The Correlation Of Survivin,Caspase-3 And VEGF In Tongue Squamous Cell Carcinoma And The Antitumor Role Of Paclitaxel In Vitro

Introduction and ObjectiveTongue squamous cell carcinoma (TSCC) is the most common malignant tumor of the oral cavity in our country, presenting severe malignancy, rapid and extensive invasive growth, which may involves tongue muscles and lead to disability of eating, swallowing and speech. Cervical lymph node metastasis is often observed in the early stage of TSCC, and blood metastasis, which is common in lung, may occur in late stage. Despite refinement of surgical techniques and adjuvant therapies, the prognosis for patients remains poor, with a 5-year survival rate of 5-10%.Survivin is the latest discovery member in the family of inhibitor of apoptosis proteins (IAP), and is by far the most strong apoptosis inhibitor. Survivin distribution is tissue specific, with high expression in most of the malignant tumors, and is correlated with tumor biology and clinical pathological features. In the signal pathway of apoptosis, cysteinyl aspartate-specific protease-3 (Caspase-3) is in the downstream as a major effector. Survivin directly or indirectly inhibits Caspase-3 and promotes carcinogenesis.In recent years, support for the relationship between over-expression of Survivin and vascular endothelial growth factor (VEGF) and tumor angiogenesis and metastasis has derived from several studies. Survivin has been suggested to be involved in tumor angiogenesis by protecting vascular endothelial and controlling several vascular growth factors.Paclitaxel is a kind of chemical compound that is isolated from ormasia China fir. Paclitaxel belongs to inhibitor of mitosis, and may stabilize cytoplasmic microtubules, block cell division, arrest cells in the G2 / M phase of the cell cycle, and induce the apoptosis. This compound is proved obvious inhibition to ovary carcinoma cell line in vitro. In clinical, paclitaxel has demonstrated a unique ability to palliate the symptoms of many types of advanced cancers, including carcinomas of the ovary, lung, breast and esophagus.Currently, the mechanism of paclitaxel anti-TSCC and the regulation of paclitaxel to Survivin expression are not fully understood, and the study about the action of Survivin, Caspase-3 and VEGF in apoptosis and angiogenesis of TSCC is seldom reported. In the present study, we detected the expression of Survivin mRNA and Caspase-3 mRNA by hybridization in situ and discussed the correlation between them and their relations with clinical pathological features of TSCC. In addition, Immunohistochemistry of pv-9000 method was used to detect the expression of Survivin and VEGF proteins, their relationships with angiogenesis and metastasis and clinical pathological features of TSCC were investigated. On the other hand, flow cytometry was employed to detect the action of paclitaxel to cell cycle and apoptosis. Reverse transcription polymerase chain reaction (RT-PCR) is employed to detect the influence of paclitaxel on Survivin mRNA expression. The purpose of the study is to provide theoretical basis to carcinogenesis, progress, metastasis, angiogenesis and clinical chemotherapy of paclitaxel on TSCC.Materials and Methods45 TSCC samples and 10 normal tongue mucosa (NTM) samples were collected. The tissues were fixed in 10% formalin and embedded in paraffin. No patients had received preoperative radiotherapy and chemotherapy. In situ hybridization was used to detect the expression of Survivin mRNA and Caspase-3 mRNA; Immunohistochemistry was used to detect the expression of Survivin and VEGF. Human Tca8113 cell line was cultured in RPMI 1640 medium containing 10% fetal bovine serum, which was treated by paclitaxel with different concentrations. Light microscope was employed to observe morphological changes of the cell; CCK-8 was used to detect cell proliferation; Flow cytometry was employed to detect cell cycle and apoptosis; RT-PCR was employed to detect the changes of Survivin mRNA. Statistical analyses were performed by using the SPSS 13.0 software package (SPSS, Inc, Chicago, IL). Continuous Data are reported as (?)±s. Continuous variables were compared by ANOVA(LSD), or t-test as appropriate, whereas categorical variables were compared by x~2 Test or Fisher's Exact Test as appropriate. Spearman rank correlation was analyzed. Size of test was 0.05.Results1. The Survivin mRNA positive rate in TSCC was 55.6%, and zero in normal tongue mucosa. The result showed a significant difference between the two groups (P＜0.01). In highly, moderately and poorly differentiated TSCCs, the positive rates of Survivin mRNA expression were 38.5%, 50.0% and 83.3%, respectively, and a statistical significance was only observed for Survivin mRNA between the highly and poorly differentiated groups(P＜0.05). In the TNM stages, the positive rates of Survivin mRNA expression inⅢ,Ⅳgroup andⅠ,Ⅱgroup were 75.0% and 33.3%. The result showed a significant difference between the two groups (P＜0.01). The positive rate of Survivin mRNA expression in the TSCC with cervical lymph node metastasis was 81.3%, higher than that without cervical lymph code metastasis(P＜0.05). The results showed no significant difference between Survivin mRNA expression and the patients' gender and ages (P＞0.05).2. The Caspase-3 mRNA positive rate in normal tongue mucosa was 80.0%, and 42.2% in TSCC. The result showed a obviously significant difference between the two groups (P＜0.01). In highly, moderately and poorly differentiated TSCCs, the positive rates of Caspase-3 mRNA expression were 61.5%%, 45.0% and 16.7%, respectively, and a statistical significance was only observed for Caspase-3 mRNA between the highly and poorly differentiated groups(P＜0.05). In the TNM stages, the positive rates of Caspase-3 mRNA expression inⅢ,Ⅳgroup andⅠ,Ⅱgroup were 16.7% and 76.2%, and the result showed a significant difference between the two groups (P＜0.01). The positive rates of Caspase-3 mRNA expression in the TSCC with and without cervical lymph node metastasis were 18.8 % and 55.2%, respectively, and there was a significant difference between the groups(P＜0.05). The results showed no significant difference between Caspase-3 mRNA expression and the patients' gender and ages (P＞0.05).3. Among the 25 positive expressions of Survivin mRNA in TSCC, the ratio of 6 positive expressions of Caspase-3 mRNA was 24.0%, and it was 65.0% among the 20 negative expressions of Survivin mRNA. There was a negative relation between them, and the Spearman correlation coefficient r=-0.412.4. The positive rate of Survivin protein in TSCC was 68.9%, and zero in normal tongue mucosa. A significant difference was observed between the two groups (P＜0.01). In highly, moderately and poorly differentiated TSCCs, the positive expression rates of Survivin were 46.2%, 70.0% and 91.7%, respectively, and a statistical significance was observed for Survivin between the highly and poorly differentiated groups(P＜0.05). In the TNM stages, the positive expression rates of Survivin inⅢ,Ⅳgroup andⅠ,Ⅱgroup were 83.3 % and 52.4 %. The result showed a significant difference between the two groups(P＜0.05). The positive rate of Survivin in the TSCC with cervical lymph node metastasis was 93.8%, higher than 55.2% in that without cervical lymph node metastasis(P＜0.01).The results showed no significant difference between Survivin expression and the patients' gender and ages (P＞0.05).5. The positive rate of VEGF protein in TSCC was 64.4%, and none in normal tongue mucosa. The result showed a obviously significant difference between the two groups (P＜0.01). The positive expression rates of VEGF inⅢ,Ⅳgroup andⅠ,Ⅱgroup were 79.2% and 47.6%. The result showed a significant difference between the two groups (P＜0.05). The positive rate of VEGF in the TSCC with cervical lymph code metastasis was 87.5%, higher than 51.7% in that without cervical lymph code metastasis (P＜0.05).The results showed no significant difference between VEGF expression and the differentiation, the patients' gender and ages (P＞0.05).6. Among the 31 positive expressions of Survivin in TSCC, the ratio of 24 positive expressions of VEGF was77.4%, and it was 35.7% among the 14 negative expressions of Survivin. There was a positive relation between them, and the Spearman correlation coefficient r=0.403.7. After treated with different concentrations of paclitaxel, Tca8113 cells' proliferation was markedly inhibited, and the inhibition rates were dose-dependent and time-dependent. When Tca8113 cells were treated with 0.1μmol/L paclitaxel for 48 hours, the ratio of cell in G2/M was 41.68±5.93 %, and there was a significant difference with control group and other groups (P＜0.01); When Tca8113 cells were treated with 0.1μmol/L paclitaxel for 12,24 and 48 hours, the apoptosis rates were 1.45±0.21%,3.59±0.42 %,7.83±1.03%, respectively, and there was a significant difference with control group (P＜0.01). After 12 hours treatment with 0.1μmol/L paclitaxel, Survivin mRNA increased, higher than that in control group; Survivin mRNA began to decrease after 24 hours, and remarkably decreased after 48 hours.Conclusions1. The high expression of Survivin mRNA in TSCC correlates closely with carcinogenesis and progress, probably by inhibiting Caspase-3 mRNA expression.2. The high expressions of Survivin and VEGF proteins were cooperated in the carcinogenesis, progress, metastasis and angiogenesis of TSCC.3. Paclitaxel may induce apoptosis of Tca8113 cell line by inhibiting Survivin mRNA.