LBH589或联合多烯紫杉醇对人卵巢癌OVCAR-3细胞增殖和凋亡的研究

  目的  探讨新型组蛋白去乙酰化酶抑制剂LBH589或联合多烯紫杉醇（DTX）对人卵巢癌OVCAR-3细胞增殖和凋亡的影响。  方法  采用不同浓度LBH589、DTX或两者联合处理OVCAR-3细胞, 用四甲基偶氮唑蓝（MTT）法检测细胞增殖活力, 台盼兰、吖啶橙溴化乙啶（AO/EB）双染色法检测细胞凋亡; Western blot检测聚腺苷二磷酸核糖聚合酶（PARP）、caspase-3、caspase-7、bcl-2、bax蛋白水平。  结果  LBH589、DTX均能抑制OVCAR-3细胞增殖, 诱导凋亡, 小剂量LBH589联合DTX作用更强。经Calcusyn软件分析证明两者联合具有明显的协同作用。Western blot检测发现caspsae-3、PARP-85kD剪切蛋白增加, bax表达增加, bcl-2表达减少。caspase-7无明显变化。  结论  LBH589、DTX能抑制OVCAR-3细胞增殖, 诱导凋亡, 两者联合有明显的协同作用。      

MYB down-regulation enhances sensitivity of U937 myeloid leukemia cells to the histone deacetylase inhibitor LBH589 in vitro and in vivo

The effect of combining MYB suppression with the histone deacetylase inhibitor LBH589 was studied in human myeloid leukemia cell lines. MYB knockdown inhibited proliferation and induced apoptosis in U937 and K562 cells in vitro, and also sensitized both to the pro-apoptotic effect of LBH589. This was accompanied by enhanced expression of the pro-apoptotic BCL2 family members BOK and BIM. U937 cells carrying inducible MYB shRNA were also transplanted into NOD/SCID mice. The combination of MYB knockdown and LBH589 prolonged survival compared to either treatment alone, suggesting that further development of such combinations might lead to effective and safe leukemia therapies.     

The histone deacetylase inhibitor LBH589 enhances the anti-myeloma effects of chemotherapy in vitro and in vivo

Panobinostat (LBH589) is a potent histone deacetylase inhibitor (HDACi) that has shown anti-tumor activity in preclinical studies in both solid and hematological malignancies. We evaluated the anti-multiple myeloma (MM) effects of LBH589 alone and with melphalan or doxorubicin using MM cell lines and our human MM xenograft model LAGλ-1. LBH589 treatment resulted in increased acetylation of histones, induction of caspase cleavage, inhibition of cell proliferation and synergistic anti-MM effects with melphalan or doxorubicin in vitro. LBH589 with melphalan or doxorubicin also showed significantly enhanced anti-myeloma activity in vivo. These findings provide the basis for clinical development of these combination therapies.     

IL 6经PI3K/Akt通路诱导卵巢癌细胞对他莫西芬耐药

目的:探讨IL-6诱导卵巢癌细胞对他莫西芬(tamoxifen,TAM)耐药的分子机制.方法:构建内源性过表达IL-6的人卵巢癌A2780细胞系和内源性抑制IL-6表达的人卵巢癌CAOV-3细胞,50 ng/ml外源性IL-6预处理A2780细胞(A2780/preIL-6细胞),Western blotting检测内/外源IL-6对卵巢癌细胞ERα Ser167位磷酸化水平的影响;IL-6与PI3K抑制剂Wort-mannin单独或联合作用于A2780细胞,Western blotting检测其对A2780细胞Akt磷酸化和ERα磷酸化的影响;MTT法检测Wortmannin和内/外源IL-6对A2780细胞TAM敏感性的影响;荧光素酶报告基因检测卵巢癌细胞ERα的转录活性,并分析其可能涉及的信号通路.结果:外源性及内源性过表达IL-6可明显促进A2780细胞ERα Ser167位点磷酸化水平(均P＜0.01),而内源性抑制IL-6表达则可降低CAOV-3细胞ERα Ser167位点的磷酸化水平(P＜0.01);Wortmannin可阻断IL-6诱导的A2780细胞对TAM的耐药及ERα的磷酸化(P ＜0.05);IL-6可促进细胞ERα的转录活性(P＜0.01),而Wortmannin并不能阻断IL-6对ERα的转录活性的影响(P＞0.05).结论:IL-6可经PI3 K/Akt通路引起ERα磷酸化从而活化ER信号通路,进而诱导卵巢癌细胞对TAM耐药.     

Inhibition of PI3K/Akt/mTOR signaling pathway enhances the sensitivity of the SKOV3/DDP ovarian cancer cell line to cisplatin in vitro

The activation of the PI3K/AKT/m TOR pathway plays a key role in ovarian cancer tumorigenesis, progression and chemotherapy resistance. This study aimed to explore the possible mechanism that PI-103, a dual inhibitor of phosphatidylinositide 3-kinase and m TOR, enhances the sensitivity of SKOV3/DDP ovarian cancer cell line to cisplatin chemotherapy. The results showed that PI-103 could significantly increase the sensitivity of SKVO3/DDP cells to cisplatin through inhibiting the activation of PI3K/Akt/m TOR signaling pathway and inducing cell cycle arrest and apoptosis.     

Midkine as a potential diagnostic marker in epithelial ovarian cancer for cisplatin/paclitaxel combination clinical therapy

The paclitaxel/cisplatin combination therapy commonly is used as the first-line treatment for advanced ovarian cancer patients. Midkine (MK), known as a novel tumor biomarker, has been elevated in the serum of patients with epithelial ovarian cancer (EOC). In this study, we aimed to detect the expression of MK in EOC tissues and evaluate clinical value of MK in diagnosis and therapy of EOC. We perform immunohistochemistry analysis to detect MK in EOC sample with postoperative platinum/paclitaxel combination therapy, we found that 71.4% (85 in 119 samples) of these samples were MK positive (> 10% of the cells were stained), and the expression of MK was significantly associated with disease histology (P = 0.038) as well as differentiation grade (P < 0.001). Moreover, MK positive samples show much more sensitive to cisplatin/paclitaxel combination therapy, compared with MK negative samples (P = 0.029). Those results indicated that MK expression might correlate with paclitaxel and/or cisplatin cytotoxicity in clinical therapy of EOC. Then, we evaluated the sensitivity to cisplatin and paclitaxel in 5 ovarian cancer cell lines (ES2, A2870, HO-8910, SKOV3 and SW626), and ES2, the highest MK expression among those cell lines, show the most sensitive to paclitaxel and cisplatin. Further, we confirmed this correlation between MK and paclitaxel and/or cisplatin cytotoxicity with the gain- and lost- of function. Finally, we demonstrated that MK enhanced the cytotoxicity of paclitaxel and/or cisplatin by accumulated cisplatin and paclitaxel through inhibited the expression of multidrug resistance-associated protein 3 (MRP3). In conclusion, MK could be an effective biomarker in diagnosis and therapy of EOC, especially for the drug selection at the time of initial diagnosis.     

Melittin对非小细胞肺癌增殖、凋亡及PI3K／Akt信号通路的影响

目的 探讨Melittin对非小细胞肺癌（NSCLC）细胞增殖、凋亡及PI3K／Akt信号通路的影响。方法 分别采用0、10、20、50、100 μmol／L Melittin处理NSCLC细胞株A549、SPC-A1及人肺上皮细胞株16HBE 24、48、72和96 h后,采用四甲基偶氮唑盐（MTT）比色法检测各细胞株的增殖抑制率变化,同时采用流式细胞术Annexin-FITC／PI双染法及PI单染法检测不同浓度Melittin 处理24、48 h后的A549细胞凋亡及细胞周期情况,Western blotting检测不同浓度Melittin处理48 h后的A549细胞中PI3K／Akt信号通路相关蛋白（Akt和PTEN）及凋亡促进基因（caspase-9）的表达情况。结果 在10～100 μmol／L范围内,Melittin可呈剂量和时间依赖的方式提高A549、SPC-A1细胞的增殖抑制率,差异均有统计学意义（P＜0.05）,但对16HBE无细胞毒性作用（P＞0.05）;与0 μmol／L比较,除10 μmol／L Melittin处理24 h后的晚期凋亡率和G2／M期细胞比例无统计学差异（P＞0.05）,10～100 μmol／L的早、晚期凋亡率、G0／G1期细胞比例及PTEN和caspase-9蛋白水平均升高,S期、G2／M期细胞比例及Akt水平均降低,以上差异有统计学意义（P＜0.05）,且10～100 μmol／L范围内各浓度间的差异均有统计学意义（P＜0.05）。结论 Melittin可对NSCLC细胞有毒性作用,但对正常肺上皮细胞无影响,且可诱导A549细胞凋亡及细胞周期阻滞并抑制PI3K／Akt信号通路的激活。     

LncRNA DLEU2对胃癌细胞增殖、凋亡和PI3K/Akt信号通路的影响

目的探讨长链非编码RNA淋巴细胞白血病缺失基因2(DLEU2)对胃癌细胞增殖、凋亡和磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)信号通路的影响。方法通过GEPIA在线分析来自TCGA数据库和GTEx项目的胃癌组织DLEU2水平,采用实时定量PCR(QPCR)检测正常胃黏膜上皮GES-1细胞和胃癌BGC-823细胞的DLEU2水平;脂质体法将3条靶向DLEU2的小干扰RNA(si-DLEU2-1、si-DLEU2-2和si-DLEU2-3)转染至BGC-823细胞中,筛选最佳si-DLEU2干扰序列进行实验。体外常规培养BGC-823细胞并分为转染si-DLEU2的干扰组、转染无义siRNA序列的阴性对照(NC)组和未转染的空白对照(Blank)组,采用MTT比色法和AnnexinⅤ-FITC/PI双染流式细胞术检测细胞的增殖活力和凋亡率,QPCR和Western blotting检测凋亡相关因子Bcl-2、Bax和caspase-3的水平,Western blotting检测磷酸化的PI3K调节亚基p85(p-p85)和Akt(p-Akt)水平。结果GEPIA在线分析显示胃癌组织的DLEU2水平高于正常组织(P<0.05);QPCR结果显示BGC-823细胞的DLEU2水平高于GES-1细胞(P<0.05),且3条si-DLEU2序列转染后,BGC-823细胞的DLEU2水平均降低(P<0.05),其中si-DLEU2-3序列的效果最佳,故选取si-DLEU2-3序列进行后续实验;干扰组的DLEU2水平及转染24、48 h后的增殖活力均低于Blank组和NC组(P<0.05)。干扰组的凋亡率为(21.529±1.320)%,高于Blank组的(9.032±0.536)%和NC组的(8.641±0.365)%(P<0.05)。与Blank组和NC组相比,干扰组的Bcl-2、p-p85和p-Akt水平降低,而Bax和caspase-3水平均升高(P<0.05)。Blank组和NC组上述指标的差异无统计学意义(P>0.05)。结论DLEU2在胃癌组织和细胞中均升高且发挥促癌作用,可能通过激活PI3K/Akt信号通路来增强细胞增殖能力并抑制凋亡,有望成为胃癌治疗的新靶点。     

扶正抑瘤汤通过PI3K/Akt/mTOR信号通路抑制非小细胞肺癌增殖、凋亡及自噬

目的 探讨扶正抑瘤汤对非小细胞肺癌细胞增殖、侵袭、迁移、凋亡、自噬以及PI3K/Akt/mTOR信号通路的影响。方法 采用不同浓度（12.5 mg/mL、25 mg/mL、50 mg/mL、100 mg/mL、200 mg/mL、400 mg/mL）扶正抑瘤汤体外培养非小细胞肺癌A549细胞, CCK-8法测定细胞增殖抑制率;Transwell小室侵袭实验检测细胞侵袭能力;划痕实验检测细胞迁移能力;流式细胞仪检测细胞凋亡率;Western blot检测细胞自噬相关蛋白以及PI3K/Akt/mTOR信号通路相关蛋白的表达水平;透射电镜下观察自噬溶酶体形成情况。结果 200 mg/mL和400 mg/mL扶正抑瘤汤作用后A549细胞增殖抑制率增加（均P<0.05）。200 mg/mL扶正抑瘤汤作用后A549细胞侵袭和迁移能力降低（均P<0.05）;细胞凋亡率增加（P<0.05）;Beclin-1、LC3Ⅱ/Ⅰ蛋白的表达水平升高（均P<0.05）,P62蛋白的表达水平降低（P<0.05）;A549细胞自噬溶酶体数量增加;p-PI3K/PI3K、p-Akt/Akt和p-mTOR/mTOR蛋白的表达降低（均P<0.05）。结论 扶正抑瘤汤可以抑制A549细胞增殖,并促进细胞凋亡和自噬,可能与PI3K/Akt/mTOR信号通路失活有关,是治疗非小细胞肺癌的潜在药物。     

过表达miR-141-3p通过靶向下调PTEN并激活PI3K/Akt信号通路促进卵巢癌A2780 细胞的恶性生物学行为

[摘要] 目的：探讨miR-141-3p 通过靶向PTEN并调控PI3K/Akt 通路对卵巢癌细胞增殖、侵袭和凋亡的影响。方法：收集2014 年4 月至2017 年10 月河南省人民医院妇产科收治的资料完整的28 例卵巢癌患者肿瘤组织和相应的癌旁组织,采用qPCR检测卵巢癌组织和细胞系中miR-141-3p 的表达水平,双荧光素酶报告基因实验验证miR-141-3p 和PTEN的靶向关系;过表达或敲降miR-141 及PTEN基因后,采用CCK-8、Transwell 和Annexin V-FITC/PI 双染流式术检测卵巢癌A2780 细胞增殖、侵袭和凋亡水平,WB实验进一步检测miR-141-3p 对PTEN-PI3K/Akt 信号通路的调控作用。结果：miR-141-3p 在卵巢癌组织和细胞系中高表达（P<0.05 或P<0.01）。双荧光素酶报告基因证实miR-141-3p 靶向作用于PTEN并下调其表达水平（P<0.01）。与对照组相比,敲降miR-141-3p 后A2780 细胞的增殖受到显著抑制（48 h 时,0.36±0.04 vs 0.82±0.06,P<0.05）、侵袭能力明显降低[穿膜细胞数（45.14±7.88）vs（215.32±16.04）个,P<0.01]、细胞凋亡率显著升高[ （9.29±0.65）% vs（1.85±0.26）%,P<0.01]。过表达PTEN显著抑制了A2780 细胞中p-Akt 的表达（均P<0.01）、抑制细胞增殖和侵袭能力（均P<0.01）而明显促进细胞凋亡（均P<0.01）,在过表达PTEN的同时过表达miR-141-3p 或添加IGF-1 后可逆转上述的变化。结论：miR-141-3p 能够促进A2780 细胞增殖、侵袭和诱导凋亡,其机制可能与靶向调控PTEN并激活PI3K/Akt通路有关。     

RY-2f,an isoflavone analog,overcomes cisplatin resistance to inhibit ovarian tumorigenesis via targeting the PI3K/AKT/mTOR signaling pathway

Ovarian cancer remains the leading cause of death in gynecologic malignancies partially because of resistance to chemotherapy. In the present study, we show that RY-2f, a chemically synthesized isoflavone analog, inhibited ovarian cancer cell proliferation, blocked cell cycle in G2/M phase and induced cellular apoptosis through up-regulation of p21, cyclin B1, Bax, Bad and cleaved-PARP, and suppression of cyclin A, CDK2 and Bcl-2. We also show that RY-2f could increase the chemotherapeutic efficacy of cisplatin as tested by cell proliferation and colony formation assays, indicating a synergistic effect of RY-2f and cisplatin. Mechanistic study revealed that RY-2f exerted the anti-tumor activities mainly through suppression of the PI3K/AKT/mTOR signaling. Finally, in vivo studies showed that RY-2f blocked the A2780-induced xenograft tumor growth without detectable toxicity in the animals at the therapeutic doses, and whereas RY-2f re-sensitized the cisplatin resistant cell line A2780/CDDP induced xenograft tumor to cisplatin treatment. Thus, RY-2f may be developed as a potential therapeutic agent to treat ovarian cancer.     

异莲心碱通过PI3K/Akt/mTOR信号通路影响结肠癌SW480细胞增殖、 凋亡和自噬

目的：探讨异莲心碱（Iso）通过PI3K/Akt/mTOR信号通路对结肠癌SW480细胞增殖、凋亡和自噬的影响。方法：用 10、20和40 μmol/L的Iso处理结肠癌SW480细胞,CCK-8法、流式细胞术和WB法分别检测Iso对细胞增殖活力、凋亡和自噬相关 蛋白LC3Ⅰ、LC3Ⅱ、p62表达的影响。然后,用20 μmol/L的Iso和25 μmol/L的PI3K激活剂740 Y-P分别处理SW480细胞,将细 胞分为对照组、740 Y-P组、Iso组和Iso+740 Y-P组,流式细胞术、WB法检测Iso和740 Y-P对各组细胞凋亡及细胞中LC3Ⅰ、LC3Ⅱ、 p62、PI3K、p-PI3K、 mTOR和p-mTOR蛋白表达的影响。结果：10、20和40 μmol/L的Iso处理后,SW480细胞增殖活力均显著下 降（均P<0.05）,细胞凋亡率均显著升高（均P<0.05）,LC3Ⅱ/LC3Ⅰ表达均显著上调（均P<0.05）,p26蛋白表达显著下调（P<0.05）。 Iso和740 Y-P处理后,与对照组相比,740 Y-P 组细胞凋亡率、LC3Ⅱ/LC3Ⅰ表达均显著下降（均P<0.05）,p26、p-PI3K/PI3K和 p-mTOR/mTOR 表达均显著升高（均 P<0.05）;Iso 组 细胞凋亡率、LC3Ⅱ/LC3Ⅰ表达升高（均 P<0.05）,p26、p-PI3K/PI3K和 p-mTOR/mTOR 表达均显著下降（均 P<0.05）;与 740 Y-P 组相比,Iso+740 Y-P 组细胞凋亡率、LC3Ⅱ/LC3Ⅰ表达升高（P<0.05）, p26、p-PI3K/PI3K和p-mTOR/mTOR表达均显著下降（均P<0.05）;与Iso组相比,Iso+740 Y-P组细胞凋亡率、LC3Ⅱ/LC3Ⅰ表达下 降（均P<0.05）,p26、p-PI3K/PI3K和p-mTOR/mTOR表达均显著升高（均P<0.05）。结论：Iso通过抑制PI3K/Akt/mTOR信号通路 抑制结肠癌SW480细胞增殖并诱导细胞凋亡和自噬。     

Combination of nigericin with cisplatin enhances the inhibitory effect of cisplatin on epithelial ovarian cancer metastasis by inhibiting slug expression via the Wnt/β-catenin signalling pathway

Epithelial ovarian cancer (EOC) is the most lethal cancer among female genital tumours. Standard therapies, including postoperative chemotherapy, exhibit high proportions of recurrence and resistance. Novel therapeutic strategies are combined with chemotherapy. Emerging studies have demonstrated that nigericin, an H⁺, K⁺ and Pb²⁺ ionophore, exhibits promising anticancer activity in various types of malignancy, such as colorectal and epithelial ovarian cancer. Our previous study suggested that nigericin could regulate EOC cell proliferation, migration and invasion, and may be a novel chemotherapy candidate for EOC. However, to the best of our knowledge, the effects of combined therapy with cisplatin, and the associated underlying mechanisms, are not yet fully understood. The present study aimed to clarify the effects of combined chemical therapy with nigericin and cisplatin on EOC cells and to reveal its mechanism. Wound healing, Transwell, cell viability and colony formation assays were used to measure the migration, invasion and proliferation of EOC cells. Western blotting was used to detect protein expression. A slug overexpression lentivirus was used to create a slug overexpression model in SK-OV-3 cells. Small interfering RNA was used to knock down slug expression. Nigericin combined with cisplatin enhanced the inhibitory effects of cisplatin on the migration and colony formation of EOC cells. Nigericin also enhanced the inhibitory effects of cisplatin on the expression levels of MMP7, as well as the inhibitory effects of cisplatin on the expression levels of β-catenin and GSK-3β, indicating that nigericin and cisplatin regulated in the Wnt/β-catenin signalling pathway. When slug was knocked down, the effect of nigericin was weakened. Overexpression of slug could repress the inhibitory effect of nigericin on the Wnt/β-catenin signalling pathway. Furthermore, nigericin inhibited slug expression by enhancing its modification through small ubiquitin-like modifiers (SUMOs; referred to as SUMOylation). Overall, the present results demonstrated that nigericin combined with cisplatin might serve as a novel therapeutic strategy in patients with metastatic EOC because the combined therapy had higher effectiveness than single drug use. The underlying mechanism of combined therapy maybe the enhanced inhibitory effect of slug through its nigericin-induced SUMOylation.     

醉茄素A对非小细胞肺癌A549细胞增殖、凋亡及PI3K／Akt信号通路的影响

目的 探讨醉茄素A（WFA） 对非小细胞肺癌（NSCLC）A549细胞增殖、凋亡及PI3K／Akt信号通路的影响。方法 采用0、2.5、5.0、10.0、20.0μmol／L WFA 处理A549细胞,采用四甲基偶氮唑盐（MTT）比色法检测上述浓度处理24、48、72和96h的细胞增殖抑制率,Hoechst染色和磷酯酰丝氨酸结合蛋白 异硫氢酸荧光素／碘化丙啶双染法（Annexin V-FITC／PI）检测各浓度组48h的细胞凋亡情况,流式细胞仪检测各浓度组48h的细胞周期分布情况,免疫印迹检测各浓度组48h凋亡相关基因（Bcl-2、Bax和Cleaved caspase-3）和PI3K／Akt信号通路重要蛋白Akt及其磷酸化形式p-Akt的蛋白水平。结果 WFA 可抑制细胞增殖,并呈剂量和时间依赖性（P＜0.05）;0、2.5、5.0、10.0、20.0μmol／L WFA作用48h后A549细胞的凋亡指数分别为2.75±0.64、4.61±1.36、9.75±2.78、12.92±3.42和18.68±4.31,组间差异有统计学意义（P＜0.05）。除2.5μmol／L外,其余浓度组的早、晚期凋亡率、凋亡促进基因（Bax和Cleaved caspase-3）水平及G0／G1期细胞比例均高于0μmol／L,凋亡抑制基因Bcl-2水平及S期和G2／M期细胞比例均低于0μmol／L（P＜0.05）; 2.5、5.0、10.0、20.0μmol／L的组间差异有统计学意义（P＜0.05）。随着浓度升高,p-Akt／Akt值呈降低趋势,差异有统计学意义（P＜0.05）。结论 WFA能够抑制A549细胞的增殖及凋亡,可能通过抑制PI3K／Akt通路激活实现。     

银杏内酯B通过阻抑PI3K/Akt/mTOR信号通路抑制胃癌HGC-27 细胞的恶性生物学行为

目的：探讨银杏内酯B（GKB）是否通过阻抑PI3K/Akt/mTOR信号通路抑制胃癌HGC-27细胞的增殖、凋亡、迁移及侵袭。方法：将HGC-27细胞分为对照、GKB低剂量（100 mg/L）、GKB高剂量（200 mg/L）、GKB高剂量（200 mg/L）+740Y-P（PI3K激活剂）、Ly294002（PI3K抑制剂）组。采用MTT、Edu、FCM、Transwell实验分别检测各组细胞的增殖、凋亡、迁移和侵袭能力,qPCR和WB法分别检测各组细胞中PI3K mRNA、Akt mRNA、mTOR mRNA和Ki-67、caspase-3、p-PI3K/PI3K、p-Akt/Akt、p-mTOR/mTOR蛋白的表达。构建胃癌HGC-27细胞裸鼠移植瘤模型,观察GKB对移植瘤生长的影响,WB法检测移植瘤组织中Ki-67、caspase-3、p-PI3K/PI3K、p-Akt/Akt、p-mTOR/mTOR蛋白的表达。结果：体外实验结果表明,与对照组相比,GKB低剂量组、GKB高剂量组、Ly294002组HGC-27细胞的增殖活力及细胞增殖率、迁移和侵袭细胞数,PI3K、Akt、mTOR mRNA表达,以及Ki-67、p-PI3K/PI3K、p-Akt/Akt、p-mTOR/mTOR蛋白表达均显著降低（均P<0.05）;细胞凋亡率、caspase-3蛋白表达均显著升高（均P<0.05）;740Y-P可部分逆转GKB对HGC-27细胞的抑制作用（均P<0.05）。荷瘤裸鼠实验结果显示,GKB可显著抑制HGC-27细胞裸鼠移植瘤的生长（P<0.05）,且可下调PI3K/Akt/mTOR通路相关蛋白的表达。结论：GKB可通过阻抑PI3K/Akt/mTOR信号通路而抑制胃癌HGC-27细胞增殖、迁移与侵袭并促进其凋亡。     

miR-103 靶向PTEN并激活PI3K/AKT通路促进肺癌细胞A549 对达沙替尼耐药

[摘要] 目的：探讨miR-103 靶向PTEN并激活PI3K/AKT信号通路促进肺癌细胞对达沙替尼（dasatinib,DASA）耐药的机制。方法：收集2014 年4 月至2018 年1 月昆明医科大学第一附属医院胸外科收治的资料完整的肺癌DASA耐药组织和不耐药组织各35 例。采用qPCR实验检测miR-103 在肺癌DASA耐药组织和细胞中的表达水平,同时,采用CCK-8、Transwell 和Wb实验检测敲降miR-103 对A549/DASA细胞增殖、迁移和上皮间质转化（EMT）的影响,双荧光素酶报告基因验证miR-103 与PTEN的靶向关系。进一步采用CCK-8、Transwell 和Wb实验检测miR-103 通过PTEN-PI3K/AKT信号通路对A549/DASA细胞恶性生物学行为的影响。结果：miR-103 在肺癌DASA 耐药组织和A549/DASA 细胞中均高表达（均P<0.01）。敲降miR-103 可显著抑制A549/DASA细胞的增殖、迁移和EMT（P<0.05 或P<0.01）。此外,双荧光素酶报告基因证实miR-103 靶向作用PTEN并下调其表达水平（P<0.01）。进一步实验显示,过表达miR-103 通过靶向下调PTEN并激活PI3K/AKT信号通路进而显著促进A549/DASA细胞增殖、迁移和EMT（P<0.05 或P<0.01）,从而上调A549/DASA细胞对DASA的耐药性。结论：miR-103/PTEN/PI3K/AKT信号通路与肺癌DASA耐药性存在调控关系,敲降miR-103 可逆转A549/DASA对DASA耐药。     

乳腺癌组织和细胞中低表达的LZTS2 通过PI3K/AKT信号通路抑制癌细胞增殖、迁移和EMT

[摘要] 目的：探讨亮氨酸拉链肿瘤抑制因子2（leucine zipper tumor suppressor 2, LZTS2）基因在人乳腺癌组织和细胞系中的表达及其对乳腺癌细胞增殖、迁移和EMT的影响及其作用机制。方法：收集2016 年1 月至2016 年12 月开封中心医院乳腺外科收治的50 例女性乳腺癌患者的癌及癌旁组织标本和乳腺癌细胞系MCF-7、MDA-MB-231、MDA-MB-468 以及正常人乳腺上皮细胞株HBL-100,用qPCR 和Western blotting 检测乳腺癌组织和细胞中LZTS2 mRNA和蛋白表达水平。构建pcDNA-LZTS2 真核表达载体并采用脂质体转染MCF-7 细胞,同时转染pcDNA3.1 作为阴性对照。用Western blotting 检测转染48~72 h 后MCF-7 细胞中LZTS2 蛋白表达水平;用MTT法、Transwell 小室法检测LZTS2 过表达对细胞增殖、迁移和侵袭的影响,同时用Western blotting检测细胞中EMT相关蛋白Cyclin D1、波形蛋白、神经钙黏蛋白、上皮钙黏蛋白以及PI3K/AKT信号通路中相关蛋白的表达。结果：人乳腺癌组织中LZTS2 mRNA和蛋白表达水平均明显低于癌旁组织（P<0.05 或P<0.01）;乳腺癌MCF-7、MDA-MB-231 和MDA-MB-468 细胞中LZTS2 mRNA和蛋白表达水平显著低于乳腺上皮细胞HBL-100（P<0.05 或P<0.01）。与空白对照组和pcDNA3.1组相比,pcDNA-LZTS2 组MCF-7 细胞中LZTS2 蛋白表达水平明显上调（P<0.01）,细胞增殖、迁移和侵袭能力显著受到抑制（P<0.05 或P<0.01）,同时过表达LZTS2 细胞中Cyclin D1、波形蛋白和神经钙黏蛋白表达水平均明显降低（P<0.05 或P<0.01）、上皮钙黏蛋白表达水平明显升高（均P<0.01）,显示LZTS2 过表达通过降低p-PI3K和p-AKT 表达而抑制PI3K/AKT信号通路。结论：LZTS2 在乳腺癌中低表达,过表达LZTS2 能够抑制乳腺癌细胞的增殖、迁移和侵袭能力,可能与抑制细胞EMT过程的PI3K/AKT信号通路有关。     

HER2/neu过表达通过PI3K/Akt通路对乳腺癌细胞MCF7中p53基因表达及细胞生长和放疗敏感性的影响

目的 研究HER2／neu基因过表达通过磷脂酰肌醇-3-激酶(PI3K)通路对乳腺癌细胞MCF7野生型p53基因表达、细胞增殖及对γ射线照射敏感性的影响。方法 以脂质体介导的HER2／neu基因转染MCF7细胞,用G418筛选阳性克隆。通过Western blot鉴定HER2／neu蛋白的表达,并检测p53、信号转导分子Akt和p-Akt蛋白含量的变化及PI3K通路抑制剂LY294002对上述蛋白表达水平的影响。以MTT法检测细胞增殖以及细胞对γ射线照射的敏感性。结果 共获得18个稳定转染HER2／neu基因的阳性克隆,其中1个克隆有HER2／neu基因过表达。过表达HER2／neu的MCF7细胞p-Akt蛋白含量升高,p53蛋白含量低于对照组细胞,LY294002能够抑制p-Akt蛋白和p53蛋白的变化。同时,过表达HER2／neu基因的MCF7细胞生长速度高于对照组细胞,对γ射线照射治疗的敏感性降低,而LY294002能够抑制细胞生长并增强放射治疗的敏感性。结论 MCF7细胞中HER2／neu基因的过表达,能够通过激活PI3K通路导致野生型p53蛋白含量减少、细胞增殖加快及放疗的敏感性降低,这可能是某些p53蛋白为野生型的肿瘤患者对治疗产生抗性的原因。