A small molecule compound inhibits AKT pathway in ovarian cancer cell lines

BACKGROUND AND OBJECTIVE: Overactivation of AKT1 and gene amplification of AKT2 are frequently detected in ovarian cancer. Activated AKT kinases provide a cell survival signal that may confer resistance to apoptosis induced by conventional therapies in cancer cells. Therefore, development of potent inhibitors that block AKT pathway is an attractive therapeutic strategy for treating ovarian carcinoma. METHODS: Ovarian cancer cell lines, A2780, MDAH2774, OVCAR-8, Caov-3, and normal murine fibroblasts (NIH3T3) were used. Cells were treated with different doses of a non-peptide small molecule compound, 9-methoxy-2-methylellipticinium acetate (termed API-59-OME) that potentially inhibit AKT pathway. Kinase assays and the phosphorylation of AKT, GSK-3alpha/beta, PDK1, ERK1/2, SGK, p38, FAK, EGFR, JAK2, PKC isoforms, and the cleavage of poly (ADP-ribose) polymerase (PARP) were examined in treated and untreated cell lines. Further, cells treated with API-59-OME were analyzed for induction of apoptosis using sub-G1 profile with propidium iodide staining. RESULTS: API-59-OME inhibited AKT kinase activity but did not inhibit ERK or JNK kinase activities in A2780, MDAH2774, and OVCAR-8 cell lines. API-59-OME did not reduce phosphorylation of other protein kinases in these cell lines. API-59-OME induced apoptosis and the cleavage of PARP in A2780, MDAH2774, and OVCAR-8 ovarian cancer cell lines that express elevated levels of phosphorylated AKT. In contrast, in Caov-3 and NIH3T3 cell lines, which lack constitutive AKT activity, API-59-OME only had minimal effect to induce apoptosis. CONCLUSION: These data suggest that API-59-OME may be a potent agent to target constitutively activated AKT pathway in ovarian cancer cells.     

线粒体膜间蛋白Smac和卵巢癌

Smac是存在于线粒体并调节细胞凋亡的蛋白质,其促凋亡作用是通过逆转凋亡抑制蛋白(IAPs)尤其是X连锁凋亡抑制蛋白(XIAP)的作用实现的.当细胞受到凋亡刺激时,线粒体释放Smac蛋白到细胞质中,后者与IAPs结合,使其丧失抑制半胱氨酸天冬氨酸蛋白酶(caspase)活性的作用,从而促进细胞凋亡.细胞凋亡的减少与肿瘤的发生关系密切,认为Smac蛋白分别是caspase凋亡依赖途径和非caspase凋亡依赖途径的组成部分,其结构、功能、释放及调节成为研究热点.通过调控凋亡信号转导途径相关的胞内调控因子可能影响肿瘤细胞的化疗敏感性,因此Smac在卵巢肿瘤耐药性机制中的作用也受到关注.     

Thailandepsins are new small molecule class I HDAC inhibitors with potent cytotoxic activity in ovarian cancer cells: a preclinical study of epigenetic ovarian cancer therapy

Background

New treatment strategies are emerging to target DNA damage response pathways in ovarian cancer. Our group has previously shown that the class I biased HDAC inhibitor romidepsin (FK228) induces DNA damage response and has potent cytotoxic effects in ovarian cancer cells. Here, we investigated newly discovered HDAC inhibitors, thailandepsin A (TDP-A) and thailandepsin B (TDP-B), to determine the effects on cell viability, apoptosis and DNA damage response in ovarian cancer cells.

Methods

FK228, TDP-A and TDP-B were tested in five ovarian cancer cell lines. Cellular viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Immunofluorescence assays were used to assess activated caspase 3. Western blots were performed to detect protein expression of PARP cleavage, pH2AX, P-glycoprotein and tubulin acetylation.

Results

Treatment with TDPs decreased cell viability at nanonomolar concentrations in four of the five ovarian cancer cell lines studied. Similar to FK228, both TDP compounds exerted minimal effects on NCI/ADR-RES ovarian cancer cells. Across the four cell lines sensitive to the TDPs, TDP-B consistently had a greater inhibitory effect than TDP-A on cell viability. TDP-B also had relatively greater effects on promoting cell apoptosis and induction of pH2AX (a mark of DNA damage response), than TDP-A. These antitumor effects of TDP-B were of similar magnitude to those induced by an equal concentration of FK228. Similar to FK228, the nanomolar concentrations of the TDPs had little effect on tubulin acetylation (a mark of class II HDAC6 inhibition).

Conclusions

The new small molecule HDAC inhibitors TDP-A and TDP-B are FK228 analogues that suppress cell viability and induce apoptosis at nanomolar drug concentrations. TDP-B showed the most similarity to the biological activity of FK228 with greater cytotoxic effects than TDP-A in vitro. Our results indicate that FK228-like small molecule class I HDAC-biased HDAC inhibitors have therapeutic potential for ovarian cancer.     

Glucose deprivation activates AMPK and induces cell death through modulation of Akt in ovarian cancer cells

Objectives

Upregulation of glycolysis has been demonstrated in multiple tumor types. Glucose deprivation results in diminished intracellular ATP; this is counteracted by AMPK activation during energy deficiency to restore ATP levels. We sought to determine whether glucose deprivation could induce cytotoxicity in ovarian cancer cells through activation of AMPK, and whether AMPK activators could mimic glucose deprivation induced cytotoxicity.

Methods

Sensitivity to 2DG induced cytotoxicity and glucose deprivation was determined in a panel of ovarian cancer cells. Cellular growth rate, rate of glucose uptake, and response to glucose deprivation were determined. Expression of Glut-1, HIF1-α, AMPK and Akt was determined by immunoblotting.

Results

Incubation of ovarian cancer cells with glucose-free media, 2-DG and AMPK activators resulted in cell death. The glycolytic phenotype of ovarian cancer cells was present in both normoxic and hypoxic conditions, and did not correlate with HIF1-α expression levels. Sensitivity to glucose deprivation was independent of growth rate, rate of glucose uptake, and appeared to be dependent upon constitutive activation of Akt. Glucose deprivation resulted in activation of AMPK and inhibition of Akt phosphorylation. Treatment with AMPK activators resulted in AMPK activation, Akt inhibition, and induced cell death in ovarian cancer cells.

Conclusions

Ovarian cancer cells are glycolytic as compared to normal, untransformed cells, and are sensitive to glucose deprivation. Because ovarian cancer cells are dependent upon glucose for growth and survival, treatment with AMPK activators that mimic glucose deprivation may result in broad clinical benefits to ovarian cancer patients.     

A case of pulmonary type of ovarian small cell carcinoma

Small cell carcinoma is a rare form of ovarian cancer with a poor prognosis. It is divided into two types, the hypercalcemic and the pulmonary type, of which the latter is extremely rare. A 49-year-old woman presented with an acute abdomen and was suspected to have torsion of a left ovarian tumor, which was followed up with an emergency operation. Postoperative pathological examination gave a diagnosis of the pulmonary type of ovarian small cell carcinoma. Six courses of paclitaxel and carboplatin therapy were given as adjuvant chemotherapy. The patient has survived for 36 months without recurrence. Here we present an extremely rare patient with the pulmonary type of ovarian small cell carcinoma.     

Reversal of multidrug resistance by small interfering double-stranded RNAs in ovarian cancer cells

OBJECTIVE: Cisplatin (DDP) resistance is a major barrier to overcome before chemotherapy can become curative for most patients presenting with ovarian cancer. In this study, we investigated the effect of siRNAs on expression of p-gp, GST-pi mRNA and protein in cisplatin-resistant human ovarian cancer cells in order to restore sensitivity to DDP. METHODS: Small interfering double-stranded RNAs (siRNA) were designed to target p-glycoprotein (p-gp) and glutathione S-transferases (GST) mRNA as a strategy to inhibit both resistant gene expression at the mRNA level. Using Real-Time PCR and western blotting assay the changes of the RNA and protein levels of both drug resistant genes were studied. RESULTS: Transfection of MDR-1 and GST siRNAs into human multi-drug resistance (MDR) ovarian cancer cell lines, COC1/DDP and SKOV3/DDP, resulted in a time-dependent inhibition of both gene expressions with the decline of the IC(50) values but had no effect on the expression of a-Tubulin. Inhibition of P-gp and GST expression by siRNA enhanced the intracellular accumulation of and restored sensitivity to DDP. CONCLUSIONS: These studies suggest that p-gp and GST siRNAs are effective inhibitors of MDR gene expression and reverse the resistance of ovarian carcinomas. Our studies may provide a new insight to develop siRNAs as a novel therapeutic tool for the treatment of ovarian carcinomas.     

Rab25 siRNA诱导人卵巢癌细胞凋亡及其机制的研究

目的:通过RNA干涉抑制Rab25在人卵巢上皮性癌的过表达,探讨Rab25siRNA对肿瘤细胞凋亡的生物学效应及作用机制.方法:构建针对Rab25的siRNA重组质粒,稳定转染A2780细胞,通过RT-PCR检测Rab25的表达,用Hoechst 33258染色和TUNEL法研究Rab25 siRNA诱导细胞的凋亡;用免疫组化法分析抑制Rab25的表达对凋亡相关蛋白Bcl-2和Bax表达的影响.结果:Rab25 siRNA可诱导肿瘤细胞凋亡;蛋白水平检测表明抑制Rab25表达使Bcl-2表达下降,Bax表达增强.结论:Rab25 siRNA可诱导细胞凋亡,Rab25影响卵巢癌细胞生存途径是通过调节Bcl-2/Bax表达含量发挥作用.     

Single cell sequencing reveals heterogeneity within ovarian cancer epithelium and cancer associated stromal cells

Objectives

The purpose of this study was to determine the level of heterogeneity in high grade serous ovarian cancer (HGSOC) by analyzing RNA expression in single epithelial and cancer associated stromal cells. In addition, we explored the possibility of identifying subgroups based on pathway activation and pre-defined signatures from cancer stem cells and chemo-resistant cells.

Cl-channel blockers inhibit cell proliferation and arrest the cell cycle of human ovarian cancer cells

OBJECTIVE: To investigate the role of chloride channels in cell proliferation and cell cycles of human ovarian cancer cell line A2780. METHODS: Chloride channel blockers were used to observe the effects of chloride channels on A2780 cells with MTT assay and flow cytometry. RESULTS: NPPB (100 microM) significantly inhibited the cell proliferation and affected the cell cycle, which increased the percentage of cells in the G1 phase, and reduced it in the S phase. NFA (100 microM) and TAM (30 microM) had similar inhibitory effects. Glibenclamide (100 microM), however, had no effect on cell proliferation or cycle. Moreover, chloride channel blockers could inhibit Ca2+ influx in these cells. CONCLUSION: Chloride channels, voltage-gated chloride channels, and volume-sensitive chloride channels especially, play an important role in the cell proliferation and cycle of A2780 cells. It is likely that the influence of chloride channels on cell proliferation and cell cycle is mediated by a Ca2+-dependent mechanism.     

Rab25 siRNA抑制人卵巢癌细胞粘附侵袭的实验研究

目的通过RNA干涉抑制Rab25在人卵第上皮性癌的过表达，观察Rab25基因沉默对卵巢癌A2780细胞的生物学效应，探讨Rab25基因沉默对卵巢癌侵袭及转移能力的影响。方法根据基因库上的Rab25mRNA序列，构建针对Rab25的siRNA重组质粒，稳定转染A2780细胞，通过RT-PCR检测Rab25的表达，利用粘附实验和transwell小室实验检测其对细胞的粘附力及侵袭能力的影响。结果成功构建Rab25的siRNA重组质粒，稳定转染靶细胞后可显著降低Rab25的表达，并且与对照相比，Rab25siRNA可显著影响卵巢癌细胞的侵袭粘附能力（P〈0．01）。结论成功构建Rab25siRNA显著抑制肿瘤细胞的侵袭粘附能力，对Rab25的研究有望为卵巢癌的基因治疗开辟新途径。     

Endocrine cell micronests in an ovarian mucinous cystadenofibroma: a mimic of microinvasion.

An ovarian mucinous cystadenofibroma with peculiar neuroendocrine cell micronests is described in a 59-year-old Japanese woman. Aggregates of epithelial cells resembling microinvasive carcinoma cells were scattered throughout the adenofibromatous area. These micronests were composed of small uniform cells with argentaffin and argyrophil granules. Numerous small cells with neuroendocrine granules were also seen within mucinous glands. This is the first report of neuroendocrine micronests in an ovarian neoplasm, a finding that should be distinguished from microinvasion.     

Necrotic cell death of ovarian adenocarcinoma caused by seminal plasma

Objective. From the knowledge of risk factors of epithelial ovarian cancer, we deduced a hypothesis that human seminal plasma (HSP) has a preventive role in the development of epithelial ovarian cancer. To examine whether HSP directly influences the growth of ovarian cancer, we have investigated the in vitro and in vivo effect of HSP on ovarian adenocarcinoma cell lines (SK-OV-3 and OVCAR-3) in comparison with its effects on normal ovarian surface epithelial cells (NOSE).Methods. Cell viability was determined by MTT assay. Cytotoxic effect was evaluated by flow cytometry analysis, by DNA laddering, and by morphological analysis. In vivo therapeutic effect of HSP was evaluated by the subcutaneous inoculation of SK-OV-3 cells in nude mice (BALB-c) model.Results. HSP at a final concentration of 1:50 induced a time- and dose-dependent inhibition of SK-OV-3 and OVCAR-3 growth, whereas NOSE was not affected. Flow cytometric analysis, DNA laddering, and morphological analysis indicated that HSP induced necrosis, rather than apoptosis, of both ovarian carcinoma cell lines. In in vivo experiment that used the nude mice (Balb-C) with tumor inoculation of SK-OV-3 cells, HSP induced necrosis of tumor with no detectable toxic effects on the major organs.Conclusion. These results show that HSP inhibits the growth and induces the necrosis of epithelial ovarian cancer cells and suggests that one or more components of HSP may provide a scientific basis for preventing epithelial ovarian cancer.     

Regulation of growth of normal ovarian epithelial cells and ovarian cancer cell lines by transforming growth factor-beta.

OBJECTIVE: The purpose of this study was to study the role of transforming growth factor-beta in regulation of proliferation of normal and malignant ovarian epithelial cells. STUDY DESIGN: We examined production of and responsiveness to transforming growth factor-beta in primary monolayer cultures of epithelial cells from five normal human ovaries and in five ovarian cancer cell lines. RESULTS: In normal ovarian epithelial cells, proliferation always was inhibited by transforming growth factor-beta (greater than 40%) (p less than 0.01). Among the cancer cell lines, proliferation of one was markedly inhibited (greater than 95%) (p less than 0.01), two were only modestly inhibited (15% to 20%) (p less than 0.05), and two were unaffected. In addition, we found that all of the normal ovarian epithelial cells and four of five ovarian cancer cell lines produce transforming growth factor-beta ribonucleic acid and protein. CONCLUSIONS: These data suggest that transforming growth factor-beta may act as an autocrine growth inhibitory factor for normal ovarian epithelium in vivo. Because most of the ovarian cancer cell lines are relatively resistant to the growth inhibitory effect of transforming growth factor-beta and because one cell line does not produce transforming growth factor-beta, it is possible that loss of the transforming growth factor-beta pathway may play a role in the development of some ovarian cancers.     

卵巢癌拓扑替康耐药细胞株的建立及其生物学特性

目的 :建立卵巢癌拓扑替康 (TPT)耐药细胞株 ,研究其生物学特性。方法 :用浓度梯度递增法建立卵巢癌TPT耐药细胞株。用四甲基偶氮唑蓝 (MTT)比色法检测此耐药株对多种化疗药物的耐药指数 ;用细胞计数法描绘两株细胞的生长曲线并计算群体倍增时间 ;用流式细胞仪检测两株细胞的凋亡率及周期分布 ;荧光显微镜及透射电镜下观察亲本细胞、敏感细胞及耐药细胞的超微结构。结果 :历时 6个月建立了卵巢癌TPT耐药细胞株A2 780 /TPT。此细胞株对TPT及米托蒽醌 (MX)耐药 ,耐药指数分别为 2 5及 19,对喜树碱 (CPT)轻微耐药 ,耐药指数为 3,对其他多种化疗药物不交叉耐药。耐药细胞的生长速度比亲本细胞慢 (P <0 .0 5 ) ,且细胞凋亡率及G2 M期细胞比例较亲本细胞高 (P <0 .0 5 )。敏感细胞呈典型的凋亡形态特征 ,而耐药细胞凋亡核的数量明显减少 ,凋亡程度明显减轻 ,且仍有较多的细胞核形态正常。结论 :所建立的卵巢癌TPT耐药株具有耐药细胞的基本生物学特征 ,且该耐药模型稳定。