| Head and neck squamous cell carcinoma (HNSCC) represents a seriously clinical problem with more than500,000new cases diagnosed [1]and200,000deaths worldwide annually [2].Surgery is the main treatment option for HNSCC, with radiotherapy, chemotherapy and molecular targeted therapy as adjuvant treatment options. Despite advances in diagnosis and treatment, HNSCC is still a great threat to human life with an unsatisfied5-year survival[4]. Like all solid tumors, HNSCC is thought to be initiated and progress through a series of genetic alterations. Therefore, identifying common genetic alterations which occur during HNSCC progression and novel biomarkers is an important component for early detection and further successful prevention and treatment for patients with HNSCC[5,6].iASPP, an evolutionally conserved inhibitory member of the apoptosis stimulating protein of P53(ASPP) family, can specifically inhibit P53-mediated cell apoptosis, as an new oncoprotein. Recently, it has been shown that iASPP overexpressed in several kinds of human cancers, such as breast carcinomas [7],acute leukemia[8],hepatocellular carcinoma, and ovarian cancer [10]. Elevated iASPP expression contributes to tumorigenesis by proliferative and antiapoptotic effects However, there is little information available in the literature about the role of iASPP in HNSCC. Therefore, the present study was undertaken to investigate the role of iASPP in the tumorigenesis, progression and chemosensitivity of HNSCC. Charpter1Expression of iASPP in head and neck squamous cell carcinoma and itsclinical significanceObjective:To study the expression of iASPP in head and neck squamous cell carcinoma (HNSCC) tissue and cell lines and to evaluate its clinical significance in HNSCC.Methods:The expression of iASPP in109primary HNSCC tissue specimens was examined by immunohistochemistry and its association with clinicopathological parameters and prognosis was analyzed. Additionally, expression status of iASPP in16paired HNSCC tissues and7HNSCC cell lines was evaluated by quantitative real-time PCR (qPCR) and immunoblotting.Results:The protein and mRNA expression of iASPP were increased in HNSCC tissues and cell lines. Immunohistochemical staining indicated iASPP was detected in both cytoplasm and nucleus. Importantly, overexpression of cytoplasmic and nuclear iASPP was significantly associated with T classification (p=0.002and p=0.033, respectively), clinical stage (p<0.001and p=0.004), lymph node metastasis (p=0.001and p<0.001) and recurrence (both p<0.001). Survival analysis demonstrated high iASPP expression significantly correlated with shorter disease-free survival (DFS)(both p<0.001for cytoplasmic and nuclear expression) and overall survival (OS)(both p <0.001for cytoplasmic and nuclear expression). Multivariate analysis revealed cytoplasmic iASPP was the only independent prognostic factor for HNSCC patients.Conclusion:iASPP expression is elevated in HNSCC tissues and cell lines, which suggests iASPP may contribute to the malignant progression of HNSCC, and sever as a novel prognostic marker in HNSCC. Charpter2Influences of down-regulation of iASPP expression on proliferation, apoptosis, cycle and invasiveness in HNSCC cell lineObjective:To investigate the effects of down-regulation of iASPP expression on the proliferation, cell apoptosis, cell cycle and invasion in HNSCC cell line.Methods:HNSCC cell lines Tu686was transfected with iASPP shRNA lentiviral particles to silence its iASPP gene expression. Stable clones were established by puromycin screening. qPCR and Western blotting were applied to validate iASPP gene silencing efficiency in Tu686cell line. CCK-8assay, flow cytometry, invasion assay were used to detect the proliferation, apoptosis, cell cycle and invasion, respectively.Results:1. iASPP shRNA lentiviral particles efficiently silenced the mRNA and protein expression of iASPP in HNSCC cell line Tu686. And stable iASPP silencing cell lines were obtained.2. The CCK-8showed the proliferation of Tu686was markedly inhibited, after knockdown of iASPP gene expression. The growth inhibition rate of LV-shiASPP cell line at the time of72h,96h,120h, had statistical significance (P<0.01), compared with that of LV-shNon cell line and UT cell line. However,there was no difference between LV-shNon cell line and UT cell line (P>0.05)3. Flow cytometry showed that the apoptosis ratio of LV-shiASPP cell line is9.42±0.39%, which is significantly higher than that of UT cell line (2.80±0.42%) and LV-shNon cell line (3.18±0.28%)(P<0.01). The percentage of LV-shiASPP cells in G0/G1phase was strikingly increased (74.65±1.09%vs55.19±1.02%,54.62±0.88%), whereas the percentage of cells in S phase (17.54±1.21%vs32.56±0.98%,32.50±1.17%) and G2/M phase (7.81±0.76%vs12.24±0.68%,12.87±0.31%) decreased significantly (all P<0.01). However, there was no significant difference in cell cycle distribution between UT group and LV-shNon group (P>0.05). 4. Matrigel invasiveness assay in transwell culture chambers showed invasiveness decreased significantly in LV-shiASPP group, compared with UT group and LV-shNon group. The number of cells passing through the basement membrane at48h were56±4vs111±3,105±8, respectively (P<0.01).The difference between UT group and LV-shNon group had no statistical significance (P>0.05)Conclusion:Knockdown of iASPP gene expression led to promotion of apbptosis, an arrest in G0/G1phase, inhibition of cell proliferation and invasion in vitro, suggestting that iASPP plays an important role in progression and aggressive behavior of HNSCC. Charpter3The effect of iASPP down-regulation on paclitaxel chemosensitivity in HNSCC cellsObjective:To investigation the effect of iASPP knockdown on paclitaxel chemosensitivity in HNSCC Tu686cells.Methods:Different concentration of paclitaxel were employed to treat with UT, LV-shNon, and LV-shiASPP cell lines for48h. The OD value was detected by CCK-8assay and IC50of paclitaxel was obtained. Cell apoptosis and cycle of the three groups were detected by flow cytometry, after treating with IC30of paclitaxel for24h.Results:1.The IC50of paclitaxel for LV-shiASPP, UT and LV-shNon cell lines were3.84±0.28nM/L,47.46±2.12nM/L and50.93±2.27nM/L, respectively, which demonstrated the chemosensitivity in LV-shiASPP cell lines was highly increased, compared with UT and LV-shNon cell lines (P<0.01). Nevertheless, there was no significant difference between UT and LV-shNon groups (P>0.05).2. After treatment with IC30of paclitaxel, flow cytometry showed the apopsis ratio of LV-shiASPP cell line is strikingly increased, compared with UT and LV-shNon cell lines.(18.27±1.13%vs7.31±0.33%,8.20±0.34%). Compared with UT and LV-shNon cells, the percentage of LV-shiASPP cells in G2/M phase was obviously increased (28.68±0.76%vs19.21±0.89%,17.99±0.13%)(P<0.01) whereas the percentage of cells in S phase decreased significantly (15.11±1.09%vs26.38±2.04%,26.58±0.67%)(P<0.01).For G0/G1phase, there was no obvious difference (56.21±1.82%vs54.41±1.15%,55.42±0.56%)(P>0.05). However, there was no significant difference in cell cycle distribution between UT group and LV-shNon group (P>0.05).Conclusion:Inhibition of iASPP expression can significantly promote cell apoptosis and induce cell cycle arrest, which can strengthen chemosensitivity of paclitaxel in HNSCC cell line. It suggests that iASPP may be a novel and efficient chemotherapeutic target for the treatment of HNSCC. |
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