iASPP调控头颈鳞癌生长增殖、侵袭及化疗敏感性的实验研究
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摘要
全球每年大约有50多万头颈鳞癌(Head and Neck Squamous Cell Carcinoma, HNSCC)的新发患者,而每年全球因头颈鳞癌而死亡的患者达20万左右。目前,头颈鳞癌的治疗方式主要以手术治疗为主,放疗、化疗及分子靶向治疗等其他治疗方式为辅。尽管各种治疗手段都在不断的更新和进步,但是头颈鳞癌患者的五年生存率并没有因此而得到显著的提高,依然严重危害着人类的健康和生命。那么,是什么因素促进了头颈鳞癌的发生发展,并使现有的治疗方式陷入窘境?目前认为和多种实体瘤一样,头颈鳞癌在发生发展过程中历经了一系列的基因改变,因此,从分子基因水平研究头颈鳞癌的发生发展机制,并寻找该过程中起关键作用的调控因子,从而进行针对性的预防、干预和控制,是早期诊断及有效预防和治疗头颈鳞癌的关键。
iASPP (inhibitory member of the ASPP family)是ASPP (ankyrin-repeat, SH3-domain and prolinerichdomain protein)即P53凋亡刺激蛋白(apoptosis stimulating protein of P53)家族中的抑制成员,它能竞争性地与P53结合,抑制P53的抑癌功能,是近年来发现的一种新的癌蛋白。目前研究表明,iASPP在乳腺癌[7]、白血病、肝癌[9]、卵巢癌等多种恶性肿瘤中表达升高,它通过促进肿瘤细胞的增殖并抑制其凋亡进而促进肿瘤的发生发展。然而,迄今为止,国内外尚未见iASPP在头颈鳞癌中的报道,因此,本研究拟探讨其在头颈鳞癌发生发展中的作用及对化疗敏感性的影响。
第1章iASPP在头颈鳞癌中的表达及其临床意义
目的：检测iASPP mRNA及蛋白在头颈部鳞状细胞癌(头颈鳞癌)组织及细胞株中的表达,并探讨其表达与头颈鳞癌患者临床病理特征及预后之间的关系。
方法：采用免疫组织化学技术检测iASPP蛋白在109例头颈鳞癌石蜡组织标本和15例癌旁组织标本中的表达,并统计分析iASPP蛋白的表达与头颈鳞癌患者临床病理特征及预后之间的关系；同时,运用荧光定量RT-PCR及Western blot方法检测iASPP mRNA及蛋白在16例配对头颈鳞癌组织及癌旁粘膜组织中的表达；此外还采用Western blot方法检测iASPP蛋白在7株头颈鳞癌细胞株和人口腔粘膜癌前病变细胞株DOK中的表达情况。
结果：iASPP蛋白在头颈鳞癌组织及细胞株中的表达均上调,且iASPP蛋白在肿瘤细胞胞浆和胞核中均有表达。进一步统计分析表明胞浆iASPP蛋白和胞核iASPP蛋白的表达与头颈鳞癌患者的T分级(T1+T2/T3+T4)(P=0.002, P=0.033)、临床分期(Ⅰ期+Ⅱ期/Ⅲ期+Ⅳ期)(P<0.001,P=0.004)、淋巴结转移(P=0.001,P<0.001)及复发(两者均P<0.001)密切相关；Kaplan-Meier生存分析结果显示iASPP高表达组与低表达组的5年无病生存率及5年总生存率之间的差异具有统计学意义(P均<0.001)；多因素Cox比例风险回归模型进一步表明,胞浆iASPP蛋白的表达水平是头颈鳞癌预后的独立影响因素(P<0.05)。同时,荧光定量RT-PCR及Western blot结果显示iASPP mRNA及蛋白在头颈鳞癌组织的表达显著高于其在癌旁粘膜中的表达,且差异具有统计学意义(P<0.001,P=0.002)。
结论：iASPP在头颈鳞癌组织及细胞中的表达显著升高,且其高表达与头颈鳞癌患者的T分级、临床分期、淋巴结转移、复发及预后密切相关。这些结果均提示iASPP可能在头颈鳞癌的发生、发展中发挥着重要的作用,并且有望成为早期诊断和评估头颈鳞癌患者预后的重要分子标志物。
第2章沉默iASPP基因的表达对头颈鳞癌细胞生长增殖、凋亡、周期和侵袭能力的影响
目的：观察沉默iASPP基因表达后,对头颈鳞癌细胞生长增殖、凋亡、周期及侵袭能力等生物学行为的影响。
方法：采用慢病毒介导的iASPP shRNA感染头颈鳞癌Tu686细胞,用嘌呤霉素筛选并建立稳定沉默iASPP基因的Tu686细胞,荧光定量RT-PCR和Western blot技术检测iASPP基因的沉默效果；采用CCK-8法、流式细胞术、Transwell侵袭实验检测沉默iASPP基因表达后,对头颈鳞癌Tu686细胞的体外生长增殖能力、细胞凋亡和细胞周期分布及侵袭能力的影响。
结果：1.慢病毒介导的iASPP shRNA有效地抑制了头颈鳞癌Tu686细胞中iASPP基因的表达,并成功建立了稳定沉默iASPP基因的Tu686细胞。
2.CCK-8检测结果显示iASPP基因沉默后能显著抑制Tu686细胞的体外增殖能力。自72h时间点开始(72h、96h、120h) LV-shiASPP细胞(稳定表达iASPP shRNA基因片段的Tu686细胞)的OD值显著低于对照组UT细胞(未处理的Tu686亲本细胞)和LV-shNon细胞(稳定表达Control shRNA基因片段的Tu686细胞)的OD值(P均<0.01)。而在每个检测时间点上UT细胞和LV-shNon细胞的OD值间相比均无统计学差异(P均>0.05)。
3.流式细胞术检测结果显示沉默iASPP基因表达后,LV-shiASPP细胞的凋亡率较对照组UT和LV-shNon细胞明显增多(9.42±0.39%vs2.80±0.42%,3.18±0.28%),其差异具有统计学意义(P<0.01)。同时发现LV-shiASPP细胞中处于G0/G1期的细胞比例较对照组UT和LV-shNon细胞显著增多(74.65±1.09%vs55.19±1.02%,54.62±0.88%)(P<0.01),而处于S期(17.54±1.21%vs32.56±0.98%,32.50±1.17%)(P<0.01)和G2/M期(7.81±0.76%vs12.24±0.68%,12.87±0.31%)(P<0.01)的细胞比例显著减少,而UT和LV-shNon两对照组细胞之间的周期分布无统计学差异(P>0.05)。
4.沉默iASPP基因表达后,Tu686的侵袭能力显著减弱。Transwell侵袭实验显示48小时后穿过Transwell侵袭小室聚碳酸酯膜的LV-shiASPP细胞较两对照组UT细胞和LV-shNon细胞明显减少(56±4vs111±3,105±8)(P<0.01),而两对照组间的差异不明显(P>0.05)。
结论：沉默iASPP的表达,能促进细胞凋亡,诱导G0/G1期捕获,从而抑制Tu686细胞体外生长增殖能力,并减弱其侵袭能力,提示iASPP在头颈鳞癌的发生及恶性进展中发挥了重要的作用。
第3章沉默iASPP基因的表达对头颈鳞癌细胞紫杉醇化疗敏感性的影响
目的：探讨沉默iASPP基因的表达后,对头颈鳞癌Tu686细胞紫杉醇化疗敏感性的影响。
方法：以不同浓度的紫杉醇药物作用于UT细胞、LV-shNon细胞和LV-shiASPP细胞,48小时后,采用CCK-8法检测各组细胞生长增殖情况,绘制各组细胞的生存曲线,计算各组细胞紫杉醇作用的IC50值,及紫杉醇作用UT细胞的IC30值。以紫杉醇IC30浓度处理UT细胞、LV-shiASPP细胞和LV-shNon细胞,24小时后行流式细胞术检测三组细胞凋亡和细胞周期情况。
结果：1. LV-shiASPP细胞、UT细胞和LV-shNon细胞紫杉醇作用的IC50值分别为3.84±0.28nM/L,47.46±2.12nM/L和50.93±2.27nM/L,其差异具有统计学意义(P<0.01),表明LV-shiASPP细胞对紫杉醇的化疗敏感性明显较对照组UT细胞和LV-shNon细胞增强,而UT细胞和LV-shNon细胞紫杉醇作用的IC50值相比,差异无统计学意义(P>0.05)。
2.以UT细胞紫杉醇作用的IC30浓度分别作用于三组细胞,流式细胞术检测结果显示LV-shiASPP细胞的凋亡率较UT和LV-shNon细胞明显增多(18.27±1.13%vs7.31±0.33%,8.20±0.34%),且差异具有统计学意义(P<0.01)。同时,LV-shiASPP细胞中处于G2/M期的细胞比例较UT和LV-shNon细胞显著增多(28.68±0.76%vs19.21±0.89%,17.99±0.13%)(P<0.01),而处于S期的细胞比例显著减少(15.11±1.09%vs26.38±2.04%,26.58±0.67%)(P<0.01),GO／Gl期细胞比例变化不大(56.21±1.82%vs54.41±1.15%,55.42±0.56%)(P>0.05),而UT和LV-shNon两组细胞之间的周期分布无统计学差异(P>0.05)。
结论：沉默iASPP基因的表达后能促进紫杉醇诱导Tu686细胞发生凋亡和细胞周期阻滞,从而增强头颈鳞癌Tu686细胞对紫杉醇的化疗敏感性,提示iASPP很可能成为增强头颈鳞癌紫杉醇化疗敏感性的有效的分子治疗靶点。
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 significance
Objective: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 line
Objective: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 cells
Objective: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.
iASPP (inhibitory member of the ASPP family)是ASPP (ankyrin-repeat, SH3-domain and prolinerichdomain protein)即P53凋亡刺激蛋白(apoptosis stimulating protein of P53)家族中的抑制成员,它能竞争性地与P53结合,抑制P53的抑癌功能,是近年来发现的一种新的癌蛋白。目前研究表明,iASPP在乳腺癌[7]、白血病、肝癌[9]、卵巢癌等多种恶性肿瘤中表达升高,它通过促进肿瘤细胞的增殖并抑制其凋亡进而促进肿瘤的发生发展。然而,迄今为止,国内外尚未见iASPP在头颈鳞癌中的报道,因此,本研究拟探讨其在头颈鳞癌发生发展中的作用及对化疗敏感性的影响。
第1章iASPP在头颈鳞癌中的表达及其临床意义
目的：检测iASPP mRNA及蛋白在头颈部鳞状细胞癌(头颈鳞癌)组织及细胞株中的表达,并探讨其表达与头颈鳞癌患者临床病理特征及预后之间的关系。
方法：采用免疫组织化学技术检测iASPP蛋白在109例头颈鳞癌石蜡组织标本和15例癌旁组织标本中的表达,并统计分析iASPP蛋白的表达与头颈鳞癌患者临床病理特征及预后之间的关系；同时,运用荧光定量RT-PCR及Western blot方法检测iASPP mRNA及蛋白在16例配对头颈鳞癌组织及癌旁粘膜组织中的表达；此外还采用Western blot方法检测iASPP蛋白在7株头颈鳞癌细胞株和人口腔粘膜癌前病变细胞株DOK中的表达情况。
结果：iASPP蛋白在头颈鳞癌组织及细胞株中的表达均上调,且iASPP蛋白在肿瘤细胞胞浆和胞核中均有表达。进一步统计分析表明胞浆iASPP蛋白和胞核iASPP蛋白的表达与头颈鳞癌患者的T分级(T1+T2/T3+T4)(P=0.002, P=0.033)、临床分期(Ⅰ期+Ⅱ期/Ⅲ期+Ⅳ期)(P<0.001,P=0.004)、淋巴结转移(P=0.001,P<0.001)及复发(两者均P<0.001)密切相关；Kaplan-Meier生存分析结果显示iASPP高表达组与低表达组的5年无病生存率及5年总生存率之间的差异具有统计学意义(P均<0.001)；多因素Cox比例风险回归模型进一步表明,胞浆iASPP蛋白的表达水平是头颈鳞癌预后的独立影响因素(P<0.05)。同时,荧光定量RT-PCR及Western blot结果显示iASPP mRNA及蛋白在头颈鳞癌组织的表达显著高于其在癌旁粘膜中的表达,且差异具有统计学意义(P<0.001,P=0.002)。
结论：iASPP在头颈鳞癌组织及细胞中的表达显著升高,且其高表达与头颈鳞癌患者的T分级、临床分期、淋巴结转移、复发及预后密切相关。这些结果均提示iASPP可能在头颈鳞癌的发生、发展中发挥着重要的作用,并且有望成为早期诊断和评估头颈鳞癌患者预后的重要分子标志物。
第2章沉默iASPP基因的表达对头颈鳞癌细胞生长增殖、凋亡、周期和侵袭能力的影响
目的：观察沉默iASPP基因表达后,对头颈鳞癌细胞生长增殖、凋亡、周期及侵袭能力等生物学行为的影响。
方法：采用慢病毒介导的iASPP shRNA感染头颈鳞癌Tu686细胞,用嘌呤霉素筛选并建立稳定沉默iASPP基因的Tu686细胞,荧光定量RT-PCR和Western blot技术检测iASPP基因的沉默效果；采用CCK-8法、流式细胞术、Transwell侵袭实验检测沉默iASPP基因表达后,对头颈鳞癌Tu686细胞的体外生长增殖能力、细胞凋亡和细胞周期分布及侵袭能力的影响。
结果：1.慢病毒介导的iASPP shRNA有效地抑制了头颈鳞癌Tu686细胞中iASPP基因的表达,并成功建立了稳定沉默iASPP基因的Tu686细胞。
2.CCK-8检测结果显示iASPP基因沉默后能显著抑制Tu686细胞的体外增殖能力。自72h时间点开始(72h、96h、120h) LV-shiASPP细胞(稳定表达iASPP shRNA基因片段的Tu686细胞)的OD值显著低于对照组UT细胞(未处理的Tu686亲本细胞)和LV-shNon细胞(稳定表达Control shRNA基因片段的Tu686细胞)的OD值(P均<0.01)。而在每个检测时间点上UT细胞和LV-shNon细胞的OD值间相比均无统计学差异(P均>0.05)。
3.流式细胞术检测结果显示沉默iASPP基因表达后,LV-shiASPP细胞的凋亡率较对照组UT和LV-shNon细胞明显增多(9.42±0.39%vs2.80±0.42%,3.18±0.28%),其差异具有统计学意义(P<0.01)。同时发现LV-shiASPP细胞中处于G0/G1期的细胞比例较对照组UT和LV-shNon细胞显著增多(74.65±1.09%vs55.19±1.02%,54.62±0.88%)(P<0.01),而处于S期(17.54±1.21%vs32.56±0.98%,32.50±1.17%)(P<0.01)和G2/M期(7.81±0.76%vs12.24±0.68%,12.87±0.31%)(P<0.01)的细胞比例显著减少,而UT和LV-shNon两对照组细胞之间的周期分布无统计学差异(P>0.05)。
4.沉默iASPP基因表达后,Tu686的侵袭能力显著减弱。Transwell侵袭实验显示48小时后穿过Transwell侵袭小室聚碳酸酯膜的LV-shiASPP细胞较两对照组UT细胞和LV-shNon细胞明显减少(56±4vs111±3,105±8)(P<0.01),而两对照组间的差异不明显(P>0.05)。
结论：沉默iASPP的表达,能促进细胞凋亡,诱导G0/G1期捕获,从而抑制Tu686细胞体外生长增殖能力,并减弱其侵袭能力,提示iASPP在头颈鳞癌的发生及恶性进展中发挥了重要的作用。
第3章沉默iASPP基因的表达对头颈鳞癌细胞紫杉醇化疗敏感性的影响
目的：探讨沉默iASPP基因的表达后,对头颈鳞癌Tu686细胞紫杉醇化疗敏感性的影响。
方法：以不同浓度的紫杉醇药物作用于UT细胞、LV-shNon细胞和LV-shiASPP细胞,48小时后,采用CCK-8法检测各组细胞生长增殖情况,绘制各组细胞的生存曲线,计算各组细胞紫杉醇作用的IC50值,及紫杉醇作用UT细胞的IC30值。以紫杉醇IC30浓度处理UT细胞、LV-shiASPP细胞和LV-shNon细胞,24小时后行流式细胞术检测三组细胞凋亡和细胞周期情况。
结果：1. LV-shiASPP细胞、UT细胞和LV-shNon细胞紫杉醇作用的IC50值分别为3.84±0.28nM/L,47.46±2.12nM/L和50.93±2.27nM/L,其差异具有统计学意义(P<0.01),表明LV-shiASPP细胞对紫杉醇的化疗敏感性明显较对照组UT细胞和LV-shNon细胞增强,而UT细胞和LV-shNon细胞紫杉醇作用的IC50值相比,差异无统计学意义(P>0.05)。
2.以UT细胞紫杉醇作用的IC30浓度分别作用于三组细胞,流式细胞术检测结果显示LV-shiASPP细胞的凋亡率较UT和LV-shNon细胞明显增多(18.27±1.13%vs7.31±0.33%,8.20±0.34%),且差异具有统计学意义(P<0.01)。同时,LV-shiASPP细胞中处于G2/M期的细胞比例较UT和LV-shNon细胞显著增多(28.68±0.76%vs19.21±0.89%,17.99±0.13%)(P<0.01),而处于S期的细胞比例显著减少(15.11±1.09%vs26.38±2.04%,26.58±0.67%)(P<0.01),GO／Gl期细胞比例变化不大(56.21±1.82%vs54.41±1.15%,55.42±0.56%)(P>0.05),而UT和LV-shNon两组细胞之间的周期分布无统计学差异(P>0.05)。
结论：沉默iASPP基因的表达后能促进紫杉醇诱导Tu686细胞发生凋亡和细胞周期阻滞,从而增强头颈鳞癌Tu686细胞对紫杉醇的化疗敏感性,提示iASPP很可能成为增强头颈鳞癌紫杉醇化疗敏感性的有效的分子治疗靶点。
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 significance
Objective: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 line
Objective: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 cells
Objective: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.
引文
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[8]Naumovski L, Cleary M L. The p53-binding protein 53BP2 also interacts with Bcl2 and impedes cell cycle progression at G2/M[J]. Mol Cell Biol,1996,16(7): 3884-3892.
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[17]Bergamaschi D, Samuels Y, Sullivan A, et al. iASPP preferentially binds p53 proline-rich region and modulates apoptotic function of codon 72-polymorphic p53[J]. Nat Genet,2006,38(10):1133-1141.
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[20]Cai Y, Qiu S, Gao X, et al. iASPP inhibits p53-independent apoptosis by inhibiting transcriptional activity of p63/p73 on promoters of proapoptotic genes[J]. Apoptosis,2012,17(8):777-783.
[21]Liu Z J, Cai Y, Hou L, et al. Effect of RNA interference of iASPP on the apoptosis in MCF-7 breast cancer cells[J]. Cancer Invest,2008,26(9):878-882.
[22]Lu B, Guo H, Zhao J, et al. Increased expression of iASPP, regulated by hepatitis B virus X protein-mediated NF-kappaB activation, in hepatocellular carcinoma[J]. Gastroenterology,2010,139(6):2183-2194.
[23]Lin B L, Xie D Y, Xie S B, et al. Down-regulation of iASPP in human hepatocellular carcinoma cells inhibits cell proliferation and tumor growth[J]. Neoplasma,2011,58(3):205-210.
[24]Zhang X, Wang M, Zhou C, et al. The expression of iASPP in acute leukemias[J]. Leuk Res,2005,29(2):179-183.
[25]Liu H, Wang M, Diao S, et al. siRNA-mediated down-regulation of iASPP promotes apoptosis induced by etoposide and daunorubicin in leukemia cells expressing wild-type p53[J]. Leuk Res,2009,33(9):1243-1248.
[26]Liu T, Li L, Yang W, et al. iASPP is important for bladder cancer cell proliferation[J]. Oncol Res,2011,19(3-4):125-130.
[27]Li G, Wang R, Gao J, et al. RNA interference-mediated silencing of iASPP induces cell proliferation inhibition and G0/G1 cell cycle arrest in U251 human glioblastoma cells[J]. Mol Cell Biochem,2011,350(1-2):193-200.
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[1]Samuels-Lev Y, O'Connor D J, Bergamaschi D, et al. ASPP proteins specifically stimulate the apoptotic function of p53[J]. Mol Cell,2001,8(4):781-794.
[2]Bergamaschi D, Samuels Y, O'Neil N J, et al. iASPP oncoprotein is a key inhibitor of p53 conserved from worm to human[J]. Nat Genet,2003,33(2):162-167.
[3]Yang J P, Hori M, Sanda T, et al. Identification of a novel inhibitor of nuclear factor-kappaB, RelA-associated inhibitor[J]. J Biol Chem,1999,274(22): 15662-15670.
[4]Slee E A, Gillotin S, Bergamaschi D, et al. The N-terminus of a novel isoform of human iASPP is required for its cytoplasmic localization[J]. Oncogene,2004, 23(56):9007-9016.
[5]Zhang X, Diao S, Rao Q, et al. Identification of a novel isoform of iASPP and its interaction with p53[J]. J Mol Biol,2007,368(4):1162-1171.
[6]Slee E A, O'Connor D J, Lu X. To die or not to die:how does p53 decide?[J]. Oncogene,2004,23(16):2809-2818.
[7]Flores E R, Tsai K Y, Crowley D, et al. p63 and p73 are required for p53-dependent apoptosis in response to DNA damage[J]. Nature,2002,416(6880): 560-564.
[8]Naumovski L, Cleary M L. The p53-binding protein 53BP2 also interacts with Bcl2 and impedes cell cycle progression at G2/M[J]. Mol Cell Biol,1996,16(7): 3884-3892.
[9]Llanos S, Royer C, Lu M, et al. Inhibitory member of the apoptosis-stimulating proteins of the p53 family (iASPP) interacts with protein phosphatase 1 via a noncanonical binding motif[J]. J Biol Chem,2011,286(50):43039-43044.
[10]Skene-Arnold T D, Luu H A, Uhrig R G, et al. Molecular mechanisms underlying the interaction of protein phosphatase-lc with ASPP proteins[J]. Biochem J,2013,449(3):649-659.
[11]Robinson R A, Lu X, Jones E Y, et al. Biochemical and structural studies of ASPP proteins reveal differential binding to p53, p63, and p73[J]. Structure,2008, 16(2):259-268.
[12]Yang A, Kaghad M, Wang Y, et al. p63, a p53 homolog at 3q27-29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities[J]. Mol Cell,1998,2(3):305-316.
[13]Jost C A, Marin M C, Kaelin W J. p73 is a simian [correction of human] p53-related protein that can induce apoptosis[J]. Nature,1997,389(6647):191-194.
[14]Di Como C J, Gaiddon C, Prives C. p73 function is inhibited by tumor-derived p53 mutants in mammalian cells[J]. Mol Cell Biol,1999,19(2):1438-1449.
[15]Strano S, Rossi M, Fontemaggi G, et al. From p63 to p53 across p73[J]. FEBS Lett,2001,490(3):163-170.
[16]Slee E A, Lu X. The ASPP family:deciding between life and death after DNA damage[J]. Toxicol Lett,2003,139(2-3):81-87.
[17]Bergamaschi D, Samuels Y, Sullivan A, et al. iASPP preferentially binds p53 proline-rich region and modulates apoptotic function of codon 72-polymorphic p53[J]. Nat Genet,2006,38(10):1133-1141.
[18]Laska M J, Vogel U B, Jensen U B, et al. p53 and PPP1R13L (alias iASPP or RAI) form a feedback loop to regulate genotoxic stress responses[J]. Biochim Biophys Acta,2010,1800(12):1231-1240.
[19]Bell H S, Dufes C, O'Prey J, et al. A p53-derived apoptotic peptide derepresses p73 to cause tumor regression in vivo[J]. J Clin Invest,2007,117(4):1008-1018.
[20]Cai Y, Qiu S, Gao X, et al. iASPP inhibits p53-independent apoptosis by inhibiting transcriptional activity of p63/p73 on promoters of proapoptotic genes[J]. Apoptosis,2012,17(8):777-783.
[21]Liu Z J, Cai Y, Hou L, et al. Effect of RNA interference of iASPP on the apoptosis in MCF-7 breast cancer cells[J]. Cancer Invest,2008,26(9):878-882.
[22]Lu B, Guo H, Zhao J, et al. Increased expression of iASPP, regulated by hepatitis B virus X protein-mediated NF-kappaB activation, in hepatocellular carcinoma[J]. Gastroenterology,2010,139(6):2183-2194.
[23]Lin B L, Xie D Y, Xie S B, et al. Down-regulation of iASPP in human hepatocellular carcinoma cells inhibits cell proliferation and tumor growth[J]. Neoplasma,2011,58(3):205-210.
[24]Zhang X, Wang M, Zhou C, et al. The expression of iASPP in acute leukemias[J]. Leuk Res,2005,29(2):179-183.
[25]Liu H, Wang M, Diao S, et al. siRNA-mediated down-regulation of iASPP promotes apoptosis induced by etoposide and daunorubicin in leukemia cells expressing wild-type p53[J]. Leuk Res,2009,33(9):1243-1248.
[26]Liu T, Li L, Yang W, et al. iASPP is important for bladder cancer cell proliferation[J]. Oncol Res,2011,19(3-4):125-130.
[27]Li G, Wang R, Gao J, et al. RNA interference-mediated silencing of iASPP induces cell proliferation inhibition and G0/G1 cell cycle arrest in U251 human glioblastoma cells[J]. Mol Cell Biochem,2011,350(1-2):193-200.
[28]Chen J, Xie F, Zhang L, et al. iASPP is over-expressed in human non-small cell lung cancer and regulates the proliferation of lung cancer cells through a p53 associated pathway[J]. BMC Cancer,2010,10:694.
[29]Jiang L, Siu M K, Wong O G, et al. iASPP and chemoresistance in ovarian cancers:effects on paclitaxel-mediated mitotic catastrophe[J]. Clin Cancer Res,2011, 17(21):6924-6933.
[30]Liu W K, Jiang X Y, Ren J K, et al. Expression pattern of the ASPP family members in endometrial endometrioid adenocarcinoma[J]. Onkologie,2010,33(10): 500-503.
[31]Zhang B, Xiao H J, Chen J, et al. Inhibitory member of the apoptosis-stimulating protein of p53 (ASPP) family promotes growth and tumorigenesis in human p53-deficient prostate cancer cells[J]. Prostate Cancer Prostatic Dis,2011,14(3): 219-224.
[32]Liu Z, Zhang X, Huang D, et al. Elevated expression of iASPP in head and neck squamous cell carcinoma and its clinical significance[J]. Med Oncol,2012,29(5): 3381-3388.
[33]Cao L, Huang Q, He J, et al. Elevated expression of iASPP correlates with poor prognosis and chemoresistance/radioresistance in FIGO Ibl-IIa squamous cell cervical cancer[J]. Cell Tissue Res,2013.
[34]Li S, Shi G, Yuan H, et al. Abnormal expression pattern of the ASPP family of proteins in human non-small cell lung cancer and regulatory functions on apoptosis through p53 by iASPP[J]. Oncol Rep,2012,28(1):133-140.
[35]李强,程湘,孙守勋,等.乳腺癌细胞过表达iASPP对顺铂作用的影响[J].第三军医大学学报,2008(5).
[36]Laska M J, Lowe S W, Zender L, et al. Enforced expression of PPP1R13L increases tumorigenesis and invasion through p53-dependent and p53-independent mechanisms[J]. Mol Carcinog,2009,48(9):832-842.