Guggulsterone-induced apoptosis in human prostate cancer cells is caused by reactive oxygen intermediate dependent activation of c-Jun NH2-terminal kinase

Guggulsterone, a constituent of Indian Ayurvedic medicinal plant Commiphora mukul, causes apoptosis in cancer cells but the sequence of events leading to cell death is poorly understood. We now show that guggulsterone-induced cell death in human prostate cancer cells is caused by reactive oxygen intermediate (ROI)-dependent activation of c-Jun NH(2)-terminal kinase (JNK). Exposure of PC-3 and LNCaP cells to apoptosis inducing concentrations of guggulsterone resulted in activation of JNK and p38 mitogen-activated protein kinase (p38 MAPK) in both cell lines and activation of extracellular signal-regulated kinase 1/2 (ERK1/2) in LNCaP cells. The guggulsterone-induced apoptosis in PC-3/LNCaP cells was partially but statistically significantly attenuated by pharmacologic inhibition (SP600125) as well as genetic suppression of JNK activation. On the other hand, pharmacologic inhibition of p38 MAPK activation in PC-3 or LNCaP cells (SB202190) and ERK1/2 activation in LNCaP cells (PD98059) did not protect against guggulsterone-induced cell death. The guggulsterone treatment caused generation of ROI in prostate cancer cells but not in a normal prostate epithelial cell line (PrEC), which was also resistant to guggulsterone-mediated JNK activation. The guggulsterone-induced JNK activation as well as cell death in prostate cancer cells was significantly attenuated by overexpression of catalase and superoxide dismutase. In addition, guggulsterone treatment resulted in a decrease in protein level and promoter activity of androgen receptor in LNCaP cells. In conclusion, the present study reveals that the guggulsterone-induced cell death in human prostate cancer cells is regulated by ROI-dependent activation of JNK and guggulsterone inhibits promoter activity of androgen receptor.     

Gonadotropin-releasing hormone induces apoptosis of prostate cancer cells: role of c-Jun NH2-terminal kinase, protein kinase B, and extracellular signal-regulated kinase pathways

A standard therapy used today for prostate cancer is androgen ablation by gonadotropin-releasing hormone analogs (GnRH-a). Although most patients respond to androgen ablation as an initial systemic therapy, nearly all cases will develop androgen resistance, the management of which is still a major challenge. Here, we report that GnRH-a can directly induce apoptosis of the androgen-independent prostate cancer-derived DU145 and PC3 cell lines. Using specific inhibitors, we found that the apoptotic effect of GnRH-a is mediated by c-Jun NH2-terminal kinase (JNK) and inhibited by the phosphatidylinositol 3'-kinase (PI3K)-protein kinase B (PKB) pathway. Indeed, in DU145 cells, GnRH-a activates the JNK cascade in a c-Src- and MLK3-dependent manner but does not involve protein kinase C and epidermal growth factor receptor. Concomitantly, GnRH-a reduces the activity of the PI3K-PKB pathway, which results in the dephosphorylation of PKB mainly in the nucleus. The reduction of PKB activity releases PKB-induced inhibition of MLK3 and thus further stimulates JNK activity and accelerates the apoptotic effect of GnRH-a. Interestingly, extracellular signal-regulated kinase is also activated by GnRH-a, and this occurs via a pathway that involves matrix metalloproteinases and epidermal growth factor receptor, but its activation does not affect JNK activation and the GnRH-a-induced apoptosis. Our results support a potential use of GnRH-a for the treatment of advanced prostate cancer and suggest that the outcome of this treatment can be amplified by using PI3K-PKB inhibitors.     

The activation of c-Jun NH2-terminal kinase is required for dihydroartemisinin-induced autophagy in pancreatic cancer cells

Background

c-Jun NH2-terminal kinases (JNKs) are strongly activated by a stressful cellular environment, such as chemotherapy and oxidative stress. Autophagy is a protein-degradation system in which double-membrane vacuoles called autophagosomes are formed. The autophagy-related gene Beclin 1 plays a key role in this process. We previously found that autophagy was induced by dihydroartemisinin (DHA) in pancreatic cancer cells. However, little is known about the complex relationship between ROS, JNK activation, autophagy induction, and Beclin 1 expression.

Methods

Cell viability and CCK-8 assays were carried out to determine the cell proliferation; small interfering RNAs (siRNAs) were used to knockdown c-Jun NH₂-terminal kinases (JNK1/2) genes; western blot was performed to detect the protein expression of LC3, JNK, Beclin 1, caspase 3 and β-actin; production of intracellular ROS was analyzed using FACS flow cytometry; autophagy induction was confirmed by electron microscopy.

Results

In the present study, we explored the role of DHA and Beclin 1 expression in autophagy. DHA-treated cells showed autophagy characteristics, and DHA also activated the JNK pathway and up-regulated the expression of Beclin 1. Conversely, blocking JNK signaling inhibited Beclin 1 up-regulation. JNK activation was found to primarily depend on reactive oxygen species (ROS) resulting from the DHA treatment. Moreover, JNK pathway inhibition and Beclin 1 silencing prevented the induction of DHA-induced autophagy.

Conclusions

These results suggest that the induction of autophagy by DHA is required for JNK-mediated Beclin 1 expression.     

Bone morphogenetic protein 7 protects prostate cancer cells from stress-induced apoptosis via both Smad and c-Jun NH2-terminal kinase pathways

We reported earlier that exposure to exogenous bone morphogenetic protein 7 (BMP7) could strongly inhibit serum starvation-induced apoptosis to C4-2B cell line, a variant of the LNCaP human prostate cancer cell line with propensity for bone metastasis. Whereas serum starvation suppressed the expression of survivin, a member of the inhibitor of apoptosis protein family, its expression was sustained in the presence of BMP7. In this study, we present evidence that BMP7 exposure up-regulated survivin promoter activity, an effect that was associated with activation of Smad, and could be repressed by dominant-negative Smad5. Additionally, serum starvation-induced suppression of c-jun NH2-terminal kinase (JNK) activity in C4-2B cells could be mostly restored by BMP7, and a JNK inhibitor could counteract the antiapoptotic effect of BMP7, without a significant effect on the level of survivin expression. Thus, we identified JNK pathway as another signaling mode for the antiapoptotic function of BMP7. To test the effect of endogenous up-regulation of BMP7, we genetically modulated the C4-2B cell line to overexpress BMP7 protein. Not only was this altered cell line resistant to serum starvation-induced apoptosis but it also exhibited patterns of Smad activation, survivin up-regulation, and JNK activation similar to those of the parental C4-2B cells exposed to exogenous BMP7. Consistent with these in vitro findings of BMP7 action, we acquired correlative results of Smad activation, survivin expression, and JNK activation in the progression of prostate cancer in the conditional Pten deletion mouse model, in which we first obtained the evidence of BMP7 overexpression.     

Prx1 suppresses radiation-induced c-Jun NH2-terminal kinase signaling in lung cancer cells through interaction with the glutathione S-transferase Pi/c-Jun NH2-terminal kinase complex

Radiotherapy is one of the major treatment modalities for lung cancer. Cell killing by ionizing radiation is mediated primarily through the reactive oxygen species (ROS) and ROS-driven oxidative stress. Prx1, a peroxiredoxin family member, was shown to be frequently elevated in lung cancer cells and tissues. Although the antioxidant function of Prx1 is expected to affect the radiotherapy response of lung cancer, the physiologic significance of its peroxidase activity in irradiated cells is unclear because the catalytic Cys52 is easily inactivated by ROS due to its overoxidation to sulfinic or sulfonic acid. In this study, we investigated the role of Prx1 in radiation sensitivity of human lung cancer cells, with special emphasis on the redox status of the catalytic Cys52. We found that overexpression of Prx1 enhances the clonogenic survival of irradiated cells and suppresses ionizing radiation-induced c-Jun NH2-terminal kinase (JNK) activation and apoptosis. The peroxidase activity of Prx1, however, is not essential for inhibiting JNK activation. The latter effect is mediated through its association with the glutathione S-transferase pi (GSTpi)-JNK complex, thereby preventing JNK release from the complex. Reduced JNK activation is observed when the peroxidase activity of Prx1 is compromised by Cys52 overoxidation or in the presence of the Cys52 to Ser52 mutant (Prx1C52S) lacking peroxidase activity. We show that both Prx1 and Prx1C52S interact with the GSTpi-JNK complex and suppress the release of JNK from the complex. Our study provides new insight into the antiapoptotic function of Prx1 in modulating radiosensitivity and provides the impetus to monitor the influence of Prx1 levels in the management of lung cancer.     

Docetaxel-induced apoptosis of human melanoma is mediated by activation of c-Jun NH2-terminal kinase and inhibited by the mitogen-activated protein kinase extracellular signal-regulated kinase 1/2 pathway.

PURPOSE: Our studies have shown variable sensitivity of cultured melanoma cells to docetaxel. To better understand this response, we studied the role of signal transduction pathways in modulating docetaxel-induced melanoma killing. EXPERIMENTAL DESIGN: Involvement of c-Jun NH(2)-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinase, and Akt signaling was studied by evaluating their extent of activation in melanoma cells after treatment with docetaxel. The effect of their activation on docetaxel-induced apoptosis was assessed using biochemical inhibitors of the pathways and Western blot analysis of proteins involved. RESULTS: Docetaxel induced activation of both JNK and ERK1/2 but not p38 mitogen-activated protein kinase or Akt kinases. Apoptosis was dependent on activation of JNK and mediated through activation of caspase-2 and caspase-dependent changes in Bax and Bak. The levels of activated JNK in individual lines showed a close correlation with the levels of apoptosis. In contrast, activation of ERK1/2 by docetaxel inhibited apoptosis and the levels of activation in individual lines were inversely correlated to the degree of apoptosis. Studies on the Bcl-2 family proteins seemed to reflect changes induced by activation of JNK and ERK1/2 pathways. Docetaxel-induced JNK activation was required for Bcl-2 phosphorylation as well as caspase-2-dependent activation of Bax and Bak and subsequent mitochondrial release of apoptosis-inducing factor and cytochrome c. In contrast, activation of ERK1/2 resulted in degradation of BH3-only protein Bim and phosphorylation of Bad. CONCLUSIONS: These studies provide further insights into sensitivity of melanoma cells to taxanes and provide a basis for the current rationale of combining taxanes with inhibitors of the Raf-ERK1/2 pathway.     

Cyclic GMP mediates apoptosis induced by sulindac derivatives via activation of c-Jun NH2-terminal kinase 1.

Sulindac sulfone (Exisulind) induces apoptosis and exhibits cancer chemopreventive activity, but in contrast to sulindac, it does not inhibit cyclooxygenases 1 or 2. We found that sulindac sulfone and two potent derivatives, CP248 and CP461, inhibited the cyclic GMP (cGMP) phosphodiesterases (PDE) 2 and 5 in human colon cells, and these compounds caused rapid and sustained activation of the c-Jun NH2-terminal kinase 1 (JNK1). Rapid activation of stress-activated protein/ERK kinase 1 (SEK1) and mitogen-activated protein kinase kinase kinase (MEKK1), which are upstream of JNK1, was also observed. Other compounds that increase cellular levels of cGMP also activated JNK1, and an inhibitor of protein kinase G (PKG), Rp-8-pCPT-cGMPS, inhibited JNK1 activation by the sulindac sulfone derivatives. Expression of a dominant-negative JNK1 protein inhibited CP248-induced cleavage of poly(ADP-ribose) polymerase, a marker of apoptosis. Thus, it appears that sulindac sulfone and related compounds induce apoptosis, at least in part, through activation of PKG, which then activates the MEKK1-SEK1-JNK1 cascade. These studies also indicate a role for cGMP and PKG in the JNK pathway.     

Induction of apoptosis by the garlic-derived compound S-allylmercaptocysteine (SAMC) is associated with microtubule depolymerization and c-Jun NH(2)-terminal kinase 1 activation

Epidemiological and experimental carcinogenesis studies provide evidence that components of garlic (Allium sativum) have anticancer activity. We recently reported that the garlic derivative S-allylmercaptocysteine (SAMC) inhibits growth, arrests cells in G(2)-M, and induces apoptosis in human colon cancer cells (Shirin et al., Cancer Res., 61: 725-731, 2001). Because a fraction of the SAMC-treated cells are specifically arrested in mitosis, we examined the mechanism of this effect in the present study. Immunofluorescent microscopy revealed that the treatment of SW480 cells or NIH3T3 fibroblasts with 150 micro M SAMC (the IC(50) concentration) caused rapid microtubule (MT) depolymerization, MT cytoskeleton disruption, centrosome fragmentation and Golgi dispersion in interphase cells. It also induced the formation of monopolar and multipolar spindles in mitotic cells. In vitro turbidity assays indicated that SAMC acted directly on tubulin to cause MT depolymerization, apparently because it interacts with -SH groups on tubulin. To investigate the signaling pathways involved in SAMC-induced apoptosis, we assayed c-Jun NH(2)-terminal kinase (JNK) activity and found that treatment with SAMC caused a rapid and sustained induction of JNK activity. The selective JNK inhibitor SP600125 inhibited the early phase (24 h) but not the late phase (48 h and later) of apoptosis induced by SAMC. Expression of a dominant-negative mutant of JNK1 in SW480 cells inhibited apoptosis induced by SAMC at 24 h but had no protective effect at 48 h. JNK1(-/-) mouse embryonic fibroblasts were resistant to SAMC-induced apoptosis at 24 h but not at 48 h. On the other hand, the inhibition or abrogation of JNK1 activity did not inhibit the G(2)-M arrest induced by SAMC. SAMC also activated caspase-3. The general caspase inhibitor z-VAD-fmk inhibited both early and late phases of apoptosis induced by SAMC. We conclude that the garlic-derived compound SAMC exerts antiproliferative effects by binding directly to tubulin and disrupting the MT assembly, thus arresting cells in mitosis and triggering JNK1 and caspase-3 signaling pathways that lead to apoptosis.     

Activated Ki-Ras suppresses 12-O-tetradecanoylphorbol-13-acetate-induced activation of the c-Jun NH2-terminal kinase pathway in human colon cancer cells.

Although the frequency of activated Ki-ras genes is high in human colorectal tumors, much less is known of activated Ki-ras-mediated signaling pathways. Using gene targeting, we examined HCT116 cells that contain the Gly-13-->Asp mutation of Ki-ras and activated Ki-ras-disrupted clones derived from HCT116. 12-O-Tetradecanoylphorbol-13-acetate (TPA) induced immediate early genes, such as c-Jun, c-Fos, and Egr-1 in activated Ki-ras-disrupted clones, whereas c-Jun induction was rare in HCT116. TPA induced both phosphorylation of stress-activated protein kinase kinase 1 (SEK1) and c-Jun NH2-terminal kinase (JNK) in the activated Ki-ras-disrupted clones but not in HCT116. On the other hand, TPA-induced mitogen-activated protein kinase kinase 1/2 (MEK1/2)-extracellular signal-regulated kinase (ERK) activation was equally induced between HCT116 and the Ki-ras-disrupted clones. Furthermore, TPA-induced SEK1-JNK activation was observed in a DLD-1-derived activated Ki-ras-disrupted clone but not in DLD-1. The TPA-induced SEK1-JNK activation in these disrupted clones was completely inhibited by the protein kinase C (PKC) inhibitor, GF109203X (1 microM), but not by another PKC inhibitor, H7 (50 microM), whereas TPA-induced MEK1/2-ERK activation was partially and completely inhibited by GF109203X (1 microM) and H7 (50 microM), respectively. A phosphoinositol 3-kinase inhibitor, LY294002, did not inhibit the TPA-induced SEK1-JNK activation. Taken together, these results suggest that activated Ki-Ras-mediated signals are involved in the SEK1-JNK pathway through a PKC isotype that is distinct from that involved in MEK1/2-ERK activation in human colon cancer cells and independent of phosphoinositol 3-kinase activation, and the imbalance between ERK and JNK activity caused by activated Ki-Ras may play critical roles in human colorectal tumorigenesis.     

BAD Ser128 is not phosphorylated by c-Jun NH2-terminal kinase for promoting apoptosis

The phosphorylation and regulation of the proapoptotic Bcl-2 family protein BAD by c-Jun NH2-terminal kinase (JNK) is controversial. JNK can suppress interleukin-3 withdrawal-induced apoptosis via phosphorylation of BAD at Thr201. However, it has also been reported that JNK promotes apoptosis through phosphorylation of BAD at Ser128. Here, we report that JNK is not a BAD Ser128 kinase. JNK phosphorylates murine BAD (mBAD), but not human BAD (hBAD), in which Ser91 is equivalent to Ser128 in mBAD. In contrast, Cdc2, which phosphorylates Ser128, phosphorylates both mBAD and hBAD. Replacement of Ser128 by alanine has no effects on BAD phosphorylation by JNK in vitro and in vivo. Two-dimensional phosphopeptide mapping in combination with phosphoamino acid analysis reveals that JNK does not phosphorylate BAD at Ser128. Elimination of Ser128 phosphorylation has no effects on the proapoptotic activity of BAD in apoptosis induced by UV via JNK or growth factor withdrawal. Thus, our results show that Ser128 is not phosphorylated by JNK for promoting cell death.     

Regulation of PTEN expression in intestinal epithelial cells by c-Jun NH2-terminal kinase activation and nuclear factor-kappaB inhibition

The tumor suppressor protein phosphatase and tensin homologue deleted on chromosome ten (PTEN) plays an important role in intestinal cell proliferation and differentiation and tumor suppression by antagonizing phosphatidylinositol 3-kinase. Despite its importance, the molecular mechanisms regulating PTEN expression are largely undefined. Here, we show that treatment of the colon cancer cell line HT29 with the differentiating agent sodium butyrate (NaBT) increased PTEN protein and mRNA expression and induced c-Jun NH2-terminal kinase (JNK) activation. Inhibition of JNK by chemical or genetic methods attenuated NaBT-induced PTEN expression. In addition, our findings showed a cross-talk between nuclear factor kappaB (NF-kappaB) and JNK with respect to PTEN regulation. Overexpression of the NF-kappaB superrepressor increased PTEN expression and JNK activity, whereas overexpression of the p65 NF-kappaB subunit reduced both basal and NaBT-mediated JNK activation and PTEN expression. Moreover, we showed that overexpression of PTEN or treatment with NaBT increased expression of the cyclin-dependent kinase inhibitor p27(kip1) in HT29 cells; this induction was attenuated by inhibition of PTEN or JNK expression or overexpression of p65. Finally, we show a role for PTEN in NaBT-mediated cell death and differentiation. Our findings suggest that the JNK/PTEN and NF-kappaB/PTEN pathways play a critical role in normal intestinal homeostasis and colon carcinogenesis.     

ABL-N-induced apoptosis in human breast cancer cells is partially mediated by c-Jun NH<Subscript>2</Subscript>-terminal kinase activation

Introduction  

The present study was designed to determine the possibility of acetylbritannilactone (ABL) derivative 5-(5-(ethylperoxy)pentan-2-yl)-6-methyl-3-methylene-2-oxo-2,3,3a,4,7,7a-hexahydrobenzofuran-4-yl 2-(6-methoxynaphthalen-2-yl)propanoate (ABL-N) as a novel therapeutic agent in human breast cancers.     

Identification of a novel synthetic thiazolidin compound capable of inducing c-Jun NH2-terminal kinase-dependent apoptosis in human colon cancer cells

Development of new therapeutic agents for colon cancer is highly desirable. To this end, we screened a chemical library for new anticancer agents and identified a synthetic compound, 5-(2,4-dihydroxybenzylidene)-2-(phenylimino)-1,3-thiazolidin (DBPT), which kills cancer cells more effectively than it kills normal human fibroblasts. The molecular mechanism of the antitumor action of DBPT was further analyzed in three human colorectal cancer cell lines. DBPT effectively inhibited the growth of colorectal cancer cells, independent of p53 and P-glycoprotein status, whereas normal fibroblasts were unaffected at the same IC50. Over time, DLD-1 cancer cells treated with DBPT underwent apoptosis. The general caspase inhibitor benzyloxycarbonyl-valine-alanine-aspartate-fluoromethylketone partially blocked DBPT-induced apoptosis in a dose-dependent manner. DBPT-induced apoptosis, including cytochrome c release and caspase activation, was abrogated when c-Jun NH2-terminal kinase (JNK) activation was blocked with either a specific JNK inhibitor or a dominant-negative JNK1 gene. However, constitutive JNK activation alone did not replicate the effects of DBPT in DLD-1 cells, and excessive JNK activation by adenovirus encoding MKK7 had little influence on DBPT-induced apoptosis. Our results suggested that DBPT induces apoptosis in colorectal cancer cell lines through caspase-dependent and caspase-independent pathways and that JNK activation was crucial for DBPT-induced apoptosis. DBPT and its analogues might be useful as anticancer agents.     

A dominant role for the c-Jun NH2-terminal kinase in oncogenic ras-induced morphologic transformation of human lung carcinoma cells

Oncogenic (activated) Ras is a signal transducer that activates multiple effector-mediated signaling pathways leading to altered cell morphology, growth and differentiation, and neoplastic transformation. Activating mutations of Ras family genes have been detected in many types of human cancers, including lung cancer. However, the signaling mechanisms by which oncogenic Ras controls cancer cell growth is poorly characterized. This study evaluates the role of two specific signaling pathways, the c-Jun NH2-terminal kinase (JNK) pathway, and the extracellular signal-regulated kinase (ERK) pathway, in oncogenic Ras-induced morphological transformation of NCI-H82 human small cell lung cancer cells. In the NCI-H82 cell line, oncogenic Ras causes a marked and sustained activation of JNK but only has a modest effect on activation of the ERK pathway. The persistent JNK activation is associated with Ras-induced changes in cell morphology and enhanced transforming activity. Furthermore, JNK activation correlates with the induction of c-Jun expression, c-Jun phosphorylation on serines 63 and 73, and increased AP-1 activity. Deregulation of the JNK pathway using a dominant-negative mutant of JNK1, JNK1(APF), completely reverses the oncogenic Ras-induced transformed phenotype, including morphological reversion and inhibition of anchorage-independent growth and low-serum growth. Moreover, expression of JNK1(APF) leads to a decrease in c-Jun/AP-1 activity. In contrast, inhibition of ERK activation via a pharmacological approach using a mitogen-activated protein kinase/ERK kinase-specific inhibitor 2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one is unable to reverse the Ras-induced transformed morphology and c-Jun/AP-1 induction. These results demonstrate that the JNK/c-Jun/AP-1 pathway plays an essential role in mediating oncogenic Ras function in lung carcinoma cells.     

Arsenic induces apoptosis through a c-Jun NH2-terminal kinase-dependent, p53-independent pathway.

Arsenic has been used as an effective chemotherapy agent for some human cancers, such as acute promyelocytic leukemia. In this study, we found that arsenic induces activation of c-Jun NH2-terminal kinases (JNKs) at a similar dose range for induction of apoptosis in JB6 cells. In addition, we found that arsenic did not induce p53-dependent transactivation. Similarly, there was no difference in apoptosis induction between cells with p53 +/+ or p53 -/-. In contrast, arsenic-induced apoptosis was almost totally blocked by expression of a dominant-negative mutant of JNK1. These results suggest that the activation of JNKs is involved in arsenic-induced apoptosis of JB6 cells. Taken together with previous findings that p53 mutations are involved in approximately 50% of all human cancers and nearly all chemotherapeutic agents kill cancer cells mainly by apoptotic induction, we suggest that arsenic may be a useful agent for the treatment of cancers with p53 mutation.