Induction of apoptosis by metformin in epithelial ovarian cancer: involvement of the Bcl-2 family proteins

Objective

The aims of the study were to evaluate the ability of metformin to induce apoptosis in epithelial ovarian cancer cell lines and to identify the pathways involved in this effect.

Methods

After treatment with metformin and/or cisplatin, OVCAR-3 and OVCAR-4 cellular apoptosis was assessed by flow cytometry and caspase 3/7 activity. Cell cycle analysis was also performed by flow cytometry as well. Modulation of protein expression of the Bcl-2 family after treatment with metformin and/or cisplatin was determined by Western blotting.

Results

Metformin induced apoptosis in OVCAR-3 and OVCAR-4 cell lines in an AMPK-independent manner and provoked a cell cycle arrest in the S and G2/M phase. Moreover, we established that metformin can induce apoptosis in OVCAR-3 and OVCAR-4 cells by activating caspases 3/7, down-regulating Bcl-2 and Bcl-xL expression, and up-regulating Bax and Bad expression. The induction of apoptosis by metformin was also enhanced by cisplatin and combination of these drugs did not modulate the expression of Bcl-2 family proteins in OVCAR-3 cell line, whereas the effect was enhanced in OVCAR-4 cell line.

Conclusion

Bcl-xL and Bcl-2 targeted strategies were suggested to constitute an effective therapeutic tool for the treatment of chemoresistant ovarian carcinoma, in conjunction with conventional chemotherapy. These data are relevant to ongoing translational research efforts and clinical trials exploring a possible protective effect of metformin against ovarian cancer, including Bcl-2 inhibition.     

Activation of mTOR signaling pathway associated with adverse prognostic factors of epithelial ovarian cancer

Objective

Activation of the mammalian target of rapamycin (mTOR) pathway enhances cell survival and growth by regulating the efficiency of protein translation. This study was conducted to evaluate the association of activated mTOR signaling molecules with the clinicopathologic characteristics in epithelial ovarian cancer.

Methods

Immunohistochemical staining with antibodies against p-4EBP1, p-mTOR, and p-p70S6K were performed on specimens of 103 patients with ovarian cancer. Tumors were classified as chemoresistant in cases where time to recurrence after the end of chemotherapy was shorter than 6 months.

Results

Expressions of p-mTOR, p-4EBP1, and p-p70S6K were detected in 47.6%, 85.4%, and 64.1% of all patients, respectively. p-4EBP1 overexpression was associated with advanced stage (p = 0.04), histologic grade (p < 0.01), residual mass (p < 0.01), shorter disease-free survival rate (p = 0.01) and chemoresistance (p = 0.02). p-p70S6K was associated with residual mass with marginal significance (p = 0.06). p-4EBP1 expression was correlated with p-p70S6K expression (r = 0.42, p < 0.01), whereas p-mTOR was not associated with expression of its downstream effectors or prognostic factors.

Conclusions

Our findings suggest that p-4EBP1 expression was associated with poor prognostic factors of ovarian cancer and that p-4EBP1 overexpression may be a prognostic biomarker of ovarian cancer.     

p70S6K在小鼠受精卵第一次有丝分裂周期中的表达

目的:研究p70S6K在小鼠受精卵第一次有丝分裂周期中的表达。方法:采用Westernblot和RT-PCR方法,检测在小鼠受精卵第一次有丝分裂的G1、S、G2以及M期中p70S6K在mRNA水平以及蛋白水平的表达。结果:在mRNA水平,p70S6K的表达在各期无明显变化,在蛋白水平,磷酸化状态的p70S6K在S期升高,表明从S期p70S6K的活性升高。结论:p70S6K在小鼠卵子受精后磷酸化程度增加,因而有可能参与了小鼠受精卵的早期发育。     

荷人卵巢上皮癌裸鼠冻融卵巢组织移植的效果评价

目的:获取人卵巢上皮癌裸鼠原位移植瘤癌旁正常卵巢组织,经安全筛查并冻融后移植至去势裸鼠体内,探讨移植后效果,为临床应用提供依据。方法:将人卵巢上皮癌OVCAR3细胞种植于裸鼠皮下以获取瘤源,并进行卵巢原位移植,建立卵巢癌原位移植瘤模型,解剖裸鼠获取癌旁正常卵巢组织。癌旁组:取筛选后的癌旁卵巢组织进行玻璃化冷冻,复苏后分别进行皮下及原位移植,各20例;对照组:同龄正常裸鼠卵巢组织,皮下移植及原位移植各20例;去势裸鼠组:20只同龄去势裸鼠;正常卵巢组:20只同龄正常裸鼠。移植12周后,分析各组裸鼠卵巢组织内卵泡形态及激素分泌功能。结果:40只人卵巢上皮癌裸鼠原位移植瘤模型中共获取35份活检正常的癌旁卵巢组织,获取率87.5%(35/40)。玻璃化冷冻前后卵巢组织中卵泡形态及各级卵泡比例均无显著差异(P>0.05),癌旁冷冻组织和癌旁新鲜组织的异常卵泡比例差异无统计学意义(P>0.05),冷冻组织以初级卵泡及次级卵泡等窦前卵泡为主。癌旁组皮下移植组织存活率80%(16/20),对照组皮下移植组织存活率90%(18/20),癌旁组原位移植组织存活率90%(18/20),对照组原位移植组织存活率95%(19/20)。移植后组织内卵泡以次级卵泡及窦状卵泡为主,各级卵泡的形态及构成比和未移植的同龄正常裸鼠卵巢相似(P>0.05);癌旁组卵泡数明显低于对照组(P<0.05)及正常卵巢组(P<0.01);皮下移植和原位移植组织内的各级卵泡数差异无统计学意义(P>0.05)。癌旁组卵泡刺激素水平明显低于去势裸鼠组,而雌二醇水平明显高于去势裸鼠组(P<0.01);癌旁组卵泡刺激素水平明显高于对照组(P<0.05)及正常卵巢组(P<0.01),而雌二醇水平明显低于对照组(P<0.05)及正常卵巢组(P<0.01);皮下移植和原位移植的卵泡刺激素水平和雌二醇水平差异无统计学意义(P>0.05)。结论:癌旁卵巢组织冻融移植后有正常卵泡发育及激素分泌功能;皮下移植和原位移植均可取得较好的效果;癌旁卵巢组织冻融移植有望作为卵巢上皮癌患者治疗后恢复卵巢内分泌功能的有效手段。     

Metformin targets ovarian cancer stem cells in vitro and in vivo

Purpose

Studies in non-gynecologic tumors indicate that metformin inhibits growth of cancer stem cells (CSC). Diabetic patients with ovarian cancer who are taking metformin have better outcomes than those not taking metformin. The purpose of this study was to directly address the impact of metformin on ovarian CSC.

Methods

The impact of metformin on ovarian cancer cell line growth and viability was assessed with trypan blue staining. Aldehyde dehydrogenase (ALDH) expressing CSC were quantified using FACS®. Tumor sphere assays were performed to determine the impact of metformin on cell line and primary human ovarian tumor CSC growth in vitro. In vivo therapeutic efficacy and the anti-CSC effects of metformin were confirmed using both tumor cell lines and ALDH(+) CSC tumor xenografts.

Results

Metformin significantly restricted the growth of ovarian cancer cell lines in vitro. This effect was additive with cisplatin. FACS analysis confirmed that metformin reduced ALDH(+) ovarian CSC. Consistent with this, metformin also inhibited the formation of CSC tumor spheres from both cell lines and patient tumors. In vivo, metformin significantly increased the ability of cisplatin to restrict whole tumor cell line xenografts. In addition, metformin significantly restricted the growth of ALDH(+) CSC xenografts. This was associated with a decrease in ALDH(+) CSC, cellular proliferation, and angiogenesis.

Conclusions

Metformin can restrict the growth and proliferation of ovarian cancer stem cells in vitro and in vivo. This was true in cell lines and in primary human CSC isolates. These results provide a rationale for using metformin to treat ovarian cancer patients.     

Anti-diabetic doses of metformin decrease proliferation markers in tumors of patients with endometrial cancer

Background

Metformin has been associated with reduced cancer risk. The mechanisms underlying this cancer protective effect remain unknown.

Methods

“Window of opportunity” study of metformin in women with operable endometrial cancer (EC). Eleven newly diagnosed, untreated, non-diabetic patients with EC received metformin 500 mg tid from diagnostic biopsy to surgery. Fasting plasma insulin, insulin-like growth factor 1 (IGF-1), insulin-like growth factor binding protein 1 (IGFBP-1) and insulin-like growth factor binding protein 7 (IGFBP-7) measurements were taken before and after metformin treatment. Ki-67, pAMPK, and pS6 immunohistochemistry staining was performed on the endometrial cancer before and after metformin treatment and was compared to a control group of 10 women with EC who did not receive metformin.

Results

Metformin was administered for a mean of 36.6 days. None of the patients suffered side effects requiring withdrawal from the study. The study group comprised 8 patients with endometrioid EC, and 3 non-endometrioid EC, with a mean follow-up time of 57 months. Mean plasma insulin (p = 0.0005), IGF-1 (p = 0.001), and IGFBP-7 (p = 0.0098) were significantly reduced after metformin treatment. A clear reduction in ki-67 and pS6 expression was observed by both conventional light microscope analysis and digital image analysis with a significant mean reduction in percentage of cells staining for ki-67 (9.7%, P = 0.02) and pS6 (31%, P = 0.03). In the non-treated control group expression was similar between the biopsy and the surgical specimens.

Conclusions

This pilot trial presents biological evidence consistent with anti-proliferative effects of metformin in women with EC in the clinical setting.     

Translational and pharmacological principles of hyperthermic intraperitoneal chemotherapy for ovarian cancer

The long-term survival of advanced-stage ovarian cancer patients remains poor, despite extensive cytoreductive surgery, chemotherapy, and the recent addition of poly (ADP-ribose) polymerase inhibitors (PARPi). Hyperthermic intraperitoneal chemotherapy (HIPEC) has shown survival benefit by specifically targeting peritoneal metastases, the primary site of disease recurrence. Different aspects of how HIPEC exerts its effect remain poorly understood. Improved understanding of the effects of hyperthermia on ovarian cancer cells, the synergy of hyperthermia with intraperitoneal chemotherapy, and the pharmacological and pharmacokinetic properties of intraperitoneally administered cisplatin may help identify ways to optimize the efficacy of HIPEC. This review provides an overview of these translational and pharmacological principles of HIPEC and aims to expose knowledge gaps that may direct further research to optimize the HIPEC procedure and ultimately improve survival for women with advanced ovarian cancer.     

复发性卵巢上皮癌二次细胞减灭术62例分析

目的 探讨复发性卵巢上皮癌二次细胞减灭术的意义及可行性。方法 62例复发性卵巢上皮癌接受了二次细胞减灭术。其中48例手术成功，14例仅做探查术。术式包括部分肠切除、肠修补、脾切除、残余大网膜切除及其它转移瘤切除。仅作探查术者是由于转移癌浸润大血管及胰十二指肠等重要脏器，肠系膜呈蜡肠状等。术后辅以铂剂为主的联合化疗。结果 残余瘤直径小于2cm的27例2年和5年存活率分别为40．7％及14．8％。残余瘤大于2cm的35例只有1例存活超过2年（2．9％）。结论 成功的二次肿瘤细胞减灭术能提高患者的存活率。术前作好检查筛去不适合手术病例会提高成功率。     

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.     

HDAC10 as a potential therapeutic target in ovarian cancer

Objective

We analyzed histone deacetylase 10 (HDAC10) for function in the context of the DNA damage response in BRCA1-null ovarian cancer cells as well as evaluated the potential of general HDAC inhibitors in primary ovarian carcinoma cells. HDAC10 had previously been shown to be highly stimulatory to the process of homology directed repair in HeLa cells, and in this study we investigated whether HDAC10 could impact in vitro the response to anticancer therapies. We hypothesized that the loss of HDAC10 would sensitize cells to platinum therapy.

Prevalence of cysts in epithelial ovarian cancer

Objective

Ovarian carcinomas mostly appear as large cystic masses. However, the exact prevalence of cysts in epithelial ovarian cancer (EOC) has never been documented as well as the tumor factors that are related to the presence of cysts. Demonstrating the prevalence of cysts in EOC is essential for research focused on predictive and prognostic biomarkers in ovarian cyst fluid.

Study design

From 233 patients with primary EOC who underwent surgery, pathological data were collected from pathology reports. Univariate and multivariate logistic regression were used to analyze the relationship between the presence of cysts and other tumor characteristics.

Results

Cysts in EOC were present in 83.7% of the patients and were mostly (61%) multilocular. The most common histological subtypes (serous, mucinous, endometrioid, clear cell) contained cysts in more than 85% of the cases. In univariate regression analysis, early FIGO stage, low tumor grade and a large tumor size were significantly associated with the presence of cysts (OR (95% CI) = 5.312 (1.81-15.57), 6.906 (2.31-20.66) and 1.169 (1.08-1.27), respectively). In multivariate regression analysis, apart from tumor size, only tumor grade was independently associated with the presence of cysts (adjusted OR (95% CI) = 4.234 (1.36-13.22)).

Conclusions

The large majority of all EOCs contained cysts. Histological subtype, FIGO stage, tumor necrosis and age were not associated with the presence of cystic EOC. In contrast, tumor grade and tumor size were independently related to the presence of cystic EOC. This means that cystic EOCs represent a subgroup of larger and more well-differentiated tumors. The evident relationship between the presence of cysts and differentiation grade is interesting from a clinical point of view as grading is especially important for the prognosis and treatment of patients with stage I EOC.     

二甲双胍对卵巢癌细胞侵袭迁移及MTA1表达的影响

目的:体外细胞培养实验检测二甲双胍对卵巢癌细胞SKOV3侵袭迁移能力的影响,以及其对转移相关因子1(MTA1)表达的影响。方法:用不同浓度二甲双胍处理卵巢癌细胞,采用划痕实验和transwell实验检测二甲双胍对细胞侵袭迁移能力的影响,Western blot法检测二甲双胍对细胞中MTA1蛋白表达的影响,qRT-PCR检测二甲双胍对细胞中MTA1 mRNA表达的影响。结果:与对照组(0mmol/L)相比,二甲双胍干预组的细胞迁移速度明显下降(P0.01);作用24h时,2.5mmol/L与5mmol/L组未见差异(P0.05);作用48h时,卵巢癌细胞的迁移速度随着二甲双胍浓度的增加而减弱(P0.01)。随着二甲双胍浓度的增加,穿膜细胞数减少(P0.01);二甲双胍干预组的MTA1 mRNA及MTA1蛋白表达明显降低(P0.05),不同药物浓度之间无差异。结论:二甲双胍抑制SKOV3细胞侵袭迁移,抑制作用与浓度呈正相关,其机制可能与抑制MTA1表达有关。     

胰岛素样生长因子1受体与卵巢癌SKOV3细胞顺铂耐药相关性的研究

目的:探讨胰岛素样生长因子1受体(IGF-1R)与卵巢癌SKOV3细胞顺铂耐药的相关性。方法:建立人卵巢癌细胞系SKOV3异种移植瘤模型,其中部分用顺铂处理;由此模型获得细胞分为SKOV3-P组(未化疗组)和SKOV3-R组(化疗组)。采用免疫细胞化学法和流式细胞术检测瘤细胞中IGF-1和IGF-1R的表达差异;MTT法和流式细胞术分别检测不同浓度顺铂(0、1、5、10、15、20μg/ml)和IGF-1R抑制剂NVP-AEW541(0、1、5、10、20μmol/L)单独及联合应用对瘤细胞的增殖及凋亡的影响。结果:SKOV3-P组、SK-OV3-R组细胞中均表达IGF-1和IGF-1R,且SKOV3-R组细胞的表达均显著高于SKOV3-P组细胞(P<0.05);NVP-AEW541对SKOV3-P组、SKOV3-R组细胞均表现增殖抑制,呈现剂量-时间依赖关系;NVP-AEW541联合顺铂作用瘤细胞时抑制作用显著增强,移植瘤细胞的顺铂敏感性显著增加(P<0.05);流式细胞术检测联合用药组细胞(10μmol/LNVP-AEW541+10μg/ml顺铂)凋亡率明显高于对照组和单独用药组(10μmol/L NVP-AEW541),且SKOV3-P组细胞的抑制率和凋亡率均显著高于SKOV3-R组细胞(P<0.05)。结论:IGF-1R参与了化疗耐药过程,通过NVP-AEW541抑制IGF-1R可能逆转卵巢癌细胞的顺铂耐药。     

二甲双胍通过下调IL-6抑制卵巢癌肿瘤相关成纤维细胞活性的研究

目的:探讨二甲双胍对卵巢癌肿瘤相关成纤维细胞(CAF)活性的影响,以及可能的调控机制。方法:RT-PCR法检测二甲双胍作用于SKOV3细胞系及原代CAF后炎症因子(IL-6、OPN、IL-1b、COX-2、Cyr61)mRNA水平变化;将MRC5于SKOV3-CM(condition medium)培养7~10天得到活化的MRC5即MRC5-CAF,原代CAF及MRC5-CAF经免疫荧光鉴定α-SMA表达;Western blot检测上述炎症因子蛋白水平。在TCGA数据库557例卵巢癌中验证炎症因子与CAF属性相关基因的相关性。免疫荧光验证活化的MRC5中二甲双胍对α-SMA及IL-6表达的影响。在MRC5-CAF细胞系中进一步验证了二甲双胍或IL-6对CAF属性相关基因的影响。增殖实验和Transwell实验比较二甲双胍或IL-6对MRC5-CAF促进SKOV3增殖及迁移能力的影响。胶原回缩试验比较二甲双胍或IL-6对MRC5-CAF收缩细胞外基质ECM的影响。结果:二甲双胍下调CAF中炎症因子尤其是IL-6,同时下调CAF中α-SMA水平。TCGA数据库中IL-6 mRna与CAF属性相关基因(FAP、PDGFRB、FN1、COLL6A6)呈显著正相关(P0.0001);二甲双胍、IL-6共刺激组较IL-6组,STAT3通路和α-SMA下调;二甲双胍能逆转IL-6对卵巢癌CAF促肿瘤增殖转移及收缩胶原的能力。结论:卵巢癌中IL-6可能参与维持了间质CAF细胞活性和间质属性,二甲双胍可能通过下调CAF中IL-6/P-STAT3通路从而抑制卵巢癌CAF对肿瘤细胞的支持作用。     

Cytoreductive surgery in primary advanced epithelial ovarian cancer

Epithelial ovarian cancer is one of the most common malignancy and one of the principal causes of death among gynaecological neoplasm. The majority of patients (about 70%) present with an advanced International Federation of Gynaecology and Obstetrics stage disease. The current standard treatment for these patients consists of complete cytoreduction and combined systemic chemotherapy (CT). An increasing proportion of patients undergoing complete cytoreduction to no gross residual disease (RD) is associated with progressively longer overall survival. As a counterpart, some authors hypothesized the improving in survival could be due more to a less diffused initial disease than to an increase in surgical cytoreduction rate. Moreover the biology of the tumor plays an important role in survival benefit of surgery. It’s still undefined how the intrinsic features of the tumor make intra-abdominal implants easier to remove. Adjuvant and hyperthermic intraperitoneal CT could play a decisive role in the coming years as the completeness of macroscopic disease removal increases with advances in surgical techniques and technology. The introduction of neo-adjuvant CT moreover will play a decisive role in the next years Anyway cytoreduction with no macroscopic residual of disease should always be attempted. However the definition of RD is not universal. A unique and definitive definition is needed.     

拓扑替康联合顺铂治疗卵巢上皮性癌的临床研究

目的 探讨拓扑替康联合顺铂（TP）治疗卵巢上皮性癌（卵巢癌）的疗效、毒副反应及预后。方法 将手术后经病理检查确诊为Ⅱ～Ⅳ期卵巢癌的94例患者分为3组：（1）TP组：30例,给予顺铂75mg／m^2,第1天;拓扑替康0．75mg·m^-2·d^-1,第1～5天。（2）紫杉醇＋卡铂（TC）组：31例,卡铂剂量按（血浆浓度-时间）曲线下面积（AUC）=5给予,第1天;紫杉醇135mg／m^2,第1天。（3）环磷酰胺＋顺铂（PC）组：33例,给予顺铂75mg／m^2,第1天;环磷酰胺500mg／m^2,第1天。3组患者均以21～28d为1个化疗周期,6～8个疗程后评价疗效,完全缓解（CR）加部分缓解（PR）为有效。比较3组患者的疗效、毒副反应及预后。结果 （1）化疗反应率：TP组CR为8例,PR为13例,有效率为70％（21／30）;TC组CR为10例,PR为14例,有效率为77％（24／31）;PC组CR为5例,PR为9例,有效率为42％（14／33）。TP组有效率与TC组相比,差异无统计学意义（P〉0．05）;与PC组相比,差异则有统计学意义（P〈0．05）。（2）无瘤生存率：中位随访时间25个月,TP、TC、PC组患者1年无瘤生存率分别为67％、71％、42％;2年无瘤生存率分别为57％、64％、39％。各组间1年及2年无瘤生存率分别比较,差异均无统计学意义（P〉0．05）。（3）总生存率：TP、TC、PC组1年总生存率分别为93％、97％、91％;2年总生存率分别为77％、84％、67％。各组间1年及2年总生存率分别比较,差异均无统计学意义（P〉0．05）。（4）毒副反应：发生Ⅲ～Ⅳ度骨髓抑制的患者,TP组为18例（60％）,TC组为8例（26％）,PC组为10例（30％）,TP组分别与TC、PC组比较,差异均有统计学意义（P〈0．05）;其他毒副反应,3组间比较,差异均无统计学意义（P〉0．05）。结论 作为卵巢癌的一线化疗方案,TP方案虽不能取代TC方案,但可作为临床治疗的一种选择。