYSE150 and EC9706 cells, our result demonstrated that the cells exposed to XIAP siRNA in the presence of Paclitaxel, Cisplatin, Fluorouracil, or Etoposide showed a significant decrease in IC₅₀ compared with control siRNA, mock transfection or no-treatment. As shown in Fig.6B, XIAP siRNA treatment significantly enhanced the growth inhibitory effect of Paclitaxel, Cisplatin, Fluorouracil, and Etoposide in both cell lines. The control siRNA had either no effect or only a minimal effect.But for TE10 cell, the IC₅₀ of Paclitaxel, Cisplatin, or Fluorouracil, three of the four drugs, could be decreased sightly by XIAP siRNA; and for KYSE150 cell, only the IC₅₀ of Paclitaxel could be decreased by XIAP siRNA compared with control siRNA, mock transfection or no-treatment group. Similarly, the growth inhibitory effect of Paclitaxel, Cisplatin and Fluorouracil could be enhanced slightly by XIAP siRNA in TE10 cell; and XIAP siRNA could only enhance the growth inhibitory effect of Paclitaxel in KYSE510 cell. Moreover, the degree of either the decrease of IC₅₀ or the enhancement of growth inhibitory of the drugs in TE10 or KYSE510 was much smaller than in KYSE150 or EC9706 respectively (P<0.05)Conclusion1. In this study, we detected XIAP expression using RT-PCR, Western blot and immunohistochemistry on ESCC tissues and the adjacent normal tissues. XIAP was highly expressed in ESCC tumors compared with normal tissues. Different methods showed similar results. However, there was no significant correlation between XIAP expression and clinicopathologic characteristics. Because of its significantly higher expression in ESCC compared with normal mucosa, it may be a feasible marker for the diagnosis of ESCC.2. XIAP downregulation could increase apoptosis in XIAP high expression ESCC cell lines, KYSE150 and EC9706, but could not increase apoptosis in XIAP low expression ESCC cell lines, TE10 and KYSE150. From this result we could draw a conclusion that the apoptosis was XIAP dependent. And this confirmed the role that XIAP and caspase apoptosis pathway played in pathogenesis of ESCC.3. Compared with XIAP low expression cell lines, TE10 and KYSE510, XIAP siRNA mediated decrease in XIAP expression resulted in enhanced tumor cell killing by Paclitaxel, Cisplatin, Fluorouracil, and Etoposide in greater degree in KYSE510 and EC9706. And XIAP siRNA could enhance the growth inhibitory effect of these drugs more significantly in XIAP high expression cell lines than the low expression cell lines. These data indicated that combination treatments were more effective at inducing cytotoxic effect in tumor cells, compared with treatment of either siRNA or anticancer drug alone. Furthermore, together with the result of the experiment in apoptosis detection, it is clear that the ESCC cell killing was XIAP dependent and downregulation of XIAP did sensitize ESCC cells to chemotherapeutics. These results suggest that XIAP siRNA combined with Paclitaxel, Cisplatin, Fluorouracil, and Etoposide may be a feasible strategy to enhance the effects of chemotherapy in patients with ESCC.