Mixed lineage kinase 3 connects reactive oxygen species to c-Jun NH2-terminal kinase-induced mitochondrial apoptosis in genipin-treated PC3 human prostate cancer cells

It has been reported that genipin, the aglycone of geniposide, induces apoptotic cell death in human hepatoma cells via a NADPH oxidase-reactive oxygen species (ROS)-c-Jun NH(2)-terminal kinase (JNK)-dependent activation of mitochondrial pathway. This continuing work aimed to define that mixed lineage kinase 3 (MLK3) is a key mediator, which connect between ROS and JNK in genipin-induced cell death signaling. In PC3 human prostate cancer cells, genipin stimulated MLK3 activity in concentration- and time-dependent manner. The PC3 cells stably transfected with dominant-negative form of MLK3 was less susceptible to population of the sub-G1 apoptotic cells, activation of caspase, collapse of mitochondrial membrane potential, and release of cytochrome c triggered by genipin, suggesting a crucial role of MLK3 in genipin signaling to apoptotic cell death. Diphenyleneiodonium (DPI), a specific inhibitor of NADPH oxidase, markedly inhibited ROS generation and MLK3 phosphorylation in the genipin-treated cells. Pretreatment with SP0600125, a specific inhibitor of JNK but neither U0126, a specific inhibitor of MEK1/2 nor PD169316, a specific inhibitor of p38 suppressed genipin-induced apoptotic cell death. Notably, both the phosphorylation of JNK and induction of c-Jun induced by genipin were markedly inhibited in PC3-EGFP-MLK3 (K144R) cells expressing a dominant-negative MLK3 mutant. Taken together, our observations suggest genipin signaling to apoptosis of PC3 cells is mediated via activation of ROS-dependent MLK3, which leads to downstream activation of JNK.     

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.     

Reversing chemoresistance in cisplatin-resistant human ovarian cancer cells: a role of c-Jun NH2-terminal kinase 1

To investigate the role of activation of c-Jun NH2-terminal kinase 1 (JNK1) in mediating cisplatin-induced apoptosis and the possibility of induction of JNK activity in triggering relation to DNA damage and drug resistance. We investigated the difference of cisplatin-induced activation of JNK pathway and H2O2 alteration between cisplatin-sensitive human ovarian carcinoma cell line A2780 and its resistant variant A2780/DDP. JNK, p-JNK protein, and extracellular H2O2 levels were determined in both A2780 and A2780/DDP cells which were transfected with dominant negative allele of JNK and recombinant JNK1 separately. Both A2780 and A2780/DDP were treated with CDDP, the JNK pathway was activated and a prolonged JNK activation was maintained for at least 12 h in A2780, and only a transient activation (3 h) was detected in A2780/DDP in response to cisplatin treatment. Inhibition of JNK activity by transfection with a dominant negative allele of JNK blocked CDDP-induced apoptosis significantly in A2780 cells. Selective stimulation of the JNK pathway by lipofectamine-mediated delivery of recombinant JNK1 led to activation of c-Jun and decrease of extracellular H2O2, as well as apoptosis sensitization to CDDP in A2780/DDP cells. We concluded that JNK pathway might play an important role in mediating cisplatin-induced apoptosis in A2780 cells, and the duration of JNK activation might be critical in determining whether cells survive or undergo apoptosis. The resistance to CDDP can be reversed through activating c-Jun and decreasing extracellular generation of H2O2 by pcDNA3(FLAG)-JNK1-wt transfection in A2780/DDP cells.     

Triptolide, a diterpenoid triepoxide, induces antitumor proliferation via activation of c-Jun NH2-terminal kinase 1 by decreasing phosphatidylinositol 3-kinase activity in human tumor cells

Triptolide, a diterpenoid triepoxide extracted from the Chinese herb Tripterygium wilfordii Hook f., exerts antitumorigenic actions against several tumor cells, but the intracellular target signal molecule(s) for this antitumorigenesis activity of triptolide remains to be identified. In the present study, we demonstrated that triptolide, in a dose-dependent manner, inhibited the proliferation of human fibrosarcoma HT-1080, human squamous carcinoma SAS, and human uterine cervical carcinoma SKG-II cells. In addition, triptolide was found to decrease phosphatidylinositol 3-kinase (PI3K) activity. A PI3K inhibitor, LY-294002, mimicked the triptolide-induced antiproliferative activity in HT-1080, SAS, and SKG-II cells. There was no change in the activity of Akt or protein kinase C (PKC), both of which are downstream effectors in the PI3K pathway. Furthermore, the phosphorylation of Ras, Raf, and mitogen-activated protein/extracellular signal-regulated kinase 1/2 was not modified in HT-1080 cells treated with triptolide. However, the phosphorylation of c-Jun NH(2)-terminal kinase 1 (JNK1) was found to increase in both triptolide- and LY-294002-treated cells. Furthermore, the triptolide-induced inhibition of HT-1080 cell proliferation was not observed by JNK1 siRNA-treatment. These results provide novel evidence that PI3K is a crucial target molecule in the antitumorigenic action of triptolide. They further suggest a possible triptolide-induced inhibitory signal for tumor cell proliferation that is initiated by the decrease in PI3K activity, which in turn leads to the augmentation of JNK1 phosphorylation via the Akt and/or PKC-independent pathway(s). Moreover, it is likely that the activation of JNK1 is required for the triptolide-induced inhibition of tumor proliferation.     

Requirement of c-Jun NH2-terminal kinase activation in interferon-alpha-induced apoptosis through upregulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in Daudi B lymphoma cells

Interferon alpha (IFN-alpha) inhibits growth, at least in part, through induction of apoptosis. However, the molecular mechanisms underlying IFN-alpha-induced apoptosis are not completely understood. In the present study, we found that IFN-alpha induced a sustained activation of c-Jun N-terminal kinase 1 (JNK1), but not extracellular kinases (ERKs), in Daudi B lymphoma cells, as assessed by Western blotting using phospho-specific antibodies. Several lines of evidence support the notion that the IFN-alpha-induced activation of JNK is responsible for IFN-alpha-induced apoptosis, at least in part, through upregulation of TNF-related apoptosis-inducing ligand (TRAIL). First, pretreatment of Daudi cells with a JNK inhibitor reduced IFN-alpha-induced upregulation of TRAIL and loss of mitochondrial membrane potential (DeltaPsim) and annexin-positive cells, which was assessed by flow cytometry. Second, a dominant-negative form of JNK1 (dnJNK1) also reduced these apoptotic events, while a constitutively active form of JNK1, MKK7-JNK1beta, enhanced them. Finally, treatment with IFN-alpha enhanced the promoter activity of the TRAIL gene, which was partially abrogated by either JNK inhibitor or dnJNK1, while it was moderately enhanced by MKK7-JNK1beta. These findings are useful for understanding molecular mechanisms of IFN-alpha-induced apoptosis and also for development of treatment modalities of some tumors with IFN-alpha.     

Mitogen-activated protein kinases (p38 and c-Jun NH2-terminal kinase) are differentially regulated during cardiac volume and pressure overload hypertrophy

Chronic pressure overload (PO) and volume overload (VO) result in morphologically and functionally distinct forms of myocardial hypertrophy. However, the molecular mechanism initiating these two types of hypertrophy is not yet understood. Data obtained from different cell types have indicated that the mitogen-activated protein kinases (MAPKs) comprising c-Jun NH₂-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 play an important role in transmitting signals of stress stimuli to elicit the cellular response. We tested the hypothesis that early induction of MAPKs differs in two types of overload on the heart and associates with distinct expression of hypertrophic marker genes, namely ANF, α-myosin heavy chain (α-MHC), and β-MHC. In rats, VO was induced by aortocaval shunt and PO by constriction of the abdominal aorta. The PO animals were further divided into two groups depending on the severity of the constriction, mild (MPO) and severe pressure overload (SPO), having 35 and 85% aortic constriction, respectively. Early changes in MAPK activity (2–120 min and 1 to 2 d) were analyzed by the in vitro kinase assay using kinase-specific antibodies for p38, JNK, and ERK2. The change in expression of hypertrophy marker genes was examined by Northern blot analysis. In VO hypertrophy, the activity of p38 was markedly increased (10-fold), without changing the activity of ERK and JNK. However, during PO hypertrophy, the activity of JNK was significantly increased (two-to sixfold) and depended on the severity of the load. The activity of p38 was not changed in MPO hypertrophy, whereas it was slightly elevated (50%) in hearts with SPO. Similarly, ERK activity was not changed in hearts with MPO, but a transient rise in activity was observed in hearts with SPO. The expression of ANF and β-MHC genes was elevated in both PO and VO hypertrophy; however, this change was much greater in hearts subjected to PO than VO hypertrophy. α-MHC expression was downregulated in PO but remained unchanged in VO hypertrophy hearts. Thus, these results demonstrate differential activation of MAPKs in two types of cardiac hypertrophy and this, in part, may contribute to differential expression of cardiac muscle gene expression, giving rise to unique cardiac phenotype associated with different hemodynamic overloads.     

MPTP activates c-Jun NH(2)-terminal kinase (JNK) and its upstream regulatory kinase MKK4 in nigrostriatal neurons in vivo

The neuropathology of Parkinson's disease is reflected in experimental animals treated with the selective nigrostriatal dopaminergic neurotoxin MPTP. Neurons exposed to MPTP (MPP(+)) express morphological features of apoptosis, although the intracellular pathways that produce this morphology have not been established. The c-Jun NH(2)-terminal kinase (JNK) signaling cascade has been implicated as a mediator of MPTP-induced apoptotic neuronal death based on the ability of CEP-1347/KT-7515, an inhibitor of JNK activation, to attenuate MPTP-induced nigrostriatal dopaminergic degeneration. In these studies, MPTP-mediated activation of the JNK signaling pathway was assessed in the nigrostriatal system of MPTP-treated mice. MPTP elevated levels of phosphorylated JNK and JNK kinase (MKK4; also known as SEK1 or JNKK), by 2.5- and fivefold, respectively. Peak elevations occurred soon after administration of MPTP and coincided with peak CNS levels of MPP(+). Increased MKK4 phosphorylation, but not JNK phosphorylation, was found in the striatum, suggesting that activation of MKK4 occurs in injured dopaminergic terminals. Both JNK and MKK4 phosphorylations were attenuated by pretreatment with l-deprenyl, indicating that these phosphorylation events were mediated by MPP(+). Moreover, CEP-1347/KT-7515 inhibited MPTP-mediated MKK4 and JNK signaling at a dose that attenuates MPTP-induced dopaminergic loss. These data implicate this signaling pathway in MPTP-mediated nigrostriatal dopaminergic death and suggest that it may be activated in the degenerative process in Parkinson's disease.     

Essential role of c-Jun-NH2-terminal kinase on synergy induction of apoptosis by TRAIL plus ADM in ADM resistant MCF-7/ADM cells

Combined treatment modalities using tumor necrosis factor related apoptosis-inducing ligand L (TRAIL) and cytotoxic drugs revealed highly additive effects in some tumor cell lines. Little is known about the efficacy and underlying mechanistic effects of the modalities in chemoresistant tumor cells. The purpose of this study is to investigate the possible role of JNK in the synergistic effect in Doxorubicin (Adriamycin, ADM) resistant MCF-7/ADM cells. Here we showed that the JNK pathway was activated slightly by TRAIL in MCF-7/ADM cell lines and was enhanced by the combination of the two treatments. Inhibition of JNK activity by transfection with dominant-negative JNK blocks TRAIL plus ADM induced-apoptosis significantly, and selective stimulation of the JNK pathway sensitizes ADM resistant breast cancer cells to ADM and TRAIL co-treatment through activation of mitochondria-regulated apoptotic pathway. We conclude that the JNK pathway plays an important role in mediating TRAIL plus ADM induced-apoptosis in breast cancer cells. Fang Li and Li Meng contributed equally to this work.     

c-Jun NH2-terminal kinase (JNK)-dependent nuclear translocation of apoptosis-inducing factor (AIF) following engagement of membrane immunoglobulin on WEHI-231 B lymphoma cells

WEHI-231 B lymphoma cells have been employed for analysis of antigen-induced B cell unresponsiveness because these cells undergo cell cycle arrest in G1, accompanied by induction of apoptosis. In the present study, we examined the requirement for toxic small molecules apoptosis-inducing factor (AIF) and cytochrome c, and subsequent caspase activation in apoptotic cell death in WEHI-231 and CH31 B lymphoma cells following engagement of membrane immunoglobulin (mIg). Pan-caspase inhibitor BD-fmk blocked mIg-mediated increase in cells with sub-G1 DNA content, whereas it did not affect mIg-mediated loss of mitochondrial membrane potential and phosphatidylserine exposure on B cell membrane. Dominant-negative form of c-Jun NH2-terminal kinase1 (JNK1) blocked the translocation of AIF into the nuclei and cytosol from the mitochondria in the WEHI-231 and CH31 cells following mIg engagement, whereas constitutively active form of JNK1 enhanced it. This AIF translocation was also blocked by Bcl-xL, but not by BD-fmk. Moreover, AIF-deficient clones via small interfering RNA (siRNA)-mediated method showed small increase in loss of mitochondrial membrane potential. After mIg engagement, the AIF-deficient clones displayed an enhanced sensitivity to mIg-mediated apoptosis, concomitant with translocation of a residual AIF into the nuclei, compared with control clone. Our findings are compatible with the notion that AIF has dual role, with a proapoptotic function in the nuclei and a distinct anti-apoptotic function in the mitochondria. These observations would be valuable for analysis of B cell unresponsiveness and hopefully for treatment of diseases involving B cell dysfunction.     

Human glutamylcysteine synthetase protects HEK293 cells against UV-induced cell death through inhibition of c-Jun NH2-terminal kinase

Human glutamylcysteine ligase catalytic subunit (GCLC) is the rate-limiting enzyme for glutathione synthesis. The heavy subunit possesses all the catalytic activities. UV irradiation (UV-C, 30 J/m(2)) induced apoptosis in HEK293 cells, but the morphological changes were inhibited significantly by expression of GCLC. MTS assay and flow cytometry results also indicated that GCLC and JNK1(APF) expression enhanced cellular resistance to UV irradiation. Western blotting showed that irradiation strongly activated the c-Jun NH(2)-terminal kinases (JNKs) and caspase-3 as well as p38 in HEK293 cells. Interestingly, existing data show that GCLC blocks JNK1 phosphorylation but does not affect p38 phosphorylation. Therefore, overexpression of GCLC protected HEK293 cells against UV irradiation-induced cell death by inhibiting the phosphorylation and activation of JNK1, concomitantly with the inhibition of caspase-3 activation and p21(WAF1)-luciferase activity downstream of JNK.     

c-Jun NH2-terminal kinase decreases ubiquitination and promotes stabilization of p21(WAF1/CIP1) in K562 cell

Proteasome-dependent degradation of regulatory proteins is a known mechanism of cell cycle control. p21(WAF1/CIP1) (p21), a negative regulator of the cell division cycle, exhibits proteasome-sensitive turnover and ubiquitination. In the present study, we analyzed the regulatory effects of JNK1 on p21 protein accumulation in p53 null K562 cells. We found that JNK1 (wild type, WT) mediated H(2)O(2)-induced p21 protein up-regulation. Over-expression of JNK1 (WT) could elevate endogenous p21 protein level but did not affect p21 mRNA level and also prolong the p21 half-life as well as inhibited the p21 ubiquitination. These findings indicated that JNK1 could regulate cellular p21 level via inhibiting ubiquitination of p21, which provided a new insight for analyzing the regulatory effect of JNK after stress.     

Doxorubicin coupled to penetratin promotes apoptosis in CHO cells by a mechanism involving c-Jun NH2-terminal kinase

Doxorubicin (Dox) has demonstrated potent activity in treating malignant lymphomas but its therapeutic efficacy is hampered by induction of cardiotoxicity. This side effect is related to the ability of the drug to generate reactive oxygen species in cells. Previously, we demonstrated that coupling Dox to penetratin (Pen), a cell penetrating peptide, represent a valuable strategy to overcome drug resistance in CHO cells. In the present study, we evaluated the consequences of the conjugation of Dox to Pen in term of apoptosis induction. When tested on CHO cells, Dox-Pen generated a typical apoptotic phenotype but at lower dose that needed for unconjugated Dox. Cell death induction was associated with chromatin condensation, caspase activation, Bax oligomerisation and release of cytochrome c. By using reactive oxygen species and c-jun NH2-terminal kinase (JNK) inhibitors, we prevented Dox- and Dox-Pen-induced CHO cell death. The chimeric soluble DR5 receptor that inhibits TRAIL induced cell death does not prevent Dox or Dox-Pen-induced cytotoxicity. These observations indicate that conjugation of Dox to cell penetrating peptide does not impair the ability of the drug to trigger cell death through activation of the intrinsic pathway involving c-Jun NH2-terminal kinase but could exhibit less toxic side effects and could warrant its use in clinic.     

Phosphorylation of Bcl-2 and activation of caspase-3 via the c-Jun N-terminal kinase pathway in ursolic acid-induced DU145 cells apoptosis

There is currently no successful therapy for androgen-independent prostate cancer. Ursolic acid (UA), a pentacyclic triterpenoid compound, has been shown to have an anti-proliferative effect on various tumors. We investigated the effect of UA on cell viability in the human hormone-refractory prostate cancer cell line DU145, as well as the molecular mechanisms underlying its growth inhibiting effect. We demonstrated that UA induces apoptosis and the activation of caspase-3 in DU145 cells. UA also causes the activation of c-Jun N-terminal kinase (JNK), but has no effect on extracellular signal-regulated protein kinases (ERK1/2) and p38 MAP kinases (p38). UA-induced JNK activation could result in Bcl-2 phosphorylation (Ser70) and degradation in DU145 cells, which may be one of the molecular mechanisms by which it induces apoptosis. Although further evaluation, such as in vivo testing, is clearly needed, the present results suggest the potential utility of UA as a novel therapeutic agent in advanced prostate cancer.     

Polyamines are required for activation of c-Jun NH2-terminal kinase and apoptosis in response to TNF-alpha in IEC-6 cells

Intracellular polyamine homeostasis is important for the regulation of cell proliferation and apoptosis and is necessary for the balanced growth of cells and tissues. Polyamines have been shown to play a role in the regulation of apoptosis in many cell types, including IEC-6 cells, but the mechanism is not clear. In this study, we analyzed the mechanism by which polyamines regulate the process of apoptosis in response to tumor necrosis factor-alpha (TNF-alpha). TNF-alpha or cycloheximide (CHX) alone did not induce apoptosis in IEC-6 cells. Significant apoptosis was observed when CHX was given along with TNF-alpha, as indicated by a significant increase in the detachment of cells, caspase-3 activity, and DNA fragmentation. Polyamine depletion by treatment with alpha-difluoromethylornithine significantly reduced the level of apoptosis, as judged by DNA fragmentation and the caspase-3 activity of attached cells. Apoptosis in IEC-6 cells was accompanied by the activation of upstream caspases-6, -8, and -9 and NH2-terminal c-Jun kinase (JNK). Inhibition of JNK activation prevented caspase-9 activation. Polyamine depletion prevented the activation of JNK and of caspases-6, -8, -9, and -3. SP-600125, a specific inhibitor of JNK activation, prevented cytochrome c release from mitochondria, JNK activation, DNA fragmentation, and caspase-9 activation in response to TNF-alpha/CHX. In conclusion, we have shown that polyamine depletion delays and decreases TNF-alpha-induced apoptosis in IEC-6 cells and that apoptosis is accompanied by the release of cytochrome c, the activation of JNK, and of upstream caspases as well as caspase-3. Polyamine depletion prevented JNK activation, which may confer protection against apoptosis by modulation of upstream caspase-9 activation.     

Polypeptide Compositions and NH2-terminal Amino Acid Sequences of Proteins in Foxtail and Proso Millets

Seed protein of foxtail and proso millets were fractionated into polypeptides that were analyzed for their major protein, prolamin, and the NH₂-terminal amino acid sequences of the proteins were determined. The proteins extracted from foxtail and proso millets were 64.1% and 80.0% prolamin, respectively. The polypeptides of the prolamins were classified into two groups. The major polypeptides of 27-19 kDa were rich in leucine and alanine, whereas the 17-14 kDa polypeptides were rich in methionine and cysteine. Glutelin-like proteins that were extracted with a reducing reagent were high in proline content, the major polypeptides being 17 and 20 kDa. The NH₂-terminal amino acid sequence showed that the major polypeptides of prolamin were homologous to α-zein and a glutelin-like protein containing the Pro-Pro-Pro sequence, like the repetitive sequence of γ-zein. Although the prolamin consisted of a similar subunit to that of zein, polypeptides with various pI values were found among them.     

Inhibition of CD95-mediated apoptosis through beta 1 integrin in the HSG epithelial cell line

The HSG cell line serves as a model for salivary gland epithelial progenitor cell differentiation. In order for a progenitor cell to differentiate, the cell must maintain viability within its niche. Studies were designed to elucidate the mechanism for integrin-mediated HSG cell survival. HSG cells, grown on Matrigel®, were resistant to CD95-mediated apoptosis. Western blot analysis showed that Matrigel® induced the expression of bcl-2, bcl-xL, p63, and ΔNp63. This induction occurred by as early as 2 hrs and remained for 24 hrs. CD95-mediated apoptosis resistance was dependent, however, upon the expression of the bcl-2 family. Furthermore, Matrigel® induced bcl-2 family expression was dependent on the transactivation of the EGF receptor pathway since PD98059 and AG1478 inhibited Matrigel® induced bcl-2 family expression and caused HSG cells to be sensitive to CD95-mediated apoptosis. Activation of the EGF receptor pathway, by itself, however, was not sufficient to inhibit apoptosis. Blocking antibody showed that bcl-2 family expression was mediated through β1 integrin. These studies show that salivary progenitor epithelial cell survival is integrin dependent and involves the transactivation of the EGF receptor pathway.     

Hydrogen peroxide induces apoptosis in HeLa cells through mitochondrial pathway

Cervical cancer is the most common cancer amongst females in India and is associated with high risk HPVs, reactive oxygen species (ROS), and excessive inflammation in most cases. ROS in turn affects the expression of pro- and anti-apoptotic proteins. The objective of the present study was to elucidate the effect of hydrogen peroxide (H(2)O(2)) on apoptotic signaling molecules in vitro. HeLa cell line expresses the Human papilloma virus - 18, E6 oncoprotein which causes the ubiquitin mediated degradation of p53 protein and is thus p53 deficient. p53 is known to act as a cellular stress sensor and triggers apoptosis. p73, a member of the p53 family also induces apoptosis in response to DNA damaging agents but unlike p53, it is infrequently mutated in human tumors. We demonstrate here, that in HeLa cells, apoptosis is triggered by H(2)O(2) via the mitochondrial pathway involving upregulation of p73, and its downstream target Bax. This was accompanied by upregulation of ERK, JNK, c-Myc, Hsp-70 and down regulation of anti-apoptotic Bcl-XL, release of cytochrome c from mitochondria and activation of caspases-9 and -3.