Up-regulation of survivin in oral squamous cell carcinoma correlates with poor prognosis and chemoresistance

Vol. 110 No. 4 October 2010

ORAL AND MAXILLOFACIAL PATHOLOGY

Editor: Mark W. Lingen

Up-regulation of survivin in oral squamous cell carcinoma correlates with poor prognosis and chemoresistance Su Liping, PhD,a Wang Ying, PhD,a Xiao Mingzhen, MD,a Lin Yuan, MD,a and Yu Lei, PhD,b Xi’an, China FOURTH MILITARY MEDICAL UNIVERSITY Survivin, a key member of the inhibitor of apoptosis protein family, has been reported to be capable of regulating both cellular proliferation and apoptotic cell death. This protein is found to be overexpressed in many human cancers. The aim of this study was to evaluate the prognostic significance of survivin mRNA expression in oral squamous cell carcinoma (OSCC) and to analyze its correlation with chemoresistance. Reverse-transcription polymerase chain reaction assay was performed to detect the expression of survivin mRNA in OSCC cell lines or tissue samples. Immunohistochemistry was performed to detect the expression of survivin protein in OSCC tissues or corresponding nontumor tissues. Then the correlation between survivin mRNA expression and clinicopathologic features or prognosis of OSCC patients was analyzed. Small interfering RNA technology was used to down-regulate the expression of the survivin gene in the OSCC cell line. Methylthiazol tetrazolium and flow cytometric assays were performed to detect proliferation and apoptosis of the OSCC cell line (HSC-3). Furthermore, the effect of small interfering RNA (siRNA) targeting survivin on the sensitivity of OSCC cells to chemotherapeutic agents (cisplatin and 5-fluorouracil [5-FU]) was determined. Results showed that the levels of survivin mRNA expression were significantly higher in OSCC cells or tissues than those in normal human oral keratinocyte or corresponding noncancerous tissues. The immunostaining of survivin protein was significantly stronger in OSCC tissues than in corresponding nontumor tissues. Moreover, high survivin mRNA expression was correlated with poorer tumor differentiation, higher clinical stage, and the presence of lymph node metastasis (P ⬍ .05). Multivariate analysis showed that the status of survivin mRNA could be an independent prognostic factor for OSCC patients (hazard ratio 2.71, 95% confidence interval 1.465.10; P ⫽ .012). In addition, siRNA-mediated survivin down-regulation could significantly inhibit proliferation and induce apoptosis of OSCC cells. Suvivin down-regulation could also significantly enhance chemosensitivity of OSCC cells, which was associated with apoptosis enhancement. Thus, the status of survivin mRNA expression was a potential prognostic factor for OSCC patients, and siRNA-mediated survivin down-regulation could become a novel strategy for chemosensitization of human OSCCs. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010;110: 484-491)

Oral squamous cell carcinoma (OSCC) has become a major oncologic problem around the world.1,2 OSCC affects about 30,000 Americans each year. Over 95% smoke, drink alcohol, or both. In the U.S., 3% of cancers in men and 2% in women are OSCCs, most of

a

Department of Endodontics, College of Stomatology. Department of Urology, Xi Jing Hospital. Received for publication Jan 30, 2010; returned for revision Apr 1, 2010; accepted for publication Apr 5, 2010. 1079-2104/$ - see front matter © 2010 Published by Mosby, Inc. doi:10.1016/j.tripleo.2010.04.009 b

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which occur after the age of 50 years. OSCC patients are treated by classic therapeutic modalities consisting of surgery, radiotherapy, and/or chemotherapy.3 However, OSCC still shows significant mortality rates. If detected at an early stage and treated effectively, the 5-year survival rates of OSCC patients could reach ⬎50%. Currently, there is no proven effective systemic chemotherapy for OSCC, owing to resistance of tumor cells to cytotoxic drugs. Therefore, an understanding of the molecular mechanisms involved in OSCC transformation and progression would be helpful to attain early detection and develop more effective treatments for OSCC patients.

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Survivin, a member of the family of inhibitor of apoptosis proteins (IAPs), is a bifunctional protein that suppresses apoptosis and regulates cell division.4 IAPs are a family of proteins containing ⱖ 1 characteristic baculoviral IAP repeat (BIR) domains. These proteins have multiple biologic activities, including binding and inhibiting caspases, regulating cell cycle progression, and modulating receptor-mediated signal transduction.5 Among the several IAPs, survivin is the smallest member, a 16.5-kDa protein that contains only a single BIR and no RING domain. It has been reported that survivin is overexpressed in many human tumors,6-8 but the clinical significance of survivin expression in OSCC has not been systematically investigated. In the present study, we attempted to explore the expression of survivin gene in OSCC tissue samples and to analyze its correlation with clinicopathologic factors, prognosis, or response to chemotherapy of OSCC patients. MATERIALS AND METHODS Cell culture Four OSCC cell lines (BHY, HSC-2, HSC-3, and HN) and a normal human oral keratinocyte (NHOK) line were cultured in Dulbecco Modified Eagle Medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Sigma, St. Louis, MO), 100 units/mL penicillin G, and 100 ␮g/mL streptomycin (Gibco) at 37°C in a humidified 5% CO2/95% air atmosphere. Patients and tissue samples Sixty-eight patients with histologically verified OSCC were collected from the Department of Oral and Maxillofacial Surgery. None of the patients received chemotherapy or radiation therapy before surgery. Thirty-six patients were male and 32 patients were female. All patients were followed with clinical and radiologic examinations. Staging and grading of the tumors were performed according to World Health Organization histopathologic typing. Patient characteristics were shown in Table I. The study was approved by the Ethics Committee of Shannxi Province Institute of Medicine. Informed consent for experimental use of the samples was obtained from the patients according to the hospital ethical guidelines. All OSCC tissues and corresponding noncancerous tissues were stored in liquid nitrogen until detection. Reverse-transcription polymerase chain reaction (RT-PCR) analysis of survivin mRNA expression Total RNA from cell lines or tissue samples was prepared using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and reverse-transcribed with Expand Reverse Transcriptase (Boehringen, Mannheim, Germany) according to

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Table I. Correlation between survivin mRNA expression and clinicopathologic features Survivin mRNA expression Clinicopathologic feature Gender Female Male Age (y) ⬍60 ⱖ60 Smoker No Yes Tumor size T1/T2 T3/T4 Tumor differentiation Good Moderate Poor Clinical stage I/II III/IV Lymph node metastasis N0 N1/N2

No. of patients

Low (n ⫽ 37)

High (n ⫽ 31)

45 23

26 11

19 12

24 44

14 23

10 21

22 46

9 28

13 18

39 28

22 15

17 14

21 22 25

15 13 9

6 9 16

36 32

25 12

11 20

38 30

26 11

12 19

P value .436

.632

.122

.701

.048*

.008*

.009*

*P ⬍ .05.

the manufacturer’s instructions, using Oligo(dT)18 primers. For survivin, primers used for detection were sense 5=ATGGGTGCCCCGACGTTG-3= and reverse 5=-AGAGGCCTCAATCCATGG-3=. As an interal control, ␤-actin primers were designed as sense 5=-GTGCGTGACATTAAGGAG-3= and reverse 5=-CTAAGTCATAGTCCGCC-3=. The amplification conditions were: denaturing at 94°C for 3 minutes, and 45 cycles at 94°C for 5 seconds and at 57°C for 5 seconds. The amplification products were visualized by electrophoresis on a 1.5% agarose gel stained with ethidium bromide. Western blot analysis of survivin protein expression Protein fractions from cells were extracted using a nuclear/cytosol fraction kit (Kamiya Biomedical Co., Seattle, WA, USA) according to the manufacturer’s specification. The fractions were then separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transfered to nitrocellulose. Immunoblots were processed using a rabbit antihuman antisurvivin monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) followed by detection using enhanced chemiluminescence (ECL Western blotting reagents; Amersham Biosciences, Piscataway, NJ, USA).

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Immunostaining of survivin protein expression Tisssues were fixed in formalin, embedded in paraffin, and cut into 3-␮m sections. The sections were deparaffinized in xylene, dehydrated in a graded ethanol series, and then immersed in methanol with 0.3% hydrogen peroxide for 15 minutes to inhibit endogenous peroxidase activity. Thereafter, the sections were immunostained using 2 types of rabbit polyclonal antibody against human survivin (1:500 dilution; Santa Cruz Biotechnology), rinsed 5 times with phosphatebuffered saline solution (PBS), and then incubated with peroxidase-conjugated antimouse/rabbit immunoglobulins (Boster, Wuhan, China). The sections were then developed with 3,3=-diaminobenzidine tetrahydrochloride and counterstained with hematoxylin. Plasmid construction DNA oligonucleotides targeting human survivin gene (GenBank NM_001168) were synthesized as the following: small interfering RNA (siRNA)/survivin, sense: 5=-GATCCACTGGACAGAGAAAGAGCCTTCAAGAGAGGCTCTTTCTCTGTCCAGTTTTTTTGTCGACA-3=; siRNA/control, sense: 5=-GATCCGACTTCATAAGGCGCATGCACTTCAAGAGAGTGCATGCGCCTTATGAAGTCTTTTTTGTCGACA-3=. Then, these DNA oligonucleotides were inserted into the BamHI and HindIII sites of pSilencer4.1CMVneo according to the manufacturer’s instructions (Ambion, Inc., Austin, TX, USA). The pSil-siRNA/ control vector containing an siRNA/control insert does not suppress expression of genes expressed in human, rat,, or mouse. All of the inserted sequences were verified by DNA sequencing. Each vector (pSil-siRNA/ survivin and pSil-siRNA/control) contains the SV40 early promoter to provide G418 resistance in mammalian cells. Transfection and selection of stable transfectants Cells were transfected with different vectors (pSil-siRNA/ survivin and pSil-siRNA/control) by Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Stable transfectants, named HSC-3–siRNA/ survivin and HSC-3–siRNA/control, were selected for neomycin resistance in medium containing 700 ␮g/mL G418 and later maintained in medium with 300 ␮g/mL G418. Cell proliferation assay Cell proliferation was evaluated by methylthiazol tetrazolium (MTT) assay. Briefly, a total of 5.0 ⫻ 103 cells/well (mock HSC-3, HSC-3–siRNA/control, and HSC-3–siRNA/survivin) were seeded into five 96-well culture plates, with each plate having all 3 kinds of cells and each group consisting of 3 parallel wells. Absorp-

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tion value of 1 of 5 culture plates was determined by MTT at 490 nm. Then, the absorbance (A) of every culture plate was detected after 6 days. The protracted cell growth curve and the results of the inhibitory rates of cell growth were applied to A as follows: inhibitory rate ⫽ (1-HSC-3–siRNA/survivin A/HSC-3 A) ⫻ 100. Each experiment was performed 3 times. Flow cytometry assay Culture supernatants were collected and cells were washed 2 times with PBS, trypsinized, and lysed in a hypotonic solution containing 0.1% sodium citrate, 0.1% Triton X-100, and 50 ␮g/mL propidium iodide (Sigma). Analysis of labeled nuclei was performed on a FACS Calibur fluorescence-activated cell sorter (FACS; Becton Dickinson, Franklin Lakes, NJ, USA). The percentage of apoptotic cells was determined by measuring the fraction of nuclei with a subdiploid DNA content. Chemotherapy assay To determine the effect of pSil-siRNA/survivin on in vitro chemosensitivity, HSC-3–siRNA/control or HSC3–siRNA/survivin cells were treated with chemotherapeutic agents (cisplatin: 4 mg/mL; 5-fluorouracil [5-FU]: 2 ␮g/mL; Sigma). After 48 hours of incubation, the cells were incubated with 0.5 mg/mL MTT (Sigma). After 4 hours of incubation, the medium was replaced with 100 ␮L DMSO (Sigma) and then vortexed for 15 minutes. Absorbance at 490 nm was determined using Versamax microplate reader (Molecular Devices, Sunnyvale, CA). Cell viability was calculated as a percentage of the absence of cisplatin or 5-FU. The activity of caspase-3 was also detected by Caspase-3 Colorimetric Detection Kit (Keygen, Nanjing, China) according to the manufacturer’s instructions. Statistical analysis All statistical analyses were performed by using SPSS 15.0 (SPSS, Chicago, IL). Data were expressed as mean ⫾ SD, and categoric data were compared using the chi-squared test and Fisher exact test. Survival rates were estimated using the Kaplan-Meier method, and statistical analysis was performed using the log-rank test for equality of the survival curves. Prognostic factors were examined by univariate and multivariate analyses (Cox proportional hazards model). The criterion for significance was P ⬍ .05. RESULTS Expression of survivin mRNA in OSCC cell lines or tissues The expression of survivin mRNA in OSCC cell lines was determined by RT-PCR assay. We showed

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Fig. 1. Reverse-transcription polymerase chain reaction detection of survivin mRNA in cell lines. The expression levels of survivin mRNA in oral squamous cell carcinoma cell lines (BHY, HSC-2, HSC-3, and HN) were significantly higher than those in normal human oral keratinocytes (NHOK). *P ⬍ 0.05.

that the expression of survivin mRNA was not detected in NHOKs and showed high levels in 4 OSCC cell lines (Fig. 1). The expression of survivin mRNA in OSCC tissue samples and corresponding noncancerous tissue samples was also detected. As shown in Fig. 2, the expression of survivin mRNA in OSCC tissue was significantly higher; the relative average level of survivin mRNA was 0.94 ⫾ 0.16, but the expression of survivin mRNA was not detected in corresponding noncancerous tissues. OSCC patients with survivin mRNA expression levels in tumor tissues equal to or greater than the median value of 0.422 were considered to be a high-expression group (n ⫽ 31), whereas OSCC patients with survivin mRNA expression levels in tumor tissues ⬍0.422 were considered to be a low-expression group (n ⫽ 37).

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Fig. 2. Reverse-transcription polymerase chain reaction detection of survivin mRNA in tissue samples. Representative results showed that the expression levels of survivin mRNA in oral squamous cell carcinoma cell tissue samples (T) were significantly higher than those in corresponding noncancerous tissues (N).

Table I. The expression of survivin mRNA was significantly correlated with tumor differentiation, clinical stage, and status of lymph nodes (P values .048, .008, and .009, respectively). We could find that OSCC patients with high survivin mRNA expression showed poorer tumor differentiation, higher clinical stage, and present lymph node metastasis. However, there was no correlation between survivin mRNA expression and other clinicopathologic features, including gender, age, smoking, and tumor size (P values .436, .632, .122, and .701, respectively).

Immunostaining of survivin protein in OSCC tissues Immunohistochemistry was performed in the resected OSCC tissues and corresponding noncancerous tissues. The staining of survivin protein was mainly located in the cytoplasm of OSCC cells, but the staining of survivin protein in noncancerous tissues was not detected (Fig. 3). These data were consistent with the results from real-time RT-PCR analysis of survivin mRNA expression in tissues. Thus, survivin may play important roles in OSCC progression and development.

Prognostic value of survivin protein expression in OSCC patients To explore the prognostic value of survivin mRNA expression, the correlation between high survivin mRNA expression and overall survival was initially evaluated using Kaplan-Meier survival curves with the log-rank test and then confirmed with multivariate Cox regression models. Figure 4 shows that the 5-year overall survival rate of patients with high survivin mRNA expression (40.3%) was significantly lower than that of patients with low survivin mRNA expression (64.5%; P ⬍ 0.01 [log-rank test]). Table II presents the results of multivariate analysis. The status of survivin mRNA expression might be an independent prognostic factor for predicting overall survival of OSCC patients (hazard ratio [HR] 2.71, 95% confidence interval [CI] 1.465.10; P ⫽ .012). Lymph node metastasis was also an independent prognostic factor (HR 5.24, 95% CI 2.898.77; P ⫽ .008).

Correlation between survivin mRNA expression and clinicopathologic features The correlation between survivin mRNA and clinicopathologic features of OSCC patients was shown in

Inhibition of survivin expression by pSil-siRNA/survivin To explore the effect of siRNA targeting survivin on the expression of survivin gene, RT-PCR and Western

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Fig. 3. Immunostaining of survivin protein in tissues (⫻10). The staining of survivin was mainly located in the cytoplasm of oral squamous cell carcinoma cell cells. N, Nontumor tissues; T, tumor tissues.

Table II. Multivariate analysis of prognostic factors for oral squamous cell carcinoma patients Variable

Unfavorable/ favorable

ⱖ60/⬍60 Yes/No T3-T4/T1-T2 Moderate-poor/ good Clinical stage III⫹IV/I⫹II Lymph node metastasis N1-N2/N0 Survivin mRNA High/low expression Age Smoker Tumor size Tumor differentiation

Fig. 4. Kaplan-Meier survival curves of oral squamous cell carcinoma patients according to the expression of survivin mRNA. The patients with high survivin mRNA expression tumor showed significantly shorter survival than those with low survivin mRNA expression tumor (P ⬍ .01; log-rank test).

blot assays were performed to detect the expression of survivin mRNA and protein, respectively, in stably transfected cells. As shown in Fig. 5, the levels of survivin mRNA and protein expression in HEC-3-pSilsiRNA/survivin cells were significantly down-regulated by ⬃67.2 ⫾ 2.4% and ⬃58.3 ⫾ 1.8%, respectively (P ⬍ .05), but the expression of survivin mRNA and protein showed no difference between untransfected HSC-3 cells and HSC-3-pSil-siRNA/control cells (P ⬎ .05). Therefore, pSil-siRNA/survivin could significantly inhibit the expression of survivin gene in HSC-3 cells. Effect of pSil-siRNA/survivin on proliferation and apoptosis of HSC-3 cells To explore the effect of siRNA-mediated survivin down-regulation on the phenotypes of OSCC cells,

HR

95%CI

P value

3.47 1.88 0.92 2.12

0.54-11.2 0.33-5.24 0.68-2.47 0.98-8.23

.118 .208 .460 .089

3.12 0.44-6.23 5.24 2.89-8.77 2.71 1.46-5.10

.234 .008* .012*

HR, Hazard ratio; CI, confidence interval. *P ⬍ .05.

MTT and flow cytometry assays were performed. As shown in Fig. 6, A, the proliferation of HSC-3 cells was significantly inhibited, and the highest inhibitory rate was 46.8 ⫾ 2.7% at day 6 (P ⬍ .01). To further demonstrate the mechanism of proliferation inhibition, we detected the effect of survivin down-regulation on the apoptosis induction of HSC-3 cells. Results showed that the relative level of fragmented DNA in HSC-3– siRNA/survivin cells (24 ⫾ 4.2%) was significantly higher than that in HSC-3 or HSC-3–siRNA/control cells (3.2 ⫾ 1.8 and 4.1 ⫾ 1.3%, respectively; Fig. 6, B; P ⬍ .01). Therefore, siRNA-mediated survivin downregulation could significantly inhibit the proliferation of OSCC cells, which might be associated with apoptosis induction. Effect of survivin down-regulation on the sensitivity of HSC-3 cells to chemotherapeutic agents The untransfected or stably transfected HSC-3 cells were treated with or without 4 mg/mL cisplatin (or 2

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Fig. 5. Analyzing the effect of pSil–small interfering RNA (siRNA)/survivin on survivin expression in HSC-3oral squamous cell carcinoma cells. A, Reverse-transcription polymerase chain reaction analysis of survivin mRNA expression. B, Western blot analysis of survivin protein expression. *P ⬍ .05 vs. untransfected cells. 1, HSC-3; 2, HSC-3–siRNA/ control; 3, HSC-3–siRNA/survivin.

␮g/mL 5-FU), which was determined according to ⬃⬍10% of the proliferation suppression dose of HSC-3 cells 48 hours after cisplatin or 5-FU treatment by MTT assay (data not shown). As shown in Fig. 7, A, the cellular viability of HSC-3-pSil-siRNA/survivin cells was significantly decreased by 60.3 ⫾ 3.5% or 46.6 ⫾ 2.4% after administration of cisplatin or 5-FU (P ⬍ .05). We also analyzed the effect of survivin down-regulation combined with administration of cisplatin or 5-FU on proliferation of HSC-3 cells. Results showed that the apoptotic rate of HSC-3-pSil-siRNA/ survivin cells was significantly enhanced by 98.9 ⫾ 5.2% or 101.9 ⫾ 8.4% after addition of cisplatin or 5-FU (P ⬍ .05; Fig. 7, B). Therefore, we concluded that siRNA-mediated survivin down-regulation could synergistically enhance the cytotoxicity of chemotherapeutic agents. DISCUSSION Oral squamous cell carcinoma is the most common neoplasm in oral cavity cancer, and the incidence has recently been increasing.9 The prognosis of oral cancer remains unfavorable, with high mortality rates despite

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Fig. 6. Analyzing the effect of pSil–small interfering RNA (siRNA)/survivin on cell proliferation and apoptosis of HSC-3 oral squamous cell carcinoma cells. A, Methylthiazol tetrazolium analysis of HSC-3 cell proliferation. *P ⬍ .05; **P ⬍ .01 vs. untransfected HSC-3 cells. B, Flow cytometric analysis of HSC-3 cell apoptosis. **P ⬍ .01 vs. untransfected HSC-3 cells.

advances in diagnosis and therapy, including radical surgery, novel chemotherapy, and hyperfractionated/ conformational radiotherapy. Thus, elucidating the molecular mechanisms of OSCC tumorigenesis and progression would be helpful to finding novel strategies for both diagnosis and treatment that are far more efficient than currently available methods to improve the prognosis of OSCC patients. The initiation and progression of tumor is regulated by multiple genes. Many studies have documented the overexpression of antiapoptotic factors, such as IAPs, in a variety of solid tumors.10 Survivin, a novel member of the IAP family, which is overexpressed in tumor tissues but not in normal tissues, exists in a novel mitochondrial pool in tumor cells.11 Mitochondrial survivin orchestrates a novel pathway of apoptosis inhibition, which contributes to tumor progression. Recently, there have been many reports indicating the relationship between survivin expression and tumorigenesis or

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Fig. 7. Analyzing the effect of pSil–small interfering RNA (siRNA)/survivin on chemosensitivity of HSC-3 oral squamous cell carcinoma cells in vitro. A, HSC-3, HSC-3–siRNA/control, and HSC-3–siRNA/survivin cells were administered with vehicle (cisplatin or 5-fluorouracil [5-FU]) or chemotherapeutic agents (4 mg/mL cisplatin or 2 ␮g/mL 5-FU) for 48 hours, respectively. The cell viability (%) was shown as a percentage of untransfected HSC-3 cells in the absence of cisplatin or 5-FU. B. The apoptotic rates of cells were detected by flow cytometry. pSil-siRNA/survivin combined with cisplatin or 5-FU could synergistically induce apoptosis enhancement (P ⬍ .05).

prognosis of tumor patients. Kim et al.12 detected the expression of survivin in CIN (cervical intraepithelial nuolasia) and invasive squamous cell carcinoma (SCC) of the uterine cervix and found that survivin expression seemed to be more closely related to the progression of CIN and SCC than other molecular markers, such as Fas-ligand, Fas, and Bcl-2. Lin et al.13 reported that the expression of survivin was the essential event in the early stage of colorectal carcinogenesis and played an important role in the transition sequence and that it was not related to histologic differentiation grade of colorectal carcinoma. Span et al.14 showed that survivin demonstrated a strong independent association with poor prognosis and might be used as a new marker to stratify breast cancer patients for more optimal treatment modalities. Sarela et al.15 also showed that survivin expression provided prognostic information that might have important therapeutic implications in patients with stage II colorectal carcinomas. Therefore, survivin may play an important role in malignant transformation by maintaining normal mitosis, promoting cell proliferation, and facilitating angiogenesis. However, the biologic role of survivin mRNA in human

OSCC and its association with the sensitivity of OSCC cells to chemotherapeutic agents (cisplatin and 5-FU) is unclear. The present study first compared the expression of survivin mRNA in NHOK and OSCC cells and indicated that survivin gene was not detected in NHOKs and showed high levels in 4 OSCC cell lines. We also analyzed the expression of survivin mRNA in a quantity of OSCC tissue samples and found that the levels of survivin mRNA in OSCC tissues were significantly higher than those in corresponding noncancerous tissues. The staining of survivin protein in OSCC tissues was significantly stronger than that in nontumor tissues. Additionally, the high level of survivin mRNA in OSCC tissues was significantly correlated with poorer tumor differentiation, higher tumor stage, and present lymph node metastasis, but not with other clinicopathologic features. Patients with high survivin mRNA expression showed lower 5-year survival rates than patients with low survivin mRNA expression. Furthermore, multivariate analysis by Cox proportional hazards model suggested that the status of survivin mRNA expression might be an independent prognostic

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factor for predicting the prognosis of OSCC patients. These data were consistent with others’ studies about survivin in oral cancers. Liu et al.16 also reported that the expression of survivin was correlated with angiogenesis in OSCC. Therefore, up-regulation of survivin might be significantly correlated with OSCC tumorigenesis and development. Second, we analyzed whether the overexpression of survivin could affect phenotypes of OSCC cells. Small interfering RNA technology was used to down-regulate the expression of survivin in OSCC cells, and the down-regulation of survivin could induce proliferation inhibition and apoptosis enhancement of OSCC cells. We demonstrated that survivin down-regulation could significantly enhance cytotoxicity of chemotherapeutic agents (4 mg/mL cisplatin or 2 ␮g/mL 5-FU) in OSCC cells even at comparably low doses. Other groups also showed that expression of survivin gene shows significant correlation with chemosensitivity or radiosensitivity of esophageal cancer, pancreatic cancer, cervical carcinoma, prostate cancer, and other tumors.17-21 Therefore, survivin might be a potential molecular target for cancer therapy. In conclusion, the expression of survivin mRNA is an independent prognostic factor for OSCC patients, and this gene will become a novel molecular target for OSCC chemotherapy. Further investigations are under way to comfirm this finding in prospective clinical trial studies.

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Reprint requests: Yu Lei 169 Changle West Rd Xi’an 710033 Shaanxi China [email protected]