NK4基因转染对人卵巢癌细胞SKOV-3生物学特性的影响

目的 通过在人卵巢癌细胞SKOV-3中转染NK4基因表达片段,研究NK4对人卵巢癌的治疗作用,从而验证NK4可作为卵巢癌潜在的基因治疗新策略.方法 用NK4和荧光霉素表达质粒分别稳定转染人卵巢癌细胞SKOV-3.用West-ern blot检测细胞培养基中NK4蛋白表达,以及经不同培养基(SKOV-3、SKOV-3/LUC和SKOV-3/NK4培养上清)培养后SKOV-3细胞中c-Met和磷酸化-c-Met的表达.用MTT试剂盒绘制细胞生长曲线.用细胞划痕试验检测NK4对细胞转移的作用.结果 在SKOV-3/NK4培养基中有NK4蛋白表达,对照(SKOV-3,SKOV-3/LUC)细胞中无表达.磷酸化-c-Met在SKOV-3/NK4培养基培养的SKOV-3细胞中被抑制,而在对照(SKOV-3,SKOV-3/LUC)细胞培养基培养的SKOV-3细胞中正常表达.c-Met表达在各组差异无统计学意义.三种细胞体外生长曲线差异无统计学意义(P＞0.05).细胞划痕试验表明SKOV-3/NK4划痕区域的细胞个数明显少于SKOV-3和SKOV-3/LUC细胞.结论 NK4蛋白在SKOV-3/NK4细胞培养基中大量分泌,NK4能抑制人卵巢癌细胞c-Met受体磷酸化,并能抑制卵巢癌细胞体外活动能力,为应用NK4作为卵巢癌基因治疗方法提供了研究基础.     

NK4基因转染对裸鼠卵巢癌腹水瘤生长影响及其机制的探讨

目的 在人卵巢癌细胞SKOV-3中转染NK4cDNA片段,研究NK4在裸鼠体内对人卵巢癌的治疗作用.方法 NK4和荧光霉素(luciferase)表达质粒分别转染人卵巢癌细胞SKOV-3,稳定转染后,得到SKOV-3/NK4细胞和对照(SKOV-3/LUC)细胞.检测NK4在细胞培养基中的表达情况以及经不同培养基(SKOV-3、SKOV-3/LUC和SKOV-3/NK4培养基上清)培养后SKOV-3细胞中c-Met和磷酸化-c-Met的表达,检测三组肿瘤细胞(SKOV-3/LUC和SKOV-3/NK4)体外生长速度,建立裸鼠卵巢癌腹水瘤模型,用Kaplan-Meier方法比较SKOV-3/LUC和SKOV-3/NK4细胞接种形成腹水瘤的裸鼠的累积生存率的差异.结果 在SKOV-3/NK4培养基中有NK4蛋白表达,对照(SKOV-3和SKOV-3/LUC)细胞中无表达.磷酸化-c-Met在SKOV-3/NK4培养基培养的SKOV-3细胞中被抑制,而在对照(SKOV-3和SKOV-3/LUC)细胞培养基培养的SKOV-3细胞中正常表达.c-Met表达在各组差异无统计学意义.三种细胞体外生长曲线比较差异无统计学意义(P＞0.05).腹腔内接种SKOV-3/LUC细胞的裸鼠,均产生腹腔内种植肿瘤伴腹水形成,于50 d内相继死亡,而接种SKOV-3/NK4细胞的裸鼠生存期均大于70 d,差异有统计学意义(P＜0.01).结论 SKOV-3/NK4细胞在培养基中大量分泌NK4蛋白,NK4能抑制人卵巢癌细胞c-Met受体磷酸化.在裸鼠体内NK4通过抗血管生成作用抑制卵巢癌细胞腹腔内种植瘤的生长和腹水形成,提高累积生存率,改善预后.NK4可作为卵巢癌基因治疗的新方法.     

小干扰RNA靶向EGFR基因逆转卵巢癌细胞顺铂耐药的研究

目的：探讨载体表达的小干扰RNA（siRNA）影响卵巢癌耐药细胞株EGFR基因的表达并逆转其顺铂耐药的可行性。方法：体外构建EGFR小发卡状RNA（shRNA）的表达质粒,脂质体法介导将其转染入SKOV3/DDP细胞。实验分为正常对照组、空质粒转染组、非特异性转染组和特异性转染组。采用逆转录聚合酶链反应（RT-PCR）检测EGFR mRNA的表达;使用免疫细胞化学法（ICC）检测EGFR蛋白的表达;使用四甲基偶氮唑蓝法（MTT）测定各组细胞对顺铂的半数抑制浓度（IC50）。结果：EGFR shRNA转染组细胞EGFR mRNA的表达与其他两组相比明显减弱（P〈0.01）,EGFR蛋白表达明显下调（P〈0.01）;顺铂敏感性比正常对照组提高了约2.5倍。结论：针对EGFR合成的siRNA能够有效地抑制EGFR mRNA和蛋白的表达,并能恢复其对顺铂的敏感性。应用RNAi技术,能够逆转卵巢癌细胞对化疗药物的耐药性。     

一种新的人源化抗CD19嵌合抗原受体NK细胞的制备和对体外肿瘤细胞杀伤作用

摘要 目的：构建人源化抗CD19嵌合抗原受体NK细胞（hCAR19-NK）,并且在体外证明其对CD19 阳性血液病肿瘤细胞杀伤作用。方法：构建人源化的第二代CD19 CAR的逆转录病毒载体,使用辐照的K562-4-1BBL-mIL21细胞刺激外周血来源的NK细胞,通过逆转录病毒转导NK细胞获得hCAR19-NK细胞;采用流式细胞术和Western blot检测转导效率;采用4 h荧光杀伤实验和ELISA法检测hCAR19-NK细胞对淋巴瘤细胞的杀伤能力和IFN-γ释放水平;采用CD107a脱颗粒实验评估淋巴瘤细胞对hCAR19-NK细胞的特异性激活;比较对照组（Mock）和hCAR19-NK组细胞扩增倍数。结果：流式细胞术和Western blot 证明构建的CAR可以成功转导外周血来源的NK细胞;4 h荧光杀伤实验证明随着效靶比例升高,hCAR19-NK对Raji-GL杀伤率增加,明显高于Mock组;ELISA法检测显示Raji和K562-CD19作为靶细胞时,hCAR19-NK细胞的IFN-γ释放明显高于Mock组（P<0.01）;CD19⁺细胞（Raji和K562-CD19）可以特异性刺激hCAR19-NK细胞表达CD107a,具有统计学意义（P<0.05）;Mock组和hCAR19-NK组细胞扩增倍数无显著差异。结论：成功构建了可以杀伤CD19⁺ 肿瘤的人源化scFv的第二代hCAR19-NK细胞。     

靶向survivin的siRNA对胃癌BGC-823细胞增殖的影响

目的探讨干扰RNA沉默生存素（survivin）基因表达对人胃癌BGC-823细胞增殖和凋亡的影响。方法设计并合成3条靶向survivin的小分子干扰RNA（siRNA）,构建表达性干扰RNA质粒（shRNA）——shRNA-survivin-1、shRNA-survivin-2和shRNA-survivin-3,分别转染胃癌BGC-823细胞,实时定量PCR检测干扰RNA沉默survivin mRNA表达效果,Westernblot观察对胃癌BGC-823细胞survivin蛋白质表达的抑制,MTT（四甲基偶氮唑盐）比色法分析检测细胞生长抑制率,流式细胞计数检测各组细胞周期和凋亡率,探讨干扰RNA对胃癌BGC-823细胞生长的影响。结果在体外,shRNA-survivin-1有效沉默人胃癌BGC-823细胞survivin mRNA的表达,使sur-vivin mRNA相对水平明显降低（P〈0.05）,survivin蛋白质表达抑制,72h细胞生长抑制率达74.92%（P〈0.05）,shRNA-survivin-1使G2/M期细胞百分比明显增加,凋亡率显著增加（P〈0.05）。结论 shRNA-survivin-1可以沉默survivin基因的表达,可以显著抑制胃癌BGC-823细胞的增殖,在一定程度上诱导其自发凋亡。本研究为靶向sur-vivin的RNA干扰在胃癌的基因治疗提供了有力的理论依据和技术储备。     

TnI-fast基因表达抑制卵巢癌移植瘤生长

为探讨利用TnI-fast 基因进行卵巢癌基因治疗的有效性及其机制, 将TnI-fast基因 cDNA转染人卵巢癌细胞系SKOV3. 采用MTT法和流式细胞技术分别检测TnI-fast基因转染、空载体转染和未转染的SKOV3细胞体外生长状态. 收集3种细胞培养上清液, 检测3种培养上清液对人脐静脉内皮细胞增殖抑制效应. 3种细胞分别接种到裸鼠, 观察肿瘤生长、细胞凋亡、肿瘤血管生成和TnI-fast基因局部表达. 体外试验发现, 与空载体转染和未转染的SKOV3细胞比较, TnI-fast基因表达对肿瘤细胞自身的生长无抑制作用, 但可抑制人脐静脉内皮细胞增殖. 动物实验中, TnI-fast基因表达可显著抑制肿瘤生长, 生长抑制率达73%. 其肿瘤细胞增殖率与对照组相当, 但微血管密度显著降低, 细胞凋亡显著增加. 提示, 肿瘤自身血管生成抑制可显著延缓卵巢癌生长. 利用血管生成特异性抑制基因TnI-fast进行抗肿瘤血管生成基因治疗可作为肿瘤治疗的新策略之一.     

neuronatin shRNA真核表达载体的构建及其生物学功能

目的:获得能持续干扰neuronatin（nnat）基因表达的细胞,观察nnat基因沉默对神经细胞发育与分化的影响,为研究基因功能奠定基础。方法:构建含nnat基因短发夹RNA(shRNA)表达质粒,将质粒转染大鼠肾上腺嗜铬细胞瘤细胞PC12,RT-PCR方法筛选出最有效干扰质粒,稳定转染PC12细胞后观察细胞表型变化,免疫荧光检测nnat蛋白表达,NGF诱导观察nnat表达下调对细胞分化的影响。结果:成功构建并筛选出有效的靶向nnat基因的shRNA真核表达载体;载体稳定转染PC12细胞之后能特异性沉默nnat基因的表达,PC12细胞长出突起,向神经元方向分化,加入诱导因子NGF后能促进突起生长。结论:nnat可能是作为神经分化抑制因子在神经发育与成熟过程中发挥作用。     

肿瘤对自然杀伤细胞的杀伤敏感性研究

自然杀伤细胞（NK细胞）是机体抗肿瘤的第一道防线,是机体天然免疫的主要承担者,也是获得性细胞免疫的核心调节细胞。NK细胞对肿瘤的杀伤活性与其细胞表面受体和肿瘤细胞表面的配体密切相关,由于肿瘤细胞表面配体表达不同致使对NK细胞杀伤的敏感性有很大差异,临床疗效不一。我们简要介绍了影响肿瘤细胞对NK细胞杀伤敏感性的机制,对目前提高肿瘤细胞敏感性的策略和方法进行了总结。     

Maspin在卵巢癌中作用机制的体外研究

目的:检测卵巢癌细胞中Maspin对细胞增殖能力、VEGF和乙酰肝素酶Heparanase(HPA)表达的影响。方法:构建Maspin真核表达质粒,体外转染卵巢癌SKOV3细胞,磺基罗丹明B法((sulforhodamine B,SRB)检测转染后细胞增殖能力变化,细胞免疫化学及半定量逆转录聚合酶链反应(RT-PCR)方法检测VEGF和HPA的表达。结果:成功构建Maspin真核表达质粒并转染于卵巢癌细胞SKOV3后,证实转染目的基因的SKOV3-Maspin细胞中Maspin表达明显增强;转染Maspin cDNA的SKOV3细胞的体外增殖能力明显弱于转染空载体组和未转染组,而后两者之间差异无显著性;转染Maspin cDNA的SKOV3细胞中VEGF的表达明显弱于转染空载体组和未转染组,而后两者之间差异无显著性;但是HPA的表达无明显改变。结论:Maspin可能通过下调VEGF的表达抑制卵巢癌血管生成。HPA表达与卵巢癌的恶性生物学行为密切相关,但与Maspin无明显相关性。     

沉默MDR1基因增强急性早幼粒白血病耐药细胞HT9药物敏感性

该研究利用短发卡RNA（small hairpinin RNA,shRNA）表达载体沉默HT9急性早幼粒白血病耐药细胞MDRl基因表达,以提高细胞对三尖杉酯碱、阿霉素的敏感性。通过设计合成编码shRNA的DNA模板序列,定向克隆到pSilencer2．1－u6neo质粒,成功构建1个P－gP蛋白基因特异的shRNA表达载体,稳定电转染HT9细胞,实时荧光定量PCR分析MDRlmRYAg表达,Westem blot检测细胞P—gp蛋白表达,流式细胞术检测P—gP蛋白外排泵功能,MTT法检测细胞对药物敏感性。结果显示,成功构建了shRNA表达载体pSilencer2-1-U6neo—MDRl,转染HT9细胞后,PCR检测重组质粒整合到HT9／sh－2－1－1细胞基因组DNA,获得稳定遗传;HT9／sh－2－1—1细胞MDRlmRNA表达降低了78．84％（P〈0．01）,P—gP蛋白表达降低了48．27％（P〈0．05）,细胞内Rh0123相对荧光强度由（10．8±0．58）％升高至（73．56±1．37m;转染细胞对三尖杉酯碱、阿霉素敏感性明显增强,IC50分别由（2．06±0．15）gmol／L降至（0．57±0．01）gmol／L、（4．04±017）gmol／L降至（1．56±0．05）μtmol／L。提示shRNA干扰表达载体pSilencer2．1－U6neo—MDRl能够稳定、持久地抑制MDRI基因表达,并能有效增强HT9细胞对三尖杉酯碱、阿霉素的敏感性。     

Cosmc基因在卵巢癌中生物学功能的研究

目的:明确Cosmc基因在卵巢癌细胞中的生物学功能。方法:本研究采用慢病毒转染技术,在卵巢癌细胞A2780和SKOV3中过表达Cosmc基因,MTT实验、细胞凋亡实验以及Transwell侵袭实验对过表达Cosmc后卵巢癌细胞在生长、凋亡、侵袭等方面的影响。结果:慢病毒转染卵巢癌细胞A2780和SKOV-3后,Cosmc的蛋白表达显著提高;MTT结果显示,与空载体对照组相比,过表达Cosmc的卵巢癌细胞生长能力显著减弱;流式细胞术结果表明Cosmc过表达的卵巢癌细胞凋亡数较空载体对照组细胞显著增加;Transwell实验显示Cosmc过表达的细胞侵袭和迁移能力较空载体对照组显著减少。结论:卵巢癌细胞系中过表达Cosmc基因能抑制卵巢癌细胞的生长和侵袭并促进细胞凋亡。     

Nanog 基因在卵巢癌和卵巢肿瘤干细胞中的表达及意义

目的:探讨胚胎干细胞关键因子Nanog基因mRNA及其蛋白在卵巢癌和卵巢癌肿瘤干细胞中的表达及意义。方法:选取10例正常卵巢上皮组织、10例卵巢良性肿瘤及60例卵巢癌组织,采用逆转录酶-聚合酶链反应(RT-PCR)方法和免疫组织化学PV-6000两步法检测Nanog mRNA和蛋白表达水平;采用无血清悬浮培养法从SKOV-3卵巢癌细胞株中分离培养肿瘤干细胞,流式细胞术鉴定肿瘤干细胞CD117表达,采用RT-PCR和Western Blot方法检测SKOV-3卵巢癌细胞及肿瘤干细胞中NanogmRNA及其蛋白的表达水平。结果:Nanog mRNA在卵巢癌组织中的表达水平均高于正常卵巢组织和卵巢良性肿瘤组织(P<0.05);Nanog mRNA在不同分化程度及临床分期的卵巢癌组织中表达水平不同,低分化组高于高分化组(P<0.05);III-IV期高于I-II期(P<0.05);免疫组化结果同RT-PCR。从SKOV-3卵巢癌细胞株中成功分离出肿瘤干细胞,SKOV-3卵巢癌细胞和肿瘤干细胞Nanog mRNA相对含量分别为0.6044±0.0368,0.8736±0.0537,差异具有统计学意义(P<0.05),两种细胞Nanog蛋白相对含量分别为0.6364±0.0169 1.2788±0.0314,差别具有统计学意义(P<0.05)。结论:Nanog基因在卵巢癌组织和SKOV-3细胞系中均高表达,其在组织中的表达强度与临床分期及病理分级关系密切,且在肿瘤干细胞中表达高于一般卵巢癌细胞,其与卵巢癌的发生发展关系密切,可能是卵巢癌干细胞的表面标志物,有望成为新的标志物。     

蟾毒灵对人卵巢癌SKOV-3细胞增殖的影响

目的：探讨蟾毒灵对人卵巢癌SKOV-3细胞增殖抑制和凋亡的影响,为卵巢癌临床治疗提供依据和分子基础。方法：不同浓度蟾毒灵处理卵巢癌SKOV-3细胞后,MTT法检测细胞增殖抑制作用,细胞免疫化学染色法检测细胞的凋亡,Western Blot法检测Bax,Bcl-2,Caspase-3蛋白以及计算Bax／Bcl-2的比值。结果：蟾毒灵能够抑制SKOV-3细胞的增殖,且成时间和剂量依赖性,免疫荧光显示蟾毒灵对SKOV-3细胞具有凋亡作用,Western Blot检测发现蟾毒灵能够促进Caspase-3蛋白的活化,提高Bax／Bcl-2的比值。结论：蟾毒灵在体外能够抑制卵巢癌SKOV-3细胞的增殖和促进卵巢癌SKOV-3细胞的凋亡。     

大黄素对顺铂耐药卵巢癌细胞的逆转作用及其相关基因表达研究

为了研究大黄素对人卵巢癌耐药细胞株SKOV3/DDP细胞耐药逆转作用及其机制,本研究以卵巢癌SK-OV3和多药耐药细胞株SKOV3/DDP为研究对象,通过噻唑蓝(MTT)法测定SKOV3/DDP细胞的耐药指数和大黄素在无细胞毒浓度下对卵巢癌细胞耐受顺铂(DDP)的逆转作用;采用Real-time PCR技术检测耐药相关基因HIF-1α、STAT1、CK2α、GSTP1 mRNA表达情况。结果发现无细胞毒作用浓度的7.8 mg/L和3.9 mg/L大黄素能逆转SKOV3/DDP细胞对DDP的耐药性,对DDP的逆转倍数分别为1.91倍和1.30倍,与SKOV3比较,SKOV3/DDP细胞的HIF-1α、STAT1、CK2α、GSTP1 mRNA表达明显升高(P<0.01)。3.9 mg/L和7.8 mg/L大黄素作用均可下调HIF-1α、CK2α、STAT1 mRNA表达,存在剂量-效应依赖关系。7.8 mg/L大黄素作用可下调GSTP1 mR-NA表达,但3.9 mg/L大黄素作用不明显。7.8 mg/L和3.9 mg/L大黄素联合IC50浓度的DDP时,四个耐药相关基因的表达与单独化疗药组作用相比明显下调(P<0.01)。提示无细胞毒浓度的大黄素对卵巢癌细胞耐药逆转的作用可能是通过下调HIF-1α、STAT1、GSTP1、CK2α的表达起作用。     

Chk1反义寡核苷酸联合顺铂抑制卵巢癌侵袭转移的分子机制

目的:探究Chk1反义寡核苷酸(CHK1-ASODN)单独或联合顺铂(DDP)对卵巢癌细胞系SKOV-3侵袭转移能力的影响,并阐明其可能的分子机制。方法:体外培养人卵巢癌细胞系SKOV-3,CHK1-ASODN单独或联合DDP处理48 h后,划痕实验检测细胞迁移能力;Transwell实验检测细胞侵袭能力;显微镜下观察细胞上皮或间质表型特征;Western blot及实时定量PCR技术分别检测上皮间质转化(EMT)特异性标志物(E-cadherin、N-cadherin)以及EMT关键调控分子ZEB1的蛋白及m RNA的表达水平。结果:与对照组相比较,CHK1-ASODN单独或联合DDP均能显著抑制SKOV-3细胞的迁移及侵袭(P0.05);细胞表现为间质化表型;E-cadherin的表达显著升高(P0.05),而N-cadherin的表达则显著降低(P0.05);ZEB1的表达显著降低(P0.05)。结论:CHK1-ASODN单独或联合DDP下调ZEB1的表达进而逆转EMT可能是其抑制卵巢癌侵袭转移的重要机制之一。     

IDO1 and IDO2 are expressed in human tumors: levo- but not dextro-1-methyl tryptophan inhibits tryptophan catabolism

Objectives  Indoleamine-2,3-Dioxygenase (IDO) is an immunosuppressive molecule inducible in various cells. In addition to classic IDO (IDO1), a new variant, IDO2, has recently been described. When expressed in dendritic cells (DCs) or cancer cells, IDO was thought to suppress the immune response to tumors. A novel therapeutic approach in cancer envisages inhibition of IDO with 1-methyl-tryptophan (1MT). The levo-isoform (l-1MT) blocks IDO1, whereas dextro-1MT (d-1MT), which is used in clinical trials, inhibits IDO2. Here we analyze IDO2 expression in human cancer cells and the impact of both 1-MT isoforms on IDO activity. Methods  Surgically extirpated human primary tumors as well as human cancer cell lines were tested for IDO1 and IDO2 expression by RT-PCR. IDO1 activity of Hela cells was blocked by transfection with IDO1-specific siRNA and analysed for tryptophan degradation by RP-HPLC. The impact of d-1MT and l-1MT on IDO activity of Hela cells and protein isolates of human colon cancer were studied. Results  Human primary gastric, colon and renal cell carcinomas constitutively expressed both, IDO1 and IDO2 mRNA, whereas cancer cells lines had to be induced to by Interferon-gamma (IFN-γ). Treatment of Hela cells with IDO1-specific siRNA resulted in complete abrogation of tryptophan degradation. Only l-1MT, and not d-1MT, was able to block IDO activity in IFN-γ-treated Hela cells as well as in protein isolates of primary human colon cancer. Conclusions  Although IDO2 is expressed in human tumors, tryptophan degradation is entirely provided by IDO1. Importantly, d-1MT does not inhibit the IDO activity of malignant cells. If ongoing clinical studies show a therapeutic effect of d-1MT, this cannot be attributed to inhibition of IDO in tumor cells.     

Expression and roles of Slit/Robo in human ovarian cancer

The Slit glycoproteins and their Roundabout (Robo) receptors regulate migration and growth of many types of cells including human cancer cells. However, little is known about the expression and roles of Slit/Robo in human ovarian cancer. Herein, we examined the expression of Slit/Robo in human normal and malignant ovarian tissues and its potential participation in regulating migration and proliferation of human ovarian cancer cells using two ovarian cancer cell lines, OVCAR-3 and SKOV-3. We demonstrated that Slit2/3 and Robo1 were immunolocalized primarily in stromal cells in human normal ovaries and in cancer cells in many histotypes of ovarian cancer tissues. Protein expression of Slit2/3 and Robo1/4 was also identified in OVCAR-3 and SKOV-3 cells. However, recombinant human Slit2 did not significantly affect SKOV-3 cell migration, and OVCAR-3 and SKOV-3 cell proliferation. Slit2 also did not induce ERK1/2 and AKT1 phosphorylation in OVCAR-3 and SKOV-3 cells. The current findings indicate that three major members (Slit2/3 and Robo1) of Slit/Robo family are widely expressed in the human normal and malignant ovarian tissues and in OVCAR-3 and SKOV-3 cells. However, Slit/Robo signaling may not play an important role in regulating human ovarian cancer cell proliferation and migration.     

A comprehensive analysis of IDO1 expression with tumour‐infiltrating immune cells and mutation burden in gynaecologic and breast cancers

Gynaecologic and breast cancers share some similarities at the molecular level. The aims of our study are to highlight the similarities and differences about IDO1, an important immune‐related gene in female cancers. The NGS data from TCGA of cervical squamous cell carcinoma (CESC), ovarian serous cystadenocarcinoma (OV), uterine corpus endometrial carcinoma (UCEC), uterine carcinosarcoma (UCS) and breast invasive carcinoma (BRCA) were analysed to identify molecular features, and clinically significant and potential therapeutic targets of IDO1. We found IDO1 was significantly up‐regulated in four gynaecologic cancers and breast cancer. According to breast cancer PAM50 classification scheme, IDO1 expression was higher in tumours of basal than other subtypes and showed better survival prognosis in BRCA and OV. Through immune infiltration analysis, we found a strong correlation between IDO1 and immune cell populations especially for dendritic cells and T cells. In addition, we investigated the association between IDO1 and tumour mutation burden (TMB) and found that IDO1 was significantly correlated with TMB in BRCA and CESC. GSVA revealed that hallmarks significantly correlated with IDO1 were involved in interferon gamma response, allograft rejection and inflammatory response. We also found PD‐L1 and LAG3 were highly positive related to IDO1 in gynaecologic cancers when comparing with their corresponding normal tissues. Our results indicated that IDO1 participated in anti‐tumour immune process and is correlated with mutation burden. These findings may expand our outlook of potential anti‐IDO1 treatments.     

Glucocorticoid regulation of SLIT/ROBO tumour suppressor genes in the ovarian surface epithelium and ovarian cancer cells

The three SLIT ligands and their four ROBO receptors have fundamental roles in mammalian development by promoting apoptosis and repulsing aberrant cell migration. SLITs and ROBOs have emerged as candidate tumour suppressor genes whose expression is inhibited in a variety of epithelial tumours. We demonstrated that their expression could be negatively regulated by cortisol in normal ovarian luteal cells. We hypothesised that after ovulation the locally produced cortisol would inhibit SLIT/ROBO expression in the ovarian surface epithelium (OSE) to facilitate its repair and that this regulatory pathway was still present, and could be manipulated, in ovarian epithelial cancer cells. Here we examined the expression and regulation of the SLIT/ROBO pathway in OSE, ovarian cancer epithelial cells and ovarian tumour cell lines. Basal SLIT2, SLIT3, ROBO1, ROBO2 and ROBO4 expression was lower in primary cultures of ovarian cancer epithelial cells when compared to normal OSE (P<0.05) and in poorly differentiated SKOV-3 cells compared to the more differentiated PEO-14 cells (P<0.05). Cortisol reduced the expression of certain SLITs and ROBOs in normal OSE and PEO-14 cells (P<0.05). Furthermore blocking SLIT/ROBO activity reduced apoptosis in both PEO-14 and SKOV-3 tumour cells (P<0.05). Interestingly SLIT/ROBO expression could be increased by reducing the expression of the glucocorticoid receptor using siRNA (P<0.05). Overall our findings indicate that in the post-ovulatory phase one role of cortisol may be to temporarily inhibit SLIT/ROBO expression to facilitate regeneration of the OSE. Therefore this pathway may be a target to develop strategies to manipulate the SLIT/ROBO system in ovarian cancer.     

An Antimitotic and Antivascular Agent BPR0L075 Overcomes Multidrug Resistance and Induces Mitotic Catastrophe in Paclitaxel-Resistant Ovarian Cancer Cells

Paclitaxel plays a major role in the treatment of ovarian cancer; however, resistance to paclitaxel is frequently observed. Thus, new therapy that can overcome paclitaxel resistance will be of significant clinical importance. We evaluated antiproliferative effects of an antimitotic and antivascular agent BPR0L075 in paclitaxel-resistant ovarian cancer cells. BPR0L075 displays potent and broad-spectrum cytotoxicity at low nanomolar concentrations (IC₅₀ = 2–7 nM) against both parental ovarian cancer cells (OVCAR-3, SKOV-3, and A2780-1A9) and paclitaxel-resistant sublines (OVCAR-3-TR, SKOV-3-TR, 1A9-PTX10), regardless of the expression levels of the multidrug resistance transporter P-gp and class III β-tubulin or mutation of β-tubulin. BPR0L075 blocks cell cycle at the G2/M phase in paclitaxel-resistant cells while equal concentration of paclitaxel treatment was ineffective. BPR0L075 induces cell death by a dual mechanism in parental and paclitaxel-resistant ovarian cancer cells. In the parental cells (OVCAR-3 and SKOV-3), BPR0L075 induced apoptosis, evidenced by poly(ADP-ribose) polymerase (PARP) cleavage and DNA ladder formation. BPR0L075 induced cell death in paclitaxel-resistant ovarian cancer cells (OVCAR-3-TR and SKOV-3-TR) is primarily due to mitotic catastrophe, evidenced by formation of giant, multinucleated cells and absence of PARP cleavage. Immunoblotting analysis shows that BPR0L075 treatment induced up-regulation of cyclin B1, BubR1, MPM-2, and survivin protein levels and Bcl-XL phosphorylation in parental cells; however, in resistant cells, the endogenous expressions of BubR1 and survivin were depleted, BPR0L075 treatment failed to induce MPM-2 expression and phosphorylation of Bcl-XL. BPR0L075 induced cell death in both parental and paclitaxel-resistant ovarian cancer cells proceed through caspase-3 independent mechanisms. In conclusion, BPR0L075 displays potent cytotoxic effects in ovarian cancer cells with a potential to overcome paclitaxel resistance by bypassing efflux transporters and inducing mitotic catastrophe. BPR0L075 represents a novel microtubule therapeutic to overcome multidrug resistance and trigger alternative cell death by mitotic catastrophe in ovarian cancer cells that are apoptosis-resistant.