The kinase RSK2 modulates the sensitivity of ovarian cancer cells to cisplatin

Platinum-based chemotherapy (e.g. cisplatin, carboplatin) is standard of care for many types of cancer including ovarian cancer, however, the efficacy of treatment is hampered by the development of therapy resistance. The mechanisms behind platinum resistance are not completely understood. Here, we have investigated the role of the family of p90 Ribosomal S6 kinases (RSK), important downstream mediators of ERK1/2, in the response to cisplatin chemotherapy. Strikingly, whereas treatment with cisplatin did not alter the levels of RSK1 in response to cisplatin treatment, the structurally related RSK2 protein was downregulated in an ovarian cancer cell line (A2780). Furthermore, we found that knockdown of RSK2, in contrast to knockdown of RSK1, gave rise to enhanced cisplatin sensitivity in a cisplatin sensitive as well as a cisplatin-resistant A2780 cell line. These results indicate that RSK2 is regulated in response to cisplatin treatment, and this downregulation may contribute to the cytotoxic action of cisplatin. Since RSK2 is frequently amplified in a growing number of cancers, this may have implications for the sensitivity of these tumours to platinum-based cytotoxics.     

Modulation by lonidamine on the combined activity of cisplatin and epidoxorubicin in human breast cancer cells

The ability of lonidamine (LND), an energolytic derivativeof indazole-carboxylic acid, to modulate the cytotoxic activityof cisplatin (CDDP) and epidoxorubicin (EPI), singly orin combination, was investigated in two human breastcancer cell lines (MCF7 and T47D). A 72-hrpost-incubation with a non-cytotoxic concentration of LND (75M) increased the activity of a 1-hr CDDPtreatment as well as that of a 1to 16-hr EPI treatment. A different pattern ofinteraction among the drugs and modulator was observedas a function of the sequence of drugtreatment. Specifically, supra-additive or additive effects of thecombination were obtained in the two cell linesaccording to the different treatment schemes. In particular,the maximum potentiation was observed in MCF7 cellssimultaneously exposed to CDDP, EPI and LND for1 hr and then post-incubated with LND for72 hr, and in T47 first exposed toEPI and LND, then to CDDP and LND,and finally post-incubated with LND. Flow cytometric analysisof MCF7 cell distribution in the different cyclephases showed that combined treatment with EPI/CDDP/LND wasable to stabilize cell cycle perturbations (mainly G₂Maccumulation) induced by individual agents. The ability ofLND to potentiate CDDP and EPI cytotoxicity, andthe consideration that LND causes side effects differentfrom those caused by alkylating agents and anthracyclines,make this compound an attractive candidate for multidrugcombination therapy in breast cancer.     

Modulation of cisplatin sensitivity and resistance by buthionine sulfoximine and cyclosporin A in human esophageal cancer cells

We established a 2.4-fold cisplatin (CDDP)-resistant human esophageal cancer cell line (TE2R) from the parent TE2 line. CDDP accumulation was reduced in TE2R. The Na+, K+-ATPase inhibitor ouabain inhibited CDDP accumulation in TE2 but not TE2R, suggesting that TE2R may have alterations in the Na+, K+-ATPase and defective CDDP uptake mechanism. Buthionine sulfoximine (BSO) enhanced CDDP sensitivity of both cell lines and cyclosporin A (CsA) modified CDDP resistance in TE2R. These effects were associated with increased CDDP accumulation. Thus, BSO and CsA may be useful for modulation of CDDP sensitivity or resistance in esophageal cancer.     

The Janus face of dendritic cells in cancer

On the basis of experimental models and some human data, we can assume that tumor outgrowth results from the balance between immunosurveillance (the extrinsic tumor suppressor mechanisms) and immunosubversion dictated by transformed cells and/or the corrupted surrounding microenvironment. Cancer immunosurveillance relies mainly upon conventional lymphocytes exerting either lytic or secretory functions, whereas immunosubversion results from the activity of regulatory T or suppressor myeloid cells and soluble mediators. Although specific tools to target or ablate dendritic cells (DCs) became only recently available, accumulating evidence points to the critical role of the specialized DC system in dictating most of the conventional and regulatory functions of tumor-specific T lymphocytes. Although DC can be harnessed to silence tumor development, tumors in turn can exploit DC to evade immunity. Indeed, DCs harbor defects in their differentiation and stimulatory functions in cancer-bearing hosts and can actively promote T-cell tolerance to self-tumor antigens. In this review, we will focus on the dual role of DC during tumor progression and discuss pharmacoimmunological strategies to harness DC against cancer.     

Modulation of cell growth and cisplatin sensitivity by membrane gamma-glutamyltransferase in melanoma cells

The plasma membrane enzyme gamma-glutamyltransferase (GGT) is regarded as critical for the maintenance of intracellular levels of glutathione (GSH). GGT expression has been implicated in drug resistance through elevation of intracellular GSH. The dependence of intracellular GSH on GGT expression was not conclusively ascertained. The present study was designed to investigate the role of GGT and of intracellular GSH levels in modulating proliferation and sensitivity to cisplatin of melanoma cells. GGT transfection resulted in increased growth, both in vitro and in tumour xenografts. In addition, GGT-transfected cells exhibited reduced sensitivity to cisplatin associated with lower DNA platination. A decrease in intracellular GSH levels, rather than an increase, was observed in GGT-transfected cells; moreover, in cysteine-deficient conditions, the expression of GGT did not provide transfected cells with the ability of utilising extracellular GSH. In conclusion, these results indicate that GGT activity confers a growth advantage unrelated with intracellular glutathione supply, and are consistent with the interpretation that cisplatin resistance is the consequence of modifications of cellular pharmacokinetics as a result of extracellular drug inactivation by thiol metabolites originated by GGT-mediated GSH cleavage.     

Modulation of cisplatin resistance in human malignant melanoma cells.

Previous studies have shown that the combination of dacarbazine, carmustine, cisplatin (DDP), and tamoxifen (TAM) produced a 53% overall response rate in patients with disseminated melanoma. Deletion of TAM from the regimen resulted in a fall in the response rate to 10%, and reincorporation of TAM returned the response rate to 52%, suggesting an important role for TAM. Using the human melanoma cell line T-289, we examined the nature of the interaction between TAM and each member of this combination in clonogenic assays in soft agar. The combination of TAM with DDP was highly synergistic as demonstrated by median effect analysis, whereas TAM was antagonistic with carmustine and an activated form of dacarbazine. The mean combination index at 50% kill was 0.26 +/- 0.02 (mean +/- SD) for TAM and DDP. This marked synergism was observed at concentrations of TAM that are clinically achievable. TAM had no effect on the uptake of the DDP analogue [3H]dichloro(ethylenediamine)platinum(II). There was no effect on the formation or repair of DDP intrastrand DNA adducts. Similarly, there was no effect demonstrated on the intracellular concentrations of glutathione or metallothioneins. We conclude that the interaction between TAM and DDP is truly synergistic in this cell line and is accomplished through none of the four mechanisms commonly associated with DDP resistance.     

Modulation of cisplatin cytotoxicity by sulphasalazine.

The efficacy of cisplatin [cis-diamminedichloroplatinum (II); DDP] is hampered by acquired or de novo resistance of malignant cells to its cytotoxic effects. We have previously reported that cisplatin resistance parallels glutathione S-transferase (GST) activity in several human small-cell lung cancer cell lines. In the presently described studies, we used sulphasalazine, an inhibitor of GSTs, to evaluate the relative role of GSTs in mediating cisplatin resistance in two human small-cell lung cancer cell lines, NCI H-69 and H-2496. The H-69 cell line, which contained relatively higher GST activity than the H-2496 cell line (317 +/- 7 vs 9 +/- 1 mU mg-1 protein respectively), also displayed a greater degree of cisplatin resistance (IC50 values of 25.0 +/- 3.9 vs 4.5 +/- 1.0 microM respectively). Western blot and Northern blot analyses of purified GSTs revealed the expression of only the pi-class GST in both cell lines. Sulphasalazine inhibited the purified GSTs (IC50 of 10 microM for H-69 and 12 microM for H-2496) from both lines in a competitive manner with similar Ki values (6.5 and 7.9 microM for the H-69 and H-2496 cell lines respectively). Cytotoxicity studies revealed that sulphasalazine increased the cytotoxicity of cisplatin towards both cell lines. Isobologram analysis showed that sulphasalazine synergistically enhanced the cytotoxicity of cisplatin towards both cell lines, the magnitude of synergy being remarkably higher in H-69 cells than in H-2496 cells. Our studies indicate that clinically achievable concentrations of sulphasalazine may be useful in modulating cisplatin resistance in malignancies with increased GST-pi content.     

增强c-Jun氨基末端激酶活性可逆转卵巢癌的顺铂耐药性

目的: 探讨卵巢癌的顺铂(cisplatin,CDDP)耐药以及如何促使其逆转的机制.方法:将携带c-Jun氨基末端激酶1(c-Jun NH2-terminal kinase 1,JNK1)显性突变体的质粒pcDNA3(FLAG)-JNK1-dn及表达野生型JNK1的质粒pcDNA3(FLAG)-JNK1-wt分别转染卵巢癌A278细胞和A2780/DDP耐药细胞株.20 μmol/L CDDP作用后,检测细胞中p-JNK及下游p-c-Jun的表达,观察2株细胞的凋亡率及CDDP作用后2株细胞中Fas/FasL的表达.结果:CDDP作用后A2780细胞中p-JNK及下游p-c-Jun持续表达,而A2780/DDP细胞中仅有短暂表达.pcDNA3(FLAG)-JNK1-dn转染A2780细胞可抑制CDDP诱导的JNK1活性,并使细胞的凋亡率明显低于未转染组[(18.3±0.4)% vs (47.3±4.7)%, P <0.01].pcDNA3(FLAG)-JNK1-wt转染可使JNK1持续活化,FasL表达上调,A2780/DDP细胞凋亡率明显高于未转染组[(36.9±1.6)% vs (10.9±1.7)%, P <0.01]. 结论:JNK信号转导通路在CDDP诱导的卵巢癌细胞凋亡中起重要作用.野生型JNK1基因转染使A2780/DDP细胞对CDDP的耐药性得到逆转.     

顺铂诱导原代培养人膀胱癌细胞的凋亡

目的：探讨顺铂诱导原代培养膀胱癌细胞发生调亡变化的特征及机制。方法：以0-100umol/L浓度顺铂处理原代培养膀胱癌细胞12-72小时,应用MTT法检测细胞增殖抑制变化。分析其与凋亡指数（AI）的关系;用光镜和透射电镜分别观察膀胱癌细胞凋亡变化的特征。结果：顺铂能够明显地诱导膀胱癌细胞凋亡,具有一定范围内的作用时间和药物浓度依赖性,与细胞增殖抑制密切相关。膀胱癌细胞凋亡的镜下特征为胞膜完整,胞浆浓缩,核碎裂、萎缩或边积,胞浆外突形成凋亡小体。结论：证实顺多功能铂诱导膀胱癌细胞广泛性凋亡是它产生杀瘤效应的途径之一。     

Modulation of resistance to cisplatin by amphotericin B and aphidicolin in human larynx carcinoma cells

The aim of this study was to examine whether resistance to cisplatin [cis-diamminedichloroplatinum (II)] (CDDP) could be overcome by amphotericin B, cyclosporin A and aphidicolin in two sublines of human larynx carcinoma HEp2 cells. The sensitivity of parental and cisplatin-resistant CA3 and CK2 cells to amphotericin B, cyclosporin A and aphidicolin, and also the effects of these drugs (given in maximal nontoxic concentrations) on cisplatin sensitivity were determined by clonogenic survival assay. CA3 ad CK2 cells were sensitive to amphotericin B, and resistant to cyclosporin A and aphidicolin, compared with their parental cells. Amphotericin B increased cisplatin toxicity 2-fold in CA3 cells and 2.7-fold in CK2 cells, while it had no effect in parental HEp2 cells. Cyclosporin A did not influence the sensitivity of examined cells to cisplatin. The sensitizing effect of aphidicolin was more obvious in cisplatin-resistant cells. Cisplatin toxicity was increased by aphidicolin: 1.5-fold in HEp2 cells, 2-fold in CA3 cells, and 1.9-fold in CK2 cells. Therefore, the resistance to cisplatin in human larynx carcinoma CA3 and CK2 cells can be partially reversed by amphotericin B and aphidicolin.     

Janus kinase 2: a critical target in chronic myelogenous leukemia

The Bcr-Abl tyrosine kinase is the causative factor in most chronic myelogenous leukemia (CML) patients. We have shown that Bcr-Abl is associated with a cluster of signaling proteins, including Janus kinase (Jak) 2, growth factor receptor binding protein 2-associated binder (Gab) 2, Akt, and glycogen synthase kinase (GSK)-3beta. Treatment of CML cell lines and mouse Bcr-Abl+ 32D cells with either Jak2 short interfering RNA or Jak2 kinase inhibitor AG490 inhibited pTyr Gab2 and pSer Akt formation, inhibited the activation of nuclear factor-kappaB, and caused the activation of GSK-3beta, leading to the reduction of c-Myc. Importantly, BaF3 cells expressing T315I and E255K imatinib-resistant mutants of Bcr-Abl underwent apoptosis on exposure to AG490 yet were resistant to imatinib. Similar to wild-type Bcr-Abl+ cells, inhibition of Jak2 by Ag490 treatment resulted in decrease of pSer Akt and c-Myc in imatinib-resistant cells. These results identify Jak2 as a potentially important therapeutic target for CML.     

Down-regulation of mammalian sterile 20-like kinase 1 by heat shock protein 70 mediates cisplatin resistance in prostate cancer cells

Mammalian sterile 20-like kinase 1 (Mst1) is an ubiquitously expressed serine/threonine kinase, and its activation results in cell apoptosis. Recent studies suggest that Mst1 may function as a tumor suppressor. Here, we reported that heat shock protein 70 (Hsp70), which is thought to protect cells against cellular stress, has been identified as an Mst1-interacting protein, in a yeast two-hybrid screen of human adult prostate cDNA library with a dominant-negative Mst1 (K59R) as bait. The interaction of Mst1 with Hsp70 was confirmed by coimmunoprecipitation in both cotransfected HEK293 cells and prostate cancer cells. Hsp70 colocalized with Mst1 in the cytoplasm of LNCaP cells. The interaction sites with Mst1 consisted of NH(2)-terminal ATPase domain in Hsp70, whereas the inhibitory domain of Mst1 mediates the binding of Hsp70 in Mst1. Overexpression of Hsp70 mediates proteasomal degradation of Mst1 in a Hsp70 interacting protein (CHIP)-dependent manner. Furthermore, the proapoptotic effect of Mst1 was markedly inhibited by overexpression of Hsp70 or CHIP. Most strikingly, in response to the treatment of anticancer drug cisplatin, the induction of Hsp70 expression is higher in the androgen-independent DU145 cells compared with the androgen-dependent LNCaP cells. The higher levels of Hsp70 induction and subsequent Mst1 degradation mediate cisplatin resistance in prostate cancer DU145 cells. Moreover, overexpression of Mst1 sensitizes prostate cancer cells to cisplatin treatment. These findings implicate that Mst1, a downstream target of Hsp70, may be developed as a target for sensitizing hormone-refractory prostate cancers to chemotherapy.     

Sensitization of ovarian cancer cells to cisplatin by gold nanoparticles

Recently we reported that gold nanoparticles (AuNPs) inhibit ovarian tumor growth and metastasis in mice by reversing epithelial-mesenchymal transition (EMT). Since EMT is known to confer drug resistance to cancer cells, we wanted to investigate whether anti-EMT property of AuNP could be utilized to sensitize ovarian cancer cells to cisplatin. Herein, we report that AuNPs prevent cisplatin-induced acquired chemoresistance and stemness in ovarian cancer cells and sensitize them to cisplatin. AuNPs inhibit cisplatin induced EMT, decrease the side population cells and key stem cell markers such as ALDH1, CD44, CD133, Sox2, MDR1 and ABCG2 in ovarian cancer cells. Mechanistically, AuNPs prevent cisplatin-induced activation of Akt and NF-κB signaling axis in ovarian cancer cells that are critical for EMT, stem cell maintenance and drug resistance. In vivo, AuNPs sensitize orthotopically implanted ovarian tumor to a low dose of cisplatin and significantly inhibit tumor growth via facilitated delivery of both AuNP and cisplatin. These findings suggest that by depleting stem cell pools and inhibiting key molecular pathways gold nanoparticles sensitize ovarian cancer cells to cisplatin and may be used in combination to inhibit tumor growth and metastasis in ovarian cancer.     

Enhancement of cisplatin activity by lonidamine in human ovarian cancer cells.

The ability of lonidamine, an energolytic derivative of indazole-carboxylic acid, to modulate the cytotoxicity of cisplatin was investigated in human ovarian-cancer cell lines sensitive (A2780) or with experimentally induced resistance (A2780/cp8) to the alkylating agent. A 24-hr post-incubation with 300 microM lonidamine significantly potentiated the activity of a 1-hr cisplatin treatment in both cell lines. In particular, the cisplatin IC50 value was reduced 4-fold in the sensitive line and 5-fold in the resistant line. Flow cytometric analysis showed that, in the resistant cell line, lonidamine alone did not affect cell kinetics, but when given after cisplatin it was able to transform the temporary G2 + M cell accumulation induced by the alkylating agent to a persistent block in S/G2 + M. In the A2780/cp8 cell line, lonidamine was also able to significantly enhance the accumulation of cisplatin-induced DNA interstrand cross-links. Our results suggest that lonidamine can positively modulate the anti-tumor activity of cisplatin in ovarian cancer cells and also indicate that the drug is potentially useful in combination therapy including the alkylating agent for ovarian cancer patients.     

Celecoxib antagonizes the cytotoxic effect of cisplatin in human gastric cancer cells by decreasing intracellular cisplatin accumulation

Cisplatin is a chemotherapeutic drug widely used for the treatment of gastric cancer. The benefit of including COX-2-selective inhibitors in cisplatin-based regimens on anti-cancer effect remains uncertain. In the present study, celecoxib and SC-236 antagonized cisplatin-induced cytotoxicity and apoptosis, whereas indomethancin and nimesulide exerted no effect, implying a COX-2-independent mechanism. Celecoxib decreased whole-cell cisplatin accumulation and DNA platination, resulting from reduced influx. In addition, combined treatment did not elicit greater antitumor activity than cisplatin or celecoxib monotherapy in vivo in a gastric xenograft model. Therefore, treatment strategies with celecoxib in combination with cisplatin should act cautiously.     

PAK2通过调节ERK信号通路的活性促进肺癌细胞顺铂耐药

目的:探讨p21激活激酶2(PAK2)在非小细胞肺癌（NSCLC）中是否存在异常表达,以及是否影响肺癌细胞的顺铂敏感性。方法:采用免疫组化和Western blot（WB）方法检测NSCLC中PAK2的蛋白表达模式。利用PAK2-siRNA下调肺癌细胞系中PAK2的表达后,通过WB和细胞功能学实验等方法,明确PAK2对NSCLC细胞系增殖和侵袭能力的影响,并检测顺铂耐药前后的肺癌细胞系中PAK2的表达改变,及其对肺癌细胞顺铂敏感性的影响。结果:PAK2在NSCLC中呈现明显的胞浆强表达,并且其异常表达与肺癌的恶性表型相关;PAK2在顺铂耐药肺癌细胞中表达增强,抑制ERK活性后,PAK2的表达上调不能显著促进肺癌细胞对顺铂耐药。结论:PAK2在NSCLC中发挥着重要的促癌基因功能,并通过调节ERK信号通路的活性介导肺癌细胞系对顺铂耐药性。