桂德霉素抗肿瘤活性研究
摘要
格尔德霉素(Geldanamycin GA)是从链霉菌培养液中分离得到的第一个苯醌安莎类抗生素,研究表明GA具有多方面的生物活性。现已证实GA通过特异性的与热休克蛋白90(Hsp90)结合,抑制其分子伴侣功能并导致被陪伴分子的去稳定和降解。由于GA的作用,参与细胞增殖和生存的许多信号传递蛋白(例如跨膜酪氨酸激酶、MET和胰岛素样生长因子-1受体、信号转导蛋白、癌基因和抑癌基因蛋白、嵌合信号蛋白、甾体激素受体(雄激素雌激素孕酮受体)、细胞周期调节因子)的稳定性或功能都因Hsp90的功能被抑制而受到影响。另外,肿瘤细胞在恶性增殖的过程中经常处于应激状态,肿瘤细胞Hsp90组成型表达要与正常细胞相比高出2—10倍。因此目前针对Hsp90抑制剂GA的药物开发已受到广泛关注。
肉桂酸(Cinnamic Acid,CA)是一种广泛存在的天然产物,由于在限定范围内无毒性,美国将其列入GRAS(一般公认为是安全的)范围。故肉桂酸是一类来源广泛和安全的物质。有报道表明它具有广泛的抗实体瘤活性,包括黑色素癌、前列腺癌、肺癌、胶质细胞瘤、肝癌和白血病等。它对肿瘤细胞具有抑制其生长增殖和诱导其分化的作用,而且其衍生物肉桂酰胺类化合物亦具有抗肿瘤括性。
本研究课题通过化学方法将肉桂酸引入到GA的17位得到GA的肉桂酸衍生物,定名为桂德霉素(Guidemycin GUM),并对GUM的体外、体内抗肿瘤活性进行了初步研究。
一、GUM作用于肿瘤细胞的体外实验研究
1.MTT法检测GUM对人乳腺癌MCF-7细胞,人胰腺癌SW1990细胞,人肝癌HepG2细胞,人肺癌H460细胞,人大肠癌HCT116细胞,人骨髓瘤U266细胞的增殖抑制作用。
不同浓度的GUM作用细胞48h后,利用MTT法检测GUM对各种肿瘤细胞的增殖抑制作用。MTT结果显示,GUM对肿瘤细胞抑制作用的IC_(50)值分别为MCF-7(13.6±1.6μg/ml),SW1990(16.8±1.8μg/ml),HepG2(63.56±5.7μg/ml),H460(67.46±7.1μg/ml),HCT116(283.8±11.8μg/ml),U266(17.8±2.7μg/ml)。
2.Hocchst 33258荧光染色法和Anncxin V-FITC/PI染色结合流式细胞仪检测细胞凋亡。
不同浓度GUM作用细胞24h和48h后,细胞凋亡比率逐渐增高,呈浓度依赖性和时间依赖性。其中10μg/ml,1μg/ml,0.1μg/ml的GUM给药24h后诱导细胞凋亡的比率分别为:人乳腺癌MCF-7细胞,5-21%±0.41%、2.7%±0.21%、2.86%±0.25%、2.15%±0.16%(Control);人胰腺癌SW1990细胞,7.63%±0.56%、5.53%±0.44%、3.85%±0.31%、2.48%±0.28%(Control);人肝癌HepG2细胞,7.68%±0.59%、6.92%±0.61%、3.72%±0.33%、3.08%±0.32%(Control);人肺癌H460细胞,9.33%±0.81%、4.92%±0.52%、3.93%±0.37%、3.44%±0.31%(Control);人骨髓瘤U266细胞,11.22%±0.89%、5.75%±0.47%、4.99%±0.41%、2.63%±0.31%(Control)。三个剂量GUM给药48h后诱导细胞凋亡的比率分别为:人乳腺癌MCF-7细胞,23.16%±0.97%、7.35%±0.51%、4.96%±0.37%、3.17%±0.24%(Control);人胰腺癌SW1990细胞,20.47%±0.91%、8.1%±0.73%、4.11%±0.38%、2.71%±0.31%(Control);人肝癌HepG2细胞,27.55%±1.02%、8.38%±0.69%、3.17%±0.29%、2.8%±0.31%(Control);人肺癌H460细胞,22.21%±1.27%、12.06%±0.74%、3.9%±0.41%、2.98%±0.29%(Control);人骨髓瘤U266细胞,25.22%±1.13%、8.0%±0.63%、2.48%±0.29%、2.98%±0.24%(Control)。GUM高剂量组和中剂量组的肿瘤凋亡率显著高于对照组(P＜0.05,P＜0.001)。荧光显微镜下可见10μg/ml和1μg/ml的GUM作用后细胞出现染色质凝集、细胞核固缩、核碎裂、形成凋亡小体等典型的凋亡细胞形态学改变。
3.利用Transwell实验观察GUM对人乳腺癌MCF-7细胞、人肝癌HepG2细胞、人肺癌H460细胞、人胰腺癌SW1990细胞迁移能力的影响。实验结果显示,10μg/ml,1μg/ml,0.1μg/ml的GUM给药8h后对肿瘤细胞迁移的抑制率分别为:人乳腺癌MCF-7细胞,79.78%±4.82%、37.42%±2.14%、8.17%±1.12%;人肝癌HepG2细胞,66.62%±3.62%、31.08%±1.96%、7.69%±1.21%;人肺癌H460细胞,33.51%±2.28%、15.13%±1.41%、6.48%±1.38%;人胰腺癌SW1990细胞,72.89%±6.49%、36.25%±3.02%、8.18%±1.07%。GUM对肿瘤细胞的迁移能力有显著的抑制作用,浓度越大抑制作用越强。高剂量组GUM对肿瘤细胞迁移能力的抑制作用明显高于肉桂酸给药组。
4.Western blot检测GUM对肿瘤细胞中RAF-1、EGFR、AKT、CDK4和HER-2蛋白水平的影响。
Western blot检测结果显示GUM明显减少人乳腺癌MCF-7细胞,人肝癌HepG2细胞,人肺癌H460细胞中RAF-1、EGFR、AKT、CDK4和HER-2的蛋白水平,并且随着GUM作用浓度的增加,抑制蛋白水平的作用亦随之加强,呈现一定的剂量依赖性。实验结果提示GUM通过与细胞内Hsp90的结合抑制RAF-1、EGFR、AKT、CDK4、HER-2等蛋白因子在细胞内正确构象的形成,并促使其在细胞内被降解,从而抑制肿瘤细胞增殖。
二、GUM的体内抗肿瘤实验研究
1.利用昆明种雄性小鼠对GUM与GA的毒性进行测定。
实验结果表明不同剂量的GUM(10mg/kg、20mg/kg、40mg/kg)和GA(1.25mg/kg、2.5mg/kg、5mg/kg)连续腹腔注射给药10天后,动物存活率有明显差别。GA给药组中,高剂量(5mg/kg)给药实验动物在第7天全部死亡,中剂量(2.5mg/kg)给药实验动物在实验结束时存活1只,低剂量(1.25mg/kg)给药实验动物在实验结束时存活3只。GUM给药组中,高、中、低剂量给药组(40 mg/kg、20 mg/kg、10 mg/kg)实验动物在实验结束时无动物死亡。对照组也无动物死亡。结果显示GUM与GA比较,GUM三个剂量组的毒性明显低于GA三个剂量组。
2.GUM对小鼠移植性肝癌H22模型的抗肿瘤活性。
结果表明,GUM 3个剂量组(10 mg/kg、20 mg/kg、40 mg/kg)的实验动物在连续腹腔注射给药10天后,对H22肿瘤的生长具有明显的抑制作用。GUM高、中、低三个剂量组的抑瘤率分别为61.8%,38.9%和25.7%,与对照组相比具有显著性差异。在实验期间,各组动物的体重变化不大。实验结束时,各组动物体重与实验初期相比略有上升。GUM组的动物体重和对照组相比,没有显著性差异。
3.利用人乳腺癌MCF-7移植瘤裸鼠模型,观察不同剂量GUM对裸鼠移植性肿瘤的生长抑制作用。
GUM腹腔注射给药,给药间隔为隔天给药,给药次数共20次。GUM在三个剂量40 mg/kg、20 mg/kg和10 mg/kg作用下,整个实验过程中没有出现裸鼠死亡,表明动物能够耐受注射剂量。高、中、低三个剂量组GUM的抑瘤率分别为65.8%、45.9%、15.4%。40 mg/kg GUM在裸鼠体内对人乳腺癌MCF-7肿瘤生长具有明显的抑制作用,与对照组相比具有显著性差异。在实验期间,各组动物的体重变化不大。实验结束时,各组动物体重与实验初期相比略有上升,GUM组的动物体重与对照组相比,没有显著性差异。
综上,研究结果表明GUM能够抑制体外培养肿瘤细胞的增殖,诱导肿瘤细胞凋亡,限制肿瘤细胞迁移,降低肿瘤细胞内RAF-1、EGFR、AKT、CDK4和HER-2等Hsp90被陪伴分子的蛋白水平。而且,体内抗肿瘤活性实验结果显示GUM的毒性明显低于GA,GUM对小鼠移植性肝癌H22模型和人乳腺癌MCF-7移植瘤裸鼠模型的肿瘤生长具有明显的抑制作用。
Geldanamycin(GA) is a naturally occurring low molecular weight compound produced by microorganisms and is the fast ansamycin-derivative of benzoquinone isolated from medium of streptomycete.GA was found to possess many kinds of biological acitivity.Currently,it has been confirmed that the target of GA is heat shock protein 90(Hsp90).GA can inhibit the function of Hsp90 that required for the folding and the activity of client proteins.Many molecules involved in survival and proliferation of tumor cells such as transmembrane tyrosine kinase,signal transduction protein,oncogenic and anti-oncogenic protein,chimeric protein,steroid hormone receptor and cyclin are associated with Hsp90.Treatment of cells with GA generally induces not only the inactivation of client proteins but also the reduction in the concentrations of these proteins in the cell.In addition,the level of Hsp90 in the tumor cell is higher than that in the normal cell.Therefore,many researchers have been actively engaged in the development of specific Hsp90 inhibitor.
Cinnamic Acid(CA) is a widely existed natural product.CA has been listed in GRAS due to non-toxic in limited doses.CA can reduce the size of many solid tumors in experimental animals by inhibiting proliferation and derivation of tumor cell.
In the present study,a new derivative of GA named(GUM) was obtained by introducing the CA group into the 17-site of GA.Furthermore,the anti-cancer effects of GUM in vitro and in vivo were evaluated.
1.Effects of GUM on tumor cells cultured in vitro
Inhibition of cell proliferation was measured by the MTT assays.A remarkable difference in chemosensitivity to GUM was found in six cell lines.The IC_(50) value of GUM for MCF-7,HepG2,H460,HCT116, SW1990 and U266 cells was 13.6±1.6μg/ml,63.56±5.7μg/ml,67.46±7.1μg/ml,283.8±11.8μg/ml,16.8±1.8μg/ml and 17.8±2.7μg/ml, respectively.
Flow cytometry combined with FITC-Annexin V/PI staining showed that GUM could induce earlier apoptosis in four cell lines(MCF-7,HepG2, SW1990,U266).The rate of apoptosis incubated with 10μg/ml,1 g/ml and 0.1μg/ml GUM for 24 h was 5.21%±0.41%,2.7%±0.21%,2.86 %±0.25%and 2.15%±0.16%(Control) in MCF-7 cells,7.63%±0.56%,5.53%±0.44%,3.85%±0.31%and 2.48%±0.28% (Control) in SW1990 cells,7.68%±0.59%,6.92%±0.61%,3.72%±0.33%and 3.08%±0.32%(Control) in HepG2 cells,9.33%±0.81%, 4.92%±0.52%,3.93%±0.37%and 3.44%±0.31%(Control) in H460 cells,11.22%±0.89%,5.75%±0.47%,4.99%±0.41%and 2.63%±0.31%(Control) in U266 cells,respectively.The rate of apoptosis incubated with 10μg/ml,1μg/ml and 0.1μg/ml for 48 h was 23.16%±0.97%,7.35%±0.51%,4.96%±0.37%and 3.17%±0.24 %(Control) in MCF-7 cells,20.47%±0.91%,8.1%±0.73%,4.11%±0.38%and 2.71%±0.31%(Control) in SW1990 cells,27.55%±1.02%,8.38%±0.69%,3.17%±0.29%and 2.8%±0.31%(Control) in HepG2 cells,22.21%±1.27%,12.06%±0.74%,3.9%±0.41% and 2.98%±0.29%(Control) in H460 cells,25.22%±1.13%,8.0%±0.63%,2.48%±0.29%and 2.98%±0.24%(Control) in U266 cells, respectively.After exposure to GUM for 48 h,most cells presented typical morphologic changes of apoptosis such as chromatin condensation or shrunken nucleus by Hoechst33258 staining.Some condensed nuclei were observed when cells exposed to higher concentrations of GUM.
The effect of GUM and CA on the migration of MCF-7,HepG2,H460 and SW1990 cells was examined using a transwell cell migration chamber assay.A dose-dependent reduction in cell migration in four cell lines was found after exposure to GUM.After treatment with 10μg/ml,1μg/ml and 0.1μg/ml GUM for 8 h,the inhibit rate of tumor cell migration was 79.78 %±4.82%,37.42%±2.14%and 8.17%±1.12%in MCF-7 cells, 66.62%±3.62%,31.08%±1.96%and 7.69%±1.21%in HepG2 cells,33.51%±2.28%,15.13%±1.41%and6.48%±1.38%inH460 cells,72.89%±6.49%,36.25%±3.02%and 8.18%±1.07%in SW1990 cells.The inhibit rate of tumor cell migration showed significant differences between control and GUM groups.
The levels of RAF-1,EGFR,AKT,CDK4 and HER-2 were determined by Western blot analysis.MCF-7,HepG2 and H460 cells treated with GUM showed a dose-dependent decrease in the levels of RAF-1,EGFR,AKT, CDK4 and HER-2.The levels of RAF-1,EGFR,AKT,CDK4 and HER-2 were markedly reduced by 10μg/ml GUM.
2.In vivo antitumor activity of GUM
Toxic assay of GUM and GA.The mice were received by i.p.injection either GUM or GA once a day for 10 days.The doses of GUM were 10mg/kg,20mg/kg and 40mg/kg.The doses of GA were 1.25mg/kg, 2.5mg/kg and 5mg/kg.After 10 days injection,there were no mice survival treated with GA at the dose of 5mg/kg.The survival numbers of treatment with GA at the dose of 2.5mg/kg and 1.25mg/kg was 1 and 3,respectively. There were no mice dead treated with GUM at the dose of 10mg/kg, 20mg/kg and 10mg/kg.
Transplantable murine hepatoma22 model was used to evaluate the antitumor activity of GUM in vivo.Animals bearing s.c.tumors received by i.p.injection(200μl) either water(vehicle control) or GUM once a days for 10 days.The doses of GUM were 40 mg/kg,20 mg/kg and 10 mg/kg, respectively.The mice were weighed and tumor sizes were measured with a vernier caliper and recorded every day.Tumor volume was calculated using the formula:length×width~2×0.5.Growth of the established s.c. tumors in mice was decreased significantly when treated with GUM over a period of 10 days compared with vehicle control treated animals. Treatment with GUM at the dose of 40 mg/kg,20 mg/kg and 10 mg/kg inhibited the growth of H22 by 61.8%,38.9%and 25.7%,respectively. The antitumor activity of GUM is remarkable.The animals' weights showed no significant difference between control and treated groups.
Athymic female BALB/c(nu/nu) mice were used for MCF-7 tumor xenografts.Mice bearing s.c.tumors received by i.p.injection(200μl) either water(vehicle control) or GUM once per 2 days for 5 weeks.The doses of GUM were 40 mg/kg,20 mg/kg and 10 mg/kg,respectively.The mice were weighed and tumor sizes were measured with a vernier caliper and recorded per 5 days.Tumor volume was calculated using the formula: length×width~2×0.5.Growth of the established s.c.tumors in nude mice was decreased significantly when treated with GUM over a period of 5 weeks compared with vehicle control treated animals.Treatment with GUM at the dose of 40 mg/kg,20 mg/kg and 10 mg/kg inhibited the growth of MCF-7 xenografts by 65.8%,45.9%and 15.4%,respectively. The antitumor activity of GUM is remarkable.The animals' weights showed no significant difference between control and treated groups.
Above all,the results show that GUM can inhibit proliferation,induce apoptosis and limit migration of the tumor cells;it also can decrease the levels of Hsp90 client proteins such as RAF-1,EGFR,AKT,CDK4 and HER-2. Meanwhile,GUM inhibits the growth of tumor in H22 allografts and MCF-7 xenografts models.
肉桂酸(Cinnamic Acid,CA)是一种广泛存在的天然产物,由于在限定范围内无毒性,美国将其列入GRAS(一般公认为是安全的)范围。故肉桂酸是一类来源广泛和安全的物质。有报道表明它具有广泛的抗实体瘤活性,包括黑色素癌、前列腺癌、肺癌、胶质细胞瘤、肝癌和白血病等。它对肿瘤细胞具有抑制其生长增殖和诱导其分化的作用,而且其衍生物肉桂酰胺类化合物亦具有抗肿瘤括性。
本研究课题通过化学方法将肉桂酸引入到GA的17位得到GA的肉桂酸衍生物,定名为桂德霉素(Guidemycin GUM),并对GUM的体外、体内抗肿瘤活性进行了初步研究。
一、GUM作用于肿瘤细胞的体外实验研究
1.MTT法检测GUM对人乳腺癌MCF-7细胞,人胰腺癌SW1990细胞,人肝癌HepG2细胞,人肺癌H460细胞,人大肠癌HCT116细胞,人骨髓瘤U266细胞的增殖抑制作用。
不同浓度的GUM作用细胞48h后,利用MTT法检测GUM对各种肿瘤细胞的增殖抑制作用。MTT结果显示,GUM对肿瘤细胞抑制作用的IC_(50)值分别为MCF-7(13.6±1.6μg/ml),SW1990(16.8±1.8μg/ml),HepG2(63.56±5.7μg/ml),H460(67.46±7.1μg/ml),HCT116(283.8±11.8μg/ml),U266(17.8±2.7μg/ml)。
2.Hocchst 33258荧光染色法和Anncxin V-FITC/PI染色结合流式细胞仪检测细胞凋亡。
不同浓度GUM作用细胞24h和48h后,细胞凋亡比率逐渐增高,呈浓度依赖性和时间依赖性。其中10μg/ml,1μg/ml,0.1μg/ml的GUM给药24h后诱导细胞凋亡的比率分别为:人乳腺癌MCF-7细胞,5-21%±0.41%、2.7%±0.21%、2.86%±0.25%、2.15%±0.16%(Control);人胰腺癌SW1990细胞,7.63%±0.56%、5.53%±0.44%、3.85%±0.31%、2.48%±0.28%(Control);人肝癌HepG2细胞,7.68%±0.59%、6.92%±0.61%、3.72%±0.33%、3.08%±0.32%(Control);人肺癌H460细胞,9.33%±0.81%、4.92%±0.52%、3.93%±0.37%、3.44%±0.31%(Control);人骨髓瘤U266细胞,11.22%±0.89%、5.75%±0.47%、4.99%±0.41%、2.63%±0.31%(Control)。三个剂量GUM给药48h后诱导细胞凋亡的比率分别为:人乳腺癌MCF-7细胞,23.16%±0.97%、7.35%±0.51%、4.96%±0.37%、3.17%±0.24%(Control);人胰腺癌SW1990细胞,20.47%±0.91%、8.1%±0.73%、4.11%±0.38%、2.71%±0.31%(Control);人肝癌HepG2细胞,27.55%±1.02%、8.38%±0.69%、3.17%±0.29%、2.8%±0.31%(Control);人肺癌H460细胞,22.21%±1.27%、12.06%±0.74%、3.9%±0.41%、2.98%±0.29%(Control);人骨髓瘤U266细胞,25.22%±1.13%、8.0%±0.63%、2.48%±0.29%、2.98%±0.24%(Control)。GUM高剂量组和中剂量组的肿瘤凋亡率显著高于对照组(P＜0.05,P＜0.001)。荧光显微镜下可见10μg/ml和1μg/ml的GUM作用后细胞出现染色质凝集、细胞核固缩、核碎裂、形成凋亡小体等典型的凋亡细胞形态学改变。
3.利用Transwell实验观察GUM对人乳腺癌MCF-7细胞、人肝癌HepG2细胞、人肺癌H460细胞、人胰腺癌SW1990细胞迁移能力的影响。实验结果显示,10μg/ml,1μg/ml,0.1μg/ml的GUM给药8h后对肿瘤细胞迁移的抑制率分别为:人乳腺癌MCF-7细胞,79.78%±4.82%、37.42%±2.14%、8.17%±1.12%;人肝癌HepG2细胞,66.62%±3.62%、31.08%±1.96%、7.69%±1.21%;人肺癌H460细胞,33.51%±2.28%、15.13%±1.41%、6.48%±1.38%;人胰腺癌SW1990细胞,72.89%±6.49%、36.25%±3.02%、8.18%±1.07%。GUM对肿瘤细胞的迁移能力有显著的抑制作用,浓度越大抑制作用越强。高剂量组GUM对肿瘤细胞迁移能力的抑制作用明显高于肉桂酸给药组。
4.Western blot检测GUM对肿瘤细胞中RAF-1、EGFR、AKT、CDK4和HER-2蛋白水平的影响。
Western blot检测结果显示GUM明显减少人乳腺癌MCF-7细胞,人肝癌HepG2细胞,人肺癌H460细胞中RAF-1、EGFR、AKT、CDK4和HER-2的蛋白水平,并且随着GUM作用浓度的增加,抑制蛋白水平的作用亦随之加强,呈现一定的剂量依赖性。实验结果提示GUM通过与细胞内Hsp90的结合抑制RAF-1、EGFR、AKT、CDK4、HER-2等蛋白因子在细胞内正确构象的形成,并促使其在细胞内被降解,从而抑制肿瘤细胞增殖。
二、GUM的体内抗肿瘤实验研究
1.利用昆明种雄性小鼠对GUM与GA的毒性进行测定。
实验结果表明不同剂量的GUM(10mg/kg、20mg/kg、40mg/kg)和GA(1.25mg/kg、2.5mg/kg、5mg/kg)连续腹腔注射给药10天后,动物存活率有明显差别。GA给药组中,高剂量(5mg/kg)给药实验动物在第7天全部死亡,中剂量(2.5mg/kg)给药实验动物在实验结束时存活1只,低剂量(1.25mg/kg)给药实验动物在实验结束时存活3只。GUM给药组中,高、中、低剂量给药组(40 mg/kg、20 mg/kg、10 mg/kg)实验动物在实验结束时无动物死亡。对照组也无动物死亡。结果显示GUM与GA比较,GUM三个剂量组的毒性明显低于GA三个剂量组。
2.GUM对小鼠移植性肝癌H22模型的抗肿瘤活性。
结果表明,GUM 3个剂量组(10 mg/kg、20 mg/kg、40 mg/kg)的实验动物在连续腹腔注射给药10天后,对H22肿瘤的生长具有明显的抑制作用。GUM高、中、低三个剂量组的抑瘤率分别为61.8%,38.9%和25.7%,与对照组相比具有显著性差异。在实验期间,各组动物的体重变化不大。实验结束时,各组动物体重与实验初期相比略有上升。GUM组的动物体重和对照组相比,没有显著性差异。
3.利用人乳腺癌MCF-7移植瘤裸鼠模型,观察不同剂量GUM对裸鼠移植性肿瘤的生长抑制作用。
GUM腹腔注射给药,给药间隔为隔天给药,给药次数共20次。GUM在三个剂量40 mg/kg、20 mg/kg和10 mg/kg作用下,整个实验过程中没有出现裸鼠死亡,表明动物能够耐受注射剂量。高、中、低三个剂量组GUM的抑瘤率分别为65.8%、45.9%、15.4%。40 mg/kg GUM在裸鼠体内对人乳腺癌MCF-7肿瘤生长具有明显的抑制作用,与对照组相比具有显著性差异。在实验期间,各组动物的体重变化不大。实验结束时,各组动物体重与实验初期相比略有上升,GUM组的动物体重与对照组相比,没有显著性差异。
综上,研究结果表明GUM能够抑制体外培养肿瘤细胞的增殖,诱导肿瘤细胞凋亡,限制肿瘤细胞迁移,降低肿瘤细胞内RAF-1、EGFR、AKT、CDK4和HER-2等Hsp90被陪伴分子的蛋白水平。而且,体内抗肿瘤活性实验结果显示GUM的毒性明显低于GA,GUM对小鼠移植性肝癌H22模型和人乳腺癌MCF-7移植瘤裸鼠模型的肿瘤生长具有明显的抑制作用。
Geldanamycin(GA) is a naturally occurring low molecular weight compound produced by microorganisms and is the fast ansamycin-derivative of benzoquinone isolated from medium of streptomycete.GA was found to possess many kinds of biological acitivity.Currently,it has been confirmed that the target of GA is heat shock protein 90(Hsp90).GA can inhibit the function of Hsp90 that required for the folding and the activity of client proteins.Many molecules involved in survival and proliferation of tumor cells such as transmembrane tyrosine kinase,signal transduction protein,oncogenic and anti-oncogenic protein,chimeric protein,steroid hormone receptor and cyclin are associated with Hsp90.Treatment of cells with GA generally induces not only the inactivation of client proteins but also the reduction in the concentrations of these proteins in the cell.In addition,the level of Hsp90 in the tumor cell is higher than that in the normal cell.Therefore,many researchers have been actively engaged in the development of specific Hsp90 inhibitor.
Cinnamic Acid(CA) is a widely existed natural product.CA has been listed in GRAS due to non-toxic in limited doses.CA can reduce the size of many solid tumors in experimental animals by inhibiting proliferation and derivation of tumor cell.
In the present study,a new derivative of GA named(GUM) was obtained by introducing the CA group into the 17-site of GA.Furthermore,the anti-cancer effects of GUM in vitro and in vivo were evaluated.
1.Effects of GUM on tumor cells cultured in vitro
Inhibition of cell proliferation was measured by the MTT assays.A remarkable difference in chemosensitivity to GUM was found in six cell lines.The IC_(50) value of GUM for MCF-7,HepG2,H460,HCT116, SW1990 and U266 cells was 13.6±1.6μg/ml,63.56±5.7μg/ml,67.46±7.1μg/ml,283.8±11.8μg/ml,16.8±1.8μg/ml and 17.8±2.7μg/ml, respectively.
Flow cytometry combined with FITC-Annexin V/PI staining showed that GUM could induce earlier apoptosis in four cell lines(MCF-7,HepG2, SW1990,U266).The rate of apoptosis incubated with 10μg/ml,1 g/ml and 0.1μg/ml GUM for 24 h was 5.21%±0.41%,2.7%±0.21%,2.86 %±0.25%and 2.15%±0.16%(Control) in MCF-7 cells,7.63%±0.56%,5.53%±0.44%,3.85%±0.31%and 2.48%±0.28% (Control) in SW1990 cells,7.68%±0.59%,6.92%±0.61%,3.72%±0.33%and 3.08%±0.32%(Control) in HepG2 cells,9.33%±0.81%, 4.92%±0.52%,3.93%±0.37%and 3.44%±0.31%(Control) in H460 cells,11.22%±0.89%,5.75%±0.47%,4.99%±0.41%and 2.63%±0.31%(Control) in U266 cells,respectively.The rate of apoptosis incubated with 10μg/ml,1μg/ml and 0.1μg/ml for 48 h was 23.16%±0.97%,7.35%±0.51%,4.96%±0.37%and 3.17%±0.24 %(Control) in MCF-7 cells,20.47%±0.91%,8.1%±0.73%,4.11%±0.38%and 2.71%±0.31%(Control) in SW1990 cells,27.55%±1.02%,8.38%±0.69%,3.17%±0.29%and 2.8%±0.31%(Control) in HepG2 cells,22.21%±1.27%,12.06%±0.74%,3.9%±0.41% and 2.98%±0.29%(Control) in H460 cells,25.22%±1.13%,8.0%±0.63%,2.48%±0.29%and 2.98%±0.24%(Control) in U266 cells, respectively.After exposure to GUM for 48 h,most cells presented typical morphologic changes of apoptosis such as chromatin condensation or shrunken nucleus by Hoechst33258 staining.Some condensed nuclei were observed when cells exposed to higher concentrations of GUM.
The effect of GUM and CA on the migration of MCF-7,HepG2,H460 and SW1990 cells was examined using a transwell cell migration chamber assay.A dose-dependent reduction in cell migration in four cell lines was found after exposure to GUM.After treatment with 10μg/ml,1μg/ml and 0.1μg/ml GUM for 8 h,the inhibit rate of tumor cell migration was 79.78 %±4.82%,37.42%±2.14%and 8.17%±1.12%in MCF-7 cells, 66.62%±3.62%,31.08%±1.96%and 7.69%±1.21%in HepG2 cells,33.51%±2.28%,15.13%±1.41%and6.48%±1.38%inH460 cells,72.89%±6.49%,36.25%±3.02%and 8.18%±1.07%in SW1990 cells.The inhibit rate of tumor cell migration showed significant differences between control and GUM groups.
The levels of RAF-1,EGFR,AKT,CDK4 and HER-2 were determined by Western blot analysis.MCF-7,HepG2 and H460 cells treated with GUM showed a dose-dependent decrease in the levels of RAF-1,EGFR,AKT, CDK4 and HER-2.The levels of RAF-1,EGFR,AKT,CDK4 and HER-2 were markedly reduced by 10μg/ml GUM.
2.In vivo antitumor activity of GUM
Toxic assay of GUM and GA.The mice were received by i.p.injection either GUM or GA once a day for 10 days.The doses of GUM were 10mg/kg,20mg/kg and 40mg/kg.The doses of GA were 1.25mg/kg, 2.5mg/kg and 5mg/kg.After 10 days injection,there were no mice survival treated with GA at the dose of 5mg/kg.The survival numbers of treatment with GA at the dose of 2.5mg/kg and 1.25mg/kg was 1 and 3,respectively. There were no mice dead treated with GUM at the dose of 10mg/kg, 20mg/kg and 10mg/kg.
Transplantable murine hepatoma22 model was used to evaluate the antitumor activity of GUM in vivo.Animals bearing s.c.tumors received by i.p.injection(200μl) either water(vehicle control) or GUM once a days for 10 days.The doses of GUM were 40 mg/kg,20 mg/kg and 10 mg/kg, respectively.The mice were weighed and tumor sizes were measured with a vernier caliper and recorded every day.Tumor volume was calculated using the formula:length×width~2×0.5.Growth of the established s.c. tumors in mice was decreased significantly when treated with GUM over a period of 10 days compared with vehicle control treated animals. Treatment with GUM at the dose of 40 mg/kg,20 mg/kg and 10 mg/kg inhibited the growth of H22 by 61.8%,38.9%and 25.7%,respectively. The antitumor activity of GUM is remarkable.The animals' weights showed no significant difference between control and treated groups.
Athymic female BALB/c(nu/nu) mice were used for MCF-7 tumor xenografts.Mice bearing s.c.tumors received by i.p.injection(200μl) either water(vehicle control) or GUM once per 2 days for 5 weeks.The doses of GUM were 40 mg/kg,20 mg/kg and 10 mg/kg,respectively.The mice were weighed and tumor sizes were measured with a vernier caliper and recorded per 5 days.Tumor volume was calculated using the formula: length×width~2×0.5.Growth of the established s.c.tumors in nude mice was decreased significantly when treated with GUM over a period of 5 weeks compared with vehicle control treated animals.Treatment with GUM at the dose of 40 mg/kg,20 mg/kg and 10 mg/kg inhibited the growth of MCF-7 xenografts by 65.8%,45.9%and 15.4%,respectively. The antitumor activity of GUM is remarkable.The animals' weights showed no significant difference between control and treated groups.
Above all,the results show that GUM can inhibit proliferation,induce apoptosis and limit migration of the tumor cells;it also can decrease the levels of Hsp90 client proteins such as RAF-1,EGFR,AKT,CDK4 and HER-2. Meanwhile,GUM inhibits the growth of tumor in H22 allografts and MCF-7 xenografts models.
引文
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