Loss of caspase-8 activation pathway is a possible mechanism for CDDP resistance in human laryngeal squamous cell carcinoma, HEp-2 cells

Cisplatin (CDDP) is among the most widely used and most effective chemotherapeutic agent for many types of human cancer. Because killing cancer cells by chemotherapy is principally executed by apoptosis, a defective apoptotic program might acquire drug resistance. Flow cytometric Annexin V assay demonstrated that HEp-2 cells (human laryngeal cancer) were persistently resistant to CDDP as compared to HeLa cells (human uterine cervical cancer), despite the same histological type and wild-type p53 status. CDDP treatment caused steady induction of p53 protein in both cancer cell types, although it was more dramatic in CDDP-resistant HEp-2 cells, which was correlated well with p53 Ser15 phosphorylation, but not with the expression level of HPV type 18 E6 oncoprotein in these cells. Importantly, CDDP differently activated caspase cascades between HEp-2 and HeLa cells. CDDP activated the caspase-8 pathway through TNFR superfamily receptors such as Fas, but not caspase-9 in HeLa cells. On the other hand, the caspase-9 pathway was significantly activated in HEp-2 cells, although the activation of caspase-8 by CDDP was deficient. This different response to CDDP in caspase-8 activation was not related with the expression level of either Fas or FasL in these cells. We concluded from these results that loss of the caspase-8 activation pathway in HEp-2 cells was a possible mechanism for its resistance to CDDP-induced apoptosis. The caspase-8 pathway might play an important role in CDDP-induced apoptosis in HPV-positive human squamous cell carcinomas.     

Modulation of anti-apoptosis by endogenous IAP expression in MKN45 human gastric cancer cells

BACKGROUND: This study was designed to clarify differences in apoptotic signal transduction between gastric cancer cells and leukemia cells. MATERIALS AND METHODS: In order to study apoptotic signal transduction of gastric cancer cells, MKN45 gastric cancer cells expressing the wild-type p53 gene and U937 myeloid leukemia cells expressing a mutated p53 gene were prepared. Cisplatin (CDDP) was used to induce apoptosis. We compared apoptotic signal transduction downstream to mitochondria between those two lines. RESULTS: In contrast to U937 cells, MKN45 gastric cancer cells revealed delayed response in release of mitochondrial cytochrome c into the cytosol following caspase 3 activation. In signal pathways downstream of caspase 3 cleavage, of the three substrates detected, poly (ADP-ribose) polymerase (PARP) and PKC (protein kinase c) delta were not activated in MKN45 cells compared with U937 cells, resulting in delayed appearance of DNA ladder formation during CDDP-induced apoptosis. MKN45 constitutively expressed cLAP1, regardless of CDDP treatment, compared with no expression in U937 Drug sensitivity testing showed that MKN45 was more resistant to CDDP than U937 cells. CONCLUSION: We demonstrated that there is a delayed mitochondrial response and incomplete activation of caspase 3 in MKN45 gastric cancer cells compared with U937 leukemia cells. In addition, there was endogenous cLAP1 expression in MKN45 cells, which may be a factor in the presumed anti-apoptotic system in these human gastric cancer cells.     

CA916798 regulates multidrug resistance of lung cancer cells

Objectives: Multidrug resistance (MDR) significantly reduces the efficacy of chemotherapy for lung cancer. In this study, we characterized the significance of CA916798, a gene up-regulated in cis-dichlorodiamine platinum (CDDP)-resistant lung adenocarcinoma cells, in mediating MDR in lung cancer cells. Methods: CA916798 was stably transfected into H446 cells with low endogenous expression of CA916798, and knocked down in A549/CDDP cells with high endogenous level of CA916798. Expression was confirmed by real-time PCR, Western immunoblotting and immunocytochemistry. Subsequent effects were examined on cellular growth, apoptosis and cell cycle progression. Results: Ectopic expression of CA916798 in H446 cells confered enhanced resistance to multiple chemotherapeutic agents, while its reduction rendered A549/CDDP cells less resistant to chemotherapeutic agents tested. Further analysis revealed that CA916798 regulates CDDP-induced cell growth, apoptosis and cell cycle progression. Conclusion: CA916798 may be a novel MDR-related target for lung cancer therapy.     

Expression of kinase suppressor of Ras1 enhances cisplatin-induced extracellular signal-regulated kinase activation and cisplatin sensitivity

Kinase suppressor of Ras1 (KSR1) interacts with several mitogen-activated protein (MAP) kinase pathway components, including Raf, MAP/extracellular signal-regulated kinase (ERK) kinase (MEK), and ERK, and acts as a positive regulator of the Ras signaling cascade. Previous studies have shown that exposure of cells to the anticancer agent cisplatin (cis-diamminedichloroplatinum, CDDP) is associated with changes in multiple signal transduction pathways, including c-Jun-NH2-kinase, ERK, and p38 pathways. Moreover, ERK activation has been linked to changes in cell survival following CDDP treatment. In this report, we have examined the effects of KSR1 expression on the sensitivity of cells to CDDP-induced apoptosis. Loss of KSR1 expression in mouse embryo fibroblasts (MEFs) derived from KSR1 knockout mice (KSR-/- MEF) is associated with decreased CDDP-induced ERK activation and increased resistance to CDDP-induced apoptosis compared with wild-type MEFs (KSR+/+ MEF). Furthermore, transduction of KSR-/- MEFs and MCF-7 breast cancer cells with wild-type KSR1 resulted in enhanced ERK activation following CDDP exposure and increased sensitivity to CDDP. In addition, inhibition of ERK activation by exposing MEFs to the MEK1/2-specific inhibitors PD98059 and U0126 protected both KSR+/+ and KSR-/- MEFs cells from CDDP-induced apoptosis. These results indicate that KSR1-mediated regulation of ERK activity represents a novel determinant of CDDP sensitivity of cancer cells.     

p22phox confers resistance to cisplatin,by blocking its entry into the nucleus

Cisplatin (CDDP) is a potent chemotherapeutic agent but resistance to the drug remains a major challenge in cancer treatment. To evaluate the efficacy of CDDP in oral squamous cell carcinoma (OSCC), we found that p22phox was highly expressed in CDDP-resistant OSCC specimens. Knockdown of p22phox sensitized OSCC cell lines to CDDP (P < 0.05). Stable overexpression of p22phox augmented CDDP resistance, as evidenced by the significantly higher IC₅₀ values. This cytoprotective effect was attributed to the abrogation of CDDP-induced apoptosis. Akt phosphorylation was increased in p22phox stable lines. However, blocking PI3K/Akt pathway only partially restored CDDP-induced apoptosis. In addition, the overexpressed p22phox in OSCC cells exhibited cytoplasmic localization with enhanced perinuclear expression, consistent with the localization pattern in OSCC specimens. Remarkably, CDDP entry into the nucleus was severely impaired in p22phox-overexpressing cells (P < 0.001), and cytoplasmically accumulated CDDP was co-localized with overexpressed p22phox. This was supported by decreased CDDP-DNA adduct formation and delayed chk1-p53 signaling activation. Together, overexpression of p22phox sequestered CDDP and caused defective CDDP entry into the nucleus, significantly attenuating CDDP-induced apoptosis. Such diminished apoptosis was further abolished by p22phox-activating PI3K/Akt pathway. Our work has suggested a novel biomarker and insight into the mechanism of CDDP resistance.     

Cisplatin (CDDP) specifically induces apoptosis via sequential activation of caspase-8, -3 and -6 in osteosarcoma

Purpose: Osteosarcoma is a common malignant tumor. The first choice of treatment plan for osteosarcoma is chemotherapy. In particular, preoperative chemotherapy is most important in clinical treatment in orthopedics. In these chemotherapies, multiple anticancer drugs such as Adriamycin (ADM), CDDP, cyclophosphamide (CPM), methotrexate (MTX) and vincristine (VCR) are commonly used in combination. Recently, anticancer drugs have been shown to trigger apoptosis in various cancer cells. However, many studies on this topic have been examined using leukemia cell lines, and many kinds of cancer cells established from solid tumor are resistant to the induction of apoptosis by anticancer drugs. So in this study, we examined the effects of the anticancer drugs ADM, CDDP, CPM, MTX and VCR on osteosarcoma cells in vitro. We also examined the signaling pathways of each anticancer drug by studying the induction of apoptosis and activation of caspases in the osteosarcoma cells. Methods: We examined the effects of the anticancer drugs ADM, CDDP, CPM, MTX and VCR, which are used clinically for the treatment of osteosarcoma, on cells of the human osteosarcoma (HOS) cell line. The cytotoxic effects of the anticancer drugs were evaluated using the MTT assay. We used both flow cytometry and activation of caspases to confirm the induction of apoptosis in the HOS cells. To dissect the pathway of the caspase cascade in apoptosis in HOS cells, we used the tetrapeptides YVAD-CHO, DMQD-CHO, VEID-CHO and IETD-CHO, which selectively inhibit caspase-1, -3, -6 and -8, respectively. Results: ADM, CDDP, CPM and VCR, but not MTX, induced death of HOS cells in a dose-dependent manner. CDDP at 10 μM, CPM at 7.5 μM, ADM at 20 μM and VCR at 150 μM caused 80% cell death of HOS cells after 12 h. However, the percentages of apoptotic cells were 5.6% (medium alone), 75.9% (CDDP), 20.0% (CPM), 22.2% (ADM), 20.5% (VCR) and 13.1% (MTX). In addition, direct measurement of caspase-3 activity revealed that CDDP but not the other drugs activated caspase-3 in HOS cells. These analyses revealed that only CDDP induced apoptosis of HOS cells via activation of caspases. Furthermore, DMQD-CHO, VEID-CHO and IETD-CHO inhibited CDDP-induced apoptosis of HOS cells, suggesting that caspase-3, -6 and -8 are involved in the signaling pathway of CDDP-induced apoptosis. In contrast, none of the caspase inhibitors inhibited cell death induced by the other anticancer drugs. Conclusions: This study demonstrates that CDDP specifically induces apoptosis via activation of caspases and the other anticancer drugs induce death of HOS cells via different signaling pathways. It also demonstrates that caspase-8 is a key molecule in the earliest stage of the signaling pathway of CDDP-induced apoptosis of HOS cells, and caspase-3 works downstream of caspase-8. Received: 29 March 1999 / Accepted: 20 July 1999     

P73 regulates cisplatin-induced apoptosis in ovarian cancer cells via a calcium/calpain-dependent mechanism

P73 is important in drug-induced apoptosis in some cancer cells, yet its role in the regulation of chemosensitivity in ovarian cancer (OVCA) is poorly understood. Furthermore, if and how the deregulation of p73-mediated apoptosis confers resistance to cisplatin (CDDP) treatment is unclear. Here we demonstrate that TAp73α over-expression enhanced CDDP-induced PARP cleavage and apoptosis in both chemosensitive (OV2008 and A2780s) and their resistant counterparts (C13* and A2780cp) and another chemoresistant OVCA cells (Hey); in contrast, the effect of ΔNp73α over-expression was variable. P73α downregulation attenuated CDDP-induced PUMA and NOXA upregulation and apoptosis in OV2008 cells. CDDP decreased p73α steady-state protein levels in OV2008, but not in C13*, although the mRNA expression was identical. CDDP-induced p73α downregulation was mediated by a calpain-dependent pathway. CDDP induced calpain activation and enhanced its cytoplasmic interaction and co-localization with p73α in OV2008, but not C13* cells. CDDP increased the intracellular calcium concentration ([Ca(2+)](i)) in OV2008 but not C13* whereas cyclopiazonic acid (CPA), a Ca(2+)-ATPase inhibitor, caused this response and calpain activation, p73α processing and apoptosis in both cell types. CDDP-induced [Ca(2+)](i) increase in OV2008 cells was not effected by the elimination of extracellular Ca(2+), but this was attenuated by the depletion of internal Ca(2+) store, indicating that mobilization of intracellular Ca(2+]) stores was potentially involved. These findings demonstrate that p73α and its regulation by the Ca(2+)-mediated calpain pathway are involved in CDDP-induced apoptosis in OVCA cells and that dysregulation of Ca(2+)/calpain/p73 signaling may in part be the pathophysiology of CDDP resistance. Understanding the cellular and molecular mechanisms of chemoresistance will direct the development of effective strategies for the treatment of chemoresistant OVCA.     

survivin 及bcl-2 基因表达在胃癌细胞株MGC803 顺铂耐药中的作用

 目的 探讨胃癌细胞株MGC803对顺铂（CDDP）耐药产生的有关机制。方法 观察不同浓度CDDP（0．1、1．0、10．0mg／L）对MGC803细胞增殖、凋亡及抗凋亡基因survivin，bcl-2 mRNA表达的影响，用MTT法检测细胞生长抑制率；荧光染色检测细胞凋亡率；流式细胞仪测定细胞周期的变化；RT-PCR检测survivin、bcl-2 mRNA的表达。结果 10mg／L的CDDP对MGC803细胞有较强的抑制增殖、促进凋亡作用，而0．1、1．0mg／L的CDDP干预24h后，其抑制细胞增殖，促进细胞凋亡的作用逐渐消失；CDDP作用于细胞48h后，细胞S期增加，G2／M期下降；MGC803细胞在1．0mg／L的CDDP作用下，survivin mRNA表达自24h后逐渐增高，bcl-2 mRNA表达逐渐下降（P〈0．05）。结论 CDDP可干扰MGC803细胞周期，但该细胞对低浓度CDDP易于产生耐药，survivin mRNA表达增高可能是MGC803细胞对CDDP产生耐药原因之一。     

Calpain is involved in cisplatin-induced endothelial injury in an in vitro three-dimensional blood vessel model

To study endothelial injury in vitro, we established a three-dimensional (3-D) blood vessel model in which human umbilical vein endothelial cells were grown in the presence of basic fibroblast growth factor and vascular endothelial growth factor. We then performed comparative studies on cisplatin (cis-platinum-diammine-dichloride, CDDP)-induced endothelial injury in 3-D and monolayer cultures. In 3-D culture, CDDP induced cell death and tube breakdown without DNA damage, whereas CDDP induced apoptosis accompanied by DNA damage in monolayer culture. CDDP also induced caspase-3 activation in a concentration-dependent manner in both cultures. A broad-spectrum caspase inhibitor, zVAD-fmk, failed to prevent CDDP-induced cell death and tube breakdown in 3-D culture, whereas zVAD-fmk suppressed CDDP-induced apoptosis in monolayer culture. A calpain inhibitor, MDL28170, attenuated CDDP-induced cell death and tube breakdown in 3-D culture, but not apoptosis in monolayer culture. These results showed that calpain is involved in CDDP-induced endothelial injury in 3-D culture and there are significant differences in signaling pathways between 3-D and monolayer cultures.     

survivin反义寡核苷酸提高人肺腺癌A549耐药细胞系对顺铂敏感性的研究

目的探讨存活素反义寡核苷酸(ASODN)体外转染对耐顺铂人肺腺癌细胞A549/ CDDP凋亡及其对顺铂(CDDP)敏感性的影响。方法常规体外培养A549/CDDP细胞,以脂质体包裹的survivin ASODN转染细胞,采用RT-PCR法和免疫细胞化学法检测survivin mRNA及蛋白表达。通过形态学观察、DNA琼脂糖凝胶电泳、caspase-3酶活性及细胞凋亡率(AI)的测定,评价细胞凋亡程度。采用MTT法测定细胞存活率和生长抑制率,计算半效抑制浓度(IC_(50))及耐药倍数(RI)。结果转染组survivin mRNA及蛋白表达下调明显,分别达41．56%和0．864±0．045,与其他各组比较,差异均有统计学意义(P＜0．05)。细胞形态学显示有细胞凋亡改变,表现为细胞核固缩、核边集和核碎裂等;DNA凝胶电泳可见DNA梯形条带;ASODN转染组细胞凋亡率和caspase-3相对活性分别增高至34．03%和1．1298±0．2502,而ASODN+CDDP组增高更为明显,分别达65．85%和1．6805±0．2758,与其他各组比较,差异均有统计学意义(P＜0．05)。ASODN转染组和ASODN+CDDP组生长抑制率分别增高至59．3%和83．7%(P＜0．05);而ASODN转染组细胞对CDDP的IC_(50)由对照组的(225．03±10．59)μmol/L减低至(158．84±4．26)μmol/L,RI由11．9减至8．4。结论survivin ASODN通过下调survivin的表达,自身诱导了细胞凋亡,并逆转细胞对CDDP诱导的细胞凋亡的耐受,降低凋亡阈值,从而增强了人肺腺癌细胞对CDDP的敏感性。     

Aurora B confers cancer cell resistance to TRAIL-induced apoptosis via phosphorylation of survivin

Tumor necrosis factor-related apoptosis-induced ligand (TRAIL) induces apoptosis selectively in cancer cells while sparing normal cells. However, many cancer cells are resistant to TRAIL-induced cell death. In this study, we examined whether Aurora B, which is frequently overexpressed in cancer cells, is associated with TRAIL resistance. The protein levels of Aurora B were higher in TRAIL-resistant cancer cell lines than in TRAIL-sensitive cancer cell lines. Exogenously expressed Aurora B attenuated TRAIL-induced apoptosis in the tested TRAIL-sensitive cancer cell lines, whereas the small interfering RNA-mediated suppression of Aurora B expression stimulated TRAIL-mediated apoptosis in the tested TRAIL-resistant cancer cell lines. Furthermore, combined treatment with TRAIL and ZM447439, a specific inhibitor of Aurora B, synergistically induced apoptosis in various TRAIL-resistant cancer cells, suggesting that this combined regimen may represent an attractive strategy for effectively treating TRAIL-resistant malignant cancers. Mechanistically, the inhibition of Aurora B activity in various cancer cells commonly downregulated survivin protein levels and potentiated the activation of caspase-3. In addition, Aurora B inhibition induced mitotic catastrophe, which also contributed to the sensitization of cells to TRAIL-mediated apoptosis. Interestingly, forced overexpression of Aurora B increased the protein levels of survivin, but not those of a non-phosphorylatable survivin mutant in which threonine 117 was replaced by alanine, indicating that phosphorylation of survivin is required for this effect. Furthermore, TRAIL-induced apoptosis in MDA-MB-435S cells was attenuated by wild-type survivin but not by the non-phosphorylatable survivin mutant. Collectively, our results demonstrate that Aurora B confers TRAIL resistance to cancer cells via phosphorylation of survivin.     

Survivin基因在胃癌中的表达及临床意义

目的研究凋亡抑制基因survivin在胃癌组织中的表达。方法运用SP免疫组化技术检测survivin基因在50例胃癌组织及相应的癌旁组织中的表达。结果survivin基因在50例癌旁组织中不表达,而在50例胃癌组织中有40例表达阳性,阳性率80％,两者差异有统计学意义（P〈0．05）。survivin基因表达阳性率与胃癌组织的分化程度、淋巴结转移及TNM分期相关,而与浸润程度无相关。结论survivin在胃癌组织中的特异性高表达,说明其表达可以抑制胃癌细胞的自身凋亡过程,其在癌细胞的生长和增殖过程中起着广泛的促进作用。     

Cisplatin activates Akt in small cell lung cancer cells and attenuates apoptosis by survivin upregulation

The inhibitor of apoptosis protein (IAP) survivin is overexpressed in many tumors but is absent in most normal adult tissues. We report high levels of survivin expression in small cell lung cancer (SCLC), and describe the role of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in survivin upregulation. Moreover, the cytoprotective function of survivin in response to the anti-cancer agent cisplatin (CDDP) was investigated. Negative modulation of PI3K/Akt using pharmacological inhibitors or dominant negative Akt (DN-Akt) decreased Akt kinase activity and resulted in decreased survivin expression and phosphorylation on Thr34, whereas transfection of constitutively active Akt (CA-Akt) increased survivin expression and phosphorylation. Interestingly, we found that treatment of SCLC cells with CDDP further increased survivin expression in a cell cycle independent manner by activation of Akt. CA-Akt or lentiviral survivin also inhibited apoptosis induced by CDDP, whereas DN-Akt or survivin-specific RNA interference sensitized cells to CDDP. We identified survivin as an anti-apoptotic protein in SCLC cells that is regulated by Akt, and demonstrate that treatment with the DNA damaging agent CDDP activates the PI3K/Akt/survivin pathway that in part protects cells from drug-induced apoptosis.     

RNA干扰survivin基因对卵巢癌细胞生长凋亡及药物敏感性的影响

目的 探讨下调survivin基因对卵巢癌顺铂(DDP)耐药细胞株SKOV3/DDP细胞生长、凋亡及药物敏感性的影响.方法 构建survivin基因的小干扰RNA(siRNA)表达载体pSilencer-survivin,用脂质体方法转染SKOV3/DDP细胞株,另设未转染组和转染pSilencer-control组作为对照.显微镜下观察转染前后细胞的变化,逆转录聚合酶链反应(RT-PCR)和Western blot分别检测survivinmRNA及蛋白的表达,二苯基溴化四氮唑蓝(MTT)法检测细胞生长情况,流式细胞仪检测细胞凋亡及周期的变化.各组细胞加入DDP,检测其对DDP药物敏感性的影响.结果 survivin siRNA可明显下调SKOV3/DDP细胞中survivin mRNA及蛋白表达水平.SKOV3/DDP细胞生长受到明显抑制,生长曲线低平.转染48 h后,转染pSilencer-survivin组细胞凋亡率为19.1%,明显高于末转染组(2.6%)和转染pSilencer-control组(3.5%),G1/G0期细胞所占的比例增高,而G2/M期细胞所占的比例降低.转染pSilencer-survivin组细胞的IC50明显降低,为1.16 μg/mi.结论 survivin siRNA可下调SKOV3/DDP细胞中survivin的表达,使细胞生长减慢,凋亡增加,对DDP的药物敏感性增强.     

Exogenous morphine inhibits human gastric cancer MGC- 803 cell growth by cell cycle arrest and apoptosis induction

Morphine is not only an analgesic treating pain for patients with cancer but also a potential anticancer drug inhibiting tumor growth and proliferation. To gain better insight into the involvement of morphine in the biological characteristics of gastric cancer, we investigated effects on progression of gastric carcinoma cells and the expression of some apoptosis-related genes including caspase-9, caspase-3, survivin and NF-κB using the MGC-803 human gastric cancer cell line. The viability of cells was assessed by MTT assay, proliferation by colony formation assay, cell cycle progression and apoptosis by flow cytometry and ultrastructural alteration by transmission electron microscopy. The influences of morphine on caspase-9, caspase-3, survivin and NF-κB were evaluated by semi-quantitative RT-PCR and Western blot. Our data showed that morphine could significantly inhibit cell growth and proliferation and cause cell cycle arrest in the G2/M phase. MGC-803 cells which were incubated with morphine also had a higher apoptotic rate than control cells. Morphine also led to morphological changes of gastric cancer cells. The mechanism of morphine inhibiting gastric cancer progression in vitro might be associated with activation of caspase-9 and caspase-3 and inhibition of survivin and NF-κB.