MAPK inhibitors and siRNAs differentially affect cell death and ROS levels in arsenic trioxide-treated human pulmonary fibroblast cells

Arsenic trioxide (ATO; As2O3) induces cell death in various types of cancer cells including lung cancer via increasing reactive oxygen species (ROS) and regulating mitogen-activated protein kinase (MAPK) signaling cascades. However, little is known about the relationship between ATO and MAPK signaling in normal lung cells. Here, we investigated the effects of MAPK inhibitors and siRNAs on ATO-treated human pulmonary fibroblast (HPF) cells in relation to cell growth, cell death, ROS and glutathione (GSH) levels. ATO induced cell growth inhibition and death in HPF cells and it increased ROS levels including O2?- and GSH depleted cell number. None of the MAPK (MEK, JNK and p38) inhibitors affected cell growth inhibition and cell death by ATO. The MEK inhibitor decreased O2?- levels in ATO-treated HPF cells whereas JNK and p38 inhibitors generally increased ROS levels including O2?- in these cells. None of these inhibitors altered the ATO-induced GSH depletion. Moreover, ERK siRNA did not change HPF cell growth and death by ATO whereas JNK and p38 siRNAs enhanced cell growth inhibition and death. In addition, JNK and p38 siRNAs increased ROS levels and GSH depletion in ATO-treated HPF cells. In conclusion, MAPK inhibitors changed ROS levels in ATO-treated HPF cells, but did not affect cell growth inhibition and death. siRNAs targeting JNK and p38 showing an increase in ROS levels and GSH depletion in ATO-treated HPF cells augmented cell growth inhibition and death.     

类风湿关节炎患者血清HIF-1α、ICAM-1、VCAM-1表达与恶性肿瘤的关系

目的 探讨类风湿关节炎(RA)患者血清低氧诱导因子-1α(HIF-1α)、细胞间黏附分子-1(ICAM-1)、血管细胞黏附分子-1(VCAM-1)水平与恶性肿瘤的关系.方法 选取活动期RA患者50例为RA组,RA合并恶性肿瘤患者25例为RA合并恶性肿瘤组,健康体检者50例为健康对照组.应用ELISA法测定3组研究对象的血清HIF-1α、ICAM-1、VCAM-1水平,记录RA组与RA合并恶性肿瘤组类风湿因子(RF)、抗环瓜氨酸肽抗体(anti-CCP)水平及DAS28评分.应用受试者特异性曲线(ROC)分析血清HIF-1α、ICAM-1、VCAM-1水平在RA合并恶性肿瘤患者诊断中的应用价值.结果 RA合并恶性肿瘤组的血清HIF-1α、ICAM-1、VCAM-1水平均高于RA组及健康对照组(P﹤0.05);RA组血清HIF-1α、ICAM-1、VCAM-1水平高于健康对照组(P﹤0.05);RA合并恶性肿瘤组的RF、anti-CCP水平及DAS28评分均明显高于RA组(P﹤0.01).经ROC曲线分析可知,HIF-1α、ICAM-1、VCAM-1曲线下面积(AUC)分别为0.792、0.785、0.752.结论 RA患者血清HIF-1α、ICAM-1、VCAM-1表达与恶性肿瘤的发生有密切的关系,可作为RA合并恶性肿瘤的早期诊断指标.     

Molecular Signature of Tumors with Monoallelic 13q14 Deletion: a Case Series of Spindle Cell Lipoma and Genetically-Related Tumors Demonstrating a Link Between FOXO1 Status and p38 MAPK Pathway

Spindle cell/pleomorphic lipomas (SCLs), cellular angiofibromas (CAFs) and mammary-type myofibroblastomas (MFBs) are rare benign mesenchymal tumors with monoallelic 13q14 deletion. They are predicted to have a common pathogenic mechanism due to shared similar histological and immunohistochemical features; however, pathological consequences of monoallelic 13q14 deletion remain unknown. We previously reported a CAF case with monoallelic 13q14 deletion in which the tumor expressed decreased levels of FOXO1 and RB1, both of which were encoded in 13q14, and increased reactive oxygen species (ROS) levels. We further demonstrated the activation of p38 mitogen-activated protein kinase (p38 MAPK) pathway induced by oxidative stress. We hypothesized that SCLs, CAFs and MFBs would share common molecular signatures involving FOXO1, ROS and p38 MAPK and that their expression patterns were different from those tumors without monoallelic 13q14 deletion such as solitary fibrous tumors (SFTs). We compared the expression levels of FOXO1, RB1, ROS markers and several signal transduction factors between SCLs and SFTs. SCLs expressed decreased levels of FOXO1 and RB1, whereas SFTs showed no change. Both tumor types exhibited increased markers of ROS; however, nuclear localization of phosphorylated p38 was significantly more frequent in SCLs than that in SFTs, suggesting p38 MAPK activation by oxidative stress. SFTs showed lower p38 MAPK activity and higher β-catenin expression, implying that oxidative stress was caused by increased cellular proliferation stress. Finally, CAFs and MFBs showed changes similar to those observed in SCLs. Overall, tumors with monoallelic 13q14 deletion showed shared molecular signatures that might be associated with pathogenesis.     

Enhanced gamma-glutamylcysteine synthetase activity decreases drug-induced oxidative stress levels and cytotoxicity

Multidrug resistance of cancer cells can be intrinsic or acquired and occurs due to various reasons, including increased repair of genotoxic damage, an enhanced ability to remove/detoxify chemical agents, or reactive oxygen species (ROS), and repression of apoptosis. Human A2780/100 ovarian carcinoma cells exhibit resistance to DNA cross-linking agents, chlorambucil (Cbl), cisplatin (Cpl), melphalan (Mel), and ionizing radiation (IR) compared to the parental cell line, A2780. In the present study, we show that when A2780/100 and A2780 cells were treated with Cbl, GSH was extruded via methionine or cystathionine-inhibitable transporters of intact plasma membrane. GSH loss was followed by a rapid increase in ROS levels. The resistant, but not drug-sensitive cells normalized the intracellular GSH concentration along with ROS levels within 4-6 h after Cbl addition, and survived drug treatment. Normalization of GSH and ROS levels in A2780/100 cells correlated well with elevated gamma-glutamylcysteine synthetase (gamma-GCS) activity (10 +/- 1.8-fold over A2780 cells). Ectopic overexpression of the gamma-GCS heavy subunit in drug-sensitive cells nearly restored GSH and ROS to pre-treatment levels consequently increased cellular resistance to genotoxic agents (Cbl, Cpl, and IR), while overexpression of gamma-GCS light subunit had no such effects. Thus, in our model system, drug-resistant cells have the inherent ability to maintain increased gamma-GCS activity, reestablish physiological GSH, and cellular redox state and maintain increased cellular resistance to DNA cross-linking agents and IR.     

CL100/MKP-1 modulates JNK activation and apoptosis in response to cisplatin

Treatment of cells with cisplatin induces a sustained activation of the stress activated protein kinase SAPK/JNK and the mitogen-activated protein kinase p38. Activation of JNK by cisplatin is necessary for the induction of apoptosis. Expression of the MAPK phosphatases CL100/MKP-1 and hVH-5 selectively prevents JNK/SAPK activation by cisplatin in a dose dependent fashion and results in protection against cisplatin-induced apoptosis. In contrast, expression of the ERK-specific phosphatase Pyst1 inhibits JNK/SAPK activity only when expressed at very high levels and does not confer protection against cisplatin. Furthermore, expression of a catalytically inactive mutant of CL100 in 293 cells decreases the IC50 for cisplatin and increases the toxicity of transplatin. This effect seems to be mediated by an increase in JNK activity since p38 activity is unaffected. These results suggest that dual-specificity MAPK phosphatases may be candidate drug targets in order to optimize cisplatin based therapeutic protocols.     

Expression of intercellular adhesion molecule-1 in hepatocellular carcinoma

The expression of intercellular adhesion molecule-1 (ICAM-1) was investigated in frozen sections obtained from 40 resected liver specimens of patients with hepatocellular carcinoma using immunoperoxidase techniques and immunoelectron microscopy. ICAM-1 was expressed in 80% of the HCC specimens on the membrane of cancer cells. In noncancerous regions characterized by cirrhosis in 28 cases and chronic hepatitis in 12 cases, ICAM-1 was rarely expressed on hepatocytes but was expressed mainly on the endothelium of portal vessels and sinusoidal lining cells. These results suggest that expression of ICAM-1 in hepatocellular carcinoma may be induced by malignant transformation of hepatocytes. © 1993 Wiley-Liss, Inc.     

CD44 variant regulates redox status in cancer cells by stabilizing the xCT subunit of system xc(-) and thereby promotes tumor growth

CD44 is an adhesion molecule expressed in cancer stem-like cells. Here, we show that a CD44 variant (CD44v) interacts with xCT, a glutamate-cystine transporter, and controls the intracellular level of reduced glutathione (GSH). Human gastrointestinal cancer cells with a high level of CD44 expression showed an enhanced capacity for GSH synthesis and defense against reactive oxygen species (ROS). Ablation of CD44 induced loss of xCT from the cell surface and suppressed tumor growth in a transgenic mouse model of gastric cancer. It also induced activation of p38(MAPK), a downstream target of ROS, and expression of the gene for the cell cycle inhibitor p21(CIP1/WAF1). These findings establish a function for CD44v in regulation of ROS defense and tumor growth.     

宫颈癌ICAM-1的表达与微淋巴管密度的关系及其对预后的评估价值

目的:探讨宫颈癌细胞间黏附分子-1(intercellular adhesion molecule-1,ICAM-1)的表达与微淋巴管密度(microlymphatic vessel density,MLVD)的相关性及二者对宫颈癌预后的评估价值.方法:采用免疫组化法检测ICAM-1、D2-40在94例宫颈癌组织、癌旁组织及20例正常宫颈组织中的表达并计数MLVD,分析上述指标与预后的关系,并采用免疫组化双重染色鉴别微淋巴管和微血管.结果:宫颈癌组织ICAM-1阳性率为63.83％ (60/94).肿瘤中心处、宫颈癌旁、正常宫颈组织中MLVD分别为(2.89±0.57)、(8.59±1.38)及(7.98±1.48).非参数检验显示ICAM-1阳性表达与宫颈癌国际妇产科联盟(International Federation of Gynecology and Obstetrics,FIGO)分期及淋巴结转移有关(P＜0.05);癌组织MLVD与FIGO分期及淋巴结转移密切相关(P＜0.05);ICAM-1阳性表达者MLVD明显高于阴性表达者(P＜0.05).结论:ICAM-1与宫颈癌淋巴管的生成相关,其可能参与了宫颈癌的侵袭与转移;ICAM-1对宫颈癌预后有评估价值,ICAM-1联合MLVD的预后评估意义更大.     

NF-κB 反义核酸对胰腺癌细胞ICAM-1表达及侵袭转移能力的影响

 目的 研究核因子-KB（NF-KB）亚单位p65反义寡核苷酸（ASODN）对人胰腺癌细胞细胞间粘附分子-1（ICAM-1）表达及侵袭转移能力的影响。方法 设计合成p65ASODN，体外转染人胰腺癌细胞株PANC-1，用流式细胞仪和荧光显微镜检测转染效率，用RT-PCR检测p65及ICAM-1mRNA的表达，用四甲基偶氮唑盐（MTT）法和细胞侵袭实验分析细胞增殖能力和侵袭转移能力的改变。结果 p65ASODN可成功转染PANC-1细胞；转染后，p65及ICAM-1mRNA表达明显减弱（P〈0．01）；细胞增殖活性和侵袭转移能力明显下降（P〈0．01）。结论 脂质体介导的p65ASODN能有效抑制p65基因的表达，从而降低细胞的侵袭转移能力，p65ASODN可能主要通过下调ICAM-1基因的表达发挥抗侵袭转移的作用。     

Long-term activation of SAPK/JNK, p38 kinase and fas-L expression by cisplatin is attenuated in human carcinoma cells that acquired drug resistance

Tumor cells chronically exposed to cisplatin (cDDP) acquire cDDP resistance that impacts tumor therapy. To elucidate the mechanism of acquired cDDP resistance (ACR), we compared HeLa cells that gained ACR upon chronic cDDP treatment with the parental strain. We show that ACR is due to a lower level of induced apoptosis. Further, upon cDDP treatment, the levels of Fas, Bax and Bid remained unchanged, whereas Bcl-2 and p-Bad were reduced at late times (120 hr) after treatment. At early times, Fas ligand (fas-L) expression was significantly enhanced in sensitive compared to resistant cells and remained upregulated up to the onset of apoptosis. Thus, activation of the Fas system is critical, which is in line with the finding that in sensitive cells, caspase-8 along with caspase-9 and -3 were activated by cDDP. cDDP provoked the activation of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p38 kinase dose-dependently, with significantly lower levels in ACR cells than in the sensitive parental line. cDDP induces c-Jun and AP-1 activity, as measured by a reporter gene assay, which was again attenuated in ACR cells. Time course analysis revealed that SAPK/JNK and p38 kinase activity was sustained upregulated (> 72 hr postexposure), which occurred at much higher level in sensitive than in ACR cells. Inhibition of either JNK or p38 kinase (by JNK inhibitor II and SB 203580, respectively) attenuated cDDP-induced apoptosis, supporting the role of JNK and p38 kinase in the cDDP response. Since several independently derived cDDP-resistant cell lines displayed attenuated MAPK signaling, sustained SAPK/JNK and p38 kinase activation may be a general mechanism of cDDP-induced cell death. ACR cells displayed a reduced level of DNA damage, indicating long-term stimulation of SAPK/JNK and p38 kinase is triggered by nonrepaired cDDP-induced DNA lesions.     

Reactive oxygen species-mediated kinase activation by dihydrotanshinone in tanshinones-induced apoptosis in HepG2 cells

The role of reactive oxygen species (ROS) and p38 mitogen-activated protein kinases (MAPK) in tanshinones-induced apoptosis was investigated in HepG2 cells in this study. The major tanshinones (cryptotanshinone, dihydrotanshinone, tanshinone I, tanshinone IIA), isolated from Salvia miltiorrhiza, inhibit cell growth and induce caspase-dependent apoptosis concentration-dependently, with dihydrotanshinone being the most potent. All four tanshinones were found to induce ROS generation, but only dihydrotanshinone can induce activation of p38 MAPK. The p38 MAPK activation by dihydrotanshinone was inhibited by N-acetyl cysteine pretreatment. It is thus concluded that ROS-mediated p38 MAPK activation plays a vital role in dihydrotanshinone-induced apoptosis in HepG2 cells.     

Role of the p38 MAPK pathway in cisplatin-based therapy

p38 MAPK has been implicated in the response to cancer therapy. To determine whether the activation of p38 MAPK could be specific to cancer therapy, we investigated the activation of p38 MAPK in response to several chemotherapeutic agents, such as cisplatin, doxorubicin and taxol in several human cell lines. Activation of p38 MAPK was measured after exposure to several chemotherapeutic agents, using specific phosphoantibodies. Only cisplatin was able to activate p38 MAPK in all the cell lines tested. Furthermore, other platinum compounds such as transplatin and platinum (IV) chloride can induce activation of p38 MAPK. The kinetics of this activation is a key event in the biological role of p38 MAPK in response to cisplatin, as we conclude from the differences observed after treatment with transplatin and cisplatin. The p38 MAPK activation is independent of the origin or genetic alterations of the cell lines and seems to be mediated through both upstream activators MKK6 and MKK3. Although the isoforms alpha/beta are mainly activated, we also demonstrated that other members of the p38 MAPK family were susceptible to activation by cisplatin when they were overexpressed in 293 T. Finally, pretreatment with specific inhibitors (SB 203580 and SKF 86002) induces a resistant phenotype in response to cisplatin. Furthermore, low activation of this SAPK pathway correlates with a resistant phenotype as demonstrated in our experimental model of head and neck cancer. Therefore, we conclude that the p38 MAPK pathway is a specific target for cisplatin-based therapy with clinical implications.     

Dequalinium induces human leukemia cell death by affecting the redox balance

Dequalinium, an amphiphilic quinolinium derivative, selectively accumulates in mitochondria and displays anticancer activity in cells from different malignancies. Previous studies indicate a differential DQA-induced cytotoxicity in NB4 and K562 human leukemia cells as a consequence of an early disturbance in mitochondrial function. Results in this paper show that DQA induces a concentration-dependent oxidative stress by decreasing GSH level and increasing ROS in a cell type specific way. Inhibitors of the JNK and p38 stress regulated kinases potentiate DQA-induced NB4 cell death suggesting a protective function for these enzymes. K562 cells with relatively high GSH levels remained resistant to DQA action.     

Overexpression of ErbB2 enhances ethanol-stimulated intracellular signaling and invasion of human mammary epithelial and breast cancer cells in vitro

Both epidemiological and experimental studies indicate that ethanol is a tumor promoter and may promote metastasis of breast cancer. However, the molecular mechanisms underlying ethanol-mediated tumor promotion remain unknown. Overexpression of ErbB proteins in breast cancer patients is generally associated with poor prognosis. The ErbB proteins are a family of receptor kinases that include four closely related members: epidermal growth factor receptor (EGFR/ErbB1), ErbB2/neu, ErbB3, and ErbB4. Particularly, ErbB2 plays a pivotal role in ErbB-mediated activities. Here we demonstrated that amplification of ErbB2 expression sensitized a specific cellular response to ethanol. Human breast cancer cells or mammary epithelial cells with a high expression of ErbB2 exhibited an enhanced response to ethanol-stimulated cell invasion in vitro. Ethanol also stimulated cell proliferation; however, this stimulation was independent of ErbB2 levels. Ethanol triggered divergent intracellular signaling among cells expressing different ErbB2 levels. In the cells overexpressing ErbB2, ethanol was more effective in the activation of c-Jun NH2 terminal protein kinases (JNKs) and p38 mitogen-activated protein kinase (p38 MAPK) as well as the induction of reactive oxygen species (ROS) than the cells with normal ErbB2 expression. Blockage of either JNKs or p38 MAPK activation eliminated ethanol-mediated cell invasion. In contrast, the reduction of hydrogen peroxide concentration by catalase exposure had little effect on ethanol-induced cell invasion. These results indicated that ethanol-induced cell invasion was primarily mediated by JNKs and p38 MAPK, whereas the involvement of ROS formation might be minimal. Our study suggests that overexpression of ErbB2 may augment ethanol-elicited signaling and promote ethanol-stimulated tumor metastasis.