乌骨藤中C_(21)甾体苷诱导SACC-83和SACC-LM细胞凋亡的作用及其机制研究
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摘要
目的研究乌骨藤提取物C_(21)甾体苷对唾液腺腺样囊性癌(salivary adenoid cystic carcinomacv,SACC)低侵袭细胞株(salivaryadenoidcysticcarcinoma-83,SACC-83)和肺高转移细胞株(SACC-LM)增殖抑制和诱导凋亡的作用及其机制。方法用不同浓度(5,10,20,40,60,80,100μmol·L~(-1))C_(21)甾体苷处理SACC-83和SACC-LM细胞48 h后,MTT法检测细胞活力,并计算药物的IC_(20)和IC_(50);细胞克隆形成实验检测细胞增殖能力;流式细胞术检测SACC-83及SACC-LM细胞凋亡情况;实时荧光定量PCR和Western blot检测SACC-83和SACC-LM细胞Bcl-2、Bax、caspase 3的mRNA和蛋白的表达。结果不同浓度的C_(21)甾体苷降低SACC-83和SACC-LM细胞活力,抑制细胞增殖,并且作用于SACC-83细胞C_(21)甾体苷的IC_(20)浓度为7.49μmol·L~(-1),IC_(50)浓度为38.34μmol·L~(-1);作用于SACC-LM细胞的C_(21)甾体苷IC_(20)浓度为9.30μmol·L~(-1),IC_(50)浓度为46.04μmol·L~(-1);细胞克隆集落形成明显减少。C_(21)甾体苷IC_(20)浓度分别促进SACC-83及SACC-LM细胞凋亡,且随着给药时间延长,凋亡率增加,具有显著性差异(P<0.05,P<0.01)。经7.49,9.30μmol·L~(-1)C_(21)甾体苷分别处理SACC-83及SACC-LM细胞后,Bcl-2的mRNA及蛋白水平显著降低(P<0.01),而Bax、Caspase3的mRNA和蛋白水平显著升高(P<0.05)。结论乌骨藤C_(21)甾体苷抑制SACC-83及SACC-LM细胞增殖、促进凋亡,其作用机制可能与调控Bcl-2、Bax和Caspase 3表达有关。
OBJECTIVE To evaluate the effects and mechanism of C_(21) sterols in Marsdeniae Tenacissimae Caulis extract on inhibition of proliferation and induction of apoptosis of low invasion and lung hypermetastasis in salivary adenoid cystic carcinomacv cell line SACC-83 and SACC-LM, respectively. METHODS SACC-83 and SACC-LM cells were treated with C_(21) sterols at different doses(5, 10, 20, 40, 60, 80, 100 μmol·L~(-1)) for 48 h, then the cell viability was measured by MTT assay,and calculated the value of IC_(20) and IC_(50) for drug. The capacity of clone formation of cells were detected by clone formation assay. The apoptotic of SACC-83 and SACC-LM cells were determined by flow cytometry. The expression levels of Bcl-2, Bax and caspase-3 mRNA and proteins in SACC-83 and SACC-LM cells were examined by q-PCR and Western blot method.RESULTS C_(21) sterols at different doses decreased the viability and inhibited proliferation of SACC-83 and SACC-LM cells,and the concentration of IC_(20) was 7.49 μmol·L~(-1), and IC_(50) was 38.34 μmol·L~(-1) for SACC-83 cells. In addition, the concentration of IC_(20) was 9.30 μmol·L~(-1), and IC_(50) was 46.04 μmol·L~(-1) for SACC-LM cells. The apoptotic rate of SACC-83 and SACC-LM cells under concentration of IC_(20) with C_(21) sterols was significantly increased(P<0.05, P<0.01). The q-PCR and Western blot results showed that the expression level of Bcl-2 in the SACC-83 and SACC-LM cells was decreased significantly(P<0.01), and the expression level of Bax and Caspase-3 were increased significantly(P<0.05). CONCLUSION C_(21) sterols in Marsdeniae Tenacissimae Caulis extract can depress the proliferation and promote apoptosis of SACC-83 and SACC-LM cells, which mechanism may be related to decreasing the Bcl-2 level, and increasing the Bax and Caspase-3 level.
OBJECTIVE To evaluate the effects and mechanism of C_(21) sterols in Marsdeniae Tenacissimae Caulis extract on inhibition of proliferation and induction of apoptosis of low invasion and lung hypermetastasis in salivary adenoid cystic carcinomacv cell line SACC-83 and SACC-LM, respectively. METHODS SACC-83 and SACC-LM cells were treated with C_(21) sterols at different doses(5, 10, 20, 40, 60, 80, 100 μmol·L~(-1)) for 48 h, then the cell viability was measured by MTT assay,and calculated the value of IC_(20) and IC_(50) for drug. The capacity of clone formation of cells were detected by clone formation assay. The apoptotic of SACC-83 and SACC-LM cells were determined by flow cytometry. The expression levels of Bcl-2, Bax and caspase-3 mRNA and proteins in SACC-83 and SACC-LM cells were examined by q-PCR and Western blot method.RESULTS C_(21) sterols at different doses decreased the viability and inhibited proliferation of SACC-83 and SACC-LM cells,and the concentration of IC_(20) was 7.49 μmol·L~(-1), and IC_(50) was 38.34 μmol·L~(-1) for SACC-83 cells. In addition, the concentration of IC_(20) was 9.30 μmol·L~(-1), and IC_(50) was 46.04 μmol·L~(-1) for SACC-LM cells. The apoptotic rate of SACC-83 and SACC-LM cells under concentration of IC_(20) with C_(21) sterols was significantly increased(P<0.05, P<0.01). The q-PCR and Western blot results showed that the expression level of Bcl-2 in the SACC-83 and SACC-LM cells was decreased significantly(P<0.01), and the expression level of Bax and Caspase-3 were increased significantly(P<0.05). CONCLUSION C_(21) sterols in Marsdeniae Tenacissimae Caulis extract can depress the proliferation and promote apoptosis of SACC-83 and SACC-LM cells, which mechanism may be related to decreasing the Bcl-2 level, and increasing the Bax and Caspase-3 level.
引文
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[3]GONDIVKAR S M,GADBAIL A R,CHOLE R,et al.Adenoid cystic carcinoma:A rare clinical entity and literature review[J].Oral Oncol,2011,47(4):231-236.
[4]TANG Y,LIANG X,ZHENG M,et al.Expression of c-kit and Slug correlates with invasion and metastasis of salivary adenoid cystic carcinoma[J].Oral Oncol,2010,46(4):311-316.
[5]刘发煇,罗春英.miRNA在唾液腺腺样囊性癌中的研究进展[J].右江民族医学院学报,2018,40(5):486-489.
[6]刘廷霞,张甘,黄琴,等.萝藦科植物C21甾体成分抗肿瘤活性近5年研究进展[J].中国现代药物应用,2018,12(15):217-218.
[7]石涛.消癌平注射液临床应用1009例分析[J].江苏医药,2012,38(9):1109-1110.
[8]王艳艳,王威,李红岩,等.RP-HPLC法测定通关藤中11α-O-顺芷酰基-12β-O-乙酰基-通关藤苷元B的含量[J].药物分析杂志,2008,28(9):1409-1412.
[9]KERR J F,WYLLIE A H,CURRIE A R.Apoptosis:a basic biological phenomenon with wide-ranging implications in tissue kinetics[J].British J Cancer,1972,26(4):239-257.
[10]HE X W,GUO G L,CHEN J X,et al.Marsdenia tenacissima extract inhibits viability and induces apoptosis of melanoma cells via regulating PI3 K/AKT/mTOR signaling pathway[J].Chin J Pathophysiol(中国病理生理杂志),2018,34(12):2180-2185.
[11]WANG F,FAN Q X,WANG H H,et al.Efficacy and safety of Xiaoaiping combined with chemotherapy in the treatment of advanced esophageal cancer[J].Chin J Oncol,2017,39(6):453-457.
[12]FANG P,ZHANG N F.Effect of SHIP1 on viability and apoptosis of leukemia Jurkat cells by regulating STAT3signaling pathway[J].Chin J Pathophysiol(中国病理生理杂志),2018,34(1):87-93.
[13]MARILINA G A,ELISABET P R,MAXIMINO R.Bcl-2inhibition to overcome resistance to chemo-and immunotherapy[J].Int J Mol Sci,2018,19(12):3950.Doi:10.3390/ijms19123950
[14]WALENSKY L D.Bcl-2 in the crosshairs:tipping the balance of life and death[J].Cell Death Differentiation,2006,13(8):1339-1350.
[15]KUBOKI M,ITO A,SIMIZU S,et al.Activation of apoptosis by caspase-3-dependent specific RelB cleavage in anticancer agent-treated cancer cells:Involvement of positive feedback mechanism[J].Biochem Biophys Res Commun,2014.456(3):810-814.
[16]LI Y S,ZHAO C,HU Z.Effect of TET on xenografted tumor of human bladder cancer BIU-87 cells on nude mice[J].Chin JMod Appl Pharm(中国现代应用药学),2012,29(12):1070-1073.
[17]REN L P,Li X J,Jing S J.Influence of sophoridine on proliferation and caspase-3/bcl-2/bax signaling pathway of human pancreatic cancer cell line capan-1 cells[J].Chin JMod Appl Pharm(中国现代应用药学),2017,34(3):325-328.