Preclinical activity of a new platinum analogue, lobaplatin, in cisplatin-sensitive and -resistant human testicular, ovarian, and gastric carcinoma cell lines

Lobaplatin [1,2-diamminomethylcyclobutane-platinum(II) lactate] is a new platinum compound with interesting preclinical activity and apparently no nephro- or neurotoxicity that is currently undergoing clinical phase II studies. Little is known about the cross-resistance between cisplatin and lobaplatin. The activity of this new compound in comparison with cisplatin and carboplatin was evaluated in cisplatin-sensitive and cisplatin-resistant human testicular, gastric, and ovarian carcinoma cell lines using 96 h continuous drug exposure in a sulforhodamine-B assay. In three cisplatin-sensitive testicular carcinoma cell lines, lobaplatin and cisplatin showed comparable antitumor activity. The 50% growth-inhibitory concentrations (IC₅₀ values) determined for cisplatin ranged from 0.1 to 0.4 M, and those found for lobaplatin ranged from 0.25 to 0.5 M. Carboplatin showed markedly lower cytotoxicity in all cell lines tested. Lobaplatin was not cross-resistant to cisplatin in a 10-fold cisplatin-resistant testicular carcinoma cell line and showed only weak cross-resistance in a 20-fold cisplatin-resistant ovarian carcinoma cell line. In contrast, complete cross-resistance between cisplatin and lobaplatin occurred in two cisplatin-resistant human gastric carcinoma cell lines, which were 3.3- and 9-fold resistant to cisplatin and 3.1- and 6.5-fold resistant to lobaplatin, respectively. Furthermore, lobaplatin showed significant activity against cisplatin-resistant human ovarian and testicular carcinoma xenografts in vivo. These data indicate a high level of activity for lobaplatin at clinically achievable concentrations in drug-sensitive testicular, ovarian, and gastric carcinoma cell lines and a lack of complete cross-resistance to cisplatin. Further clinical development of lobaplatin is clearly warranted.This work was supported in part by a grant from Asta Medica, D-6000 Frankfurt 1, Germany     

Preclinical Activity of Lobaplatin as a Single Agent and in Combination with Taxanes for Ovarian Carcinoma Cells

Lobaplatin, one of the third - generation platinum compounds, has shown encouraging anticancer activityin a variety of tumor types. However, the efficacy of lobaplatin in ovarian cancer has not been systemicallyevaluated. In this study, lobaplatin as a single agent and in combination with taxanes was investigated in - vitroand in an in vitro model of ovarian carcinoma. Using the sulforhodamine B (SRB) assay, the cytotoxic effects oflobaplatin alone and in combination with taxanes were compared with cisplatin and carboplatin in seven ovariancancer cell lines. In addition, in - vitro antitumor activities were evaluated with cisplatin - sensitive and cisplatin- resistant human ovarian cancer xenografts in nude mice. The cytotoxicity of lobaplatin was similar to or higherthan that of cisplatin and carboplatin, with IC50 values from 0.9 to 13.8 μmol/L in a variety of ovarian cancercells. The combination of lobaplatin with docetaxel yielded enhanced cytotoxic activity in vitro. In addition, inplatinum - sensitive ovarian cancer xenografts, lobaplatin alone showed similar antitumor activity to cisplatinand carboplatin. Furthermore, lobaplatin alone or in combination with docetaxel exhibited significant activityin platinum - resistant ovarian cancer xenografts. These results indicate that the use of lobaplatin alone or incombination with docetaxel might be a rational and novel therapeutic strategy for ovarian cancer. Furtherclinical development of lobaplatin is clearly warranted.     

Cross-resistance and collateral sensitivity to natural product drugs in cisplatin-sensitive and -resistant rat lymphoma and human ovarian carcinoma cells

 The cytotoxicity of mitotic spindle poisons, vinca alkaloids and the anthracycline, adriamycin, against cisplatin-sensitive and -resistant rat lymphoma and human ovarian carcinoma cell lines was investigated. Interestingly, it was found that all cell lines were more sensitive to the mitotic spindle poisons, vincristine and vinblastine. Adriamycin was the least effective and taxol had intermediate activity. The Walker rat lymphoma cell line resistant to cisplatin (WR) exhibited the multiple drug resistance phenotype since it showed collateral resistance to all drugs (ranging from twofold to taxol, colcemid and colchicine and sixfold to the vinca alkaloids). Verapamil potentiated the cytotoxic activity of adriamycin and vincristine in a striking fashion with the Walker cells. P-glycoprotein was found to be present in the plasma membranes of the Walker cells with approximately a 2.5-fold increase in the WR as compared to the sensitive (WS) cells. Glutathione levels were elevated in all of the cisplatin-resistant cell lines when compared to the cisplatin-sensitive parental cell lines. A profound effect of buthionine sulfoximine pretreatment on adriamycin cytotoxicity was observed. Glutathione S-transferase (π) was present in all the human cell lines but the WS cells had markedly lower levels (almost negligible) when compared to the WR cells. These observations imply that cisplatin-resistant cells may be more sensitive to mitotic spindle poisons and vinca alkaloids, irrespective of the mechanism of platinum resistance, and that the cytotoxicity of vinca alkaloids could be further modulated by verapamil, irrespective of the presence or absence of P-glycoprotein. Received: 8 November 1994/Accepted: 22 May 1995     

洛铂诱导顺铂耐药卵巢癌SKOV3/ DDP细胞的凋亡

 目的 探讨洛铂体外诱导人卵巢癌顺铂耐药SKOV3／DDP细胞凋亡及其机制。方法 采用四甲基偶氮唑盐（MTT）法测定不同浓度和时间洛铂对SKOV3／DDP细胞的生长抑制作用，并与其亲本细胞SKOV3（敏感株）相比较；进一步通过光镜及透射电镜观察SKOV3／DDP细胞的形态学改变；并采用流式细胞仪检测细胞周期变化。结果 洛铂作用SK0V3／DDP细胞的增殖呈时间剂量依赖模式受到抑制，与SKOV3细胞相比，差异无统计学意义（P〉0．05）；一定浓度的SKOV3／DDP作用一定时间，可诱导SKOV3／DDP细胞发生凋亡；光镜和透射电镜下可见典型的凋亡细胞形态学改变；流式细胞技术分析发现洛铂作用首先引起细胞周期分布的改变，随用药时间的延长，凋亡率逐渐升高。结论 洛铂通过诱导凋亡而抑制体外培养的SKOV3／DDP细胞生长，其机制可能与细胞G0／G1期阻滞有关。     

Metallothionein gene expression and resistance to cisplatin in human ovarian cancer

Intracellular thiols have been proposed as mediators of resistance to alkylating agents and cisplatin. As metallothionein is the predominant protein thiol, we examined its relationship to cisplatin resistance in human ovarian cancer cell lines. A human ovarian carcinoma cell line, A2780, derived from an untreated patient, was treated with cisplatin in several ways and the induced resistance to cisplatin ranged from 13- to 68-fold. The degree of resistance was dependent upon the method of selection. The drug-resistant cell lines also developed low levels of cross-resistance to cadmium. Additional cell lines established from untreated patients or ovarian cancer patients refractory to cisplatin- and/or carboplatin-containing combination chemotherapy were studied. The most cisplatin-resistant cell lines, OVCAR-8 and -10, were from patients previously treated with intensive chemotherapy. OVCAR-8 was relatively cross-resistant to cadmium while OVCAR-10 appeared relatively sensitive. Cell lines were examined for expression of metallothionein mRNA to evaluate the relationship between cisplatin resistance, cadmium cross-resistance and metallothionein expression. Only two of the cell lines with in vitro-induced resistance to cisplatin, 2780E80 and 2780CP70B3, had detectable metallothionein mRNA. The other cell lines selected in vitro for cisplatin resistance, as well as the parental A2780 ovarian cancer cell line, showed no expression at our level of detection. There was variable expression of metallothionein among the OVCAR cell lines. Cell lines from untreated patients, OVCAR-5 and -7, did express metallothionein, while the most cisplatin-resistant cell lines, OVCAR-8 and -10, did not. We also examined cisplatin induction of metallothionein mRNA in the cell lines. Only 2780CP70B3 among the cell lines with in vitro-induced cisplatin resistance showed increased expression after short-term exposure to cisplatin. OVCAR-4 also had a slight increase in expression after exposure to cisplatin. Mouse C127 cells transfected with a bovine papilloma virus-metallothionein gene construct were compared for cisplatin sensitivity to the same cell type transfected with bovine papilloma virus alone. In this model system, metallothionein expression did not influence cisplatin cytotoxicity. On the basis of these studies, we conclude that there is no causal relationship between metallothionein expression and cisplatin resistance.     

Cisplatin-induced response of c-jun N-terminal kinase 1 and extracellular signal--regulated protein kinases 1 and 2 in a series of cisplatin-resistant ovarian carcinoma cell lines

The cellular response to cisplatin involves activation of multiple signal transduction pathways, including the mitogen-activated protein (MAP) kinase pathways. In this study, we compared the cisplatin-induced activation of two MAP kinases, c-jun N-terminal kinase 1 (JNK1) and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), in the cisplatin-sensitive ovarian carcinoma cell line A2780 and its derivative cisplatin-resistant cell lines CP70 and C200. Dose-dependent and time-dependent activation of JNK1 and ERK1/2 occurred in each of the three cell lines in response to cisplatin treatment. The requirement of higher concentrations of cisplatin for induction of maximum activation of JNK1 and ERK1/2 was correlated with increased levels of cisplatin resistance. In addition, inhibition of cisplatin-induced ERK activation, using the MAP/ERK kinase 1 synthetic inhibitor PD98059, resulted in enhanced sensitivity to cisplatin in all three cell lines. These results suggest that cisplatin-induced ERK1/2 activity is not responsible for the acquired cisplatin resistance in CP70 and C200 cells but rather provides a general cytoprotective effect in both cisplatin-sensitive and cisplatin-resistant cell lines. In conclusion, different patterns of cisplatin-induced JNK1 and ERK1/2 activation are observed in cell lines with different levels of cisplatin sensitivity, and inhibition of cisplatin-induced ERK1/2 activation enhances sensitivity to cisplatin in both cisplatin-sensitive and cisplatin-resistant cell lines.     

Regulation of gamma-glutamyltransferase in cisplatin-resistant and -sensitive colon carcinoma cells after acute cisplatin and oxidative stress exposures

Glutathione plays an important role in drug resistance of tumor cells and in their ability to resist oxidative stress. Improved salvage of glutathione can be obtained through increased activity of gamma-glutamyltransferase (GGT), which is of importance in the maintenance of cellular glutathione homeostasis. We investigated the regulation of GGT in 2 cisplatin-resistant and 1 cisplatin-sensitive colon carcinoma cell lines. Enzyme activity was induced in all 3 cell lines after acute exposure to cisplatin. The elevation was significantly higher in sensitive cells (3.3-fold) than in resistant (1.6- to 1.7-fold) cells. Exposure of cells to oxidative stress generated by menadione also resulted in enzyme induction but only in cisplatin-sensitive cells. Addition of anti-oxidants had different effects on the 2 inductions: N-acetylcysteine blocked the induction of both cisplatin and menadione, whereas catalase and glutathione-ester blocked only the menadione induction. Glutathione depletion alone was not sufficient to induce GGT in these cells. The data show that GGT is regulated by multiple mechanisms during anti-tumor drug treatment and oxidative stress and that reactive oxygen species were involved in the menadione, but not cisplatin, induction of the enzyme.     

A comparison of hyperthermia cisplatin sensitization in human ovarian carcinoma and glioma cell lines sensitive and resistant to cisplatin treatment

 Two pairs of human tumor cell lines (glioma and ovarian carcinoma (OvCa)) each having a parental cell line and cisplatin-resistant variant, were evaluated for (a) cisplatin response, (b) hyperthermia response, and (c) combined hyperthermia and cisplatin response. The two resistant lines had comparable resistant responses while for the parental lines, the OvCa was more sensitive than the glioma to cisplatin doses up to 14 μg/ml. For the hyperthermia response, the OvCa parental line was more resistant than the variant line at low-temperature hyperthermia (41° C or 42°C) but became more sensitive at high temperature (45°C). For the glioma, the parental line was more sensitive to hyperthermia at all temperatures tested. Hyperthermia caused sensitization to cisplatin in all cell lines but was generally greater in the glioma cell lines. In the OvCa system, hyperthermia had a slightly greater sensitizing effect on the resistant cell lines, while in the glioma the opposite was true. The degree of sensitization increased with hyperthermia temperature. In summary, the results showed that there is no cross-resistance for hyperthermia and cisplatin, that the degree of thermal sensitization is not reduced in cisplatin-resistant cell lines, and that cisplatin thermal sensitization is cell-line and temperature dependent. Thus, hyperthermia can effectively improve tumor cell response to cisplatin and may be useful in overcoming resistance to cisplatin. Received: 10 March 1995/Accepted: 21 July 1995     

Establishment and characterization of an in vitro model of acquired resistance to cisplatin in a human testicular nonseminomatous germ cell line.

Clinically, human testicular nonseminomatous germ cell tumors exhibit remarkable sensitivity to platinum-based chemotherapy. To define better the mechanistic basis for this unusual sensitivity, the biochemical determinants of platinum-induced cytotoxicity have been investigated in a human testicular tumor cell line (GCT27) established from a previously untreated patient and in an in vitro derived 5.6-fold cisplatin-resistant stable variant (GCT27cisR). Compared to 12 ovarian and 5 cervical human tumor cell lines, the parent GCT27 line was among the most sensitive to the cytotoxic effects of both cisplatin (dosage producing 50% inhibition, 0.2 microM) and carboplatin (dosage producing 50% inhibition, 2.9 microM), thus reflecting clinical data. A 4-day exposure sulforhodamine B-staining assay was used to determine that GCT27cisR was cross-resistant to carboplatin and iproplatin and the classical bifunctional alkylating agents melphalan and chlorambucil. Partial cross-resistance was observed to tetraplatin, methotrexate, and mitomycin C. No cross-resistance was observed to Adriamycin, etoposide, vinblastine, bleomycin, 1-beta-D-arabinofuranosylcytosine, and 5-fluorouracil. Intracellular cisplatin accumulation across the dose range 2.5-100 microM (for 2 h) was 1.6 +/- 0.39-fold (mean +/- SD) greater for the parent line. There was no significant difference in glutathione levels between the two lines. The acquired resistance line was 1.9-fold more resistant than the parent line to the cytotoxic effects of cadmium chloride. There was no significant difference between the two lines, however, in the total amounts of platinum bound to DNA after cisplatin exposure (25, 50, or 100 microM for 2 h). The removal of total platinum adducts from DNA was significantly faster for GCT27cisR compared to the parent line (half-times of removal, 32 and 67 h, respectively). These data suggest that the abnormal sensitivity of the parent testicular tumor cell line to platinum-containing anticancer drugs may be due predominantly to an inherent defect in the ability of these cells to remove platinum from their DNA. This defect is apparently lost in the acquired resistance counterpart. Reduced intracellular accumulation and increased cytoplasmic concentrations of metallothionein may also contribute, in part, to the acquisition of cisplatin resistance in this model.     

Differential properties of cisplatin and tetraplatin with respect to cytotoxicity and perturbation of cellular glutathione levels

Summary We compared the cytotoxicity and the effects on the levels of cellular glutathione (GSH) of cisplatin and tetraplatin in the mouse mammary tumour cell line EMT6/P and a cisplatin-resistant variant, EMT6/CPR. EMT6/CPR showed a 2.5-fold resistance to cisplatin but no cross-resistance to tetraplatin. Basal cellular GSH levels in the resistant line were 1.7-fold those in the parent cell line. On exposure of cells to cisplatin, cellular GSH levels initially dropped in both cell lines but subsequently rose. At between 1 and 4 h after the onset of drug exposure, GSH levels were up to 2-fold the baseline levels. The cisplatin-resistant subline displayed higher GSH levels than the parent cell line at all time points. Tetraplatin failed to influence cellular GSH levels in either of the cell lines.     

细胞色素c 介导的半胱天冬氨酸蛋白酶-3活性改变与人卵巢癌顺铂耐药的关系

目的：探讨人卵巢癌顺铂耐药细胞株A2780／DDP、COC1／DDP中抗凋亡基因bcl-XL、细胞色素c的表达和半胱天冬氨酰蛋白酶－3（caspase-3)活性对人卵巢癌顺铂耐药的影响。方法：采用逆转录聚合酶链反应（RT－PCR）和Western blot检测人卵巢癌顺铂敏感细胞株A2780、COC1和顺铂耐药株A2780／DDP、COC1／DDP中bcl-XL的表达,以及顺铂作用后细胞色素c的含量和caspase-3活性的变化,并应用流式细胞仪测定顺铂作用后A2780、COC1、A2780／DDP、COC1／DDP细胞的凋亡率。结果：bcl-XL在A2780/DDP、COC1／DDP细胞中的表达明显高于A2780、COC1细胞;顺铂作用后,细胞色素c在A2780／DDP、COC1／DDP细胞中的表达明显减少,caspase-3活性和凋亡率也较A2780和COC1细胞明显降低（P＜0．05）。结论：人卵巢部细胞对顺铂产生耐药可能与细胞内bcl-XL过度表达、细胞色素c释放受抑制和caspase-3活性下降有关。     

Relationship between folate-binding protein expression and cisplatin sensitivity in ovarian carcinoma cell lines.

It has been suggested that sensitivity of ovarian carcinomas to cisplatin is in part related to an endogenous folate deficiency. In this work, we investigated whether overexpression of the folate-binding protein (FBP), a receptor involved in folate transport, might be associated with cisplatin sensitivity. The results obtained on a panel of ten ovarian carcinoma cell lines that overexpress different levels of the FBP showed a statistically significant relationship between FBP overexpression and cisplatin responsiveness, with the most sensitive cell lines expressing higher FBP levels on their membrane than the less sensitive ones. The relationship was observed both in cells growing in standard medium-containing high-folate concentrations (2.3 microM) and in cells adapted to growth in low-folate (20 nM) medium. Analysis of two cisplatin-resistant cell lines derived from the cisplatin-sensitive IGROV1 ovarian carcinoma cell line indicated that resistance was associated with a significant decrease in FBP expression. However, the receptor does not appear to be directly responsible for drug sensitivity per se as different cell lines transfected with FBP cDNA did not become more sensitive to the drug. Together, the data suggest the possible predictive value of FBP in ovarian carcinoma, as higher levels of expression can be indirectly but significantly associated with increased drug sensitivity.     

Decreased pyruvate kinase M2 activity linked to cisplatin resistance in human gastric carcinoma cell lines

Resistance to anticancer drugs is a major obstacle preventing effective treatment of disseminated cancers. Understanding the molecular basis to chemoresistance is likely to provide better treatment. Cell lines resistant to cisplatin or 5-fluorouracil (5-FU) were established from human gastric carcinoma cell lines SNU-638 and SNU-620. Comparative proteomics involving 2-dimensional gel electrophoresis (2-DE) and matrix-associated laser desorption ionization-mass spectroscopy (MALDI-MS) was performed on protein extracts from these parental and drug-resistant derivative lines to screen drug resistance-related proteins. Pyruvate kinase M2 (PK-M2) was identified as a protein showing lower expression in cisplatin-resistant cells compared to parental cells. Consistent with this finding, PK-M2 activity was also lower in cisplatin-resistant cells. Suppression of PK-M2 expression by antisense oligonucleotide resulted in acquired cisplatin resistance in SNU-638 cells. Furthermore, PK-M2 activity in 11 individual human gastric carcinoma cell lines positively correlated with cisplatin sensitivity. Taken together, PK-M2 protein and activity levels were lower in cisplatin-resistant human gastric carcinoma cell lines compared to their parental cell lines. Furthermore, suppression of PK-M2 expression using antisense oligonucleotides increased cisplatin resistance. These data clearly link PK-M2 and cisplatin resistance mechanisms.     

顺铂诱导卵巢癌耐药细胞系A2780/DDP凋亡的时间和剂量依从性

目的探讨不同浓度顺铂作用不同时间诱导敏感和耐药卵巢癌细胞系发生凋亡变化规律和凋亡途径相关蛋白的表达。方法选择卵巢癌顺铂耐药细胞系A2780/DDP和敏感细胞系A2780为研究对象。在细胞培养基础上,运用MTT比色法、原位标记法、常规免疫组化和蛋白印迹等方法,观察不同剂量的顺铂(5、10、20、40μmol/L)和作用不同时间(24、48、72h)对A2780和A2780/DDP细胞凋亡的影响和此动态变化过程中半胱天冬氨酸蛋白酶2(Caspase2)和3(Caspase3)表达。结果顺铂对A2780和A2780/DDP细胞系凋亡的诱导呈时间和剂量依从性。细胞凋亡蛋白Caspase2和Caspase3的活化促进顺铂诱导的凋亡,其表达呈现出对顺铂作用时间和剂量的依从性。结论(1)在顺铂耐药机理中,药物在体内分布和代谢,对血药有效浓度的影响和细胞对药物排斥引起顺铂进入细胞存在一定的阻碍可能是降低顺铂敏感性的因素。(2)Caspase2和caspase3蛋白活化延后和活性降低可能是影响耐药A2780/DDP细胞凋亡的原因。     

Comparative cytotoxicity of CI-973, cisplatin, carboplatin and tetraplatin in human ovarian carcinoma cell lines

The clinical efficacy of cisplatin-based chemotherapy for ovarian cancer is frequently compromised by drug resistance or dose-limiting renal and neurologic toxicities. CI-973 (NK-121), a 2-methyl-1,4-butanediamine analogue of carboplatin, has shown little nephro- and neuro-toxicity in pre-clinical model systems and in phase-I trials. Its in vitro spectrum of activity against ovarian cancer cell lines has not been previously characterized. The in vitro activities of CI-973, cisplatin, carboplatin and tetraplatin were compared in several platinum-sensitive and -resistant human ovarian carcinoma cell lines. Cytotoxicity was assessed by inhibition of clonogenic survival in soft agar with continuous drug exposure. On a molar basis, cisplatin and tetraplatin were the most potent analogues, while carboplatin was consistently less potent. Cisplatin, carboplatin and CI-973 elicited a very similar response pattern by Spearman rank correlation, distinct from that seen with tetraplatin. The magnitude of resistance to CI-973 was comparable to cisplatin in 5 cell lines but was substantially lower in the highly cisplatin-resistant 2780-CP70 and OVCAR-10 cell lines. These results suggest that CI-973 and tetraplatin may have potential utility in some cases of cisplatin-resistant ovarian cancer. In addition, our data are consistent with the existence of at least 2 platinum-resistance phenotypes--one with moderate levels of resistance to cisplatin, carboplatin and CI-973 but highly resistant to tetraplatin, the other highly resistant to cisplatin and carboplatin but only partially cross-resistant with tetraplatin and CI-973. The recognition of different resistance phenotypes may facilitate the study of cellular resistance mechanisms to cisplatin and newer platinum analogues.     

Treatment with paclitaxel alone rather than combination with paclitaxel and cisplatin may be selective for cisplatin-resistant ovarian carcinoma

BACKGROUND: We have previously reported that paclitaxel (taxol) results in cisplatin sensitization to human ovarian cancer cells with cisplatin resistance in vitro. This study was designed to determine effects of taxol and its combination with cisplatin on growth of cisplatin-sensitive cell line (KF28) and the cisplatin-resistant counterpart (KFr13) in nude mice. METHODS: From 14 days after tumor inoculation treatment was initiated. Taxol (3 mg/kg) and cisplatin (2 mg/kg) were administered i.p. once a week for 5 weeks. RESULTS: In nude mice bearing cisplatin-sensitive cells (KF28), taxol followed by cisplatin and cisplatin plus taxol inhibited significantly (P < 0.05) the tumor growth rate compared with that in nude mice treated with cisplatin alone or taxol alone and cisplatin followed by taxol. On the other hand, in nude mice bearing cisplatin-resistant KFr13 cells, treatment with taxol alone inhibited completely the tumor growth rate, whereas no schedule-dependent interaction of taxol with cisplatin was observed. CONCLUSION: These results suggest that treatment with taxol alone may be superior to combination of taxol with cisplatin in patients with cisplatin-resistant ovarian carcinoma.     

洛铂对鼻咽癌抗肿瘤作用的体外实验研究

目的 探讨洛铂对鼻咽癌的体外抗肿瘤作用及其可能的机制,为洛铂用于鼻咽癌的临床治疗提供实验依据。方法 在体外将不同浓度洛铂分别作用于低分化鼻咽癌细胞株CNE2、HONE1和SUNE1,采用CCK-8法检测细胞活性;碘化丙啶（PI）染色分析细胞中DNA的含量,流式细胞仪检测细胞周期情况;采用Annexin V-FITC／PI双染法检测细胞凋亡情况;采用蛋白印迹法（Western blotting）检测细胞周期和细胞凋亡相关蛋白的表达。结果 在体外洛铂作用3种鼻咽癌细胞株48h具有明显的细胞抑制作用,并表现出浓度依赖性。洛铂作用鼻咽癌细胞株CNE2、HONE1、SUNE1的半数抑制浓度（IC₅₀）分别为（4.05±0.49）μmol/L、（4.32±1.17）μmol/L和（2.51±0.15）μmol／L。洛铂作用3种鼻咽癌细胞株48h后,在较低浓度（0.125倍IC₅₀）时将细胞周期阻滞在G₂期,同时伴随G₂期相关蛋白Cyclin B1和phospho-cdc2（Tyr15）表达增高;而在较高浓度（0.5倍IC₅₀）时则诱导细胞呈Caspase依赖性细胞凋亡,并具有浓度依赖性。结论 洛铂对鼻咽癌细胞具有明显的细胞毒作用,且呈浓度依赖性;其机制表现为双重作用,即在较低浓度时阻滞细胞于G₂期和在较高浓度时诱导细胞凋亡,这为洛铂用于鼻咽癌的临床治疗提供可能性。     

Relationship of cellular glutathione to the cytotoxicity and resistance of seven platinum compounds.

The role of glutathione (GSH) in the effectiveness of and resistance to 7 platinum compounds [5 Pt(II) and 2 Pt(IV) drugs] was evaluated in a 8.6-fold cisplatin (CDDP)-resistant human small cell lung cancer cell line (GLC4/CDDP), the parent GLC4 line, a 3.7-fold CDDP-resistant human embryonal carcinoma cell line (Tera-CP), and the parent Tera line (NTera2/D1). Resistance factors for both CDDP-resistant cell lines were determined after continuous incubation (4 days) with CDDP. Continuous incubation with the other studied platinum drugs revealed complete cross-resistance for carboplatin (CBDCA) and zeniplatin but less for enloplatin (ENLO) and iproplatin in both models. Tetraplatin and lobaplatin showed, respectively, partial and complete cross-resistance in GLC4/CDDP but no cross-resistance in Tera-CP. GSH level, but not glutathione S-transferase activity, of the 4 cell lines correlated with platinum drug concentrations inhibiting cell survival by 50% after continuous incubation (r = 0.86, P < 0.05). GSH depletion by DL-buthionine-S,R-sulfoximine (BSO) increased sensitivity, as measured after a 4-h exposure to the drugs, of GLC4/CDDP for CDDP 2.0-fold, for CBDCA 1.7-fold, for zeniplatin 1.7-fold, and almost to the level of the sensitive GLC4 for ENLO, whereas no effect was observed for lobaplatin and the Pt(IV) compounds iproplatin and tetraplatin. BSO-modulating effect was higher in the sensitive GLC4 line for most compounds; therefore reduction of resistance could be achieved only for CDDP and ENLO. In contrast to GLC4, no modulation occurred in Tera. In Tera-CP BSO increased sensitivity for CDDP 1.5-fold, for CBDCA 1.9-fold, and for zeniplatin 1.2-fold; no effect was observed for ENLO, lobaplatin, and the Pt(IV) compounds. Reduction of CDDP resistance by BSO was known to occur with identical cellular platinum levels and higher Pt-DNA binding in GLC4/CDDP. However, pretreatment with BSO followed by 4 h ENLO incubation increased cellular platinum levels in both GLC4 and GLC4/CDDP while Pt-DNA binding remained unchanged. In conclusion, GSH reflected sensitivity to platinum-containing drugs. However, since the involvement of GSH differed between the models and the various platinum drugs, the effect of modulation with BSO was unpredictable.     

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.     

Dihydrodiol dehydrogenases regulate the generation of reactive oxygen species and the development of cisplatin resistance in human ovarian carcinoma cells

We have previously demonstrated that overexpression of dihydrodiol dehydrogenase isoform 1 (DDH1) or DDH2 leads to the induction of drug resistance to platinum based drugs in human ovarian, lung, cervical and germ cell tumor cell lines. DDH belongs to a family of aldoketo reductases that are involved in the detoxification of several endogenous and exogenous substrates. DDH1 and DDH2 in particular have been shown to be involved in the detoxification (activation?) of polycyclic aromatic hydrocarbons (PAH). Based on the involvement of DDH in the detoxification of electrophilic PAH intermediates, the effect of DDH on the production of reactive oxygen species (ROS) in a cisplatin-sensitive and -resistant human ovarian carcinoma cell line was investigated in the current study. In addition to the overexpression of DDH1 and DDH2, increased expression of DDH3 was demonstrated in the cisplatin-resistant 2008/C13* cells, compared to the parental 2008 cells. However, as assessed by RT-PCR, neither cell line expressed DDH4. The 2008/C13* cells were eightfold resistant to cisplatin, and transfection experiments utilizing cisplatin-sensitive 2008 cells suggest that this could be mediated by overexpression of either DDH1, DDH2, or DDH3. The 2008/C13* cells had lower basal intracellular ROS level as compared to the 2008 cells and ROS production was decreased in the recombinant 2008 cells with forced, constitutive overexpression of either, DDH1, DDH2, or DDH3. Transfection of siRNA against DDH1 or DDH2 in the cisplatin-resistant 2008/C13* cells not only significantly decreased their cisplatin-resistance index (as assayed by MTT and colony formation assay) but also led to an increase in the basal levels of ROS production (although transfection of siRNA against DDH3 resulted in cell death). The 2008/C13* cells were found to be cross-resistant to the cytotoxic effects of hydrogen peroxide and tert-butyl hydroperoxide and knockdown of either DDH1 or DDH2 expression (using siRNA) resulted in sensitization of the resistant cells to these agents. These results support the conclusion that the increased levels of DDH in the 2008/C13* cells are directly responsible for the reduced production of ROS and that this may play a role in the development of cisplatin resistance.