蛋白酶體抑制劑MG-132對喉鱗癌Hep-2細胞作用的實驗研究
發(fā)布時間：2018-09-03 14:04
【摘要】： 喉癌(laryngeal carcinoma)是頭頸部常見的惡性腫瘤,其發(fā)病率及死亡率均呈逐年上升的趨勢,嚴重威脅著人類的健康與生命。雖然手術為主,放療與化療為輔的綜合治療方法,使患者的5年生存率有所提高。但對于那些晚期或復發(fā)、轉移的患者預后仍舊很差,手術喪失時機,而傳統(tǒng)放化療又由于其嚴重的毒、副作用常使得患者不能耐受,從而使患者的治療受到極大的限制。因此,積極尋求喉癌治療的新策略成為廣大耳鼻咽喉科研究者面臨的新課題。 泛素-蛋白酶體通路(Ubiquitin-Proteasome Pathway,UPP)是真核細胞中內源性蛋白質選擇性降解的重要途徑,是調節(jié)細胞多種生物學過程如細胞周期運轉、增殖、凋亡、信號傳遞、基因轉錄等的關鍵機制。它與人類惡性腫瘤的發(fā)生、發(fā)展密切相關。阻斷UPP可以促使與腫瘤細胞增殖、分化、凋亡密切相關的多種蛋白質代謝發(fā)生紊亂,從而誘導細胞凋亡。于是蛋白酶體抑制劑作為一種新型抗腫瘤靶向治療藥物受到人們的廣泛關注。硼替佐米作為第一進入臨床的蛋白酶體抑制劑已經(jīng)被美國食品藥品監(jiān)督局(FDA)批準用于難治性及復發(fā)性多發(fā)性骨髓瘤及套細胞淋巴瘤。關于肺癌及淋巴瘤的研究也已經(jīng)進入臨床試驗階段,但蛋白酶體抑制劑在喉癌中的研究甚少,其作用機制尚不完全清楚。信號轉導與轉錄激活因3(STAT3,signal transducer and activator of transcription-3)是信號轉導通路中的重要因子,是EGFR、IL-6/JAK、Src等多個致癌性酪氨酸激酶信號通道的匯聚的焦點,與人類惡性腫瘤的發(fā)生、發(fā)展和演進關系密切。有報道指出蛋白酶體抑制劑可誘導結直腸癌細胞中STAT3的激活。在喉癌中,蛋白酶體抑制劑對STAT3的影響尚無相關報道。我們前期的研究表明STAT3的激活與喉癌的發(fā)生、發(fā)展密切相關,抑制STAT3的激活可抑制喉癌細胞的惡性增殖及誘導凋亡。于是本研究以體外培養(yǎng)的人喉鱗癌Hep-2細胞株為研究對象,應用四甲基偶氮唑藍(MTT)法、流式細胞儀、Western Blot、基因轉染等技術探討了蛋白酶體抑制劑對喉癌細胞的作用、可能機制及對STAT3的影響,并進一步聯(lián)合pshSTAT3及常規(guī)化療藥物順鉑誘導喉癌細胞凋亡作用的觀察,為尋找更加有效的分子靶向、多靶點聯(lián)合治療途徑以及喉癌的臨床治療提供理論基礎。本實驗分為以下三部分: 第一部分蛋白酶體抑制劑MG-132誘導喉癌細胞凋亡及對STAT3的影響 目的:通過檢測不同濃度及不同作用時間的蛋白酶體抑制劑MG-132對體外培養(yǎng)的人喉鱗癌Hep-2細胞株的增殖、周期分布、凋亡的作用及相關蛋白表達的情況,探討蛋白酶體抑制劑對喉鱗癌細胞的作用、相關機制及對STAT3的影響。 方法: 1.以Hep-2細胞為實驗對象,四甲基偶氮唑藍(MTT)法檢測不同濃度(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后細胞的增殖情況。 2.流式細胞技術(FCM)檢測不同濃度(0、1、2.5μmol/L)MG-132作用48h的細胞凋亡及2.5μmol/L MG-132作用0h、12h、24h、48h、72h后細胞凋亡及周期分布情況。 3. Western blot檢測MG-132(2.5μmol/L)作用0h、12h、24h、48h后,Hep-2細胞的p21、cyclinD1、CDK4、Bcl-2、STAT3、p-STAT3蛋白表達水平。 結果: 1. MTT檢測顯示:MG-132可以有效抑制Hep-2細胞的增殖,在本實驗范圍內呈濃度(24h:r=0.925,P0.01;48h:r=0.944,P0.01)及時間依賴性(r=0.945,P0.01);24h的半數(shù)抑制量(IC50)=20μmol/L;48h的半數(shù)抑制量(IC50)=2.5μmol/L。 2. FCM結果顯示:細胞凋亡率隨MG-132作用時間的延長及濃度的遞增而上升,同樣具有量效(r=0.842,P0.01)及時效性(r=0.888,P0.01)。Hep-2細胞的周期分布發(fā)生變化,處于G0/G1和G2/M期的細胞比例上升,S期的細胞比例下降(P0.01)。 3. Western blot:p21蛋白水平隨著MG-132作用時間的延長而表達顯著增強,cyclinD1有中等程度的下調。MG-132作用12h時,可觀察到p-STAT3蛋白表達明顯增強且維持在一個穩(wěn)定的水平,Bcl-2蛋白也隨之逐漸增強,CDK4及STAT3蛋白水平在不同時間點無明顯變化。 結論: 1.蛋白酶體抑制劑可以有效抑制人喉鱗癌Hep-2細胞在體外的增殖并且誘導其發(fā)生凋亡,隨作用濃度及時間的不同而變化,呈濃度及時間依賴性。 2.蛋白酶體抑制劑可以誘導Hep-2細胞發(fā)生G0/G1和G2/M期的雙重阻滯。 3.蛋白酶體抑制劑對喉癌細胞作用的機制之一與p21蛋白水平的上調及cyclinD1蛋白水平的下調相關。 4.蛋白酶體抑制劑在發(fā)揮有效作用的同時導致了關鍵信號分子STAT3蛋白的激活及抗凋亡家族成員Bcl-2蛋白水平的上調,從而在一定的程度上降低了藥物的效能。 第二部分蛋白酶體抑制劑聯(lián)合pshSTAT3對喉癌細胞增殖與凋亡的影響 目的:研究短發(fā)夾RNA沉默STAT3能否增強蛋白酶體抑制劑MG-132對喉癌細胞的抗腫瘤作用。 方法: 1.構建及擴增pshSTAT3,并經(jīng)酶切和測序鑒定。 2.研究分五組:⑴細胞對照組:未經(jīng)任何處理的Hep-2細胞;⑵陰性對照質粒組:轉染陰性對照質粒的Hep-2細胞;⑶MG-132組:經(jīng)2.5μmol/L MG-132處理的Hep-2細胞;⑷聯(lián)合組:陽性重組質粒轉染+MG-132(2.5μmol/L)處理的Hep-2細胞;⑸pshSTAT3組:轉染陽性重組質粒的Hep-2細胞。 3.利用MTT和FCM兩種方法分別檢測不同實驗組Hep-2細胞的增殖活性及凋亡率的情況。 4. Western blot檢測不同實驗組Hep-2細胞p-STAT3蛋白表達的情況。 結果: 1.經(jīng)酶切及測序結果證實,成功構建陽性重組質粒pshSTAT3。 2.重組質粒轉染Hep-2細胞情況的觀察:真核表達質粒和陰性對照質粒轉染Hep-2細胞后6h即可在熒光顯微鏡下觀察到綠色熒光,轉染后24h,綠色熒光逐漸增多增強,熒光倒置顯微鏡下觀察細胞轉染效率在80%以上,FCM檢測轉染效率為85.76%。 3. pshSTAT3增強了MG-132對Hep-2細胞的增殖抑制作用:將陽性重組質�；蜿幮詫φ召|粒轉染Hep-2細胞,轉染后24h,聯(lián)合或不聯(lián)合2.5μmol/L MG-132,再繼續(xù)培養(yǎng)48h,MTT法檢測各組的細胞增殖抑制率。結果發(fā)現(xiàn)陰性對照質粒組和細胞對照組細胞的體外增殖活性無明顯差異(P0.05);聯(lián)合組能顯著抑制Hep-2細胞的增殖活性(抑制率:62.23±1.25%),與MG-132組、pshSTAT3組及陰性對照質粒組相比,差異均有統(tǒng)計學意義(P0.01)。 4. pshSTAT3增強了MG-132對Hep-2細胞誘導凋亡的作用:細胞轉染后24h加2.5μmol/L MG-132,再繼續(xù)培養(yǎng)48h,FCM檢測各組的細胞凋亡率,結果顯示聯(lián)合組細胞出現(xiàn)明顯的凋亡峰,其凋亡率(55.80±3.12%)顯著高于MG-132組(41.20±3.21%)和pshSTAT3組(22.13±1.84%),差異具有統(tǒng)計學意義(P0.01);陰性對照質粒組和細胞對照組間無明顯差異(P0.05)。 5. pshSTAT3與MG-132對p-STAT3表達的影響:Western blot檢測顯示細胞對照組與陰性對照質粒組兩組之間的p-STAT3蛋白水平無明顯差異;MG-132組p-STAT3蛋白表達顯著增強;聯(lián)合組及pshSTAT3組p-STAT3蛋白表達明顯減弱。 結論: pshSTAT3可以抑制MG-132誘導的STAT3蛋白的激活,從而提高了蛋白酶體抑制劑MG-132對喉鱗癌細胞的抗腫瘤作用。第三部分蛋白酶體抑制劑MG-132聯(lián)合化療誘導喉癌細胞凋亡的觀察目的:探討蛋白酶體抑制劑MG-132能否增強常規(guī)化療藥物順鉑(DDP)對喉鱗癌細胞的毒性作用。 方法:取對數(shù)期生長的Hep-2細胞分為四組:對照組、MG-132組、DDP組、DDP+ MG-132組。利用MTT和FCM兩種方法分別檢測不同實驗組細胞的增殖及凋亡情況。 結果: 1. MG-132聯(lián)合DDP對Hep-2細胞增殖活性的影響:不同濃度DDP(10、20、40、80μmol/L)單用或聯(lián)合1μmol/L MG-132作用于Hep-2細胞48h后結果顯示:在每個濃度水平上DDP組與DDP+MG-132組比較均有統(tǒng)計學意義(P0.01,P0.05)。10μmol/L DDP+MG-132組與20μmol/L DDP組、20μmol/L DDP+MG-132組與40μmol/L DDP組比較均具有統(tǒng)計學差異(P0.01,P0.05)。DDP單獨作用的IC50=62.22μmol/L;當與1umol/L MG-132聯(lián)合作用時,其IC50下降至27.79μmol/L。 2. MG-132聯(lián)合DDP誘導Hep-2細胞凋亡作用的觀察:10μmol/L DDP單用或聯(lián)合1μmol/L MG-132作用于Hep-2細胞48h后,FCM檢測結果顯示DDP+MG-132組誘導細胞凋亡作用最強,與對照組、DDP組、MG-132組相比均具有顯著性差異(P0.01)。 結論:蛋白酶體抑制劑能顯著增強常規(guī)化療藥物順鉑對喉鱗癌Hep-2細胞株的毒性作用,兩者聯(lián)合可以在有效作用發(fā)揮的同時降低DDP的應用劑量,從而減輕臨床上常見的毒副作用。
[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.
【學位授予單位】：河北醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R739.65
本文編號：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.
【學位授予單位】：河北醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R739.65
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