MicroRNA 506 regulates expression of PPAR alpha in hydroxycamptothecin-resistant human colon cancer cells

Chemotherapeutic drug resistance remains a major obstacle to the successful treatment of colon cancer. Here, we show that 77 differentially expressed miRNAs were identified in SW1116/HCPT versus SW1116, and over-expressed miR-506 in SW1116/HCPT cells was validated. Then it was indicated that PPARα is a common target of miR-506 by using a luciferase reporter assay. Our results also demonstrated that cytotoxic ability of HCPT requires the concomitant presence of PPARα, and that loss of PPARα expression imparts resistance to HCPTs anti-tumor effects. All together, our studies indicate that miR-506 over-expression in established HCPT-resistant colon cancer cell line confers resistance to HCPT by inhibiting PPARα expression, then providing a rationale for the development of miRNA-based strategies for reversing resistance in HCPT-resistant colon cancer cells.     

Annexin A3 depletion overcomes resistance to oxaliplatin in colorectal cancer via the MAPK signaling pathway

Colorectal cancer (CRC) is a common disease with high mortality and morbidity. Annexin A3 (ANXA3) belongs to the structurally homologous family of Ca²⁺ and phospholipid-binding proteins. This study aimed to investigate the effects and potential mechanisms of ANXA3 on oxaliplatin (Ox) resistance in CRC. We generated two human CRC cell lines (HCT116/Ox and SW480/Ox) with acquired Ox resistance and determined their resistance properties. ANXA3 expression and cell apoptosis, migration and invasion also were evaluated. We found that cell viability of HCT116/Ox and SW480/Ox was higher than that in parental cells in the presence of Ox. ANXA3 was highly expressed in HCT116/Ox and SW480/Ox cells. ANXA3 downregulation diminished cell survival, migration and invasion, while increased the apoptosis of HCT116 and SW480 with or without Ox. Moreover, depletion of ANXA3 reduced cell viability and BrdU incorporation, increased cell apoptosis and c-caspase 3 expression in HCT116/Ox with or without Ox. A transwell assay determined that knockdown of ANXA3 impeded the migration and invasion of HCT116/Ox and SW480/Ox cells. Additionally, phosphorylation of extracellular signal–regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) decreased upon ANXA3 depletion in HCT116/Ox cells, and ANXA3 silencing suppressed Ox-induced activation of ERK and JNK signaling pathway. ANXA3 downregulation reduced Ox resistance in CRC, and treatment with the ERK inhibitor PD098059 or JNK inhibitor SP600125 contributed to this process. These results indicate that silencing ANXA3 could overcome Ox resistance in CRC via the mitogen-activated protein kinase signaling pathway.     

Inhibition of cell proliferation and cell cycle progression by specific inhibition of basal JNK activity: evidence that mitotic Bcl-2 phosphorylation is JNK-independent

The c-Jun NH(2)-terminal kinase (JNK) subgroup of mitogen-activated protein kinases has been implicated largely in stress responses, but an increasing body of evidence has suggested that JNK also plays a role in cell proliferation and survival. We examined the effect of JNK inhibition, using either SP600125 or specific antisense oligonucleotides, on cell proliferation and cell cycle progression. SP600125 was selective for JNK in vitro and in vivo versus other kinases tested including ERK, p38, cyclin-dependent protein kinase 1 (CDK1), and CDK2. SP600125 inhibited JNK activity and KB-3 cell proliferation with the same dose dependence, suggesting that inhibition of proliferation was a direct consequence of JNK inhibition. Inhibition of proliferation by SP600125 was associated with an increase in the G(2)-M and apoptotic fractions of cells but was not associated with p53 or p21 induction. Antisense oligonucleotides to JNK2 but not JNK1 caused highly significant inhibition of cell proliferation. Wild-type mouse fibroblasts responded similarly with proliferation inhibition and apoptosis induction, whereas c-jun(-/-) fibroblasts were refractory to the effects of SP600125, suggesting that JNK signaling to c-Jun is required for cell proliferation. Studies in synchronized KB-3 cells indicated that SP600125 delayed transit time through S and G(2)-M phases. Correspondingly, JNK activity increased in late S phase and peaked in late G(2) phase. During synchronous mitotic progression, cyclin B levels increased concomitant with phosphorylation of c-Jun, H1 histone, and Bcl-2. In the presence of SP600125, mitotic progression was prolonged, and c-Jun phosphorylation was inhibited, but neither H1 nor Bcl-2 phosphorylation was inhibited. However, the CDK inhibitor roscovitine inhibited mitotic Bcl-2 phosphorylation. These results indicate that JNK, and more specifically the JNK2 isoform, plays a key role in cell proliferation and cell cycle progression. In addition, conclusive evidence is presented that a kinase other than JNK, most likely CDK1 or a CDK1-regulated kinase, is responsible for mitotic Bcl-2 phosphorylation.     

Butyrate induces cell apoptosis through activation of JNK MAP kinase pathway in human colon cancer RKO cells

Butyrate has been shown to display anti-cancer activity through the induction of apoptosis in various cancer cells. However, the underlying mechanism involved in butyrate-induced apoptosis is still not fully understood. Here, we investigated the cytotoxicity mechanism of butyrate in human colon cancer RKO cells. The results showed that butyrate induced a strong growth inhibitory effect against RKO cells. Butyrate also effectively induced apoptosis in RKO cells, which was characterized by DNA fragmentation, nuclear staining of DAPI, and the activation of caspase-9 and caspase-3. The expression of anti-apoptotic protein Bcl-2 decreased, whereas the apoptotic protein Bax increased in a dose-dependent manner during butyrate-induced apoptosis. Moreover, treatment of RKO cells with butyrate induced a sustained activation of the phosphorylation of c-jun N-terminal kinase (JNK) in a dose- and time-dependent manner, and the pharmacological inhibition of JNK MAPK by SP600125 significantly abolished the butyrate-induced apoptosis in RKO cells. These results suggest that butyrate acts on RKO cells via the JNK but not the p38 pathway. Butyrate triggered the caspase apoptotic pathway, indicated by an enhanced Bax-to-Bcl-2 expression ratio and caspase cascade reaction, which was blocked by SP600125. Taken together, our data indicate that butyrate induces apoptosis through JNK MAPK activation in colon cancer RKO cells.     

SP600125, a selective JNK inhibitor, protects ischemic renal injury via suppressing the extrinsic pathways of apoptosis

Accumulating evidence suggests that c-Jun N-terminal kinase (JNK) signaling pathway plays a critical role in renal ischemia/reperfusion injury. However, the downstream mechanism that accounts for the proapoptotic actions of JNK during renal ischemia/reperfusion has not been elucidated. We report that SP600125, a potent, cell-permeable, selective, and reversible inhibitor of c-Jun N-terminal kinase (JNK), potently decreased renal epithelial tubular cell apoptosis induced by renal ischemia/reperfusion via suppression of the extrinsic pathway. This corresponds to the decrease in JNK phosphorylation at 20 min and c-Jun phosphorylation (Ser63/73) at 3 h after renal ischemia. Additionally, SP600125 attenuated the increased expression of FasL induced by ischemia/reperfusion at 3 h. The administration of SP600125 prior to ischemia was also protective. Thus, our findings imply that SP600125 can inhibit the activation of the JNK-c-Jun-FasL pathway and protect renal tubular epithelial cells against ischemia/reperfusion-induced apoptosis. Taken together, these results indicate that targeting the JNK pathway provides a promising therapeutic approach for renal ischemia/reperfusion injury.     

SP600125 inhibits cap-dependent translation independently of the c-Jun N-terminal kinase pathway

We investigated the effects of SP600125 (formerly called c-Jun N-terminal kinase (JNK) inhibitor II) on translation using cultured mouse cells. SP600125 (50 μM) treatment rapidly repressed overall protein synthesis, accompanied by a reduction in the mRNAs for housekeeping genes such as glyceraldehyde-3-phosphate dehydrogenase in the polysomal fraction. SP600125 decreased polysomes with a concomitant increase in free ribosomal subunits in the cytoplasm, suggesting that global translation was inhibited at the initiation step. A reporter analysis using exogenous mRNAs showed that SP600125 inhibited cap-dependent but not internal ribosome entry site-dependent translation. SP600125 significantly attenuated phosphorylation of components in the mTOR pathway, which is responsible for cap-dependent translation. In contrast to SP600125, short hairpin RNAs for JNK1 and JNK2 failed to affect overall protein synthesis. Collectively, SP600125 inhibits cap-dependent translation, independent of the JNK pathway.     

Activation of c-Jun NH2-terminal kinase is required for gemcitabine's cytotoxic effect in human lung cancer H1299 cells

Although gemcitabine is a potent therapeutic agent in the treatment of human non-small cell lung cancer (NSCLC), resistance to gemcitabine is common. In this study, we investigated the molecular mechanisms involved in acquired gemcitabine resistance against NSCLC cells. Gemcitabine-resistant NSCLC H1299 cells (H1299/GR) were selected by long-term exposure of parental H1299 cells to gemcitabine. The median inhibitory concentrations of gemcitabine in H1299 and H1299/GR cells were 19.4 and 233.1 nM, respectively. Gemcitabine induced activation of c-Jun NH2-terminal kinase (JNK) in parental H1299 cells but not in H1299/GR cells after 48 h. Blocking JNK activation by pretreatment with SP600125, a specific JNK inhibitor, or by transfection with dominant-negative JNK vectors abrogated gemcitabine-induced apoptosis in parental H1299 cells as evidenced by interruption of caspase activation. Transient transfection with a JNKK2-JNK1 plasmid expressing constitutive JNK1 partially restored the effect of gemcitabine in H1299/GR cells. Our results indicate that gemcitabine-induced apoptosis in human NSCLC H1299 cells requires activation of the JNK signaling pathway. Attenuated JNK activation may contribute to development of acquired gemcitabine resistance in cancer cells.     

P-glycoprotein down-regulates expression of breast cancer resistance protein in a drug-free state

This study investigated whether P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) are linked in terms of expression. RT-PCR and Western blot analyses showed that the lung cancer cell line SK-MES-1/WT expressed BCRP. In a drug-free state, BCRP expression was significantly down-regulated in doxorubicin-resistant SK-MES-1/DX1000 cells overexpressing Pgp. Pharmacological inhibitors (PSC833 or verapamil) or siRNA for Pgp inhibited the down-regulation of BCRP, which was confirmed by confocal microscopy. PSC833 induced the phosphorylation of c-Jun NH2-terminal kinase (JNK) and c-Jun, while the JNK inhibitor SP600125 inhibited this effect. Dominant negative c-Jun decreased the expression of BCRP, but increased that of Pgp. These results indicate that Pgp down-regulates BCRP expression in a drug-free state in which JNK/c-Jun is involved.     

Bufalin induces autophagy-mediated cell death in human colon cancer cells through reactive oxygen species generation and JNK activation

Colorectal cancer is the second most common cause of cancer death in the world and about half of the patients with colorectal cancer require adjuvant therapy after surgical resection. Therefore, the eradication of cancer cells via chemotherapy constitutes a viable approach to treating patients with colorectal cancer. In this study, the effects of bufalin isolated from a traditional Chinese medicine were evaluated and characterized in HT-29 and Caco-2 human colon cancer cells. Contrary to its well-documented apoptosis-promoting activity in other cancer cells, bufalin did not cause caspase-dependent cell death in colon cancer cells, as indicated by the absence of significant early apoptosis as well as poly(ADP-ribose) polymerase and caspase-3 cleavage. Instead, bufalin activated an autophagy pathway, as characterized by the accumulation of LC3-II and the stimulation of autophagic flux. The induction of autophagy by bufalin was linked to the generation of reactive oxygen species (ROS). ROS activated autophagy via the c-Jun NH₂-terminal kinase (JNK). JNK activation increased expression of ATG5 and Beclin-1. ROS antioxidants (N-acetylcysteine and vitamin C), the JNK-specific inhibitor SP600125, and JNK2 siRNA attenuated bufalin-induced autophagy. Our findings unveil a novel mechanism of drug action by bufalin in colon cancer cells and open up the possibility of treating colorectal cancer through a ROS-dependent autophagy pathway.     

二氢杨梅素对人胃癌MKN45细胞迁移和侵袭的影响及其机制*

目的:探讨二氢杨梅素(DHM )对人胃癌MKN45细胞迁移和侵袭的作用及其分子机制。方法:培养人低分化胃癌MKN45细胞,用不同浓度的DHM(0,10,20,30,40,50 μmol/L)分别处理细胞24及48 h,每组实验重复3次,采用CCK8实验检测癌细胞增殖活力;划痕实验检测细胞迁移能力;Transwell小室检测细胞侵袭能力;免疫印迹分析细胞迁移和侵袭相关蛋白表达情况。结果:不同浓度DHM干预可降低MKN45细胞活力。20,30及40 μmol/L的DHM处理48 h可明显抑制细胞的迁移能力(P＜0.01)和侵袭能力(P＜0.05及0.01)。20及30 μmol/L的DHM处理48 h可增加E-cadherin蛋白表达(P＜0.01)、降低Vimentin表达(P＜0.01),从而逆转EMT过程;10,20及30 μmol/L的DHM处理48 h可明显降低pJNK的活性表达水平(P＜0.05及0.01),及MMP-2蛋白表达(P＜ 0.01);JNK通路特异性抑制剂SP600125预处理可明显促进DHM对癌细胞侵袭能力的抑制作用(P＜0.01)及降低MMP-2表达(P＜0.01)。结论:DHM具有抑制人胃癌MKN45细胞的迁移及侵袭的作用,其机制可能与通过JNK通路下调MMP-2蛋白表达水平、逆转上皮间质转化有关。     

Differentiation-associated genes regulated by TPA-induced c-Jun expression via a PKC/JNK pathway in KYSE450 cells

A group of potential differentiation-associated genes had been identified by microarray analysis as c-Jun/AP-1 target genes essential for epithelial differentiation program. Our previous study showed that c-Jun/AP-1 could bind and activate these gene promoters in vivo using chromatin immunoprecipitation. To further understand how the mitogen-activated protein kinase signaling pathways regulate AP-1 activity and expression of c-Jun target genes, our strategy was based on the use of 12-o-tetradecanoylophorbol-13-acetate (TPA) and pharmacological reagents to induce or block c-Jun expression. The mRNA and protein expression of these genes increased in response to TPA-induced c-Jun/AP-1 expression. Inhibitors of JNK (SP600125) and PKC (GF109203X) mainly blocked expression and phosphorylation of c-Jun, while inhibition of MEK-ERK activity with PD98059 (an inhibitor of MEK) had little effect. Expression of involucrin and keratin 4 in response to TPA was attenuated by pretreatments with GF109203X and SP600125, but not PD98059, suggesting involvement of PKC and JNK in this response. Taken together, these results suggested that differentiation-associated genes were regulated by TPA-induced c-Jun/AP-1 mainly via a PKC/JNK pathway in esophageal cancer cell line KYSE450.     

Transient and sustained oxidative stress differentially activate the JNK1/2 pathway and apoptotic phenotype in H9c2 cells

The aim of this study was to investigate the activation of JNK1/2 signalling pathway and the respective cellular phenotype of H9c2 cardiac myoblasts during two distinct types of oxidative insult. We examined the dose- and time-dependent activation of JNK1/2 pathway by exogenous H₂O₂, both under transient and sustained stimulation. At 2 h of either sustained or transient treatment, maximal phosphorylation of c-Jun was observed, coincidently with the activation of nuclear JNK1/2; under sustained stress, these phosphorylation levels remained elevated above basal for up to 6 h, whereas under transient stress they declined to basal ones within 4 h of withdrawal. Furthermore, the JNK1/2 selective inhibitor SP600125 abolished the c-jun phosphorylation induced by oxidative stress. Our results using cell viability assays and light microscopy revealed that sustained H₂O₂ stimulation significantly and time-dependently decreased H9c2 viability, in contrast to transient stimulation; SP600125 (10 μM) abolished cell death induced by sustained as well as cell survival induced by transient oxidative stress. Hoechst staining showed an increase in DNA condensation during sustained, but not during transient stimulation. Moreover, from the antioxidants tested, catalase and superoxide dismutase prevented oxidative stress-induced cell death. Flow cytometry studies reconfirmed that sustained oxidative stress induced apoptosis, whereas transient resulted in the recovery of cardiac myoblasts within 24 h. We conclude that in H9c2 myoblasts, sustained activation of JNK1/2 signalling pathway during oxidative stimulation is followed by an apoptotic phenotype, while transient JNK1/2 activation correlates well with cell survival, suggesting a dual role of this signalling pathway in cell fate determination.     

Activation of the JNK pathway by nanosecond pulsed electric fields

Nanosecond pulsed electric fields (nsPEFs) are increasingly recognized as a novel and unique tool in various life science fields, including electroporation and cancer therapy, although their mode of action in cells remains largely unclear. Here, we show that nsPEFs induce strong and transient activation of a signaling pathway involving c-Jun N-terminal kinase (JNK). Application of nsPEFs to HeLa S3 cells rapidly induced phosphorylation of JNK1 and MKK4, which is located immediately upstream of JNK in this signaling pathway. nsPEF application also elicited increased phosphorylation of c-Jun protein and dramatically elevated c-jun and c-fos mRNA levels. nsPEF-inducible events downstream of JNK were markedly suppressed by the JNK inhibitor SP600125, which confirmed JNK-dependency of these events in this pathway. Our results provide novel mechanistic insights into the mode of nsPEF action in human cells.     

MAPKs抑制剂对大鼠肝细胞GSH代谢相关酶的影响

目的：观察丝裂原活化的蛋白激酶（MAPKs）抑制剂对大鼠肝细胞谷胱甘肽（GSH）代谢的影响,确定哪条途径与GSH代谢相关。方法：体外培养BRL大鼠肝细胞,以c-Jun NH2-末端激酶（JNK）途径抑制剂SP600125、p38途径抑制剂SB203580、细胞外信号调节激酶1/2（ERK1/2）途径抑制剂PD98659处理24 h,采用MTT法测定细胞活力,高效液相色谱法测定细胞内GSH含量,Luminex法测定JNK和磷酸化JNK （p-JNK）的蛋白表达,采用试剂盒测定GSH代谢酶活性。结果：SP600125浓度>5 μmol/L,SB203580浓度>20 μmol/L,PD98659浓度>40 μmol/L时,细胞活力受抑制;SP600125能显著减少大鼠肝细胞内还原型GSH的含量,SB203580和PD98659作用不明显;SP600125显著减少磷酸化JNK （p-JNK）蛋白表达,显著增强谷胱甘肽过氧化物酶（GSH-Px）的活力。结论：JNK MAPK途径参与了大鼠肝细胞GSH的代谢。     

Pro-survival effects of JNK and p38 MAPK pathways in LPS-induced activation of BV-2 cells

Identifying MAPK pathways and understanding their role in microglial cells may be crucial for understanding the pathogenesis of neurodegenerative diseases since activated microglia could contribute to the progressive nature of neurodegeneration. In this study we show that the JNK pathway plays an important role in the survival of resting microglia BV-2 cells, as evidenced by Annexin-V positive staining and caspase-3 activation in cells treated with the specific JNK inhibitor SP600125. During LPS-induced activation of BV-2 cells inhibition of the p38 and JNK pathways with SB203580 and SP600125, respectively, results in apoptosis as detected by apoptotic markers. In the presence SP600125 the phosphorylation of p38 was significantly increased both in control and LPS-activated BV-2 cells. This suggests that the pro-survival role of JNK is possible due to its abrogation of a potentially apoptotic signal mediated by p38 MAPK pathway. Furthermore, inhibition of the p38 MAPK pathway during LPS-induced activation of BV-2 cells resulted in an increased phosphorylation of c-Jun, suggesting that the pro-survival effect of p38 MAPK during inflammatory conditions involves the JNK pathway. In conclusion, the results of this study demonstrate that both the JNK and p38 MAPK pathways possess anti-apoptotic functions in the microglial cell line BV-2 during LPS-induced activation.