Lung cancer is a worldwide leading cause of cancer deaths, with gradually increasing incidence and mortality. Despite the improvements in lung cancer diagnostic imaging techniques and advances in the treatments for lung cancer during the past decade, little progress has been made in the improvement of its survival rates. Apoptosis is known to be a fundamental process for the eradication of defective or potentially damaged cells, providing a defense mechanism against malignant transformation.1 Apoptosis has been considered as a potential target for cancer therapy at various stages of cancer development.
Several potent endogenous proteins that inhibit apoptosis have been identified, including the inhibitor of apoptosis (IAP) families in mammalian cells.2 IAP genes encode a family of proteins that bind and inactivate the key caspases involved in the initiation (Caspase-9) and execution (Caspase-3 and -7) of this cascade.3 An elevated expression level of X-linked inhibitor of apoptosis (XIAP), the most potent member of the mammalian IAPs, is associated with tumor resistance to cell death cues.4,5 XIAP-associated factor 1 (XAF1) is previously identified as a XIAP binding partner,6 and it directly interacts with endogenous XIAP and results in XIAP sequestration in nuclear inclusions. XAF1 antagonizes the anticaspase activity of XIAP and reverses the protective effect of XIAP overexpression in cell lines.6 Reduced or absent expression of XAF1 is a frequent event in human cancer cell lines7,8 and human cancer tissues.8-10 Loss of XAF1 expression correlated strongly with tumor staging.11 The restoration of XAF1 expression induces apoptosis and autophagy, and inhibits tumor growth in gastric, colon, liver, pancreatic and prostate cancers,12-16 implicating that XAF1 functions as a tumor suppressor. In this study, we investigated the expression of XAF1 and its role in apoptosis induction in squamous cell lung cancer. We found that the low expression of XAF1 was associated with tumor grade, disease stage, differentiation status and lymph node metastasis in squamous cell lung cancer tissues. The restoration of XAF1 expression mediated by Ad5/F35-XAF1 virus significantly inhibited cell proliferation and induced apoptosis in a dose- and time-dependent manner. Thus, our results document that XAF1 is a potential prognosis biomarker and a target for squamous cell lung cancer therapy.
Patients and tissue samples
Tumor samples were obtained from squamous cell lung cancer patients who underwent surgeries from 2008 to 2009 in the Second Affiliated Hospital of Soochow University. All cases were staged according to the guidelines of University-Industry Cooperation Committee (UICC2009). All clinicopathological data were collected and shown in Table 1. The study was approved by the Ethical Review Board of the Second Affiliated Hospital, Soochow University.
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Table 1. Relationship of XAF1 expression with clinicopathologic parameters in squamous cell lung cancer (n (%))
Formalin-fixed paraffin-embedded samples from the squamous cell lung cancer tissues were reviewed by a pathologist. At least two core tissue biopsies were taken from morphologically representative regions of each lung tumor. Cancer tissues, cancer adjacent tissues (less than 2 cm away from the cancer tissues) and normal lung tissues (more than 5 cm away from the cancer tissues) were collected from 51 squamous cell lung cancer patients. Tissue sections were dewaxed in xylene, rehydrated in ethanol with descending concentration, and incubated with anti-XAF1 (1:500, Abcam, England) or XIAP (1:100, BD Biosciences, USA) antibodies. Then sections were incubated with horseradish peroxidase-labeled secondary antibody. The immunostaining images and the staining scores were assessed by two independent observers without prior knowledge of the clinicopathologic characteristics. A consensus score was reached for each core. Overall intensity was assessed on a four-point scale: negative (0), lung cancer cells with no positive staining; weak (1), lung cancer cells stained less intensely than normal lung tissues; moderate (2), lung cancer cells stained as intensively as normal lung tissues; strong (3), lung cancer cells more intensively stained than normal lung tissues. The low expression XAF1 protein was defined as a weak XAF1 staining compared to XAF1 staining in normal tissues.
RNA was extracted from cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer instructions. Cellular RNA (2 µg) was reverse-transcripted with Moloney murine leukemia virus RT (Promega, USA) using random hexamer primers. XAF1 mRNA was determined by PCR using XAF1-specific primers as follows: forward primer: 5′-GAGGAGATAAAGCAGCCTATGAC-3′; reverse primer: 5′-CTCTTGCCTGATTGCTGTGG-3′. PCR products were separated on a 1% (w/v) agarose gel and stained with ethidium bromide. Gel images were captured on a digital camera. The low expression XAF1 mRNA was defined as a reduction of XAF1 expression in tumor tissue compared to adjacent normal tissues.
Generation of adeno-XAF1 virus
XAF1 full-length cDNA was cloned into a pDC316 carrier plasmid (Benyuanzhengyang, Beijing, China) to generate pDC316-XAF1 plasmid. The plasmid pDC316-XAF1 and the skeleton plasmid pBHG-fiber5/35 (Benyuanzhengyang) were co-transfected into HEK293-T cells (a human embryonic kidney cells that contain the SV40 Large T-antigen) using Polyfect (Qiagen, Hilden, Germany). The co-transfection generated the recombinant Ad5/F35-XAF1 virus. Successful recombination was confirmed by observation of cytotoxicity. The Ad5/F35 control virus was generated using the same protocol.
Cell culture and gene transduction
Human squamous cell lung cancer cell lines SK-MES-1 were maintained in DMEM (Invitrogen) supplemented with 10% fetal bovine serum under 37°C, 95% humidified air and 5% CO2. For gene transduction, cells were incubated with adenoviral aliquots at different multiplicity of infection (MOI) (50, 100 and 150) for 24 hours before the addition of culture medium. Twenty-four, 48 and 72 hours after infection, cells were then harvested for cell proliferation and apoptosis analysis and measurements of XAF1 mRNA and protein.
Cell viability analysis
Cells were seeded at 1×104 per well into 96-well plates. Cell viability was determined by 3-(4,5- dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT; Sigma, USA) assay according to the standard protocol. The rate of inhibition was calculated according to following formation: the rate of cell inhibition = (1–ODAd5/F35 /ODcontrol) ×100%. Each treatment was repeated at least three times and the proliferation values were expressed as mean ± standard error (SE).
Apoptosis was measured using an Annexin V-FITC Apoptosis Detection Kit (BD Pharmingen, USA) by flow cytometry assay. In brief, cells were resuspended in binding buffer, and had been incubated with 5 μl of FITC-conjugated Annexin V for 15 minutes. An additional 5 μl of PI was added, and then the cells were analyzed by flow cytometry. Each treatment was run in triplicate and the results were expressed as mean ± standard error (SE).
Western blotting analysis
Cells were lysed in RIPA buffer and protein samples were subjected to SDS gel electrophoresis, transferred to polyvinylidene fluoride membranes (Bio-Rad, USA), and the blot was incubated with primary antibodies at the indicated dilutions: XAF1 (1:1000, Santa Cruz, USA), XIAP (1:1000, BD Pharmingen) and beta-actin (1:5000, Sigma), and then incubated with a horseradish peroxidase-conjugated secondary antibody. The antigen-antibody complexes were visualized by the enhanced chemiluminescence system (ECL, Amersham Pharmacia Biotech, England).
Statistical analysis was conducted using SPSS17.0 software. The chi-squeare test was used to evaluate the correlation between XAF1 expression and clinicopathologic characters. One-way analysis of variance (ANOVA) was used to compare values of test and control samples. P values of less than 0.05 were considered to be statistically significant.
XAF1 is less expresssed in squamous cell lung cancer tissues than in normal tissues
We determined the expression of XAF1 in 51 squamous cell lung cancer tissues by immunohistochemical staining. Low expression of XAF1 was observed in 23 of 51 (45.1%) squamous cell lung cancer tissues, 11 of 51 (21.6%) cancer adjacent tissues and 7 of 51 (13.3%) normal lung tissues. Statistical analysis showed that the expression of XAF1 in cancer tissues was significantly lower than those in cancer adjacent tissues and normal lung tissues (P=0.045). No significant difference in XAF1 expression was observed between normal lung tissues and cancer adjacent tissues (P=0.087) (Figure 1). In addition, we also observed high expression of XIAP in squamous cell lung cancer tissues compared to normal lung tissues (P=0.031) (Figure 1).
We further determined the expression of XAF1 mRNA in 3 squamous cell lung cancer tissues. The levels of XAF1 mRNA in cancer tissues were lower than those in cancer adjacent tissues and normal lung tissues (P=0.006) (Figures 2 and 3). The levels of XIAP mRNA in cancer tissues were significantly higher than those in cancer adjacent tissues and normal lung tissues (P=0.004) (Figures 2 and 3). The results suggest that low expression of XAF1 and high expression of XIAP may be associated with the development of squamous cell lung cancer. However, we did not find the correlation between XAF1 and XIAP expressions in squamous cell lung cancer.
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Figure 1. The expressions of XAF1 and XIAP proteins in squamous cell lung cancer tissues, cancer adjacent tissues and normal lung tissues were determined by immunohisto- chemical staining (original magnifications ×200).
Figure 2. The expression of XAF1 mRNA in squamous cell lung cancer tissues. The expression levels of XAF1 and XIAP mRNA were determined by RT-PCR in normal lung tissues, cancer adjacent tissues and squamous cell lung cancer tissues.
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Figure 3. The intensity of XAF1 and XIAP mRNA expression in the normal lung tissues, cancer adjacent tissues and squamous cell lung cancer tissues. *,†P <0.01 vs. normal tissues, respectively.
Low expression of XAF1 correlates with poor prognosis of squamous cell lung cancer
We then determined the correlations between XAF1 expression and clinicopathologic variable. The XAF1 expression was not associated with these clinical characteristics including gender, age, and smoke (Table 1). However, low expression of XAF1 in squamous cell lung cancer tissues was related with tumor grade, disease stage, differentiation and lymph node metastasis (Table 1). The 84.6% (11 of 13) of the cases in stage I showed a strong expression of XAF1. The 48.0% (12 of 25) of the cases in stage II exhibited a moderate expression of XAF1, and only 38.5% (5 of 13) of the cases in stage III presented a weak expression. Statistical analysis showed that the disease stage was significantly associated with low expression of XAF1 (P=0.041). The lung squamous cell carcinoma patients with lymph node metastasis (N1-3) had a significantly lower level of XAF1 expression (26.3%, 5 of 19) compared to lung squamous cell carcinoma patients without lymph node metastasis (N0) (P=0.002) (Table 1). Furthermore, XAF1 expression was significantly lower in poorly differentiated cancers (27.3%, 3 of 11) compared to that in moderately or well differentiated cancers (62.5%, 25 of 40) (P=0.038) (Table 1). The results indicate that low expression of XAF1 correlates with poor prognosis of squamous cell lung cancer.
Restoration of XAF1 expression inhibits cell proliferation in squamous cell lung cancer cell lines
Next, we determined the effect of restoration of XAF1 on lung cancer cell growth. We chose a squamous cell cancer cell line SK-MES-1 with low expression of XAF1. The SK-MES-1 cells were treated with Ad5/F35-XAF1 virus and control virus at different MOI (10, 50, 100, 150) and different time (24 hours, 48 hours, 72 hours). The results showed that the infection of Ad5/F35-XAF1 markedly increased the expressions of XAF1 mRNA and protein (Figure 4), and significantly reduced cell viability in a dose- and time-dependent manner (P=0.034, Table 2, Figures 5 and 6). The results suggest that the restoration of XAF1 inhibits cell proliferation in squamous cell lung cancer cells.
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Figure 4. The transduction of Ad5/F35-XAF1 virus increases mRNA expression (A) and protein expression (B) of XAF1 in squamous cell lung cancer cell lines determined by RT-PCR (A) and Western blotting (B), respectively.
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Table 2. Effect of the restoration of XAF1 expression on cell growth of squamous cell lung cancer cell lines (mean ± SE, %)
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Figure 5. The restoration of XAF1 expression inhibits cell growth of squamous cell lung cancer cells in a dose- and time-dependent manner. Cell viability was determined by MTT method.
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Figure 6. Treatment of Ad5/F35-XAF1 virus induces apoptotic morphological alterations in SK-MES-1 lung cancer cells. A: original magnification ×100. B: original magnification×200.
Restoration of XAF1 expression induces apoptosis in squamous cell lung cancer cell line
To examine whether the decrease in cell viability was accompanied by apoptosis, we determined the apoptosis using flow cytometry analysis. The apoptotic rate was significantly increased in the cells infected with Ad5/F35-XAF1 virus compared to those infected with control virus (P=0.0041) (Table 3 and Figure 7). The infection of Ad5/F35-XAF1 virus induced apoptosis in a dose-dependent manner. The results suggest that the restoration of XAF1 expression induces apoptosis in squamous cell lung cancer cells.
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Table 3. Effect of restoration of XAF1 expression on apoptosis of squamous cell lung cancer cell lines (mean±SE, %)
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Figure 7. The Ad5/F35-XAF1 virus infection induces apoptosis of cells in squamous cell lung cancer detected by FACS analysis. *P <0.05 vs. Ad5/F35-EGFP MOI 50. †P <0.01 vs. Ad5/F35-EGFP at the same MOI.
Cancer is a heterogeneous disease. Despite its complexity, there are fundamental characteristics shared by all cancers. The inhibition of apoptosis is one of these fundamental and requisite alterations that occur in all cancer cells, regardless of origin.17 Apoptosis is the primary means by which radiotherapy and chemotherapy can kill cancer cells. Intense efforts are underway to identify new approaches that directly target apoptosis pathways.18-20 The process of apoptosis is regulated at multiple levels by several regulatory mechanisms, such as the IAPs family that bind and inhibit the activity of caspase.21-23 Induction of apoptosis has been recognized as a new approach for cancer therapy.
The accumulating studies have shown that XAF1 is a potential tumor suppressor.24-26 XAF1 can dramatically sensitize cancer cells to apoptotic triggers, such as TNF-related apoptosis-inducing ligand (TRAIL), etoposide, 5-fluorouracil, H2O2, γ-irradiation, ultraviolet.14,15,22,27 XAF1 is therefore believed to play an important role in the induction of apoptosis. The loss of XAF1 has been observed in a variety of cancer cell lines and human cancers,28,29 and is associated with the development and progression of malignant tumors.11,12 The loss of XAF1 is due, at least in part, to epigenetic alterations such as DNA methylation at the CpG sites within the promoter region.11,13 Reactivation of XAF1 by DNA methylation inhibitors restores the sensitivity of cancer cells to apoptosis-inducing agents.30 Therefore, the enhancement of XAF1 levels will provide a promising strategy for cancer therapy.
In this study, we investigated the expression of XAF1 and its role in induction of apoptosis in squamous cell lung cancer. We found that the expression of XAF1 was reduced or absent in human squamous cell lung cancer tissue and cell lines compared to that in cancer adjacent tissues and normal lung tissues. The low levels of XAF1 expression were associated with tumor grade, disease stage, differentiation status and lymph node metastasis in squamous cell lung cancer. The other clinical characteristics including gender, age and smoke, did not reveal any significant association with the expression of XAF1. Our result is consistent with previous reports7-10 that the low expression of XAF1 is a prognostic biomarker in several cancers including gastric, colon, pancreatic and bladder cancers. Our results suggest that XAF1 is a poor prognosis biomarker in squamous cell lung cancer.
In this study, we found that the XIAP expression was significantly increased in squamous cell lung cancer tissues compared that in cancer adjacent and normal tissues. XIAP has been shown to be an independent prognostic factor in certain types of cancer. However, we did not find the correlation between XAF1 and XIAP expression in squamous cell lung cancer, suggesting that XAF1 may not directly modulate XIAP expression and its function in other mechanisms independently of XIAP. The role of XIAP expression in squamous cell lung cancer needs to be further investigated.
It has reported that the restoration of XAF1 expression induces apoptosis and inhibits tumor growth in gastric, colon, pancreatic cancer.14-16 In this study, we found that the restoration of XAF1 expression in squamous cell lung cancer cells could inhibit cell proliferation and induce apoptosis. The results indicate that the loss expression of XAF1 may play an important role in the development of squamous cell lung cancer. XAF1 may be a valuable biomarker for prognosis and a target for therapy of squamous cell lung cancer.
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(Received May 30, 2011)
Edited by WANG Mou-yue and LIU Huan