芪竹方对结肠癌耐药株HCT-8/V及肝癌耐药株Bel/Fu细胞耐药体外逆转作用的观察

[目的]研究芪竹方对结肠癌耐药株HCT-8/V、肝癌耐药株Bel/FU体外逆转作用.[方法]血清药理法制备芪竹方含药血清,MTT法检测芪竹方及其含药血清对HCT-8/V及Bel/FU细胞耐药逆转作用.[结果]芪竹方250 μg/ml、50μg/ml、芪竹方20％含药血清、10％含药血清均可逆转HCT-8/V细胞的耐药性（P＜0.05）,其逆转倍数分别为14.65、6.20、6.53、3.01;芪竹方及其含药血清对Bel/FU细胞耐药性无逆转作用.[结论]芪竹方可体外逆转结肠癌耐药细胞株HCT-8/V耐药性,而对Bel/FU细胞无体外逆转作用.     

至真方对人大肠癌多药耐药细胞株HCT-8/VCR中核因子-κB及P糖蛋白表达的影响

[目的]从核因子-κB（NF-κB）途径研究至真方对大肠癌多药耐药（MDR）细胞株———人大肠癌细胞株及长春新碱细胞株（HCT-8/VCR）的逆转作用及P糖蛋白（P-gp）表达的影响。[方法]CCK-8法测定HCT-8/VCR的MDR性及至真方含药血清对HCT-8/VCR细胞株的逆转作用;Real-time PCR及Western blot检测NF-κB/p65、MDR1mRNA,NF-κB/p65、P-gp蛋白的表达。[结果]至真方含药血清可明显抑制HCT-8/VCR细胞生长。不同浓度至真方含药血清作用24h后,HCT-8/VCR细胞NF-κB/p65、MDR1mRNA及NF-κB/p65、P-gp蛋白表达均降低,且呈浓度依赖性,与阴性对照组相比差异均有统计学意义（P〈0.05）。[结论]至真方对人大肠癌MDR细胞株HCT-8/VCR的逆转作用可能与其下调NF-κB、P-gp的表达相关。     

从Akt/COX-2/P-gp信号通路研究丹参酚酸B逆转HCT-8/VCR细胞多药耐药的作用及机制

[目的]探讨丹参酚酸B(Sal-B)对人大肠癌多药耐药细胞HCT-8/VCR耐药的逆转作用,并从Akt/COX-2/P-gp信号通路探讨Sal-B逆转HCT-8/VCR细胞多药耐药的可能有效机制。[方法]1 MTT法鉴定HCT-8/VCR细胞的多药耐药性并检测Sal-B对HCT-8/VCR细胞的耐药逆转作用;2RT-PCR检测细胞中Akt、COX-2、MDR1基因表达情况;3Western blot检测细胞中Akt、p-Akt、COX-2、P-gp蛋白表达情况。[结果]HCT-8/VCR细胞有明显多药耐药性;Sal-B干预HCT-8/VCR细胞48h后,能增加其对不同化疗药物VCR、5-Fu、CDDP、TAXOL的敏感性,各化疗药物的半数抑制浓度IC50均明显下降(P0.05);Sal-B干预HCT-8/VCR细胞48h后,使pAkt、COX-2、P-gp蛋白相对表达量均明显下调(P0.05),而对Akt蛋白表达无影响;Sal-B组COX-2、P-gp基因相对表达量均明显减少(P0.05),而对Akt基因表达无影响。[结论]Sal-B能有效改善HCT-8/VCR细胞的多药耐药性,其机制可能是通过抑制Akt磷酸化,下调COX-2的表达,进而减少MDR1/P-gp的表达。     

从NF-κB信号通路研究至真方对HCT-8/VCR细胞多药耐药的影响及机制

[目的]观察至真方含药血清对人大肠癌耐长春新碱细胞株HCT-8/VCR多药耐药的影响,并从NF-κB信号通路探讨其作用机制。[方法]用8%、16%、32%体积分数的至真方含药血清干预人大肠癌敏感细胞株HCT-8与耐长春新碱细胞株HCT-8/VCR,CCK8法检测细胞多药耐药性及存活率;ELISA法检测NF-κB活性;AnnexinV/PI流式细胞术检测细胞凋亡率;Western blot检测NF-κB/p65、IκB-α和Caspase-3蛋白的表达。[结果]体积分数8%时,高、中、低剂量含药血清组对HCT-8/VCR细胞的抑制率与空白血清组比较差异均有统计学意义(P0.01),且呈浓度递增趋势;8%、16%、32%体积分数的中剂量组至真方含药血清作用于HCT-8/VCR细胞24h后,NF-κB活性较HCT-8/VCR阴性对照组明显降低(P0.01);至真方含药血清可促进HCT-8/VCR细胞凋亡,且在一定范围内细胞凋亡率呈浓度依赖性;经至真方含药血清处理后,HCT-8/VCR细胞NF-κB/p65蛋白表达较用药前明显下降(P0.05),IκB-α蛋白表达明显上调(P0.05),Caspase-3蛋白表达明显上调(P0.05)。[结论]至真方含药血清可降低HCT-8/VCR细胞NF-κB的活性,一定程度上逆转了大肠癌细胞的多药耐药,其机制可能与抑制该通路分子开关IκB-α的磷酸化,下调NF-κB/p65蛋白的表达,从而上调通路下游的Caspase-3蛋白的表达,诱导HCT-8/VCR细胞凋亡有关。     

芹菜素通过下调MDR-1/P-gp表达逆转结肠癌耐药细胞HCT-8/5FU多药耐药性的作用及机制

目的探讨芹菜素逆转结肠癌耐药细胞HCT-8/5FU多药耐药性作用及其机制。方法运用免疫细胞化学技术和逆转录多聚酶链式反应(RT-PCR)测定耐药蛋白P-糖蛋白(P-gp)及耐药基因MDR-1mRNA的表达情况。CCK-8法测定芹菜素对耐药细胞耐药逆转作用;荧光显微镜观察芹菜素、5-FU及二者联合作用时耐药细胞凋亡情况。结果芹菜素能够降低耐药蛋白P-gp糖蛋白及耐药基因MDR-1 mRNA的表达,同时能够逆转耐药细胞的多药耐药性,诱导更多细胞凋亡。结论菜素能够通过下调MDR-1/P-gp表达逆转HCT-8/5FU多药耐药性,从而有可能成为临床多药耐药逆转剂。     

至真方调控Hedgehog信号通路对HCT-8/VCR多药耐药的影响

[目的]探讨至真方含药血清对人大肠癌耐长春新碱细胞株HCT-8/VCR多药耐药性的逆转作用,及对Hedgehog信号通路中Smo、Gli1表达的影响。[方法]CCK-8法测定细胞多药耐药性及存活率;Real-time PCR及Western blot分别从基因及蛋白水平检测P-gp和Hedgehog信号通路中Smo、Gli1的表达。[结果]至真方含药血清作用24h、48h后,HCT-8/VCR生存率明显降低;至真方含药血清作用24h后,HCT-8/VCR细胞P-gp(P-Glycoprotein)、Smo、Gli1基因及蛋白表达均降低,与阴性对照组相比差异有统计学意义(P0.05),与阳性对照组相比差异无统计学意义(P0.05)。[结论]至真方对HCT-8/VCR的逆转作用可能与其抑制Hedgehog信号通路,下调该通路相关蛋白表达进而下调P-gp表达,增加HCT-8/VCR对化疗药物的敏感性有关。     

ERK信号通路参与至真方逆转人大肠癌多药耐药细胞HCT-8/VCR耐药作用机制的研究

[目的]探讨至真方是否通过影响ERK通路活性,下调P-gp蛋白及ERK、MDR1mRNA表达,逆转人大肠癌多药耐药细胞HCT-8/VCR多药耐药。[方法]细胞增殖-毒性检验CCK-8(cell counting kit-8)法鉴定HCT-8/VCR细胞株的多药耐药性及存活率;流式细胞仪测定细胞内罗丹明123(Rh123)的平均荧光强度;Real-time PCR及Western blot检测ERK、p-ERK、P-gp的基因和蛋白的表达。[结果]HCT-8/VCR为多药耐药细胞;至真方含药血清可明显抑制HCT-8/VCR细胞生长;HCT-8/VCR细胞经8%、16%、32%至真方含药血清干预48h后,细胞内Rho123外排减少,波峰右移,细胞内荧光强度明显增强(P0.01);HCT-8/VCR细胞ERK、MDR1mRNA的表达水平均有所下降,且呈浓度依赖性;p-ERK表达较用药前(0.764±0.001)明显下降(P0.01),分别为(0.513±0.002)、(0.498±0.001)、(0.471±0.12);ERK表达较用药前(0.771±0.204)分别下降至(0.437±0.004)、(0.413±0.002)、(0.398±0.001),(P0.01)。P-gp表达较用药前(0.94±0.014)下降至(0.701±0.01)、(0.663±0.01)、(0.508±0.02),(P0.01)。[结论]至真方可逆转人大肠癌多药耐药细胞株HCT-8/VCR的耐药作用,其机制与降低P-gp外排功能、降低ERK通路活性、下调P-gp蛋白及基因表达有关。     

健脾理气方药物血清对肝癌细胞端粒酶活性及凋亡的影响

目的 观察健脾理气方药物血清对肝癌细胞SMMC-7721端粒酶活性的影响以及诱导肿瘤细胞凋亡的作用,探讨其抗肿瘤治疗的作用机制.方法 采用端粒重复序列扩增(TRAP)结合非变性聚丙烯酰胺凝胶电泳银染法,利用健脾理气方中药灌服新西兰兔后制备含药血清,观察健脾理气方药物血清对体外培养的人肝癌细胞株SMMC-7721端粒酶活性的影响,同时利用流式细胞仪检测和荧光显微镜观察对细胞凋亡的影响.结果 健脾理气药物血清在d 4开始对肿瘤细胞的端粒酶活性有抑制作用,电泳条带光密度明显降低;并能诱导肿瘤细胞的凋亡,凋亡的比例随作用时间的延长而升高,流式细胞术检测d2,d4,d 6的凋亡比例分别为0.81%,5.16%,8.45%.结论 对肿瘤细胞端粒酶活性的抑制和诱导肿瘤细胞凋亡是健脾理气中药抗肿瘤治疗作用机制的一部分.     

至真方调控Hedgehog/ABCG2信号途径改善大肠癌细胞的多药耐药性

[目的]探讨至真方醇提物(ZZR)对大肠癌多药耐药细胞HCT-8/5-FU细胞耐药性的改善作用,并从Hedgehog/ABCG2信号途径探讨至真方改善大肠癌多药耐药的可能作用机制。[方法]高效液相色谱法鉴定至真方醇提物中的5种主要成分;CCK-8法检测大肠癌多药耐药细胞HCT-8/5-FU的多药耐药性及至真方对其耐药性的改善作用;应用Real-Time PCR和western blot方法分别从基因和蛋白水平检测至真方对Hedghog/ABCG2信号途径的调节作用。[结果]HCT-8/5-FU细胞有明显多药耐药性,至真方醇提物干预HCT-8/5-FU细胞48h后,能增加其对不同化疗药物(5-FU、L-OHP、Taxol)的敏感性,且Hedgehog通路上关键因子-Gli1与ABCG2的mRNA相对表达量均明显下调(P0.05),蛋白表达下降(P0.05)。[结论]至真方醇提物能够有效改善HCT8/5-FU细胞多药耐药,其机制可能与抑制Hedgehog信号通路活性从而降低ABCG2蛋白表达有关。     

SIRT1表达与结肠癌细胞多药耐药性的关系

目的建立结肠癌耐药细胞系,初步研究SIRT1表达与结肠癌细胞多耐药性的关系。方法建立耐药细胞系HCT-8/VCR。MTT法测其交叉耐药性。RT-PCR法和免疫细胞化学法分别检测SIRT1基因的mRNA水平和蛋白水平的表达,初步探讨其与肿瘤细胞多药耐药的关系。结果建立对长春新碱高度耐药的结肠癌细胞系,RT-PCR法和免疫细胞化学法表明SIRT1在HCT-8/VCR的表达增强。结论 SIRT1基因的表达与HCT-8/VCR的耐药性有一定的关系,耐药细胞SIRT1的表达明显比亲本细胞中的高。     

人大肠癌HCT-8/5-FU耐药细胞株的建立及P-gp测定

目的：建立人大肠癌多药耐药细胞株HCT-8/5- FU及并对其耐药机制进行探讨．方法：先采用较大剂量间歇诱导法进行筛选,再采用浓度梯度递增法作用,历时7mo,至HCT8细胞可长期在5-FU浓度为2．0mg/L的细胞培养液中稳定生长．电镜、HE染色观察2种细胞形态结构差异．体外细胞毒性实验观察他对5-FU ADM,DDP的耐药性．绘制亲本细胞和耐药细胞的体外生长曲线．罗丹明染色法检测其两种细胞P-gp功能表达．结果：HCT-8细胞株经7mo诱导,可在5-FU 2．0 g/L的培养液中稳定增殖,具有多药耐药性,命名为HCT-8/5-FU,该细胞株对5-FU的耐药指数为16．6,并对ADM,DDP有交叉耐药性．该细胞株体外群体倍增时间与亲本细胞差别不显著．HE染色观察耐药细胞胞体较亲本细胞大,细胞核不规则,可见双核、多形核,细胞形态不规则,呈多角形、细长形改变,可见巨核细胞．透射电镜下耐药细胞胞质内线粒体、内质网、溶酶体增多．流式细胞仪罗丹明染色法观察荧光强度曲线左移,提示耐药细胞有过度P-gp表达．结论：成功建立HCT-8/5-FU多药耐药细胞株．先采用较大剂量间歇诱导进行筛选,再采用浓度梯度递增法作用是诱导大肠癌耐药细胞株的较好方式．HCT-8/5-FU细胞株的耐药机制与P-糖蛋白表达有关．     

抑制MET对RAS突变型结肠癌细胞的抗肿瘤作用

目的探讨抑制MET对于RAS突变结肠癌的体内体外抗肿瘤作用。 方法选取4种常见RAS突变型结肠癌细胞（HCT-116、DLD-1、Lovo和HCT-15）,采用siRNA敲除MET蛋白表达,或者加入特异性MET抑制剂PHA-665752,采用MTT及集落形成实验观察抑制MET对结肠癌细胞增殖抑制的作用,应用Western Blotting检测PHA-665752对MET/AKT/ERK信号通路的作用。另外,应用HCT-116细胞系构建裸鼠皮下成瘤模型,给予PHA-665752腹腔注射,观察MET抑制剂对体内肿瘤的抑制作用。 结果RAS突变的4种结肠癌细胞系均有MET蛋白表达。应用siRNA敲除MET蛋白表达对HCT-116和Lovo细胞的增殖抑制作用分别为19.6±4.5%和27.8±5.8%,而应用MET特异性抑制剂PHA-665752对两种细胞的增殖抑制作用呈剂量依赖性,细胞克隆形成实验证实PHA-665752可有效抑制细胞克隆形成。另外,应用HCT-116细胞构建裸鼠皮下成瘤模型,给予PHA-665752单药证实MET抑制剂可明显抑制HCT-116细胞的皮下移植瘤（第四周肿瘤体积：PHA-665752组为300±72 mm³,对照组为608±59 mm³,t=5.731,P=0.005）。Western Blotting证实应用PHA-665752预处理能明显抑制HGF激活的p-MET、p-AKT和p-ERK。 结论抑制MET对于RAS突变型结肠癌具有抗肿瘤作用,靶向MET可能成为RAS突变型结肠癌的有效治疗。     

Estrogen receptor β potentiates the antiproliferative effect of raloxifene and affects the cell migration and invasion in HCT-116 colon cancer cells

Background

Estrogen receptor β (ERβ) is the predominant ER in the colorectal epithelium, whose expression is greatly reduced in colorectal cancer compared with normal colon tissue. Recent in vitro studies suggested that ERβ may suppress tumor growth. No research was reported whether ERβ can be used as therapeutic agent for colon cancer.

Methods

In this study, ERβ gene constructed into adenoviral (Ad) vectors was used to treat colon cancer HCT-116 cells alone or in combination with raloxifene. In vitro and in vivo studies were conducted to investigate the therapeutic effects of ERβ and raloxifene in HCT-116 cells.

Results

Our results indicated that, although Ad-ERβ alone had no effect on the proliferation of HCT-116 cells, the combination of Ad-ERβ with raloxifene significantly inhibited the proliferation of HCT-116 cells. The apparently apoptotic induction effects may partly explain the cytotoxicity of the two agents. The results of the study of ERβ on migration and invasion of HCT-116 cells demonstrated that overexpression of ERβ significantly decreased cell migration and increased invasion of cells. The antitumor efficacies of ERβ as well as raloxifene were further investigated on HCT-116 tumor bearing mice. Results demonstrated that both Ad-ERβ and raloxifene individually inhibited tumor growth. The combination group showed the highest inhibitory efficiency compared with other three groups.

Conclusion

These findings demonstrated that combined administration of Ad-ERβ with raloxifene represents a promising colon cancer therapeutic strategy.     

RNAi对结肠癌HCT-8细胞桩蛋白表达的沉默作用观察

目的观察RNA干扰（RNAi）对结肠癌HCT-8细胞桩蛋白（Paxillin）表达的沉默作用。方法构建针对Paxillin基因序列的短发卡RNA（shRNA）表达载体,并转染至结肠癌HCT-8细胞,72 h后分别用实时定量PCR和Western blot法测定转染细胞中Paxillin mRNA及蛋白。结果根据针对Paxillin基因设计了小干扰RNA（siRNA）序列,构建了shRNA表达载体,并成功转染至HCT-8细胞。以空白对照组作均一化,转染Paxillin-shRNA_1、Paxillin-shRNA_2、Paxillin-shRNA_3、Paxillin-shRNA_NC的细胞及空白对照组细胞Paxillin mRNA相对表达量分别为0.66±0.28、0.31±0.11、1.00±0.22、1.05±0.32、1.00±0.29,Paxillin蛋白相对表达量分别为0.62±0.11、0.39±0.13、0.74±0.21、0.94±0.09、1.00±0.16。结论 RNAi可沉默结肠癌HCT-8细胞Paxillin的表达。     

Modulation of multidrug resistance by andrographolid in a HCT‐8/5‐FU multidrug‐resistant colorectal cancer cell line

OBJECTIVE To develop an HCT‐8/5‐FU multidrug‐resistant colorectal cancer cell line and to elucidate the effect of Andrographolid (AG), an extract from Andrographis paniculate, a medicinal herb on the HCT‐8/5‐FU multidrug‐resistant colorectal cancer cell line. METHODS An HCT‐8 colorectal cancer cell line was used and a high concentration of 5‐Fluorouracid (5‐FU) was introduced at the beginning to induce drug resistance, then the concentration of 5‐FU was increased in gradients. Approximately 7 months later, the cells grew stably in 2.0 µg/mL of 5‐FU, and the cell line was named HCT‐8/5‐FU multidrug‐resistant colorectal cancer cell line. The resistant index of HCT‐8/5‐FU cells to 5‐FU, adriamycin (ADM), cisplatin (DDP) was checked by MTT test, and a growth curve was drawn. The morphological changes were observed by both light and electron microscope. The function of P‐170 was detected by rhodamine staining. After the application of AG and co‐administration of 5‐FU, ADM and DDP, the growth curves and inhibition rate as well as apoptosis rate of HCT‐8/5‐FU at different concentrations of AG were evaluated by MTT and flow cytometry. Rhodamine staining was used to investigate the possible mechanism involved by AG. RESULTS The resistance index of HCT‐8/5‐FU to 5‐FU was 16.6, and a cross‐resistance to ADM and DDP was noticed. Compared with parental cells, HCT‐8/5‐FU cell's growth rate did not change significantly but the cell's morphology was remarkably changed as compared with parental cells. Overexpression of P‐170 by HCT‐8/5‐FU cell was indicated through rhodamine staining. AG at a low concentration showed weak inhibitory effect on HCT‐8/5‐FU. However, a remarkable inhibitory and apoptosis rate was shown when AG was co‐administered with 5‐FU, ADM and DDP, respectively. Interestingly AG alone could not induce apoptosis and change the cell cycles. AG might affect the expression of P‐170, which was indicated by rhodamine staining. CONCLUSIONS The HCT‐8/5‐FU multidrug‐resistant colorectal cancer cell line has been successfully developed and because it has cross‐resistance to 5‐FU, ADM and DDP, it might serve as an ideal multidrug resistance (MDR) model for colorectal cancer research. The mechanism of HCT‐8/5‐FU resistance to chemotherapeutic agents might be related to the overexpression of P‐170. Low concentrations of AG alone have no significant inhibition on HCT‐8/5‐FU and fail to induce apoptosis and to change cell cycles. AG might act as a chemosensitizer when co‐administered with 5‐FU, ADM and DDP, and the mechanism of reversal modulation of multidrug resistance by AG in the HCT‐8/5‐FU resistant cell line might be related to its downregulation of overexpression of P‐170.