蛋白酶体抑制剂MG-132对喉鳞癌Hep-2细胞作用的实验研究
发布时间：2018-09-03 14:04
【摘要】： 喉癌(laryngeal carcinoma)是头颈部常见的恶性肿瘤,其发病率及死亡率均呈逐年上升的趋势,严重威胁着人类的健康与生命。虽然手术为主,放疗与化疗为辅的综合治疗方法,使患者的5年生存率有所提高。但对于那些晚期或复发、转移的患者预后仍旧很差,手术丧失时机,而传统放化疗又由于其严重的毒、副作用常使得患者不能耐受,从而使患者的治疗受到极大的限制。因此,积极寻求喉癌治疗的新策略成为广大耳鼻咽喉科研究者面临的新课题。 泛素-蛋白酶体通路(Ubiquitin-Proteasome Pathway,UPP)是真核细胞中内源性蛋白质选择性降解的重要途径,是调节细胞多种生物学过程如细胞周期运转、增殖、凋亡、信号传递、基因转录等的关键机制。它与人类恶性肿瘤的发生、发展密切相关。阻断UPP可以促使与肿瘤细胞增殖、分化、凋亡密切相关的多种蛋白质代谢发生紊乱,从而诱导细胞凋亡。于是蛋白酶体抑制剂作为一种新型抗肿瘤靶向治疗药物受到人们的广泛关注。硼替佐米作为第一进入临床的蛋白酶体抑制剂已经被美国食品药品监督局(FDA)批准用于难治性及复发性多发性骨髓瘤及套细胞淋巴瘤。关于肺癌及淋巴瘤的研究也已经进入临床试验阶段,但蛋白酶体抑制剂在喉癌中的研究甚少,其作用机制尚不完全清楚。信号转导与转录激活因3(STAT3,signal transducer and activator of transcription-3)是信号转导通路中的重要因子,是EGFR、IL-6/JAK、Src等多个致癌性酪氨酸激酶信号通道的汇聚的焦点,与人类恶性肿瘤的发生、发展和演进关系密切。有报道指出蛋白酶体抑制剂可诱导结直肠癌细胞中STAT3的激活。在喉癌中,蛋白酶体抑制剂对STAT3的影响尚无相关报道。我们前期的研究表明STAT3的激活与喉癌的发生、发展密切相关,抑制STAT3的激活可抑制喉癌细胞的恶性增殖及诱导凋亡。于是本研究以体外培养的人喉鳞癌Hep-2细胞株为研究对象,应用四甲基偶氮唑蓝(MTT)法、流式细胞仪、Western Blot、基因转染等技术探讨了蛋白酶体抑制剂对喉癌细胞的作用、可能机制及对STAT3的影响,并进一步联合pshSTAT3及常规化疗药物顺铂诱导喉癌细胞凋亡作用的观察,为寻找更加有效的分子靶向、多靶点联合治疗途径以及喉癌的临床治疗提供理论基础。本实验分为以下三部分: 第一部分蛋白酶体抑制剂MG-132诱导喉癌细胞凋亡及对STAT3的影响 目的:通过检测不同浓度及不同作用时间的蛋白酶体抑制剂MG-132对体外培养的人喉鳞癌Hep-2细胞株的增殖、周期分布、凋亡的作用及相关蛋白表达的情况,探讨蛋白酶体抑制剂对喉鳞癌细胞的作用、相关机制及对STAT3的影响。 方法: 1.以Hep-2细胞为实验对象,四甲基偶氮唑蓝(MTT)法检测不同浓度(0、1、2.5、5、10、20μmol/L)MG-132作用24h、48h及2.5μmol/L MG-132作用0h、12h、24h、48h、72h后细胞的增殖情况。 2.流式细胞技术(FCM)检测不同浓度(0、1、2.5μmol/L)MG-132作用48h的细胞凋亡及2.5μmol/L MG-132作用0h、12h、24h、48h、72h后细胞凋亡及周期分布情况。 3. Western blot检测MG-132(2.5μmol/L)作用0h、12h、24h、48h后,Hep-2细胞的p21、cyclinD1、CDK4、Bcl-2、STAT3、p-STAT3蛋白表达水平。 结果: 1. MTT检测显示:MG-132可以有效抑制Hep-2细胞的增殖,在本实验范围内呈浓度(24h:r=0.925,P0.01;48h:r=0.944,P0.01)及时间依赖性(r=0.945,P0.01);24h的半数抑制量(IC50)=20μmol/L;48h的半数抑制量(IC50)=2.5μmol/L。 2. FCM结果显示:细胞凋亡率随MG-132作用时间的延长及浓度的递增而上升,同样具有量效(r=0.842,P0.01)及时效性(r=0.888,P0.01)。Hep-2细胞的周期分布发生变化,处于G0/G1和G2/M期的细胞比例上升,S期的细胞比例下降(P0.01)。 3. Western blot:p21蛋白水平随着MG-132作用时间的延长而表达显著增强,cyclinD1有中等程度的下调。MG-132作用12h时,可观察到p-STAT3蛋白表达明显增强且维持在一个稳定的水平,Bcl-2蛋白也随之逐渐增强,CDK4及STAT3蛋白水平在不同时间点无明显变化。 结论: 1.蛋白酶体抑制剂可以有效抑制人喉鳞癌Hep-2细胞在体外的增殖并且诱导其发生凋亡,随作用浓度及时间的不同而变化,呈浓度及时间依赖性。 2.蛋白酶体抑制剂可以诱导Hep-2细胞发生G0/G1和G2/M期的双重阻滞。 3.蛋白酶体抑制剂对喉癌细胞作用的机制之一与p21蛋白水平的上调及cyclinD1蛋白水平的下调相关。 4.蛋白酶体抑制剂在发挥有效作用的同时导致了关键信号分子STAT3蛋白的激活及抗凋亡家族成员Bcl-2蛋白水平的上调,从而在一定的程度上降低了药物的效能。 第二部分蛋白酶体抑制剂联合pshSTAT3对喉癌细胞增殖与凋亡的影响 目的:研究短发夹RNA沉默STAT3能否增强蛋白酶体抑制剂MG-132对喉癌细胞的抗肿瘤作用。 方法: 1.构建及扩增pshSTAT3,并经酶切和测序鉴定。 2.研究分五组:⑴细胞对照组:未经任何处理的Hep-2细胞;⑵阴性对照质粒组:转染阴性对照质粒的Hep-2细胞;⑶MG-132组:经2.5μmol/L MG-132处理的Hep-2细胞;⑷联合组:阳性重组质粒转染+MG-132(2.5μmol/L)处理的Hep-2细胞;⑸pshSTAT3组:转染阳性重组质粒的Hep-2细胞。 3.利用MTT和FCM两种方法分别检测不同实验组Hep-2细胞的增殖活性及凋亡率的情况。 4. Western blot检测不同实验组Hep-2细胞p-STAT3蛋白表达的情况。 结果: 1.经酶切及测序结果证实,成功构建阳性重组质粒pshSTAT3。 2.重组质粒转染Hep-2细胞情况的观察:真核表达质粒和阴性对照质粒转染Hep-2细胞后6h即可在荧光显微镜下观察到绿色荧光,转染后24h,绿色荧光逐渐增多增强,荧光倒置显微镜下观察细胞转染效率在80%以上,FCM检测转染效率为85.76%。 3. pshSTAT3增强了MG-132对Hep-2细胞的增殖抑制作用:将阳性重组质粒或阴性对照质粒转染Hep-2细胞,转染后24h,联合或不联合2.5μmol/L MG-132,再继续培养48h,MTT法检测各组的细胞增殖抑制率。结果发现阴性对照质粒组和细胞对照组细胞的体外增殖活性无明显差异(P0.05);联合组能显著抑制Hep-2细胞的增殖活性(抑制率:62.23±1.25%),与MG-132组、pshSTAT3组及阴性对照质粒组相比,差异均有统计学意义(P0.01)。 4. pshSTAT3增强了MG-132对Hep-2细胞诱导凋亡的作用:细胞转染后24h加2.5μmol/L MG-132,再继续培养48h,FCM检测各组的细胞凋亡率,结果显示联合组细胞出现明显的凋亡峰,其凋亡率(55.80±3.12%)显著高于MG-132组(41.20±3.21%)和pshSTAT3组(22.13±1.84%),差异具有统计学意义(P0.01);阴性对照质粒组和细胞对照组间无明显差异(P0.05)。 5. pshSTAT3与MG-132对p-STAT3表达的影响:Western blot检测显示细胞对照组与阴性对照质粒组两组之间的p-STAT3蛋白水平无明显差异;MG-132组p-STAT3蛋白表达显著增强;联合组及pshSTAT3组p-STAT3蛋白表达明显减弱。 结论: pshSTAT3可以抑制MG-132诱导的STAT3蛋白的激活,从而提高了蛋白酶体抑制剂MG-132对喉鳞癌细胞的抗肿瘤作用。第三部分蛋白酶体抑制剂MG-132联合化疗诱导喉癌细胞凋亡的观察目的:探讨蛋白酶体抑制剂MG-132能否增强常规化疗药物顺铂(DDP)对喉鳞癌细胞的毒性作用。 方法:取对数期生长的Hep-2细胞分为四组:对照组、MG-132组、DDP组、DDP+ MG-132组。利用MTT和FCM两种方法分别检测不同实验组细胞的增殖及凋亡情况。 结果: 1. MG-132联合DDP对Hep-2细胞增殖活性的影响:不同浓度DDP(10、20、40、80μmol/L)单用或联合1μmol/L MG-132作用于Hep-2细胞48h后结果显示:在每个浓度水平上DDP组与DDP+MG-132组比较均有统计学意义(P0.01,P0.05)。10μmol/L DDP+MG-132组与20μmol/L DDP组、20μmol/L DDP+MG-132组与40μmol/L DDP组比较均具有统计学差异(P0.01,P0.05)。DDP单独作用的IC50=62.22μmol/L;当与1umol/L MG-132联合作用时,其IC50下降至27.79μmol/L。 2. MG-132联合DDP诱导Hep-2细胞凋亡作用的观察:10μmol/L DDP单用或联合1μmol/L MG-132作用于Hep-2细胞48h后,FCM检测结果显示DDP+MG-132组诱导细胞凋亡作用最强,与对照组、DDP组、MG-132组相比均具有显著性差异(P0.01)。 结论:蛋白酶体抑制剂能显著增强常规化疗药物顺铂对喉鳞癌Hep-2细胞株的毒性作用,两者联合可以在有效作用发挥的同时降低DDP的应用剂量,从而减轻临床上常见的毒副作用。
[Abstract]:Laryngeal carcinoma is a common malignant tumor of the head and neck. Its incidence and mortality are increasing year by year. It is a serious threat to human health and life. The prognosis is still very poor and the operation is lost. However, the traditional radiotherapy and chemotherapy are often intolerable because of their serious toxicity, which limits the treatment of laryngeal cancer.
Ubiquitin-Proteasome Pathway (UPP) is an important pathway for the selective degradation of endogenous proteins in eukaryotic cells. It is also a key mechanism for regulating many biological processes such as cell cycle operation, proliferation, apoptosis, signal transduction and gene transcription. Breaking UPP can induce apoptosis by disrupting the metabolism of many proteins closely related to proliferation, differentiation and apoptosis of tumor cells. So proteasome inhibitors, as a new type of anti-tumor targeted therapy drugs, have attracted wide attention. The study on lung cancer and lymphoma has also entered the clinical trial stage, but the research on proteasome inhibitors in laryngeal cancer is very little, and the mechanism of action is still unclear. Transducer and activator of transcription-3) are important factors in signal transduction pathways, focusing on the convergence of EGFR, IL-6/JAK, Src and other carcinogenic tyrosine kinase signaling pathways. It has been reported that proteasome inhibitors can induce STAT3 in colorectal cancer cells. In laryngeal carcinoma, the effect of proteasome inhibitor on STAT3 has not been reported. Our previous studies showed that STAT3 activation is closely related to the occurrence and development of laryngeal carcinoma. Inhibition of STAT3 activation can inhibit the malignant proliferation and induce apoptosis of laryngeal carcinoma cells. PARTICIPANTS: The effects of proteasome inhibitors on laryngeal cancer cells were investigated by MTT assay, flow cytometry, Western Blot and gene transfection. The possible mechanisms and effects of proteasome inhibitors on STAT3 were also investigated. The apoptosis of laryngeal cancer cells induced by pshSTAT3 and cisplatin were further observed in order to find a more effective method. Molecular targeting, multi-target combination therapy and clinical treatment of laryngeal cancer provide theoretical basis.
Part I: proteasome inhibitor MG-132 induces apoptosis of laryngeal carcinoma cells and its effect on STAT3.
AIM: To investigate the effect of proteasome inhibitor MG-132 on proliferation, cell cycle distribution, apoptosis and expression of proteasome-related proteins in human laryngeal squamous cell carcinoma Hep-2 cell line in vitro, and to explore the mechanism of proteasome inhibitor on laryngeal squamous cell carcinoma and its effect on STAT3.
Method:
1. Hep-2 cells were treated with different concentrations (0,1,2.5,5,10,20 micromol/L) of MG-132 for 24 hours, 48 hours and 2.5 micromol/L MG-132 for 0 hours, 12 hours, 24 hours, 48 hours and 72 hours. The proliferation of Hep-2 cells was detected by MTT assay.
2. Flow cytometry (FCM) was used to detect the apoptosis and cell cycle distribution of MG-132 treated with different concentrations (0,1,2.5 micromol/L) for 48 hours and 2.5 micromol/L MG-132 for 0, 12, 24, 48 and 72 hours.
3. Western blot was used to detect the expression of p21, cyclin D1, CDK4, Bcl-2, STAT3 and p-STAT3 proteins in Hep-2 cells at 0, 12, 24 and 48 h after MG-132 treatment.
Result:
1. MTT assay showed that MG-132 could effectively inhibit the proliferation of Hep-2 cells in the range of concentration (24h: r = 0.925, P 0.01; 48h: r = 0.944, P 0.01) and time-dependent (r = 0.945, P 0.01); half inhibitory dose (IC50) = 20 micromol/L for 24h; half inhibitory dose (IC50) = 2.5 micromol/L for 48h.
2. FCM results showed that the apoptosis rate increased with the prolongation of MG-132 action time and the increase of MG-132 concentration, and also had dose-effect (r = 0.842, P 0.01) and timeliness (r = 0.888, P 0.01). The cycle distribution of Hep-2 cells changed, the proportion of cells in G0/G1 and G2/M phase increased, and the proportion of cells in S phase decreased (P 0.01).
3. Western blot: The expression of p21 protein increased significantly with the prolongation of MG-132 treatment time, and cyclin D1 decreased moderately. After 12 hours of MG-132 treatment, the expression of p-STAT3 protein was significantly increased and maintained at a stable level, and the expression of Bcl-2 protein was gradually increased, while the levels of CDK4 and STAT3 protein were not obvious at different time points. Change.
Conclusion:
1. Proteasome inhibitors can effectively inhibit the proliferation of human laryngeal squamous cell carcinoma Hep-2 cells in vitro and induce apoptosis, which varies with the concentration and time, in a concentration-and time-dependent manner.
2. proteasome inhibitor can induce double blockade of G0/G1 and G2/M phases in Hep-2 cells.
3. One of the mechanisms of proteasome inhibitors on laryngeal cancer cells is related to the up-regulation of p21 protein and the down-regulation of cyclin D1 protein.
4. Proteasome inhibitors play an effective role in the activation of STAT3 protein, a key signal molecule, and the up-regulation of Bcl-2 protein, a member of the anti-apoptosis family.
The second part is the effect of proteasome inhibitor combined with pshSTAT3 on the proliferation and apoptosis of laryngeal carcinoma cells.
AIM: To investigate whether STAT3 silencing with short hairpin RNA can enhance the antitumor effect of proteasome inhibitor MG-132 on laryngeal cancer cells.
Method:
1. pshSTAT3 was constructed and amplified, and identified by restriction enzyme digestion and sequencing.
2. The study was divided into five groups: (1) control group: Hep-2 cells without any treatment; (2) negative control plasmid group: Hep-2 cells transfected with negative control plasmid; (3) MG-132 cells treated with 2.5 micromol/L MG-132; (3) combined group: Hep-2 cells transfected with positive recombinant plasmid + MG-132 (2.5 micromol/L); and (3) pshSTAT3 cells transfected with positive recombinant plasmid; Plasmid Hep-2 cells.
3. MTT and FCM were used to detect the proliferation activity and apoptosis rate of Hep-2 cells in different experimental groups.
4. Western blot was used to detect the expression of p-STAT3 protein in Hep-2 cells of different experimental groups.
Result:
1. by enzyme digestion and sequencing, the recombinant plasmid pshSTAT3. was successfully constructed.
2. Observation of Hep-2 cells transfected with recombinant plasmid: Green fluorescence was observed under fluorescence microscope 6 hours after transfection with eukaryotic expression plasmid and negative control plasmid. The green fluorescence increased gradually 24 hours after transfection. The transfection efficiency was over 80% under fluorescence inversion microscope, and 85.76% by FCM.
3. PShSTAT3 enhanced the inhibitory effect of MG-132 on the proliferation of Hep-2 cells: Hep-2 cells were transfected with positive recombinant plasmids or negative control plasmids. 24 hours after transfection, the cells were co-cultured or not co-cultured with 2.5 micromol/L MG-132, and then cultured for 48 hours. MTT assay was used to detect the inhibitory rate of cell proliferation in each group. There was no significant difference in proliferative activity (P 0.05); the combined group could significantly inhibit the proliferation activity of Hep-2 cells (inhibition rate: 62.23 (+ 1.25%) compared with MG-132 group, pshSTAT3 group and negative control plasmid group, the difference was statistically significant (P 0.01).
4. PShSTAT3 enhanced the apoptosis-inducing effect of MG-132 on Hep-2 cells: cells were transfected with 2.5 micromol/L MG-132 at 24 hours and then cultured for 48 hours. FCM detected the apoptotic rate of Hep-2 cells in each group. The results showed that the apoptotic peak appeared in the combined group, and the apoptotic rate was significantly higher than that in MG-132 group (41.20+3.21%) and pshSTAT3 group (22.13+1.84%). The difference was statistically significant (P0.01); there was no significant difference between the negative control plasmid group and the cell control group (P0.05).
5. The effect of pshSTAT3 and MG-132 on the expression of p-STAT3: Western blot showed that there was no significant difference in the expression of p-STAT3 protein between the cell control group and the negative control plasmid group; the expression of p-STAT3 protein in the MG-132 group was significantly increased; the expression of p-STAT3 protein in the combined group and the pshSTAT3 group was significantly decreased.
CONCLUSION: PShSTAT3 can inhibit the activation of STAT3 protein induced by MG-132, thereby enhancing the antitumor effect of proteasome inhibitor MG-132 on laryngeal squamous cell carcinoma. Part III Proteasome inhibitor MG-132 combined with chemotherapy induces apoptosis of laryngeal cancer cells OBJECTIVE: To investigate whether proteasome inhibitor MG-132 can enhance the effect of conventional chemotherapy drugs. The toxic effect of cisplatin (DDP) on laryngeal squamous cell carcinoma.
Methods: Hep-2 cells were divided into four groups: control group, MG-132 group, DDP group and DDP+MG-132 group.
Result:
1. Effects of MG-132 combined with DDP on the proliferation of Hep-2 cells: Different concentrations of DDP (10,20,40,80 micromol/L) alone or combined with 1 micromol/L MG-132 on Hep-2 cells for 48 hours showed that at each concentration level, DDP group and DDP+MG-132 group were significantly different (P 0.01,P 0.05). 10 micromol/L DDP+MG-132 group and 20 micromol/L DDP group, 20 micromol/L D D group, 20 micromol/L D D group, 20 micromol/L D D group. There was significant difference between DP+MG-132 group and 40 micromol/L DDP group (P 0.01, P 0.05). The IC50 of DDP alone was 62.22 micromol/L; when combined with 1 umol/L MG-132, the IC50 of DDP decreased to 27.79 micromol/L.
2. Observation on the apoptosis of Hep-2 cells induced by MG-132 and DDP: After 48 hours of treatment with 10 micromol/L DDP alone or combined with 1 micromol/L MG-132, FCM results showed that DDP+MG-132 group had the strongest effect on inducing apoptosis of Hep-2 cells, which was significantly different from that of control group, DDP group and MG-132 group (P 0.01).
CONCLUSION: Proteasome inhibitors can significantly enhance the toxicity of cisplatin to laryngeal squamous cell carcinoma Hep-2 cell line. The combination of the two drugs can reduce the dosage of DDP and reduce the common side effects in clinic.
【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R739.65
本文编号：2220188
[Abstract]:Laryngeal carcinoma is a common malignant tumor of the head and neck. Its incidence and mortality are increasing year by year. It is a serious threat to human health and life. The prognosis is still very poor and the operation is lost. However, the traditional radiotherapy and chemotherapy are often intolerable because of their serious toxicity, which limits the treatment of laryngeal cancer.
Ubiquitin-Proteasome Pathway (UPP) is an important pathway for the selective degradation of endogenous proteins in eukaryotic cells. It is also a key mechanism for regulating many biological processes such as cell cycle operation, proliferation, apoptosis, signal transduction and gene transcription. Breaking UPP can induce apoptosis by disrupting the metabolism of many proteins closely related to proliferation, differentiation and apoptosis of tumor cells. So proteasome inhibitors, as a new type of anti-tumor targeted therapy drugs, have attracted wide attention. The study on lung cancer and lymphoma has also entered the clinical trial stage, but the research on proteasome inhibitors in laryngeal cancer is very little, and the mechanism of action is still unclear. Transducer and activator of transcription-3) are important factors in signal transduction pathways, focusing on the convergence of EGFR, IL-6/JAK, Src and other carcinogenic tyrosine kinase signaling pathways. It has been reported that proteasome inhibitors can induce STAT3 in colorectal cancer cells. In laryngeal carcinoma, the effect of proteasome inhibitor on STAT3 has not been reported. Our previous studies showed that STAT3 activation is closely related to the occurrence and development of laryngeal carcinoma. Inhibition of STAT3 activation can inhibit the malignant proliferation and induce apoptosis of laryngeal carcinoma cells. PARTICIPANTS: The effects of proteasome inhibitors on laryngeal cancer cells were investigated by MTT assay, flow cytometry, Western Blot and gene transfection. The possible mechanisms and effects of proteasome inhibitors on STAT3 were also investigated. The apoptosis of laryngeal cancer cells induced by pshSTAT3 and cisplatin were further observed in order to find a more effective method. Molecular targeting, multi-target combination therapy and clinical treatment of laryngeal cancer provide theoretical basis.
Part I: proteasome inhibitor MG-132 induces apoptosis of laryngeal carcinoma cells and its effect on STAT3.
AIM: To investigate the effect of proteasome inhibitor MG-132 on proliferation, cell cycle distribution, apoptosis and expression of proteasome-related proteins in human laryngeal squamous cell carcinoma Hep-2 cell line in vitro, and to explore the mechanism of proteasome inhibitor on laryngeal squamous cell carcinoma and its effect on STAT3.
Method:
1. Hep-2 cells were treated with different concentrations (0,1,2.5,5,10,20 micromol/L) of MG-132 for 24 hours, 48 hours and 2.5 micromol/L MG-132 for 0 hours, 12 hours, 24 hours, 48 hours and 72 hours. The proliferation of Hep-2 cells was detected by MTT assay.
2. Flow cytometry (FCM) was used to detect the apoptosis and cell cycle distribution of MG-132 treated with different concentrations (0,1,2.5 micromol/L) for 48 hours and 2.5 micromol/L MG-132 for 0, 12, 24, 48 and 72 hours.
3. Western blot was used to detect the expression of p21, cyclin D1, CDK4, Bcl-2, STAT3 and p-STAT3 proteins in Hep-2 cells at 0, 12, 24 and 48 h after MG-132 treatment.
Result:
1. MTT assay showed that MG-132 could effectively inhibit the proliferation of Hep-2 cells in the range of concentration (24h: r = 0.925, P 0.01; 48h: r = 0.944, P 0.01) and time-dependent (r = 0.945, P 0.01); half inhibitory dose (IC50) = 20 micromol/L for 24h; half inhibitory dose (IC50) = 2.5 micromol/L for 48h.
2. FCM results showed that the apoptosis rate increased with the prolongation of MG-132 action time and the increase of MG-132 concentration, and also had dose-effect (r = 0.842, P 0.01) and timeliness (r = 0.888, P 0.01). The cycle distribution of Hep-2 cells changed, the proportion of cells in G0/G1 and G2/M phase increased, and the proportion of cells in S phase decreased (P 0.01).
3. Western blot: The expression of p21 protein increased significantly with the prolongation of MG-132 treatment time, and cyclin D1 decreased moderately. After 12 hours of MG-132 treatment, the expression of p-STAT3 protein was significantly increased and maintained at a stable level, and the expression of Bcl-2 protein was gradually increased, while the levels of CDK4 and STAT3 protein were not obvious at different time points. Change.
Conclusion:
1. Proteasome inhibitors can effectively inhibit the proliferation of human laryngeal squamous cell carcinoma Hep-2 cells in vitro and induce apoptosis, which varies with the concentration and time, in a concentration-and time-dependent manner.
2. proteasome inhibitor can induce double blockade of G0/G1 and G2/M phases in Hep-2 cells.
3. One of the mechanisms of proteasome inhibitors on laryngeal cancer cells is related to the up-regulation of p21 protein and the down-regulation of cyclin D1 protein.
4. Proteasome inhibitors play an effective role in the activation of STAT3 protein, a key signal molecule, and the up-regulation of Bcl-2 protein, a member of the anti-apoptosis family.
The second part is the effect of proteasome inhibitor combined with pshSTAT3 on the proliferation and apoptosis of laryngeal carcinoma cells.
AIM: To investigate whether STAT3 silencing with short hairpin RNA can enhance the antitumor effect of proteasome inhibitor MG-132 on laryngeal cancer cells.
Method:
1. pshSTAT3 was constructed and amplified, and identified by restriction enzyme digestion and sequencing.
2. The study was divided into five groups: (1) control group: Hep-2 cells without any treatment; (2) negative control plasmid group: Hep-2 cells transfected with negative control plasmid; (3) MG-132 cells treated with 2.5 micromol/L MG-132; (3) combined group: Hep-2 cells transfected with positive recombinant plasmid + MG-132 (2.5 micromol/L); and (3) pshSTAT3 cells transfected with positive recombinant plasmid; Plasmid Hep-2 cells.
3. MTT and FCM were used to detect the proliferation activity and apoptosis rate of Hep-2 cells in different experimental groups.
4. Western blot was used to detect the expression of p-STAT3 protein in Hep-2 cells of different experimental groups.
Result:
1. by enzyme digestion and sequencing, the recombinant plasmid pshSTAT3. was successfully constructed.
2. Observation of Hep-2 cells transfected with recombinant plasmid: Green fluorescence was observed under fluorescence microscope 6 hours after transfection with eukaryotic expression plasmid and negative control plasmid. The green fluorescence increased gradually 24 hours after transfection. The transfection efficiency was over 80% under fluorescence inversion microscope, and 85.76% by FCM.
3. PShSTAT3 enhanced the inhibitory effect of MG-132 on the proliferation of Hep-2 cells: Hep-2 cells were transfected with positive recombinant plasmids or negative control plasmids. 24 hours after transfection, the cells were co-cultured or not co-cultured with 2.5 micromol/L MG-132, and then cultured for 48 hours. MTT assay was used to detect the inhibitory rate of cell proliferation in each group. There was no significant difference in proliferative activity (P 0.05); the combined group could significantly inhibit the proliferation activity of Hep-2 cells (inhibition rate: 62.23 (+ 1.25%) compared with MG-132 group, pshSTAT3 group and negative control plasmid group, the difference was statistically significant (P 0.01).
4. PShSTAT3 enhanced the apoptosis-inducing effect of MG-132 on Hep-2 cells: cells were transfected with 2.5 micromol/L MG-132 at 24 hours and then cultured for 48 hours. FCM detected the apoptotic rate of Hep-2 cells in each group. The results showed that the apoptotic peak appeared in the combined group, and the apoptotic rate was significantly higher than that in MG-132 group (41.20+3.21%) and pshSTAT3 group (22.13+1.84%). The difference was statistically significant (P0.01); there was no significant difference between the negative control plasmid group and the cell control group (P0.05).
5. The effect of pshSTAT3 and MG-132 on the expression of p-STAT3: Western blot showed that there was no significant difference in the expression of p-STAT3 protein between the cell control group and the negative control plasmid group; the expression of p-STAT3 protein in the MG-132 group was significantly increased; the expression of p-STAT3 protein in the combined group and the pshSTAT3 group was significantly decreased.
CONCLUSION: PShSTAT3 can inhibit the activation of STAT3 protein induced by MG-132, thereby enhancing the antitumor effect of proteasome inhibitor MG-132 on laryngeal squamous cell carcinoma. Part III Proteasome inhibitor MG-132 combined with chemotherapy induces apoptosis of laryngeal cancer cells OBJECTIVE: To investigate whether proteasome inhibitor MG-132 can enhance the effect of conventional chemotherapy drugs. The toxic effect of cisplatin (DDP) on laryngeal squamous cell carcinoma.
Methods: Hep-2 cells were divided into four groups: control group, MG-132 group, DDP group and DDP+MG-132 group.
Result:
1. Effects of MG-132 combined with DDP on the proliferation of Hep-2 cells: Different concentrations of DDP (10,20,40,80 micromol/L) alone or combined with 1 micromol/L MG-132 on Hep-2 cells for 48 hours showed that at each concentration level, DDP group and DDP+MG-132 group were significantly different (P 0.01,P 0.05). 10 micromol/L DDP+MG-132 group and 20 micromol/L DDP group, 20 micromol/L D D group, 20 micromol/L D D group, 20 micromol/L D D group. There was significant difference between DP+MG-132 group and 40 micromol/L DDP group (P 0.01, P 0.05). The IC50 of DDP alone was 62.22 micromol/L; when combined with 1 umol/L MG-132, the IC50 of DDP decreased to 27.79 micromol/L.
2. Observation on the apoptosis of Hep-2 cells induced by MG-132 and DDP: After 48 hours of treatment with 10 micromol/L DDP alone or combined with 1 micromol/L MG-132, FCM results showed that DDP+MG-132 group had the strongest effect on inducing apoptosis of Hep-2 cells, which was significantly different from that of control group, DDP group and MG-132 group (P 0.01).
CONCLUSION: Proteasome inhibitors can significantly enhance the toxicity of cisplatin to laryngeal squamous cell carcinoma Hep-2 cell line. The combination of the two drugs can reduce the dosage of DDP and reduce the common side effects in clinic.
【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R739.65
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