姜黄素联合顺铂对人卵巢癌细胞株SKOV3细胞增殖的抑制作用

目的探讨姜黄素及其联合顺铂对体外培养人卵巢癌细胞株SKOV3增殖的抑制作用。方法将培养的人卵巢癌SKOV3细胞分别单独(姜黄素组或顺铂组)及联合(姜黄素与顺铂联用组)加入不同浓度姜黄素(5、10、20、40μmol/L)、顺铂(2.5 mg/L)作用后,采用四甲基偶氮唑盐比色法(MTT法)检测SKOV3细胞增殖情况。结果不同浓度姜黄素组随着药物浓度增加、作用时间延长,细胞的增殖抑制率上升,差异有统计学意义(P<0.05);联合用药组明显高于单一用药组(P<0.05),两药联合应用具有协同作用。结论姜黄素对卵巢癌SKOV3细胞增殖具有抑制作用,在一定范围内,这种抑制作用呈剂量-时间依赖性;姜黄素与顺铂联合应用具有协同作用,可提高SKOV3细胞对顺铂的敏感性,是一种理想的化疗增敏剂。     

姜黄素逆转非小细胞肺癌吉非替尼耐药的研究

目的 研究姜黄素对吉非替尼耐药非小细胞肺癌细胞株PC9/G2耐药性的影响及作用机制。方法 MTT法测定姜黄素对PC9/G2的无毒剂量及对PC9/G2对吉非替尼耐药的逆转效应,实时荧光定量法测定姜黄素对EGFR下游通路中PI3K表达水平的影响,分光光度法测定姜黄素对凋亡蛋白Caspase-3活性的影响。结果 姜黄素对PC9/G2的无毒剂量为4 μmol/L,姜黄素联合吉非替尼组细胞存活率比单用吉非替尼组降低13%~22%,姜黄素使PI3K的表达水平明显下降,Caspase-3凋亡蛋白活性显著提高。结论 姜黄素对PC9/G2具有耐药逆转作用,作用机制可能是通过下调PI3K的表达水平,诱导更多的凋亡蛋白Caspase-3而实现。     

LY294002联合姜黄素对人膀胱癌EJ细胞的体外抑制作用

目的 研究磷脂酰肌醇-3激酶/蛋白激酶B(PI3K/Akt)特异性抑制剂LY294002与姜黄素联合对体外培养的人膀胱癌EJ细胞的抑制作用.方法 用MTT法,检测姜黄素单独或联合PI3K/Akt特异性抑制剂LY294002对人膀胱癌EJ细胞的抑制率;用流式细胞技术及Western blotting法,检测药物单独或联合...     

拉帕替尼联合顺铂对人卵巢癌细胞SKOV3生长的影响

目的探讨新型靶向治疗药物拉帕替尼联合顺铂(DDP)对人卵巢腺癌细胞株SKOV3的增殖抑制作用以及对磷酸AKT蛋白表达的影响。方法体外培养人卵巢癌细胞株SKOV3,采用MTT方法检测不同浓度拉帕替尼、DDP单用或者合用对细胞增殖活力的改变;用流式细胞仪进行细胞周期分析;用流式细胞仪检测SKOV3应用拉帕替尼前后磷酸化AKT(P-AKT)蛋白的表达。结果拉帕替尼及顺铂单独使用即对人卵巢癌细胞株SKOV3细胞有增殖抑制作用,不同浓度的拉帕替尼与顺铂联合应用对人卵巢癌细胞株SKOV3细胞的增殖抑制作用较单药组增强(P<0.05),与对照组相比有统计学意义(P<0.05);应用拉帕替尼前后P-AKT蛋白的表达有显著不同(P<0.05)。结论拉帕替尼联合顺铂可显著抑制SKOV3细胞株的增殖,其作用可能与P-AKT的表达有关,为其在卵巢癌上的应用提供了新的靶点和一定的理论基础。     

microRNA-21促进人肺腺癌细胞A549对顺铂耐药性的研究

目的 研究microRNA-21(miR-21)对人肺腺癌细胞A549对于顺铂耐药性的影响及分子机制.方法 MTT法检测A549和顺铂耐药株A549/CDDP对于顺铂的敏感性,以及在A549细胞中过表达miR-21和在A549/CDDP细胞中抑制miR-21表达后对顺铂敏感性的影响;RT-PCR方法检测A549和A549/CDDP中miR-21表达情况;Western blotting检测PI3K/AKT,核因子-κB (NF-κB)信号通路的激活情况以及在过表达miR-21和抑制miR-21表达后细胞中AKT信号通路的活性变化情况.结果 相对于A549细胞,A549/CDDP对顺铂的敏感性有明显降低,而细胞中miR-21表达则明显增多.Western blotting结果表明:与A549细胞相比,A549/CDDP细胞PI3K/AKT通路被激活,NF-κB通路没有明显变化.过表达miR-21可以激活PI3K/AKT信号通路并提高A549细胞对顺铂的耐药性,相反地,抑制miR-21表达抑制了A549/CDDP细胞中的AKT通路的活化情况并降低了A549/CDDP细胞对顺铂的耐药性.结论 miR-21可以促进肺癌细胞A549对顺铂的耐药性,其机制与PI3K/AKT信号通路的激活有关.     

RNA沉默PI3K、AKT基因对人卵巢癌SKOV3细胞增殖和凋亡的比较

目的探讨RNA技术分别沉默PI3K、AKT基因表达对人卵巢癌SKOV3细胞增殖与凋亡的比较,从而为卵巢癌的基因治疗提供有效的理论依据。方法体外合成PI3Kp110α、AKT序列特异性双链RNA(dsRNA),用lipofectamin2000转染人卵巢癌SKOV3细胞,采用MTT法检测细胞活性,流式细胞仪法检测细胞凋亡率。结果 MTT法检测siRNA-AKT组细胞增殖能力降低大于siRNA-PI3K组,差异有统计学意义(P<0.05),而流式细胞术检测siRNA-AKT组细胞凋亡率则高于siRNA-PI3K组,差异有统计学意义(P<0.05)。结论体外合成靶向AKT的siRNA更能有效地抑制人卵巢癌SKOV3细胞增殖及诱导细胞凋亡。PI3K通路并非AKT激活的唯一途径。     

PI3K/AKT/mTOR信号通路抑制剂在卵巢癌治疗中的研究进展

卵巢癌是女性生殖系统最致命的恶性肿瘤。目前,针对卵巢癌的规范治疗方案是肿瘤细胞减灭术辅以紫杉醇/铂类联合化疗,然而大多数晚期卵巢癌患者最终因对化疗药物耐药而复发。PI3K/AKT/m TOR信号通路作为一条重要的原癌基因通路,在卵巢癌中激活并在卵巢癌的增殖、侵袭、细胞周期进程、血管形成及耐药中发挥着重要的作用,抑制该通路是卵巢癌的一个潜在治疗方法。对PI3K/AKT/m TOR信号通路抑制剂在卵巢癌治疗中的研究进展进行综述。     

AKT抑制剂NTQ1062与Olaparib在卵巢癌细胞中的联用研究

目的： 探讨AKT抑制剂NTQ1062和PARP抑制剂Olaparib的对卵巢癌细胞的联用增效可行性及作用机制。方法：研究NTQ1062和Olaparib联用对三种人卵巢癌细胞系（OVCAR-3、TOV-21G、A2780）增殖的影响,并通过Western blot技术检测PI3K/AKT信号通路及同源重组修复、DNA损伤修复相关蛋白的表达,最后使用Annexin V-FITC双染法检测细胞凋亡。结果：实验结果显示,NTQ1062对TOV-21G、A2780细胞株具有较强的增殖抑制作用,对OVCAR-3细胞增殖的抑制作用较弱,并且联合处理会增加三株卵巢癌细胞的增殖抑制作用;Western blot实验中NTQ1062增加卵巢癌细胞中AKT的磷酸化、抑制S6RP的磷酸化、增加γH2AX蛋白表达、降低RAD51蛋白表达,并发现联合处理具有协同作用;进一步研究发现,联合用药会降低TOV-21G细胞中BRCA1蛋白的表达,但对A2780细胞影响不显著;细胞凋亡实验中,NTQ1062增加细胞凋亡群,并且联合处理显著增加了细胞凋亡效果。结论：NTQ1062可抑制OVCAR-3、TOV-21G、A2780细胞增殖,与Olaparib联用具有协同作用,通过抑制PI3K/AKT通路、DNA损伤、减少同源重组修复、降低BRCA1蛋白表达、增加凋亡来发挥作用。本研究可为临床制订AKT抑制剂NTQ1062与PARP抑制剂联合应用于卵巢癌治疗策略提供理论和技术依据。     

PARP1基因抑制对卵巢癌细胞增殖及耐药性的影响

目的研究PARP-1基因在卵巢癌化疗耐药发生中的作用以及PARP-1小干扰RNA对紫杉醇耐药卵巢癌细胞增殖的影响。方法使用紫杉醇刺激紫杉醇耐药卵巢癌细胞SKOV3/TAX及敏感细胞SKOV3,应用小干扰RNA(siRNA)技术抑制PARP-1基因的表达;应用四甲基偶氮唑蓝(MTT)方法检测紫杉醇单独以及联合PARP siRNA对卵巢癌细胞增殖的影响。结果 SKOV3/TAX细胞中PARP-1表达高于SKOV3细胞;紫杉醇可以减少SKOV3细胞中PARP-1的表达和抑制细胞的增殖,而对SKOV3/TAX细胞无明显作用;PARP-1 siRNA可以明显增强紫杉醇对卵巢癌细胞增殖的抑制作用,逆转SKOV3/TAX细胞对紫杉醇的耐药性。结论 PARP-1在卵巢癌细胞表达水平与耐药性密切相关;PARP-1基因抑制可以增强紫杉醇对SKOV3增殖的抑制,逆转SKOV3/TAX对紫杉醇的耐药性。     

红杉黄酮通过下调PI3K/AKT信号通路抑制胃癌细胞增殖侵袭等干细胞特性

目的：探究红杉黄酮影响胃癌细胞的分子机制。方法：通过梯度浓度红杉黄酮处理胃癌细胞系AGS细胞，再使用PI3K/AKT信号通路激活剂对细胞进行诱导。采用CCK-8检测红杉黄酮对AGS细胞的最佳抑制浓度及时间，使用平板克隆、Transwell、细胞划痕实验检测细胞的增殖、迁移、侵袭能力变化。Western blot检测PI3K/AKT信号通路关键蛋白p-PI3K、PI3K、p-AKT、AKT。结果：红杉黄酮呈浓度依赖抑制AGS细胞，其半抑制浓度为0.5 mmol/L，最佳处理时间为48 h。红杉黄酮处理AGS细胞后，p-PI3K与p-AKT表达下调，AGS细胞增殖减少，迁移、侵袭能力降低；PI3K/AKT信号通路激活剂处理后，p-PI3K与p-AKT表达被部分逆转，增殖、迁移、侵袭能力降低也得到部分改善。结论：红杉黄酮通过失活PI3K/AKT信号通路抑制胃癌细胞增殖、迁移、侵袭等恶性行为。     

Curcumin inhibits cervical cancer cell proliferation,migration and invasion by suppressing the PI3K/AKT signaling pathway via the miR-29b/KDM2A axis

In this study, we aimed to examine whether curcumin exerted its anti-tumor effects by regulating miR-29b/KDM2A in cervical cancer cells. The cell viability, migration and invasion were estimated in HeLa cervical cancer cells treated with curcumin. The effects of microRNA-29b (miR-29b) on biological behaviors of HeLa SiHa cells were also assessed. Potential target genes of miR-29b were predicted and confirmed using a luciferase reporter assay, and the effects of curcumin and miR-29b on the PI3K/AKTsignaling pathway were analyzed. Curcumin treatment inhibited cell proliferation, migration and invasion of HeLa cells (P<0.05). The miR-29b expression was promoted by curcumin treatment in HeLa cells (P<0.01), and miR-29b depletion could restore the effects of curcumin on cell proliferation, migration and invasion of HeLa cells (P<0.05). KDM2A was proved as a direct target gene of miR-29b, and the activity of the PI3K/AKT signaling could be regulated by curcumin and miR-29b (P<0.05). All the data revealed that curcumin played a protective role in cervical cancer. The proliferation, migration and invasion of cervical cancer cells were inhibited by curcumin through the miR-29b/KDM2A/PI3K/AKT pathway.     

叶酸受体在食管癌细胞中的表达及其与食管癌生物学行为的关系

目的研究PI3K/AKT信号通路靶向抑制剂LY294002对食管癌细胞EC109中FR的表达的影响,从而了解食管癌生物学行为与叶酸受体的关系。方法将LY294002作用于食管癌细胞株EC109,四甲基偶氮唑蓝（MTT）法测定细胞增殖抑制率,流式细胞术检测食管癌细胞FR的表达。结果随LY294002浓度的增加及作用时间（12h、24h）的延长,实验组EC109细胞的增殖抑制率逐渐增大,LY294002抑制作用与浓度及作用时间呈正相关。LY294002干预食管癌细胞24小时后,实验组叶酸受体表达的表达较对照组明显降低。结论阻断PI3K/AKT信号通路可以改变食管癌细胞株EC109中FR的表达,并能明显抑制细胞增殖,其机制可能与其抑制AKT磷酸化,从而改变食管癌细胞的生物学行为有关。     

葛根素对人骨肿瘤细胞株 MG63 的增殖抑制和诱导凋亡作用

目的 探讨葛根素对人骨肉瘤细胞株MG63的作用及其机制。方法 取对数生长期的MG63细胞分别用不同浓度的葛根素处理(0,25,50,100 mmol·L^-1)。细胞培养48 h后,利用MTT检测MG63细胞增殖水平;流式细胞术检测细胞凋亡水平,RT-PCR检测PI3K、AKT、Bax、Bcl-2、p53 和 p21 mRNA水平。Western blotting 检测PI3K、AKT、Bax、 Bcl-2、p53和p21表达水平。结果 葛根素浓度依赖性的诱导MG63细胞增殖抑制、凋亡和 Bax 表达,减少PI3K、AKT、Bcl-2、p53和p21表达。结论 葛根素可通过抑制PI3K/AKT/p53信号通路而诱导MG63细胞增殖抑制和凋亡。     

KIF4A knockdown inhibits tumor progression and promotes chemo-sensitivity via induction of P21 in lung cancer cells

KIF4A has been demonstrated to play a crucial function in the pathogenesis of a broad number of tumors and have close association with PI3K/AKT pathway. The aim of this study was to explore the potential function of KIF4A in lung cancer progression by targeting PI3K/AKT pathway and P21 combination with doxorubicin. A549 cell lines were transfected with siRNA against KIF4A and negative control siRNA (si-NC). MTT assay and trypan blue staining were used to evaluate the effect of si-KIF4A on the doxorubicin cytotoxicity. The mRNA and protein expression levels of KIF4A and p21 were assessed by qRT-PCR and Western blotting. Apoptosis was measured by cell death ELISA kit. Our result revealed that KIF4A silencing decreased cellular proliferation in A549 lung cancer cells. Doxorubicin in combination with si-KIF4A led to significant reduction in the survival rate of A549 cell. KIF4A silencing upregulated p21. In conclusion, our results demonstrate that KIF4A inhibition sensitizes A549 cells to doxorubicin by targeting p21 and PI3K/AKT pathway, indicating a significant role for KIF4A in lung cancer chemotherapy.     

Low-dose BBR3610 toxicity in colon cancer cells is p53-independent and enhanced by inhibition of epidermal growth factor receptor (ERBB1)-phosphatidyl inositol 3 kinase signaling

We have examined the mechanisms by which the multinuclear platinum chemotherapeutic BBR3610 kills human colon cancer cells. BBR3610 more efficiently killed HCT116, DLD1, SW480, and HT29 cells than BBR3464, cisplatin, or oxaliplatin. The amount of platinum uptake per cell and its incorporation into DNA were identical for BBR3464 and BBR3610. BBR3610 lethality (IC(75)) was unaltered comparing HCT116 wild-type and p53-/- cells, was reduced in p21-/- cells, and was enhanced in K-RAS D13 null cells. Small molecule or molecular inhibition of epidermal growth factor receptor (ERBB1) or phosphatidyl inositol 3 kinase (PI3K) enhanced BBR3610 toxicity in HCT116, DLD1, and SW480 cells. Small molecule or molecular inhibition of caspase 8 function abolished the toxicity of BBR3610 and of BBR3610 + ERBB1 inhibitor treatments, whereas inhibition of caspase 9 suppressed the ability of ERBB1 inhibitors to enhance BBR3610 lethality. Treatment with BBR3610 reduced AKT activity; the expression of dominant-negative AKT enhanced and expression of constitutively active AKT suppressed, respectively, the toxicity of BBR3610 and of BBR3610 + ERBB1 inhibitor treatments. Treatment with BBR3610 reduced expression of c-FLIP-s and MCL-1, levels that were maintained in cells expressing constitutively active AKT. Overexpression of c-FLIP-s or loss of BID function suppressed BBR3610 toxicity, whereas overexpression of XIAP or Bcl-xL suppressed the potentiation of cell killing by ERBB1 inhibitors. Collectively, our data argue that BBR3610 promotes cell killing via a caspase 8-dependent mechanism, which can be enhanced by ERBB1/PI3K inhibitors that promote additional BBR3610-dependent cell killing via activation of BAX and caspase 9.     

Dual inhibitors of PI3K/mTOR or mTOR-selective inhibitors: which way shall we go?

The phosphatidylinositol-3-kinase (PI3K)/AKT/mTOR signaling pathway is a central regulator in cell proliferation, growth, and angiogenesis. Inhibition of this pathway therefore is a major strategy for cancer chemotherapy. In order to induce the maximal therapeutic outcome in cancer treatment, vertical inhibition of the PI3K/AKT/mTOR pathway or horizontal inhibition of PI3K/AKT/mTOR and other kinases has been reported. In this review, we discuss the drug design and clinical development of dual inhibitors of PI3K and mTOR as well as the mTOR-selective inhibitors, classified based on the mechanism of action and the chemical structures. Structural determinants for increasing selectivity toward PI3Kα or mTOR are revealed from the structure-activity relationship of the reported inhibitors. Current clinical development in combination therapy of inhibitors involving in the PI3K/AKT/mTOR pathway is also discussed.     

Intracellular signaling pathways involved in the relaxin-induced proliferation of rat Sertoli cells

Regulation of Sertoli cell number is a key event to determine normal spermatogenesis. We have previously shown that relaxin and its G-protein coupled receptor RXFP1 are expressed in rat Sertoli cells, and that relaxin stimulates Sertoli cell proliferation. This study examined the mechanisms underlying the mitogenic effect of relaxin in a primary culture of Sertoli cells removed from testes of immature rats. Stimulation with exogenous relaxin increased Sertoli cell number and the expression of the proliferating cell nuclear antigen (PCNA), but did not affect the mRNA level of the differentiation markers cadherins 1 and 2. Relaxin-induced Sertoli cell proliferation was blocked by inhibition of MEK/ERK1/2 or PI3K/AKT pathways, but not by inhibition of PKC or EGFR activity. Relaxin induced a rapid and transient activation of ERK1/2 phosphorylation, which was MEK and SRC-dependent, and involved upstream activation of G(i). AKT activation could be detected 5min after relaxin stimulation, and was still detected after 24h of stimulation with relaxin. Relaxin-induced AKT phosphorylation was G(i)- but not PKA-dependent, and it was blocked by both PI3K and MEK inhibitors. In conclusion, the mitogenic effect of relaxin in Sertoli cell involves coupling to G(i) and activation of both MEK/ERK1/2 and PI3K/AKT pathways.     

三种蛋白在上皮性卵巢癌中的表达及意义

目的 检测PIK3CA、AKT2、FFEN在上皮性卵巢癌中的表达情况,分析其与临床病理参数的关系,探讨三者在上皮性卵巢癌发生发展中的可能作用机制.方法 采用免疫组织化学S-P法及半定量分析法检测29例正常卵巢及64例上皮性卵巢癌组织中PIK3CA、AKT2、PTEN蛋白的表达情况,分析在上皮性卵巢癌中三者与临床病理特征之间的关系以及三者之间的相关性.结果 PIK3CA蛋白表达部位主要在细胞质内.在正常卵巢组织及上皮性卵巢癌组织中灰度值分别为(85.21±4.74)、(46.68±10.22),差异有统计学意义(P<0.01).AKT2蛋白表达部位主要在细胞质内.在正常卵巢组织及上皮性卵巢癌组织中灰度值分别为(85.72±4.29)、(46.17±9.29),差异有统计学意义(P<0.01).PTEN阳性染色主要定位于细胞质,有少量定位于细胞核.在正常卵巢组织及上皮性卵巢癌组织中灰度值分别为(41.49±4.67)、(73.74±11.33),差异有统计学意义(P<0.01).PIK3CA与AKT2蛋白在上皮性卵巢癌中的表达均高于正常卵巢组织,PTEN蛋白表达低于正常卵巢组织,差异均有统计学意义(P<0.01);上皮性卵巢癌中,PIK3CA与AKT2蛋白在中低分化及Ⅲ～Ⅳ期组织中表达高于高分化及Ⅰ～Ⅱ期,差异有统计学意义(P<0.05),PTEN蛋白在中低分化及Ⅲ～Ⅳ期组织中表达低于高分化及Ⅰ～Ⅱ期,差异有统计学意义(P<0.05),三者在患者的年龄、组织学类型分组中表达差异无统计学意义(P>0.05);上皮性卵巢癌中,PIK3CA与AKT2蛋白表达呈正相关(r=0.674,P<0.01),PIK3CA与PTEN表达呈负相关(r=-0.514,P<0.01),AKT2与PTEN表达呈负相关(r=-0.589,P<0.01).结论 PIK3CA、AKT2蛋白的表达升高和PTEN蛋白表达降低在卵巢癌的发生、发展中起重要作用,P13K/AKT信号通路参与卵巢癌的演进过程.联合检测PIK3CA、AKT2、PTEN有助于卵巢癌的临床早期诊断和治疗,为生物靶向治疗提供新的线索.

Abstract：

Objective To investigate the expression of PIK3CA, AKT2 and PTEN in epithelial ovarian cancer, to analyze their relation to clinical and pathologic features and prognosis of epithelial ovarian cancer. Methods The expression of PIK3CA, AKT2 and PTEN protein were studied by immunohisto chemical methods in 29 normal o-vary tissues and 64 epithelial ovarian carcinoma. The correlation between clinical and pathologic features in ovarian cancer was also analyzed. Results The expression of PIK3CA and AKT2 protein in epithelial ovarian cancer was significantly higher than that in normal ovary tissues ( P <0. 01). In epithelial ovarian cancer, the expression of PIK3CA and AKT2 protein in mid and low histological grade and Ⅲ-Ⅳ clinical stage was significantly higher than that in high histological grade and Ⅰ-Ⅱ clinical stage (P <0. 05). In epithelial ovarian cancer, the expressions were not related with age and histological type(P >0.05). The expression of PIK3CA protein was positively correlated with the expression of AKT2 protein (r =0.674, P <0.01). The expression of PTEN was negatively correlated with the expression of PIK3CA(r= -0.514, P<0.01)and the expression of PTEN was negatively correlated with the expression of AKT2 (r =- 0.589, P < 0.01). Conclusions The high expression of PIK3CA and AKT2 protein as well as the low expression of PTEN protein are closely related to the occurrence and development of ovarian cancer. PI3K/AKT signal pathway takes part in the evolvement process of ovarian cancer. To-investigate the expression of PIK3CA, AKT2 and PTEN protein are helpful for the clinical diagnosis and treatment of ovarian cancer.