葫芦素B抑制骨肉瘤143B细胞的增殖和侵袭并促进其凋亡
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摘要
目的 :研究葫芦素B(cucurbitacin B,CuB)对骨肉瘤143B细胞增殖、凋亡和侵袭的影响,并探讨其可能的作用机制。方法 :采用不同浓度的CuB处理143B细胞,结晶紫染色和克隆形成实验检测细胞的增殖能力;Transwell小室法检测细胞的侵袭能力;FCM法检测细胞周期及凋亡率的变化。实时荧光定量PCR和蛋白质印迹法分别检测143B细胞中上皮-间质转化相关标志物基质金属蛋白酶9(matrix metalloproteinase-9,MMP-9)、波形蛋白(Vimentin)以及Snail mRNA和蛋白的表达水平;蛋白质印迹法检测凋亡相关蛋白剪切型聚腺苷二磷酸-核糖聚合酶(cleaved-polyadenosine diphosphate ribose polymerase,cleavedPARP)、剪切型caspase3(cleaved-caspase3)和Bad以及细胞周期相关蛋白cyclin B1的表达水平;以及蛋白激酶B(protein kinase B,PKB,又称Akt)信号通路中Akt、磷酸化Akt(phospho-Akt,p-Akt)、第10号染色体缺失的磷酸酶和张力蛋白同源基因(phosphatase and tensin homolog deletedonchromosometen,PTEN)、磷酸化PTEN(phospho-PTEN,p-PTEN)蛋白的表达水平。结果 :与空白对照组相比,用不同浓度的CuB处理后,143B细胞的增殖和侵袭能力受到不同程度的抑制(P值均<0.05);30和40 nmol/L CuB浓度组中,细胞的凋亡率明显增加(P值均<0.01),细胞周期被阻滞在G2/M期(P值均<0.01)。上皮-间质转化相关标志物MMP-9、Vimentin以及Snail mRNA和蛋白表达水平下调(P值均<0.05);凋亡相关蛋白Bad、cleaved-caspase3和cleaved-PARP的表达上调(P值均<0.05),细胞周期相关蛋白cyclin B1的表达水平明显下调(P值均<0.01);143B细胞中p-Akt蛋白的表达水平降低(P值均<0.05),且伴有p-PTEN蛋白的表达水平增高(P值均<0.05)。结论 :CuB能抑制骨肉瘤143B细胞的增殖和侵袭,并促进其凋亡;这一作用可能与CuB抑制143B细胞中Akt信号通路的活性及上皮-间质转化的发生有关。
Objective: To investigate the effects of cucurbitacin B(CuB) on the proliferation, invasion and apoptosis of osteosarcoma 143B cells, to explore the related molecular mechanisms.Methods: 143B cells were treated with different concentrations of CuB. The proliferation ability of 143B cells was detected by crystal violet staining and colony formation assay. The invasion ability of 143B cells was detected by Transwell chamber assay. The apoptosis rate and cell cycle distribution were detected by flow cytometry(FCM) method. The expression levels of epithelial-mesenchymal transition(EMT)-related markers including Vimentin, Snail and matrix metalloproteinase-9(MMP-9) mRNAs and proteins were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The expression levels of apoptosis-related proteins [Bad, cleaved-caspase 3, cleaved-polyadenosine diphosphate ribose polymerase(PARP) and cyclin B1] and protein kinase B(PKB, Akt) signal pathwayrelated proteins [Akt, phospho-Akt(p-Akt), phosphatase and tensin homolog deleted on chromosome ten(PTEN) and phospho-PTEN(p-PTEN)] were detected by Western blotting.Results: Compared with the untreated control group, the proliferation and invasion of 143B cells treated with different concentrations of CuB were significantly inhibited(all P < 0.05). After treatment with 30 or 40 nmol/L CuB, the apoptosis rate of 143B cells was increased(both P < 0.05), the cell cycle was blocked in G2/M phase(both P < 0.01). The expression levels of MMP-9, Vimentin and Snail mRNAs and proteins were remarkably down-regulated(all P < 0.05) in 143B cells treated with CuB. The expression levels of apoptosis-related Bad, cleavedcaspase 3 and cleaved-PARP proteins were remarkably up-regulated(all P < 0.05) in 143B cells treated with CuB, the expression of cell cycle-related cyclin B1 protein was down-regulated(all P < 0.01) in 143B cells treated with CuB. At the same time, CuB reduced the expression level of p-Akt protein and increased the expression level of p-PTEN protein(both P < 0.05).Conclusion: CuB can inhibit the proliferation and invasion of osteosarcoma 143B cells, and promote apoptosis. These effects may be related to Akt pathway impediment and EMT processing attenuation.
Objective: To investigate the effects of cucurbitacin B(CuB) on the proliferation, invasion and apoptosis of osteosarcoma 143B cells, to explore the related molecular mechanisms.Methods: 143B cells were treated with different concentrations of CuB. The proliferation ability of 143B cells was detected by crystal violet staining and colony formation assay. The invasion ability of 143B cells was detected by Transwell chamber assay. The apoptosis rate and cell cycle distribution were detected by flow cytometry(FCM) method. The expression levels of epithelial-mesenchymal transition(EMT)-related markers including Vimentin, Snail and matrix metalloproteinase-9(MMP-9) mRNAs and proteins were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The expression levels of apoptosis-related proteins [Bad, cleaved-caspase 3, cleaved-polyadenosine diphosphate ribose polymerase(PARP) and cyclin B1] and protein kinase B(PKB, Akt) signal pathwayrelated proteins [Akt, phospho-Akt(p-Akt), phosphatase and tensin homolog deleted on chromosome ten(PTEN) and phospho-PTEN(p-PTEN)] were detected by Western blotting.Results: Compared with the untreated control group, the proliferation and invasion of 143B cells treated with different concentrations of CuB were significantly inhibited(all P < 0.05). After treatment with 30 or 40 nmol/L CuB, the apoptosis rate of 143B cells was increased(both P < 0.05), the cell cycle was blocked in G2/M phase(both P < 0.01). The expression levels of MMP-9, Vimentin and Snail mRNAs and proteins were remarkably down-regulated(all P < 0.05) in 143B cells treated with CuB. The expression levels of apoptosis-related Bad, cleavedcaspase 3 and cleaved-PARP proteins were remarkably up-regulated(all P < 0.05) in 143B cells treated with CuB, the expression of cell cycle-related cyclin B1 protein was down-regulated(all P < 0.01) in 143B cells treated with CuB. At the same time, CuB reduced the expression level of p-Akt protein and increased the expression level of p-PTEN protein(both P < 0.05).Conclusion: CuB can inhibit the proliferation and invasion of osteosarcoma 143B cells, and promote apoptosis. These effects may be related to Akt pathway impediment and EMT processing attenuation.
引文
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[16]ZHANG M,SUN C,SHAN X,et al.Inhibition of pancreatic cancer cell growth by cucurbitacin B through modulation of signal transducer and activator of transcription 3 signaling[J].Pancreas,2010,39(6):923-929.
[17]BEERLING E,SEINSTRA D,DE WIT E,et al.Plasticity between epithelial and mesenchymal states unlinks EMT from metastasis-enhancing stem cell capacity[J].Cell Rep,2016,14(10):2281-2288.
[18]GUPTA P,SRIVASTAVA SK.HER2 mediated de novo production of TGFβleads to SNAIL driven epithelial-to-mesenchymal transition and metastasis of breast cancer[J].Molecular Oncology,2014,8(8):1532-1547.
[19]YOUSIF NG.Fibronectin promotes migration and invasion of ovarian cancer[J].Cell Biol Int,2014,38(1):85-91.
[20]JIANG C,MA S,HU R,et al.Effect of CXCR4on apoptosis in osteosarcoma cells via the PI3K/Akt/NF-κB signaling pathway[J].Cell Physiol Biochem,2018,46(6):2250-2260.
[2]LONGHI A,ERRANI C,DE PAOLIS M,et al.Primary bone osteosarcoma in the pediatric age:state of the art[J].Cancer Treat Rev,2006,32(6):423-436.
[3]王春燕,汤丽娜,胡蓓蓓,等.骨肉瘤肺转移患者不同治疗方法的生存分析[J].肿瘤,2014,34(10):935-940.
[4]HATTINGER CM,FANELLI M,TAVANTI E,et al.Advances in emerging drugs for osteosarcoma[J].Expert Opin Emerg Drugs,2015,20(3):495-514.
[5]KHAN N,JAJEH F,KHAN MI,et al.Sestrin-3modulation is essential for therapeutic efficacy of cucurbitacin B in lung cancer cells[J].Carcinogenesis,2017,38(2):184-195.
[6]JAYAPRAKASAM B,SEERAM NP,NAIRMG.Anticancer and antiinflammatory activities of cucurbitacins from cucurbita andreana[J].Cancer Lett,2003,189(1):11-16.
[7]ATEBA SB,MVONDO MA,NGEU ST,et al.Natural terpenoids against female breast cancer:a 5-year recent research[J].Current Medicinal Chemistry,2018,25(27):3162-3213.
[8]MAROSTICA LL,ALB DB,OLIVEIRA J,et al.Antitumor effectiveness of a combined therapy with a new cucurbitacin Bderivative and paclitaxel on a human lung cancer xenograft model[J].Toxicol Appl Pharmacol,2017,329:272-281.
[9]KIM SH,KANG JG,KIM CS,et al.Doxorubicin has a synergistic cytotoxicity with cucurbitacin B in anaplastic thyroid carcinoma cells[J].Tumour Biol,2017,39(2):1010428317692252.
[10]CHAN KT,LI K,LIU SL,et al.Cucurbitacin B inhibits STAT3 and the Raf/MEK/ERKpathway in leukemia cell line K562[J].Cancer Letters,2010,289(1):46-52.
[11]YASUDA S,YOGOSAWA S,IZUTANI Y,et al.Cucurbitacin B induces G2 arrest and apoptosis via a reactive oxygen speciesdependent mechanism in human colon adenocarcinoma SW480 cells[J].Mol Nutr Food Res,2010,54(4):559-565.
[12]SANDBERG AA.Cytogenetics and molecular genetics of bone and softtissue tumors[J].Am J Med.Genet,2002,115(3):189-193.
[13]SCHWARTZ CL,GORLICK R,TEOT L,et al.Multiple drug resistance in osteogenic sarcoma:INT0133 from the Children’s Oncology Group[J].J Clin Oncol,2007,25(15):2057-2062.
[14]HORIE T,ONO K,NISHI H,et al.Acute doxorubicin cardiotoxicity is associated with miR-146a induced inhibition of the neuregulin-Erb B pathway[J].Cardiovasc Res,2010,87(4):656-664.
[15]DUANGMANO S,SAELIM P,SUKSAMRARNA,et al.Cucurbitacin B inhibits human breast cancer cell proliferation through disruption of microtubule polymerization and nucleophosmin/B23 translocation[J].BMC Complement Altern Med,2012,12:185.
[16]ZHANG M,SUN C,SHAN X,et al.Inhibition of pancreatic cancer cell growth by cucurbitacin B through modulation of signal transducer and activator of transcription 3 signaling[J].Pancreas,2010,39(6):923-929.
[17]BEERLING E,SEINSTRA D,DE WIT E,et al.Plasticity between epithelial and mesenchymal states unlinks EMT from metastasis-enhancing stem cell capacity[J].Cell Rep,2016,14(10):2281-2288.
[18]GUPTA P,SRIVASTAVA SK.HER2 mediated de novo production of TGFβleads to SNAIL driven epithelial-to-mesenchymal transition and metastasis of breast cancer[J].Molecular Oncology,2014,8(8):1532-1547.
[19]YOUSIF NG.Fibronectin promotes migration and invasion of ovarian cancer[J].Cell Biol Int,2014,38(1):85-91.
[20]JIANG C,MA S,HU R,et al.Effect of CXCR4on apoptosis in osteosarcoma cells via the PI3K/Akt/NF-κB signaling pathway[J].Cell Physiol Biochem,2018,46(6):2250-2260.
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