Combinatorial treatment of ovarian cancer cells with harringtonine and cisplatin results in increased cisplatin-DNA adducts

The current studies represent the first step in assessing the utility of harringtonine in combination with cisplatin as an improved approach for treating ovarian cancer. Three ovarian cancer cell lines, platinum-sensitive A2780, and platinum-resistant A2780/CP70 and OvCar-3, were exposed to their respective IC(50) dose of cisplatin for 1 h with or without a 24-h pretreatment with harringtonine. The level of platinum-DNA adducts was determined by atomic absorption spectrometry (AAS). These studies show for the first time that harringtonine pretreatment significantly increased the amount of platinum-DNA adducts in all ovarian cancer cell lines by 2-4 fold, immediately following 1-h exposure to cisplatin. Moreover, the level of cisplatin-DNA adducts in harringtonine-pretreated cells remained elevated by 3-4.7-fold for at least 6 h after cisplatin was removed, relative to cells only exposed to cisplatin. In all three cell lines the removal (repair) of platinum-DNA adducts was not significantly altered by harringtonine. In addition, the extent to which harringtonine altered the expression of select DNA damage response genes (p53, P16, ERCC1 and XPB) was determined using RT-PCR and Southern hybridization in A2780 and A2780/CP70 cells. The expression of ERCC1 and XPB RNAs were only modestly altered by harringtonine, consistent with a lack of effect of harringtonine on repair of cisplatin-DNA damage. However, harringtonine altered expression of p53 and P16 RNAs in both cell lines, although the down-regulation of p53 and P16 RNAs by harringtonine were more pronounced in A2780 cells. The novel observation that harringtonine augments platinum-DNA adducts in both platinum-sensitive and -resistant ovarian cancer cells indicates this combination of drugs may have utility in treating ovarian cancer and may be especially useful in managing platinum-resistant cancers. Additional studies are required to determine which sequence of these drugs is most beneficial, as well as the mechanism by which harringtonine increases cisplatin-DNA damage in ovarian cancer cells.     

PG545 enhances anti-cancer activity of chemotherapy in ovarian models and increases surrogate biomarkers such as VEGF in preclinical and clinical plasma samples

BackgroundDespite the utility of antiangiogenic drugs in ovarian cancer, efficacy remains limited due to resistance linked to alternate angiogenic pathways and metastasis. Therefore, we investigated PG545, an anti-angiogenic and anti-metastatic agent which is currently in Phase I clinical trials, using preclinical models of ovarian cancer.MethodsPG545’s anti-cancer activity was investigated in vitro and in vivo as a single agent, and in combination with paclitaxel, cisplatin or carboplatin using various ovarian cancer cell lines and tumour models.ResultsPG545, alone, or in combination with chemotherapeutics, inhibited proliferation of ovarian cancer cells, demonstrating synergy with paclitaxel in A2780 cells. PG545 inhibited growth factor-mediated cell migration and reduced HB-EGF-induced phosphorylation of ERK, AKT and EGFR in vitro and significantly reduced tumour burden which was enhanced when combined with paclitaxel in an A2780 model or carboplatin in a SKOV-3 model. Moreover, in the immunocompetent ID8 model, PG545 also significantly reduced ascites in vivo. In the A2780 maintenance model, PG545 initiated with, and following paclitaxel and cisplatin treatment, significantly improved overall survival. PG545 increased plasma VEGF levels (and other targets) in preclinical models and in a small cohort of advanced cancer patients which might represent a potential biomarker of response.ConclusionOur results support clinical testing of PG545, particularly in combination with paclitaxel, as a novel therapeutic strategy for ovarian cancer.     

野生型p53基因转染对卵巢癌细胞生长及药物敏感性影响

目的：探讨野生型p53基因转染对人卵巢癌细胞株SKOV-3的体外生长及裸鼠体内致瘤性抑制作用。并了解p53基因与SKOV-3细胞对顺铂敏感性之间的关系。方法：利用脂质体介导,将含有人野生型p53cDNA的真核表达质粒pCB6-p53导入SKOV-3细胞中,观察该细胞体外生长和裸鼠体内致瘤性改变;应用流式细胞仪检测细胞周期及凋亡情况,MTT法检测细胞药物敏感性改变。结果：免疫组化法证实外源性p53基因在阳性细胞克隆稳定存在;转染野生型p53基因的SKOV-3细胞体外生长速率下降,裸鼠体内致瘤性丧失;G1/G0期细胞百分比（81.5%)和凋亡细胞百分比（11%）增高;p53表达阳性的SKOV-3细胞对顺铂的敏感性明显增强。结论：野生型p53基因转染能使SKOV-3细胞出现生长抑制和对顺铂的化疗敏感性增强。     

Characterisation of the P53 status,BCL-2 expression and radiation and platinum drug sensitivity of a panel of human ovarian cancer cell lines

The P53gene is frequently mutated in late stage ovarian cancer and has been proposed as a determinant of radiation and chemosensitivity. We have therefore determined the p53 functional status, P53sequence, radiation sensitivity and cytotoxicity of cisplatin and the novel platinum analogue, AMD473, in a panel of 6 human ovarian cancer cell lines. Constitutive p53 protein levels were low in A2780, CH1, LK1, LK2 and PA1 but were markedly induced following irradiation. In OV1P, constitutive p53 protein was readily detectable and levels were induced slightly following irradiation. P21^WAF1/CIP1 and MDM-2 mRNA were constitutively expressed in all the cell lines and expression was induced markedly following irradiation. There was marked radiation induced G₁/S arrest in A2780 but only partial arrests in CH1, LK1, LK2, PA1 and OV1P lines. No mutations were found in A2780, CH1, LK1, LK2 and PA1 cells by single-strand conformational polymorphism (SSCP) analysis but a heterozygous point mutation was found in exon 5 of OV1P. All the cell lines were radiation sensitive and also relatively sensitive to cisplatin; however, OV1P was the most resistant being consistent with its heterozygous P53 status. AMD473 was less potent than cisplatin but a similar pattern of drug sensitivity was observed with the exception of LK2, which was resistant. CH1, LK1, LK2 and PA1 all expressed BCL-2 protein but there was no expression in A2780 and OV1P. Our results suggest an overall association between wild type P53 and radiation and platinum drug sensitivity in these ovarian cancer cell lines. Int. J. Cancer 77:913–918, 1998.© 1998 Wiley-Liss, Inc.     

Effects of trans-(±)-kusunokinin on chemosensitive and chemoresistant ovarian cancer cells

Ovarian cancer ranks eighth in cancer incidence and mortality among women worldwide. Cisplatin-based chemotherapy is commonly used for patients with ovarian cancer. However, the clinical efficacy of cisplatin is limited due to the occurrence of adverse side effects and development of cancer chemoresistance during treatment. Trans-(±)-kusunokinin has been previously reported to inhibit cell proliferation and induce cell apoptosis in various cancer cell types, including breast, colon and cholangiocarcinoma. However, the potential effects of (±)-kusunokinin on ovarian cancer remains unknown. In the present study, chemosensitive ovarian cancer cell line A2780 and chemoresistant ovarian cancer cell lines A2780cis, SKOV-3 and OVCAR-3 were treated with trans-(±)-kusunokinin to investigate its potential effects. MTT, colony formation, apoptosis and multi-caspase assays were used to determine cytotoxicity, the ability of single cells to form colonies, induction of apoptosis and multi-caspase activity, respectively. Moreover, western blot analysis was performed to determine the proteins level of topoisomerase II, cyclin D1, CDK1, Bax and p53-upregulated modulator of apoptosis (PUMA). The results demonstrated that trans-(±)-kusunokinin exhibited the strongest cytotoxicity against A2780cis cells with an IC₅₀ value of 3.4 µM whilst also reducing the colony formation of A2780 and A2780cis cells. Trans-(±)-kusunokinin also induced the cells to undergo apoptosis and increased multi-caspase activity in A2780 and A2780cis cells. This compound significantly downregulated topoisomerase II, cyclin D1 and CDK1 expression, but upregulated Bax and PUMA expression in both A2780 and A2780cis cells. In conclusion, trans-(±)-kusunokinin suppressed ovarian cancer cells through the inhibition of colony formation, cell proliferation and the induction of apoptosis. This pure compound could be a potential targeted therapy for ovarian cancer treatment in the future. However, studies in an animal model and clinical trial need to be performed to support the efficacy and safety of this new treatment.     

MDM2-p53反馈环增强卵巢癌对顺铂敏感性的体内实验研究

  目的 探讨MDM2-p53反馈环对卵巢癌裸鼠移植瘤药物敏感性的影响及其作用机制。方法 将p53 为野生型的人类卵巢癌细胞A2780种植于裸鼠背部皮下形成移植瘤模型，将动物随机分为5组，瘤内注射pCMV-MDM2-脂质体＋顺铂为治疗组，仅注射顺铂为对照Ⅰ组，注射pCMV-MDM2-脂质体为对照Ⅱ组，注射空载体pCMV-脂质体为对照Ⅲ组，注射RPMI1640为对照Ⅳ组；每组6只。观察裸鼠一般情况、肿瘤生长速度，测量瘤体大小及重量；取肿瘤组织进行免疫组化染色，Western blot进行MDM2、p53蛋白表达检测，流式细胞技术进行细胞周期检测。结果 治疗组肿瘤体积[（0.321±0.086）cm3]显著小于对照Ⅰ组[（1.832±0.165）cm3]、对照Ⅱ组[（3.251±0.179）cm3]（t值分别为21.213、26.317，P值均＜0.05）；重量[（0.513±0.089）g]也显著小于对照Ⅰ组[（1.412±0.134）g]、对照Ⅱ组[（2.665±0.153）g]（t值分别为12.543、15.312，P值均<0.05）；对照Ⅰ组与对照Ⅱ组之间差异有统计学意义（P＜0.05）；而对照Ⅱ、Ⅲ、Ⅳ组间差异无统计学意义。治疗组可见明显MDM2蛋白表达，而无p53蛋白表达，并且出现明显S期阻滞，而对照I组主要出现G2／M期阻滞。结论 MDM2基因转染可增强卵巢癌裸鼠移植瘤对顺铂的敏感性，这是通过抑制p53表达并使癌细胞G1检查点丧失而实现的。     

Repeated cisplatin treatment can lead to a multiresistant tumor cell population with stem cell features and sensitivity to 3-bromopyruvate

Cisplatin is used in treatment of several types of cancer, including epithelial ovarian carcinoma (EOC). In order to mimic clinical treatment and to investigate longterm effects of cisplatin in surviving cancer cells, two EOC cell lines were repeatedly treated with low doses. In the SKOV-3 cell line originating from malignant ascites, but not in A2780 cells from a primary tumor, this led to emergence of a stable population (SKOV-3-R) which in the absence of cisplatin showed increased motility, epithelial-mesenchymal transition (EMT) and expression of cancer stem cell markers CD117, CD44 and ALDH1. Accordingly, the cells formed self-renewing spheres in serum-free stem cell medium. Despite upregulation of mitochondrial mass and cytochrome c, and no upregulation of Bcl-2/Bcl-xL, SKOV-3-R were multiresistant to antineoplastic drugs. Cancer stem cells, or tumor-initiating cells (TICs) are highly chemoresistant and are believed to cause relapse into disseminated and resistant EOC. Our second aim was therefore to target resistance in these TIC-like cells. Resistance could be correlated with upregulation of hexokinase-II and VDAC, which are known to form a survival-promoting mitochondrial complex. The cells were thus sensitive to 3-bromopyruvate, which dissociates hexokinase-II from this complex, and were particularly sensitive to combination treatment with cisplatin at doses down to 0.1 x IC₅₀. 3-bromopyruvate might thus be of use in targeting the especially aggressive TIC populations.     

Synergism from sequenced combinations of curcumin and epigallocatechin-3-gallate with cisplatin in the killing of human ovarian cancer cells

Drug resistance remains an on-going challenge in ovarian cancer chemotherapy. The objective of this study was to determine the effect on synergism in activity from the sequenced combinations of cisplatin (Cis) with curcumin (Cur) and epigallocatechin-3-gallate (EGCG) in the human ovarian cancer cell lines. The drugs were added in binary combinations: Cis combined with Cur, and Cis combined with EGCG to the human ovarian A2780 and A2780(cisR) cancer cell lines, using five different sequences of administration: 0/0 h, 4/0 h, 0/4 h, 24/0 h and 0/24 h. The combination index (CI) was used to assess the combined action of the drugs. CIs <1, =1 and >1 indicated synergism, additiveness and antagonism respectively. Cellular accumulation of platinum and platinum-DNA binding levels from Cis and its combination with the phytochemicals were determined using graphite furnace atomic absorption spectrometry. Addition of Cis 4 h before Cur and EGCG (0/4 h combination) produced the most synergistic outcomes in both the A2780 and A2780(cisR) cell lines. The cellular accumulations of platinum and platinum-DNA binding resulting from the 0/4 h combinations were greater as compared to the values using Cis alone, thus providing an explanation for the synergistic action. When sequenced combinations of Cis with Cur and with EGCG are applied to human ovarian A2780 and A2780(cisR) cancer cell lines, lower concentrations and shorter time gap between the two additions seem to produce a higher cytotoxic effect.     

Trail activity in human ovarian cancer cells: potentiation of the action of cytotoxic drugs

The ability of the TRAIL ligand to induce cell killing in three ovarian cancer cell lines was investigated using a glutathione-S-transferase (GST)-TRAIL fusion protein. One of the three lines was sensitive to TRAIL, which induced cell killing in a range of concentrations similar to those necessary to kill the TRAIL-sensitive leukaemic cell line Jurkat. The relative mRNA expression of the four TRAIL receptors did not explain the different sensitivities of the three ovarian cancer cell lines to TRAIL treatment. The TRAIL-sensitive IGROV-1 cell line expressed slightly lower levels of the anti-apoptotic protein FLIP than the two TRAIL-insensitive cell lines (A2780 and SKOV-3). Nevertheless, although TRAIL did not significantly reduce cell growth in the A2780 and SKOV-3 cells it did enhance the activity of paclitaxel and cisplatin (DDP), the two most widely used drugs for the treatment of ovarian cancer, increasing their ability to induce apoptosis. The use of TRAIL in combination with classical anticancer agents might thus boost the apoptotic response, improving the activity of DDP and paclitaxel in ovarian cancer.     

蛋白激酶B调控的p53蛋白直接线粒体转移与卵巢癌细胞顺铂耐药的关系

目的 探讨蛋白激酶B(PKB,又称Akt)调控的p53蛋白直接线粒体转移在顺铂诱导的卵巢癌细胞凋亡中的作用及与卵巢癌顺铂耐药的关系.方法 应用Western blot检测顺铂作用前后卵巢癌顺铂敏感细胞OV2008、A2780s和顺铂耐药细胞C13*、A2780cp中线粒体和全细胞中p53含量.分离纯化线粒体,检测卵巢癌敏感细胞与耐药细胞p53直接线粒体作用的差异;应用Hoechst染色法检测卵巢癌细胞的凋亡率,将Akt1/2 siRNA导入耐药细胞C13*细胞中,观察p53线粒体转移作用和细胞凋亡率的改变.结果 顺铂能引起p53进入OV2008和A2780s细胞的线粒体中,但不能进入C13*和A2780cP细胞的线粒体.过度表达活化的Akt2能抑制p53进入A2780s细胞的线粒体;下调Akt后,可使p53进入C13*细胞的线粒体,同时C13*细胞对顺铂的敏感性增加.结论 顺铂能引起卵巢癌敏感细胞中p53转移至线粒体,Akt导致的卵巢癌化疗耐药在一定程度上是Akt对p53直接线粒体作用的抑制.下调Akt的表达能使卵巢癌化疗耐药细胞的p53直接线粒体作用增强,耐药性得到一定程度的逆转.

Abstract：

Objective To explore Akt-regulated direct p53 mitochondrial translocation in cisplatininduced apoptosis in ovarian cancer cells and the relationship between this and chemoresistance in ovarian cancer. Methods Chemosensitive ovarian cancer cell lines(0V2008 and A2780s) and chemoresistant cells(C13* and A2780cp) were treated with cisplatin and whole cell and mitochondrial p53 contents were determined by Western blot.The p53 accumulation in mitochondria was determined in purified mitochondrial fractions in cisplatin-sensitive and -resistant ovarian cancer cells.Akt1/2 siRNA were transfected into C13*cells.Cisplatin-induced apoptosis was measured by Hoechst staining and p53 translocation was determined by Western blot.ResultsCisplatin induced mitochondrial p53 accumulation and apoptosis in chemosensitive cells(P ＜0.05 ),but not in resistant cells(P ＞ 0.05 ).Over-expression of active Akt2 inhibited p53 directly translocate to mitochondria,and downregulation of Akt by Akt1/2 siRNA increased p53 mitochondrial accumulation and sensitize C13* cells to cisplatin treatment. Conclusions Cisplatin induces direct p53 mitochondrial accumulation in chemosensitive cells,and Akt confers resistance in ovarian cancer cells,in part,by regulating the direct action of p53 in mitochondrial death pathway.     

β-Elemene Effectively Suppresses the Growth and Survival of Both Platinum-sensitive and -resistant Ovarian Tumor Cells

The development of cisplatin drug resistance remains a chief concern in ovarian cancer chemotherapy. β-Elemene is a natural plant product with broad-spectrum antitumor activity towards many types of carcinomas. This study aimed to define the biological and therapeutic significance of β-elemene in chemoresistant ovarian cancer. In the present study, β-elemene significantly inhibited cell growth and proliferation of both the cisplatin-sensitive human ovarian cancer cell line A2780 and its cisplatin-resistant counterpart A2780/CP. β-Elemene also suppressed the growth of several other chemosensitive and chemoresistant ovarian cancer cell lines, including ES-2, MCAS, OVCAR-3, and SKOV-3, with the half maximal inhibitory concentration (IC(50)) values ranging from 54 to 78 μg/ml. In contrast, the IC(50) values of β-elemene for the human ovarian epithelial cell lines IOSE-386 and IOSE-397 were 110 and 114 μg/ml, respectively, which are almost two-fold those for the ovarian cancer cell lines. Cell cycle analysis demonstrated that β-elemene induced a persistent block of cell cycle progression at the G(2)/M phase in A2780 and A2780/CP cells. This was mediated by alterations in cyclin and cyclin-dependent kinase expression, including the down-regulation of CDC2, cyclin A, and cyclin B1, and the up-regulation of p21(WAF1/CIP1) and p53 proteins. Moreover, β-elemene triggered apoptosis and irreversible cell death in both sensitive and resistant ovarian cancer cells via the activation of caspase-3, -8 and 9; the loss of mitochondrial membrane potential (δΨm); the release of cytochrome c into the cytosol; and changes in the expression of BCL-2 family proteins. All of these molecular changes were associated with β-elemene-induced growth inhibition and cell death of ovarian cancer cells. Our results demonstrate that β-elemene has antitumor activity against both platinum-sensitive and resistant ovarian cancer cells, and thus has the potential for development as a chemotherapeutic agent for cisplatin-resistant ovarian cancer.     

Increased accumulation of p53 protein in cisplatin-resistant ovarian cell lines

We have examined p53 protein levels in cell lines selected for resistance to the chemotherapeutic drug cis-diamminedichloroplatinum (II), cisplatin. The majority of the independent cisplatin-resistant clones isolated by a single selection with cisplatin from the ovarian tumour cell line A2780 showed increased levels of p53 protein compared to the parental cell line. Elevated p53 protein levels were also observed in cisplatin-resistant ovarian human tumour lines isolated after multiple exposures to cisplatin (A2780/cp70 and OVIP/DDP). Direct PCR sequencing of p53 cDNAs showed that both the A2780/cp70 and the parental A2780 cell lines had a wild-type p53 gene sequence. The OVI P and OVI P/DDP lines both had a heterozygous mutation at codon 126. Cell-cycle analysis after gamma-irradiation or cisplatin treatment showed evidence of a G1/S and G2/M cell-cycle checkpoint in both A2780/cp70 and the sensitive parental cell lines. However, the resistant cell line A2780/cp70 showed less inhibition of DNA synthesis after gamma-irradiation than the sensitive cell line. Transfection of a mutant p53 gene construct (containing a mutation at codon 143, val to ala) into the A2780/cp70 resistant cells conferred a significantly increased sensitivity to cisplatin, suggesting that p53 is a direct determinant of cisplatin resistance in these cells. However, expression of this mutant p53 in the A2780 cells did not affect sensitivity.     

Synthesis of trans-bis-(2-hydroxypyridine)dichloroplatinum(II) and its activity in human ovarian tumour models

We report the synthesis and in vitro activity of trans-bis-(2-hydroxypyridine)dichloroplatinum(II) (coded as DH3) in humour ovarian tumour models. The compound is less active than cisplatin against the parent cell line A2780 but more so against the cisplatin-resistant A2780(cisR) cell line, thus indicating that it is better able to overcome mechanisms of resistance operating in the A2780(cisR) cell line. DH3 is marginally less active than cisplatin against ZD0473-resistant A2780(ZD0473R) cell line but with a much lower resistance factor than cisplatin. DH3 has higher platinum-DNA binding levels than cisplatin in the A2780 and A2780(ZD0473) cell lines and a lower value in the A2780(cisR) cell line. Even though DH3 has a lower activity than cisplatin, the higher platinum-DNA binding levels observed for DH3 than cisplatin in A2780 and A2780(ZD0473R) cell lines may not be entirely unexpected when we note that the two compounds are likely to differ in their nature of binding with DNA. Whereas cisplatin binds with DNA forming mainly intrastrand 1,2-Pt(GG) and 1,2-Pt(AG) adducts, DH3 is expected to form more 1,2-interstrand Pt(GG) and monofunctional adducts. The higher activity of DH3 than cisplatin in the A2780(cisR) cell line despite its lower level of platinum-DNA binding can also be seen to indicate the complexity of the situation. Although platinum-DNA binding may be an essential requirement for apoptosis, it is not sufficient to cause cell death that is actually brought about by downstream processes in the cycle. The results of interaction with pBR322 plasmid DNA combined with BamH1 digestion show that DH3 is less able to prevent BamH1 digestion than is cisplatin, indicating that cisplatin causes a greater conformational change in the DNA than DH3. CONCLUSION: DH3 is less active than cisplatin against the parent cell line A2780, but more so against the cisplatin-resistant A2780(cisR) cell line, thus indicating that it is better able to overcome mechanisms of resistance operating in the A2780(cisR) cell line.     

外源性p53基因增加卵巢癌细胞对顺铂的敏感性

 目的 探讨外源性ｐ５３基因转染对人卵巢癌细胞株的化疗敏感性的影响。方法用脂质体介导的转染技术，将人野生型ｐ５３基因的真核表达载体导入不表达ｐ５３的卵巢癌ＳＫＯＶ－３细胞中，经Ｇ４１８筛选，Ｎｏｒｔｈｅｒｎｂｌｏｔ及Ｗｅｓｔｅｒｎｂｌｏｔ鉴定后，观察其对顺铂作用后的 ＳＫＯＶ－３细胞的集落形成及凋亡的影响。结果 外源性Ｐ５３基因在转染细胞中有效表达，并增强了顺铂对ＳＫＯＶ－３细胞集落形成的抑制作用及促进了顺铂诱导的细胞凋亡。结论 外源性ｐ５３基因能增加卵巢癌细胞对顺铂的敏感性，两者联合作用能更大程度地杀灭肿瘤细胞。     

沉默S100A10基因对卵巢癌SKOV-3和A2780细胞周期的影响

目的 探讨沉默S100钙结合蛋白A10(S100A10)对卵巢癌SKOV-3和A2780细胞周期的影响及其相关分子机制.方法 将针对S100A10基因的shRNA转染至SKOV-3和A2780细胞中,获S100A10敲降稳转SKOV-3和A2780细胞系,采用CCK8方法检测细胞增殖能力,采用流式细胞技术检测细胞周期变...     

Retention of activity by the new generation platinum agent AMD0473 in four human tumour cell lines possessing acquired resistance to oxaliplatin

Four models of acquired resistance to the clinically-used platinum drug, oxaliplatin, have been established using human tumour cell lines in vitro; two colon (HCT116 and HT29) and two ovarian (A2780 and CH1). Levels of acquired resistance ranged from 3.0- to 15.8-fold with levels of resistance higher in the colon relative to the ovarian carcinoma cell lines. Notably, the platinum analogue, AMD0473, currently undergoing clinical evaluation, exhibited superior circumvention of acquired oxaliplatin resistance in comparison to either cisplatin or the trinuclear platinum BBR3464. Resistance in the two colon cell lines was unique to oxaliplatin itself among the platinum drugs studied. Acquired oxaliplatin resistance was not due to either reduced drug membrane transport or increased levels of glutathione in any of the four resistant lines. Following exposure to oxaliplatin, a lower level of platinum-DNA adducts was present in acquired oxaliplatin-resistant HT29 cells. In the remaining resistant lines, there was no change in the levels of platinum-DNA adducts relative to the parent lines. There was no change in hMLH1 DNA mismatch repair gene status in any of the four cell line pairs. However, in an A2780 subline where loss of hMLH1 and a p53phe172 mutation occurred, 5-fold resistance to cisplatin was observed, but only 1.7-fold resistance to oxaliplatin and no resistance to AMD0473 were observed. Re-introduction of hMLH1 into these cells caused no significant change in the sensitivity to cisplatin, oxaliplatin or AMD0473. These data show that acquired resistance to oxaliplatin may occur in cell lines (and therefore probably in the clinic) and in the four independent cell lines studied this was circumvented by AMD0473. Alongside previously described models of acquired resistance to cisplatin, these oxaliplatin-resistant cell line models may be useful in the evaluation of further novel platinum agents.     

Modulation of cisplatin cytotoxicity due to its combination with bortezomib and the nature of its administration

The widely used anticancer drug cisplatin (CS) is believed to cross the cell membrane by passive diffusion, carrier-mediated transport and pinocytosis. One carrier involved in the transport of CS into the cell is the copper transporter CTR1. However, CS is found to trigger the down-regulation of CTR1 and its proteasomal degradation. The proteasome inhibitor bortezomib (Bort) has been reported to block CS-induced down-regulation of CTR1 so that in the presence of Bort, the cellular uptake of CS may be increased. Increased platinum accumulation may result in increased platinum-DNA binding so that CS in combination with Bort may produce pronounced cell kill. In this study, synergism in activity from the sequenced combination of CS and Bort in human ovarian A2780, A2780(cisR) and A2780(ZD0473R) cancer cell lines was studied. We also investigated the effect on cell kill due to the administration of CS in two aliquots with a time gap. Addition of Bort 2 h before CS was found to produce greater cell kill than the converse and the bolus, especially in the resistant A2780(cisR) and A2780(ZD0473R) cell lines, in line with increased platinum accumulation and platinum-DNA binding levels. Thus, the prevention of CTR1 degradation by Bort may play a significant role, especially in the resistant cell lines. Administration of CS in two aliquots with a time gap was also found to maximise the cell kill in the ovarian cancer cell lines. If such findings are found to be true in vivo, the results may be significant clinically.     

Twist下调影响卵巢癌细胞株A2780顺铂敏感性的研究

目的:探讨Twist对卵巢癌细胞株A2780顺铂敏感性及细胞增殖凋亡的影响。方法:通过Western blot筛选细胞株,构建小干扰RNA,qPCR筛选Twist-siRNA,将构建成功的Twist-siRNA逆转录于上皮性卵巢癌细胞株A2780中,通过CCK-8细胞术及流式细胞术,研究下调Twist对卵巢癌细胞株的增殖、凋亡及对化疗药物顺铂的影响。结果:选择人卵巢癌细胞株A2780,引入小干扰RNA后,Twist表达降低;细胞的增殖能力下降,凋亡增加（P＜0.05）,差异有统计学意义。si-Twist组相比对照组Twist基因的表达量明显降低, 且呈现一定的顺铂浓度依懒性差异(P<0.05）。结论:下调Twist可抑制卵巢癌细胞A2780的增殖,促进其凋亡;下调Twist卵巢癌细胞A2780对顺铂的敏感性增加。