吉非替尼对子宫内膜癌Ishikawa细胞增殖和凋亡的影响

目的:探讨EGFR酪氨酸激酶抑制剂吉非替尼(Gefitinib)对子宫内膜癌Ishikawa细胞增殖和凋亡的影响,并探讨其内在作用机制。方法:MTT法检测不同浓度吉非替尼作用后细胞的增殖抑制率。采用流式细胞术检测吉非替尼作用48h后的细胞凋亡率及细胞周期分布,并用倒置显微镜对细胞进行形态学观察。Western blot法检测不同浓度吉非替尼作用细胞48h后细胞内p-Akt、CyclinD1蛋白水平表达的变化。结果:吉非替尼能显著抑制Ishikawa细胞的增殖,促进细胞凋亡,且呈时间-剂量依赖性(P0.05),并使细胞周期中G0/G1期比例升高(P0.05),而对S期和G2/M期比例无明显影响(P0.05)。吉非替尼作用48h后,Ishikawa细胞中p-Akt、CyclinD1蛋白表达随着吉非替尼浓度的增加逐渐下降(P0.05)。结论:吉非替尼对人子宫内膜癌细胞Ishikawa的体外生长具有明显的抑制作用,并能诱导Ishikawa的凋亡,导致G0/G1期细胞阻滞,其分子机制可能与吉非替尼下调p-Akt蛋白及CyclinD1蛋白的表达有关。     

Genistein对人卵巢癌细胞系TC-1增殖抑制和诱导凋亡作用的研究

目的:研究植物雌激素染料木黄酮(genistein)对人卵巢癌细胞系TC-1细胞增殖的抑制作用和凋亡的诱导作用,探讨其抗癌作用的机制。方法:应用MTT法检测不同浓度genistein对TC-1细胞的生长抑制作用,电镜观察药物作用后细胞超微结构的改变,流式细胞仪检测细胞周期、定量分析凋亡,DNA琼脂糖凝胶电泳法确定凋亡。结果:TC-1细胞经不同浓度genistein处理后,细胞生长明显受到抑制,且这种抑制作用呈时间及浓度依赖性,5mg/L和10mg/L genistein作用72h后,抑制率分别达到89·84%和97·99%。genistein阻断TC-1细胞生长于G2/M期,在G0/G1前出现典型的亚二倍体凋亡峰,且凋亡呈时间依赖性。电镜观察到用药后凋亡细胞典型的形态学特征。DNA凝胶电泳可观察到细胞凋亡特异的DNA梯形带。结论:genistein对TC-1细胞生长的抑制作用呈明显的时间及浓度依赖性,并诱导其发生凋亡。     

The mechanism of anticancer activity of the new synthesized compound - 6,7-Methylenedioxy-4-(2,4-dimethoxyphenyl)quinolin −2(1H)-one(12e) in human ovarian cancer cell lines

ObjectiveOvarian cancer is the most lethal of the gynecologic malignancies. Most women have advanced disease at diagnosis and require extensive debulking surgery and aggressive chemotherapy. Induction of apoptosis in cancer cells has been used as an important approach for cancer therapy. We examined the anticancer effect of 6,7-methylenedioxy-4-(2,4-dimethoxyphenyl)quinolin-2(1H)-one (12e) in human ovarian cancer cell line.Materials and methodsThe 6,7-methylenedioxy-4- (2,4-dimethoxyphenyl) quinolin-2 (1H) -one (12e) was synthesized and provided by Dr. Li-Jiau Huang of China Medical University. Cell viability analysis showed that 12e inhibited cell growth and induced cell death in time- and dose-dependent manners. In order to study the underlying cell death mechanism, 2774 and SKOV3 cells treated with 12e were studied by morphology, DAPI/TUNEL double staining, DNA gel electrophoresis. To search the mechanisms of anti-proliferative effect of 12e, cell cycle analysis was performed. Changes in proteins related to cell death were analyzed by Western blot.Results12e significantly induced apoptosis evidenced by morphological changes, TUNEL-DAPI double-staining and DNA fragmentation. Western blot analysis demonstrated that the protein level of Bcl-2 was decreased after treatment with 12e, while the level of p53 and Bax was increased. 12e treatment resulted in G2/M arrest through down modulation of cyclin B1 and cdk1.ConclusionThese results suggested that 12e -induced growth inhibition was associated with cell cycle arrest and apoptosis.     

Epigenetic silencing of miR-130b in ovarian cancer promotes the development of multidrug resistance by targeting colony-stimulating factor 1

Objective

The purpose of this study was to investigate the role of miR-130b in the development of multidrug-resistant ovarian cancer.

Methods

The expression of miR-130b was assessed in ovarian tissues and cell lines by qRT-PCR. In vitro, miR-130b level was manipulated by transfection with mimics or inhibitors. Methylation level of miR-130b was evaluated by quantitative methylation-specific PCR (qMSP). CSF-1 expression in ovarian tissues and cells was determined by qRT-PCR, immunohistochemistry and ELISA, respectively. CSF-1 regulated by miR-130b was detected using Dual Luciferase Reporter system.

Results

Down-regulation of miR-130b in ovarian cancer was associated with FIGO III-IV clinical stages and poorer histological differentiation. MiR-130b was downregulated in multidrug resistant ovarian cancer cells. Restoration of miR-130b expression could sensitize these cells to anticancer drugs. MiR-130b hypermethylation was found in ovarian cancer tissues as well as in drug resistant cell lines and the methylation level was negatively correlated with its expression. Demethylation with 5-aza-CdR led to reactivation of miR-130b expression in drug resistant ovarian cancer cell lines concomitant with increase of sensibility to cisplatin and taxol. CSF-1 expression was negatively associated with miR-130b level in ovarian tissues and cell lines. Luciferase assay validated CSF-1 is a direct target of miR-130b. Knock-down of CSF-1 sensitized ovarian cancer cells to anticancer drugs and could partially attenuate the resistance inducing effect of miR-130b inhibitors.

Conclusions

Downregulation of miR-130b promotes the development of multidrug resistant ovarian cancer partially by targeting the 3′-UTR of CSF-1, and the silencing of miR-130b may be mediated by DNA methylation.     

High-mobility group protein B1 (HMGB1) is a novel biomarker for human ovarian cancer

Background

High mobility group box l (HMGB1), a nuclear and extracellular protein, is implicated in some physiologic and pathologic conditions. In this study, we investigated the expression and function of HMGB1 in ovarian cancer.

Methods

cDNA microarray analysis was performed to compare gene expression profiles of the highly invasive and the low invasive subclones derived from the SKOV3 human ovarian cancer cell line. Immunohistochemistry (IHC) staining was performed to investigate HMGB1 expression in a total of 100 ovarian tissue specimens. In functional assays, effects of HMGB1 knockdown on the biological behavior of ovarian cancer cells were investigated.

Results

HMGB1 was overexpressed in the highly invasive subclone compared with the low invasive subclone. High HMGB1 expression was associated with poor clinicopathologic features. Knockdown of HMGB1 expression significantly suppressed ovarian cancer cell proliferation accompanied by decreased cyclin D1 and PCNA expression, and inhibited cell migration and invasion accompanied by decreased MMP2 and MMP9 activities.

Conclusion

HMGB1 is a newly identified gene overexpressed in ovarian cancer and associated with poor clinicopathologic features. HMGB1 may serve as a new biomarker and a therapeutic target for ovarian cancer in the future.     

Effect of ovarian stimulation with human menopausal gonadotropin and recombinant follicle stimulating hormone on the expression of integrins alpha(3), beta(1) in the rat endometrium during the implantation period

Objective

To investigate the effect of exogenous ovarian stimulation with human menopausal gonadotropin (hMG) and recombinant follicle stimulating hormone (rFSH) on the expression of integrins alpha(3), beta(1) in the rat endometrium during implantation.

Study design

Following three successive normal estrous cycles the animals were divided into five groups: Group I (n = 10, control group) received no medication; Group II (n = 10) received 10 units of hMG; Group III (n = 10) received 20 units of hMG; Group IV (n = 10) received 10 units of rFSH; Group V (n = 10) received 20 units of rFSH at midday of middiestrous. The rats were then mated with fertile males. The animals were sacrificed on the day of implantation. The uterine horns were placed in fixative and paraffin blocks of the tissue were cut in 5 μm sections. The tissues were stained with primary antibodies; monoclonal anti-integrin alpha(3) and monoclonal anti-integrin beta(1) using immunohistochemical methods. The staining intensities of alpha(3) and beta(1) integrins were calculated separately for epithelium and stroma in each group.

Results

Staining intensities of alpha(3) and beta(1) integrins in both the epithelium and the stroma were significantly lower in the treatment groups than the control group (p < 0.05).

Conclusion

Ovarian stimulation by low and high doses of HMG and rFSH may have an effect on endometrial receptivity, possibly via a decrease in expression of integrins in the endometrium during the implantation period.     

CD_(80)基因导入人卵巢癌细胞系及其体外诱导细胞毒T淋巴细胞的研究

目的　用逆转录病毒载体将CD80 基因导入人卵巢癌细胞系TYK ,观察表达CD80 基因的TYK(TYK hCD80 )细胞体外诱导细胞毒T淋巴细胞 (CTL)的增殖及其杀瘤活性。方法　用逆转录病毒载体将CD80 基因导入TYK细胞 ,流式细胞仪测定其CD80 基因的表达。用TYK hCD80 细胞在抗CD3 单克隆抗体 (单抗 )存在下刺激外周血单个核细胞 (PBMC) ,诱导CTL ,3 H 胸腺嘧啶核苷掺入法测定其增殖活性 ,四甲基偶氮唑蓝法测定CTL的杀瘤活性。结果　转染后经G418(geneticin ,是一种氨基糖甙类抗生素 )筛选 ,流式细胞仪检测 ,CD80 基因表达率最高为 84 9%。TYK hCD80 、TYK在抗CD3 单抗存在下刺激PBMC增殖时 ,3 H 胸腺嘧啶核苷掺入量分别为 (4 0 6 0 4± 842 )、(80 0 0± 5 94)cpm ,两者比较 ,差异有显著性 (P <0 0 5 )。TYK hCD80 体外诱导的CTL较TYK诱导者对TYK细胞的杀伤率显著增高(P <0 0 5 )。结论　表达CD80 基因的卵巢癌细胞在抗CD3 单抗存在下能诱导产生CTL ,增殖快且有较高的杀伤活性 ,可能为卵巢癌的免疫基因治疗提供实验依据     

A novel retinoid, 4-[3,5-bis (trimethylsilyl) benzamido] benzoic acid (TAC-101), induces apoptosis of human ovarian carcinoma cells and shows potential as a new antitumor agent for clear cell adenocarcinoma

OBJECTIVES: A novel retinobenzoic acid derivative, 4-[3,5-bis (trimethylsilyl) benzamido] benzoic acid (TAC-101), was reported to suppress the growth and invasion of human gastric cancer or hepatocellular carcinoma by induction of apoptosis. We examined the antitumor activity of TAC-101 against human ovarian carcinoma cell lines. METHODS: Apoptosis of human epithelial ovarian carcinoma-derived cell lines (RMG-I, RMG-II, RTSG, RMUG-S, RMUG-L, and KF) was investigated by detecting DNA laddering and was quantified by an enzyme-linked immunosorbent assay. Inhibition of apoptosis was also examined using a caspase inhibitor. Furthermore, TAC-101 (8 mg kg(-1) day(-1) orally for 30 days) was investigated in nude mice with subcutaneous RMG-II tumors. A prominent apoptotic response to TAC-101 was observed. The antitumor effects of cisplatin (7 mg/kg intravenously on day 1) and paclitaxel (36 mg/kg intravenously on days 1 and 5) were also assessed for comparison. RESULTS: Apoptosis occurred in all of the cell lines (except KF) in a concentration-dependent manner after exposure to TAC-101 and was markedly induced in RMG-I and RMG-II cells (derived from ovarian clear cell adenocarcinomas). A caspase inhibitor blocked the induction of apoptosis by TAC-101. The maximum inhibition of RMG-II tumor growth in nude mice by TAC-101, cisplatin, and paclitaxel was 45%, 34%, and 47%, respectively. CONCLUSION: Oral TAC-101 shows potential as a novel antitumor agent for ovarian carcinoma, especially ovarian clear cell adenocarcinoma.     

异搏定等药物对耐阿霉素人卵巢癌细胞系逆转耐药性的研究

目的：探讨异搏定、潘生丁、丹参、黄体酮、环孢菌素Ａ和他莫西芬等药物对耐阿霉素人卵巢癌细胞系Ａ２７８０／ＡＤＭ逆转的耐药性。方法：用ＭＴＴ法和高效液相色谱仪测定异搏定等６种药物分别对阿霉素耐药细胞的敏感性和耐药细胞内阿霉素的剂量。结果：单用异搏定等６种药物对Ａ２７８０／ＡＤＭ几乎无体外抑制作用。异搏定和环孢菌素Ａ对Ａ２７８０／ＡＤＭ有明显的增效作用，可增加ＡＤＭ在Ａ２７８０／ＡＤＭ细胞内的潴留浓度。结论：除丹参外，其它５种药物均能增强ＡＤＭ对Ａ２７８０／ＡＤＭ耐药细胞细胞毒作用，并克服ＡＤＭ的耐药性，可作为ＡＤＭ的增效剂用于临床治疗卵巢癌患者     

Notch1 induces cell cycle arrest and apoptosis in human cervical cancer cells: involvement of nuclear factor kappa B inhibition

Notch signaling can serve as a tumor suppressor or tumor promoter in the same kind of cancer, such as human papillomavirus-positive cervical cancer cells. However, the exact mechanisms remain poorly characterized. Our studies demonstrated that constitutively overexpressed active Notch1 via stable transfection with exogenous intracellular domain of Notch1 (ICN) resulted in growth inhibition of the human cervical cancer cell line HeLa by inducing G(2)-M arrest and apoptosis. Moreover, the growth inhibition was correlated with inhibition of nuclear factor kappa B (NF-kappaB) p50 activation, accompanied by a decrease in the nuclear expression of NF-kappaB p50 and an increase in the cytosolic expression of IkappaBalpha. Consistent with these results, downregulation of cyclin D1 and Bcl-2, which are both the downstream genes of NF-kappaB, were observed in ICN-overexpressed cells. Overall, our results suggest that NF-kappaB inhibition may contribute partially to cell cycle arrest and apoptosis induced by Notch1 activation in human cervical cancer cells.     

肿瘤转移抑制基因kai1对卵巢癌细胞增殖及侵袭能力的影响

目的 :探讨肿瘤转移抑制基因kai1对卵巢癌细胞增殖及侵袭能力的影响。方法 :脂质体介导kai1cDNA转染高转移性人卵巢癌细胞系HO 891 0PM ,免疫细胞化学S P法检测转染前后细胞内kai1蛋白的表达。用四甲基偶氮唑蓝 (MTT)比色法、平板克隆形成实验观察kai1基因对细胞增殖能力的影响 ,经细胞粘附实验、体外侵袭力实验比较转染前后细胞侵袭能力的变化。结果 :转染kai1cDNA后HO 891 0PM细胞内可检测到kai1蛋白的稳定表达 ,细胞增殖能力较前无明显变化 ,侵袭能力明显下降。结论 :外源性肿瘤转移抑制基因kai1可通过脂质体有效转染高转移性人卵巢癌细胞系HO 891 0PM ,降低其侵袭能力 ,有望成为卵巢癌基因治疗的新的候选基因     

Synergistic cytotoxic effects of recombinant human tumor necrosis factor and Etoposide (VP16) or Doxorubicin on A2774 human epithelial ovarian cancer cell line

Recombinant human tumor necrosis factor (rHuTNF) is a cytokine, with some antitumor activity, released by stimulated monocytes-macrophages. In vivo and in vitro cytotoxicity studies testing the effectiveness of rHuTNF alone or in combination with chemotherapeutic agents have been carried out. We have evaluated the direct cytotoxic effect of rHuTNF on a human epithelial ovarian cancer cell line in vitro (A2774), alone or in combination with Etoposide (VP16) or Doxorubicin (Doxo), some topoisomerase II (Topo II) targeted drugs, or in combination with Cisplatin (CDDP), a not Topo II interactive drug. Our results suggest that rHuTNF is directly cytotoxic and that it is also able to induce a potentiation of VP16- or Doxo-cytotoxicity, but it is unable to potentiate CDDP-cytotoxicity. These data represent a reasonable basis for combining rHuTNF with Topo II inhibitors within phase I studies. The combination regimen could be tested in ovarian cancer patients.     

Expression of the cell-cycle regulatory proteins (pRb,cyclin D1, p16INK4A and cdk4) in human endometrial cancer: correlation with clinicopathological features

Introduction. Derailments of the control mechanisms in the G1/S phase of the cell cycle play a fundamental role in the initiation and progression of cancer. However, only a few reports have addressed the issue of simultaneously occurring abnormalities of Rb-pathway components in malignant endometrial tumors. Methods. Currently, we assessed the expression of cell-cycle regulatory proteins (pRb, cyclin D1, p16^INK4A and cdk4) in 48 sporadic endometrial cancers, and investigated these tumors for a possible relationship between aberrant protein staining and clinicopathological variables of cancer and RB-LOH. Results. There was abnormal pRb, cyclin D1, p16^INK4A and cdk4 immunoreactivity in 2%, 50%, 6% and 25% of cases, respectively. Altogether, 33 of 48 (69%) endometrial malignant tumors showed abnormal expression of at least one Rb-pathway protein immunohistochemically. However, there was significant correlation neither between the cell-cycle regulators nor between the frequency of pRb, p16^INK4A and cyclin D1 abnormalities and clinicopathological variables of cancer, but a significant correlation did exist between cdk4 staining and the clinical stage of disease (P<0.05, Fisher's exact test). Moreover, an inverse relationship was also demonstrated between cdk4 expression and patient age (r=-0.367; P=0.01). However, none of the cell-cycle regulatory proteins, except for pRb, was related to loss of heterozygosity at locus 13q14. Conclusion. As a conclusion, derailments of the Rb-pathway components, cyclin D1 and cdk4 in particular, seems to participate in the endometrial cancer development in humans. Overexpression of cdk4 was related to the progression of neoplastic disease and corresponds with age of onset, suggesting a major role of altered cdk4 immunoreactivity in the progression of endometrial cancer.     

雷公藤内酯醇对耐顺铂人卵巢癌细胞株(COC1/DDP)体外活性影响机制的初步探讨

目的:通过研究雷公藤内酯醇(TP)对COC1/DDP体外活性的影响,初步探讨雷公藤内酯醇促进COC1/DDP细胞凋亡的机制,为TP成为治疗晚期或耐顺铂卵巢癌药物提供实验依据。方法:采用MTT法检测顺铂(DDP)、TP及两者联用对COC1/DDP的生长抑制作用;用透射电镜观察TP作用24h后细胞超微结构的变化;采用流式细胞术分析不同浓度TP对COC1/DDP细胞周期和细胞凋亡的影响;用DNA电泳分析用药后细胞基因组DNA断裂状况;以免疫组化分析TP对Caspase-7蛋白表达的影响。结果:DDP在24h、48h和72h三个时间段对COC1/DDP细胞的半数抑制量(50%concentration of inhibi-tion,IC50)分别为5.567μg/ml、2.866μg/ml和1.161μg/ml,其对COC1/DDP细胞增殖表现出浓度-时间依赖性的抑制作用(P<0.05);TP可抑制COC1/DDP细胞增殖,其抑制率呈浓度-时间依赖性(P<0.05);TP作用细胞24h后,电镜下可见凋亡小体形成;流式细胞术分析表明,各浓度TP组G1期细胞明显升高,细胞凋亡率呈浓度依赖性(P<0.05);DNA电泳分析可见细胞凋亡特有的DNA断裂形成的"梯形"条带;免疫组化表明Caspase-7参与了TP诱导COC1/DDP细胞凋亡过程。结论:TP对COC1/DDP具有明显的杀伤和促凋亡作用,凋亡机制与Caspase-7蛋白表达上调有关。     

Expression and cytokine mediated modulation of adhesion/costimulation molecules ICAM-1(CD54) and LFA-3(CD58) in human ovarian cancer

Previous studies have shown that paracrine delivery of helper cytokines by transduced tumor cells can bypass the lack of the B7 costimulation pathway, which when engaged induces secretion of a number of cytokines, most notably IL-2 and which results in clonal T-cell proliferation and effector function. However, other costimulation signals such as those mediated by ICAM-1 and LFA-3 molecules remain crucial to increase antigen independent conjugate formation and therefore synergize with TcR activation pathways. In this study we have analyzed tumor cells from eight freshly isolated and histologically similar human ovarian tumors for their surface antigen expression of the adhesion/costimulation molecules ICAM-1 and LFA-3 before and after exposure to the cytokines TNF-α plus IFN-γ. All eight fresh tumors expressed variable levels of ICAM-1 antigens and these levels were markedly upregulated after exposure to TNF-α plus IFN-γ. Similarly, LFA-3 antigens were shown to be expressed in all fresh tumor evaluated. However, in contrast to the ICAM-1/LFA-1 pathway of costimulatory molecules, LFA-3/CD2 pathway was shown to be resistant to modulation with these cytokines. Such findings suggest that fresh ovarian tumor cells pretreated with TNF-α plus IFN-γ could significantly increase their adhesion/costimulation activity for T cell recognition when admixed with genetically manipulated tumor cells to be used as tumor vaccines.