Biologic sequelae of c-Jun NH(2)-terminal kinase (JNK) activation in multiple myeloma cell lines

Although c-Jun NH(2)-terminal kinase (JNK) is activated by treatment with therapeutic agents, the biologic sequelae of inhibiting constitutive activation of JNK has not yet been clarified. In this study, we examine the biologic effect of JNK inhibition in multiple myeloma (MM) cell lines. JNK-specific inhibitor SP600125 induces growth inhibition via induction of G1 or G2/M arrest in U266 and MM.1S multiple myeloma cell lines, respectively. Neither exogenous IL-6 nor insulin-like growth factor-1 (IGF-1) overcome SP600125-induced growth inhibition, and IL-6 enhances SP600125-induced G2/M phase in MM.1S cells. Induction of growth arrest is mediated by upregulation of p27(Kip1), without alteration of p53 and JNK protein expression. Importantly, SP600125 inhibits growth of MM cells adherent to bone marrow stromal cells (BMSCs). SP600125 induces NF-kappaB activation in a dose-dependent fashion, associated with phosphorylation of IkappaB kinase alpha (IKKalpha) and degradation of IkappaBalpha. In contrast, SP600125 does not affect phosphorylation of STAT3, Akt, and/or ERK. IKK-specific inhibitor PS-1145 inhibits SP600125-induced NF-kappaB activation and blocks the protective effect of SP600125 against apoptosis. Our data therefore demonstrate for the first time that inhibiting JNK activity induces growth arrest and activates NF-kappaB in MM cells.     

Moscatilin induces apoptosis in human colorectal cancer cells: a crucial role of c-Jun NH2-terminal protein kinase activation caused by tubulin depolymerization and DNA damage

PURPOSE: To study the effect of moscatilin (purified from the stem of orchid Dendrobrium loddigesii) on the proliferation of human colorectal cancer HCT-116 cells in vitro and in vivo. EXPERIMENTAL DESIGN: The growth inhibition of moscatilin was screened on several human cancer cell lines. The effect of moscatilin on tubulin was detected in vitro. Following moscatilin treatment on HCT-116 cells, c-Jun NH(2)-terminal protein kinase (JNK) and caspase activation was studied by Western blot analysis, and DNA damage was done by Comet assay. Specific JNK inhibitor SP600125 was cotreated to reverse moscatilin-induced apoptosis. Tumor growth inhibition of moscatilin was done on HCT-116 xenograft models. RESULTS: Moscatilin induced a time-dependent arrest of the cell cycle at G(2)-M, with an increase of cells at sub-G(1). Moscatilin inhibited tubulin polymerization, suggesting that it might bind to tubulins. Moscatilin also induced the phosphorylation of JNK1/2. SP600125 significantly inhibited the activation of caspase-9 and caspase-3 and the subsequent moscatilin-induced apoptosis. The data suggest that JNK activation may contribute to moscatilin-mediated apoptosis signaling. A parallel experiment showed that SP600125 significantly inhibits Taxol- and vincristine-induced HCT-116 cell apoptosis. This suggests that the JNK activation may be a common mechanism for tubulin-binding agents. Moreover, moscatilin induces DNA damage, phosphorylation of H2AX and p53, and up-regulation of p21. Our HCT-116 xenograft models show the in vivo efficacy of moscatilin. CONCLUSIONS: In summary, our results suggest that moscatilin induces apoptosis of colorectal HCT-116 cells via tubulin depolymerization and DNA damage stress and that this leads to the activation of JNK and mitochondria-involved intrinsic apoptosis pathway.     

Triptolide induces Bcl-2 cleavage and mitochondria dependent apoptosis in p53-deficient HL-60 cells

Triptolide, a bioactive component of the Chinese medicinal herb Tripterygium wilfordii Hook F., induces p53-mediated apoptosis in cancer cells. This study demonstrated that triptolide activated an alternative p53-independent apoptotic pathway in HL-60 cells. In the absence of an intact p53 and without changing Bax level, at nM range triptolide induced apoptosis with concomitant DNA fragmentation, S phase cell cycle arrest, mitochondrial cytochrome c release and the activation of caspases. Besides, both caspases 8 and 9 were activated and the simultaneous inhibition of both was required to completely block triptolide's apoptotic effect. Importantly, triptolide induced the appearance of a truncated 23kD Bcl-2 which was inhibited by the general caspase inhibitor Z-VAD-FMK. In the MCF-7 cells that possessed the wild type p53 but lacked caspases 3, triptolide induced cell death with an increase in p53 but Bcl-2 remained unaltered. On the other hand, transfected cells overexpressing the 28kD Bcl-2 became more resistant to triptolide and upon triptolide treatment accumulated in the G(1) instead of S phase. After 36h treatment, triptolide activated JNK pathways, at the same time inactivated the ERK and p38 pathways. However, SP600125, a specific JNK inhibitor, could not inhibit the triptolide-mediated cleavage of caspase 3, indicated that activation of JNK might not be related to the apoptotic effects of triptolide. Our data suggest that in the absence of an intact p53 and without altering Bax level triptolide induces apoptosis activates a positive amplification loop involving caspase-mediated Bcl-2 cleavage/activation, mitochondrial cytochrome c release and further activation of caspases.     

Inhibition of JNK reduces G2/M transit independent of p53, leading to endoreduplication, decreased proliferation, and apoptosis in breast cancer cells

c-Jun N-terminal kinase (JNK) is activated by diverse cell stimuli, including stress, growth factors, and cytokines. Traditionally, activation of JNK by stress treatment is thought to induce cell death. However, our recent data indicate that JNK's ability to sensitize cells to apoptosis may be, in part, cell cycle dependent. Here, we show that the majority of both paclitaxel- and UV-induced apoptosis can be inhibited by the pharmacological JNK inhibitor, SP600125, in MCF-7 cells. However, inhibition of JNK does little to reverse doxorubicin-induced apoptosis in MCF-7 cells or doxorubicin- and UV-mediated death in MDA MB-231 cells. SP treatment causes G2/M arrest of three breast cancer cell lines and results in the endoreduplication (cellular DNA content >4N) of MCF-7 and MDA MB-231 cells. These effects on cell cycle and apoptosis are not significantly altered by the inhibition of p53, indicating that JNK is functioning independently of p53. Lastly, inhibition of JNK using both SP and antisense oligonucleotides targeted to JNK1 and JNK2 reduced proliferation of all three breast cancer cell lines. Taken together, these results suggest that the activation of JNK is important for the induction of apoptosis following stresses that function at different cell cycle phases, and that basal JNK activity is necessary to promote proliferation and maintain diploidy in breast cancer cells.     

c-Jun NH2-terminal kinase-mediated up-regulation of death receptor 5 contributes to induction of apoptosis by the novel synthetic triterpenoid methyl-2-cyano-3,12-dioxooleana-1, 9-dien-28-oate in human lung cancer cells

Death receptor (DR) 4 or 5, on binding to its ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), triggers apoptosis via activating the caspase-8-mediated caspase cascade. Certain anticancer drugs up-regulate the expression of these receptors and thereby induce apoptosis or enhance TRAIL-induced apoptosis. In this study, we explored the ability of methyl-2-cyano-3,12-dioxooleana-1,9-dien-28-oate (CDDO-Me) to activate the extrinsic DR-mediated apoptotic pathway in human lung cancer cells. We found that CDDO-Me not only activated caspase-8 but also induced expression of DRs, particularly DR5, in a p53-independent mechanism. Correspondingly, CDDO-Me augmented TRAIL-induced apoptosis in these cells regardless of p53 status as evidenced by enhanced DNA fragmentation and activation of caspase cascades, suggesting that CDDO-Me-induced DRs are functionally active. Moreover, silencing of DR5 expression using small interfering RNA suppressed apoptosis induced by CDDO-Me alone or by combination of CDDO-Me and TRAIL, indicating that DR5 up-regulation is required for induction of apoptosis by CDDO-Me and for enhancement of TRAIL-induced apoptosis by CDDO-Me. CDDO-Me rapidly activated c-Jun NH(2)-terminal kinase (JNK) before DR up-regulation and caspase-8 activation. Moreover, application of the JNK-specific inhibitor SP600125 blocked CDDO-Me-induced increases in JNK activation, DR up-regulation, caspase-8 activation, and DNA fragmentation. These results show that activation of JNK pathway results in CDDO-Me-induced DR up-regulation, caspase-8 activation, and apoptosis. Collectively, we conclude that CDDO-Me induces apoptosis via the JNK-mediated DR up-regulation in human lung cancer cells.     

Involvement of c-Jun N-terminal kinase in G2/M arrest and FasL-mediated apoptosis induced by a novel indoloquinoline derivative, IQDMA, in K562 cells

N'-(11H-Indolo[3,2-c]quinolin-6-yl)-N,N-dimethylethane-1,2-diamine (IQDMA), an indoloquinoline derivative, synthesized in our laboratory, has been demonstrated to be an effective anti-tumor agent in human leukemia cells. Treatment of K562 cells with IQDMA resulted in G2/M phase cell cycle arrest, presumably involving the concomitant up-regulation of p21 and apoptosis through up-regulation of FasL and sequential activation of caspase-8 and caspase-3. In contrast to the lack of appreciable effect on the phosphorylation of ERK and p38 MAPK, activation of JNK was noted when K562 cells were exposed to IQDMA. Moreover, IQDMA-mediated G2/M phase arrest and apoptosis were reversed after treatment with the JNK-specific inhibitors, SP600125 and JNK inhibitor 1. Further investigation showed that SP600125 reduced the activation of FasL, caspase-3, caspase-8, and led to a marked decline of p21. Taken together, our data show that JNK plays an important role in IQDMA-mediated G2/M arrest and apoptosis of K562 cancer cells.     

Bcl-2 overexpression attenuates SP600125-induced apoptosis in human leukemia U937 cells

SP600125 is a specific inhibitor of c-Jun N-terminal kinase (JNK) that is known to strongly induce apoptosis and block cell cycle progression in G₂/M phase. In this study, we demonstrated that treatment of U937 cells with SP600125 resulted in significant G₂/M cell cycle arrest that was due to decreased cyclin B1 and cdc25c protein levels. Moreover, SP600125 promoted LDH release and DNA fragmentation that was associated with caspase-3 activation and degradation of its substrates. In contrast, overexpression of the antiapoptotic protein Bcl-2 rendered leukemia cells resistant to SP600125-induced apoptosis, but more sensitive to G₂/M phase arrest and endoreduplication (>4N DNA). Overexpression of Bcl-2 significantly inhibited SP600125-induced caspase-3 activation and degradation of its substrates, and sustained expression levels of the IAP-2 proteins following SP600125 treatment. The inhibitory effect of Bcl-2 on apoptosis was attenuated by treatment with the small molecule Bcl-2 inhibitor, HA14-1. These data provide important mechanistic insights related to Bcl-2-mediated resistance to SP600125-induced apoptosis, and induction of G₂/M phase arrest and endoreduplication.     

Proteasome inhibitor PS-341 induces growth arrest and apoptosis of non-small cell lung cancer cells via the JNK/c-Jun/AP-1 signaling

Proteasome inhibitor PS-341 induces growth arrest and apoptosis of multiple myeloma (MM) cells via inactivation of NF-κB in vitro and has afforded some objective responses in individuals with relapsed, refractory MM. However, the activity of PS-341 against non-hematological malignancies remains to be fully elucidated. In this study, we found that PS-341 induced growth arrest and apoptosis of NCI-H520 and -H460 non-small cell lung cancer (NSCLC) cells in conjunction with markedly up-regulated levels of p21^waf1 and p53, and down-regulation of bcl-2 protein in these cells. Also, PS-341 caused phosphorylation of c-Jun NH₂-terminal kinase (JNK) and c-Jun, and enhanced AP-1/DNA binding activities in these cells as measured by western blotting and enzyme-linked immunosorbent assay (ELISA), respectively. Interestingly, when the JNK/ c-Jun/AP-1 signal pathway was disrupted by the JNK inhibitor SP600125, the ability of PS-341 to inhibit the growth of NSCLC cells and to up-regulate the levels of p21^waf1 in these cells was blunted, but the expression of p53 was sustained at a high level, suggesting that the JNK/c-Jun/AP-1 signal pathway might mediate the anti-lung cancer effects of PS-341, with p21^waf1 playing the central role. Thus, PS-341 might be useful for the treatment of individuals with NSCLC.     

Silibinin activates p53-caspase 2 pathway and causes caspase-mediated cleavage of Cip1/p21 in apoptosis induction in bladder transitional-cell papilloma RT4 cells: evidence for a regulatory loop between p53 and caspase 2

Silibinin, a natural flavonolignan, induces apoptosis in human bladder transitional-cell papilloma RT4 cells both in vitro and in vivo; however, mechanisms of such efficacy are not completely identified. Here, we studied the mechanisms involved in silibinin-induced apoptosis of RT4 cells having intact p53. Silibinin increased p53 protein level together with its increased phosphorylation at serine 15, activated caspase cascade and caused Bid cleavage for apoptosis. Silibinin-caused p53 activation was mediated via ATM-Chk2 pathway, which in turn induced caspase 2-mediated apoptosis. Pifithrin-alpha, a p53 inhibitor, reversed silibinin-induced caspase activation including caspase 2; however, caspase 2 inhibitor also reversed p53 phosphorylation suggesting a bidirectional regulation between them. Further, silibinin caused a rapid translocation of p53 and Bid into mitochondria leading to increased permeabilization of mitochondrial membrane and cytochrome c release into the cytosol. JNK1/2 activation was observed as a connecting link for p53-mediated caspase 2 activation. Interestingly, silibinin-induced apoptosis was mediated, in part, via Cip1/p21 cleavage by caspase, which was reversed by Cip1/p21 siRNA. Together, these results suggested the novel mechanisms for apoptosis induction by silibinin involving p53-caspase 2 activation and caspase-mediated cleavage of Cip1/p21.     

MAPK regulation and caspase activation are required in DMNQ S-52 induced apoptosis in Lewis lung carcinoma cells

6-(1-Hydroxyimino-4-methylpentyl)5,8-dimethyoxy 1,4-naphthoquinone S-52 (DMNQ S-52) was reported to have cytotoxic activity against L1210 leukemia cells. In the present study, we investigated the apoptotic mechanism of DMNQ S-52 in vitro and in vivo in murine solid cancer cells. DMNQ S-52 exerted cytotoxicity against Lewis lung carcinoma (LLC) cells (IC50=12.3 microM). DMNQ S-52 increased Annexin V positive cell population in a concentration-dependent manner. DMNQ S-52 also induced apoptosis through caspase-mediated pathway, including activation of caspase-3, cleavage of Poly(ADP-ribose) polymerase (PARP) and decreased expression of Bcl-2 in LLC cells in a time and concentration-dependent fashion. DMNQ S-52 activated the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 as well as abrogated the expression of extracellular signal-regulated kinase (ERK) in a time-dependent manner at 10 microM. Similarly, cell proliferation inhibition by DMNQ S-52 was masked by caspase inhibitor Z-Asp-Glu-Val-Asp-fluoromethylketone (Z-VAD-FMK), JNK inhibitor SP600125 and p38 inhibitor SB203580, but not by MEK inhibitor U0126. Furthermore, i.p. administration of DMNQ S-52 at 5 mg/kg resulted in a potent inhibition of the growth of LLC cells implanted on the right flank of C57BL/6 mice compared to untreated control. Immunohistochemical analysis revealed the decreased tumor cell proliferation and increased tumor cell apoptosis in DMNQ S-52 treated tumor sections using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) and proliferation cell nuclear antigen (PCNA). Taken together, these findings demonstrate that DMNQ S-52 may exhibit anti-tumor activity by inducing apoptosis via caspases and mitogen activated protein (MAP) kinase-dependent pathways.     

Dicoumarol potentiates cisplatin-induced apoptosis mediated by c-Jun N-terminal kinase in p53 wild-type urogenital cancer cell lines

3-3'-Methylene-bis [4-hydroxycoumarin] (dicoumarol), an inhibitor of NADPH:quinone oxidoreductase 1, has been reported to possess potential antineoplastic effects and the ability to abrogate p53 protein. In the present study, we investigated the cytotoxic effects of dicoumarol in combination with cisplatin (CDDP), using four bladder (RT112, 253J, J82 and UMUC3) and two prostate (LNCap and PC3) cancer cell lines. Single treatment with 100 microM dicoumarol suppressed cell proliferation but did not induce apoptosis at 24 h in all cell lines examined. On the other hand, pretreatment with dicoumarol enhanced cytotoxicity of CDDP in three cell lines with wild type of p53 (RT112, 253J and LNCap), but not in three other cell lines with mutant p53 or in RT112 stable transfectants with a dominant-negative mutant of p53. In RT112 and LNCap, CDDP induced p53 and p21 expression, while pretreatment of dicoumarol suppressed induction of p53/p21 and resulted in sequential activation of c-Jun N-terminal kinase (JNK) in a time-dependent manner. Furthermore, inhibition of JNK, using SP600125, completely suppressed activity of caspases and poly-(ADP-ribose) polymerase cleavage, leading to suppression of enhancement of CDDP-mediated apoptosis by dicoumarol. These results suggested that dicoumarol could enhance cytotoxicity of CDDP in urogenital cancer cells with wild-type p53 through the p53/p21/JNK pathways.     

Simvastatin induces apoptosis in human breast cancer cells: p53 and estrogen receptor independent pathway requiring signalling through JNK

The effect of simvastatin, a widely used statin for the treatment of hypercholesterolemia, was investigated in the estrogen receptor (ER)-positive MCF-7, and the ER-negative MDA-MB 231 human breast cancer cell lines. Simvastatin induced cell cycle arrest and apoptosis in both cells. These effects of simvastatin were not altered by 17-β-estradiol treatment.

MCF-7 cells express wild-type tumor suppressor protein p53, whereas MDA-MB 231 cells carry a p53 mutation. However, no alteration in the level or localisation of p53 was observed with simvastatin treatment in either cell line. On the other hand, simvastatin strongly stimulated phosphorylation of c-jun which was completely abolished by the c-jun NH₂-terminal kinase (JNK) inhibitor SP600125, which also significantly reduced the antiproliferative and apoptotic effects of simvastatin in these cells.

In conclusion, we describe here that simvastatin induces apoptosis via involvement of JNK in breast cancer cells independent of their ER or p53 expression status. These findings indicate a great potential for statins for the treatment of cancers resistant to currently used drugs, and target the JNK signalling pathway for a novel approach of breast cancer treatment.     

Overcoming the radioresistance of prostate cancer cells with a novel Bcl-2 inhibitor

Bcl-2 overexpression is an important mechanism underlying the aggressive behavior of prostate cancer cells and their resistance to radio- or chemotherapy. HA14-1, a recently discovered organic Bcl-2 inhibitor, potently induces apoptosis in various human cancer cells. Sequential exposure of radioresistant LNCaP (wild-type (wt) p53), LNCaP/Bcl-2 (wt p53) and PC3 (mutant p53) prostate cancer cells to a minimally cytotoxic concentration of 10 microM HA14-1 for 1 h followed by 1-6 Gy gamma radiation, resulted in a highly synergistic (combination index <1.0) induction of cell death as determined by an apoptosis assay at 72 h, and a clonogenicity assay at 12 days, after the initial treatment. The reverse treatment sequence did not cause a synergistic induction of cell death. When compared to individual treatments, cell death induced by the combined treatment was associated with dramatically increased reactive oxygen species (ROS) generation, c-Jun N-terminal kinase (JNK) activation, Bcl-2 phosphorylation, cytochrome c release, caspase-3 activation and DNA fragmentation. Exposure to either 200 microg/ml of the antioxidant alpha-tocopherol or 10 microM JNK inhibitor SP600125 before the combined treatment resulted in decreased activation of JNK and caspase-3 as well as decreased DNA fragmentation. However, treatment with the pancaspase inhibitor carbobenzoxyl-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone before the combined treatment inhibited apoptosis without affecting JNK activation, and this inhibitory effect was enhanced in the presence of alpha-tocopherol or SP600125. Taken together, our results indicate that HA14-1 potently sensitizes radioresistant LNCaP and PC3 cells to gamma radiation, regardless of the status of p53. ROS and JNK are important early signals that trigger both caspase-dependent and -independent cell death pathways and contribute to the apoptotic synergy induced by the combined treatments.     

Icariin,a natural flavonol glycoside,induces apoptosis in human hepatoma SMMC-7721 cells via a ROS/JNK-dependent mitochondrial pathway

In this study, the anticancer effect of icariin, a natural flavonol glycoside, against human hepatoma SMMC-7721 cells and the underlying mechanisms were investigated. Icariin triggered the mitochondrial/caspase apoptotic pathway indicated by enhanced Bax-to-Bcl-2 ratio, loss of mitochondrial membrane potential, cytochrome c release, and caspase cascade. Moreover, icariin induced a sustained activation of the phosphorylation of c-Jun N-terminal kinase (JNK) but not p38 and ERK1/2, and SP600125 (an inhibitor of JNK) almost reversed icariin-induced apoptosis in SMMC-7721 cells. In addition, icariin provoked the generation of reactive oxygen species (ROS) in SMMC-7721 cells, while the antioxidant N-acetyl cysteine almost completely blocked icariin-induced JNK activation and apoptosis. Taken together, these findings suggest that icariin induces apoptosis through a ROS/JNK-dependent mitochondrial pathway.     

A link between benzyl isothiocyanate-induced cell cycle arrest and apoptosis: involvement of mitogen-activated protein kinases in the Bcl-2 phosphorylation

In the present study, we clarified the molecular mechanism underlying the relationship between benzyl isothiocyanate (BITC)-induced cell cycle arrest and apoptosis and the involvement of mitogen-activated protein kinases (MAPKs). The exposure of Jurkat human T-cell leukemia cells to BITC resulted in the inhibition of the G(2)-M progression that coincided with the apoptosis induction. The experiment using the phase-specific synchronized cells demonstrated that the G(2)-M phase-arrested cells are more sensitive to undergoing apoptotic stimulation by BITC than the cells in other phases. We also confirmed that BITC activated c-Jun N-terminal kinase (JNK) and p38 MAPK, but not extracellular signal-regulated kinase, at the concentration required for apoptosis induction. An experiment using a JNK-specific inhibitor SP600125 or a p38 MAPK inhibitor SB202190 indicated that BITC-induced apoptosis might be regulated by the activation of these two kinases. Conversely, BITC is likely to confine the Jurkat cells in the G(2)-M phase mainly through the p38 MAPK pathway because only the p38 MAPK inhibitor significantly attenuated the accumulation of inactive phosphorylated Cdc2 protein and the G(2)-M-arrested cell numbers. We reported here for the first time that the antiapoptotic Bcl-2 protein was phosphorylated by the BITC treatment without significant alteration of the Bcl-2 total protein amount. This was abrogated by a JNK specific inhibitor SP600125 at the concentration required for specific inhibition of the c-Jun phosphorylation. Moreover, the spontaneous phosphorylation of antiapoptotic Bcl-2 in the G(2)-M synchronized cells was enhanced synergistically by the BITC treatment. Involvement of the MAPK activation in the Bcl-2 phosphorylation and apoptosis induction also was observed in HL-60 and HeLa cells. Thus, we identified the phosphorylated Bcl-2 as a key molecule linking the p38 MAPK-dependent cell cycle arrest with the JNK activation by BITC.     

TPA-induced p21 expression augments G2/M arrest through a p53-independent mechanism in human breast cancer cells

The tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), has a differential role on the regulation of the cell cycle in a variety of tumor cells. The mechanism between TPA and the cell cycle in breast cancer is not fully understood. Therefore, we investigated the regulatory mechanism of TPA on control of the cell cycle of breast cancer cells. Our results showed that TPA increased the level of p21 expression in MCF-7 cells with wild-type p53 and MDA-MB-231 cells with mutant p53 in a dose-dependent manner. In contrast, TPA decreased the expression of p53 in MCF-7 cells, but did not affect MDA-MB-231 cells. We next examined the regulatory mechanism of TPA on p21 and p53 expression. Our results showed that the TPA-induced up-regulation of p21 and down-regulation of p53 was reversed by UO126 (a MEK1/2 inhibitor), but not by SP600125 (a JNK inhibitor) or SB203580 (a p38 inhibitor), although TPA increased the phosphorylation of ERK and JNK in MCF-7 cells. In addition, the TPA-induced arrest of the G2/M phase was also recovered by UO126 treatment. To confirm the expression of p21 through the MEK/ERK pathway, cells were transfected with constitutively active (CA)-MEK adenovirus. Our results showed that the expression of p21 was significantly increased by CA-MEK overexpression. Taken together, we suggest that TPA reciprocally regulates the level of p21 and p53 expression via a MEK/ERK-dependent pathway. The up-regulation of p21 in response to TPA is mediated through a p53-independent mechanism in breast cancer cells.     

Loss of p21 disrupts p14 ARF-induced G1 cell cycle arrest but augments p14 ARF-induced apoptosis in human carcinoma cells

The human INK4a locus encodes two structurally unrelated tumor suppressor proteins, p16 INK4a and p14 ARF (p19 ARF in the mouse), which are frequently inactivated in human cancer. Both the proapoptotic and cell cycle-regulatory functions of p14 ARF were initially proposed to be strictly dependent on a functional p53/mdm-2 tumor suppressor pathway. However, a number of recent reports have implicated p53-independent mechanisms in the regulation of cell cycle arrest and apoptosis induction by p14 ARF. Here, we show that the G1 cell cycle arrest induced by p14 ARF entirely depends on both p53 and p21 in human HCT116 and DU145 carcinoma cells. In contrast, neither loss of p53 nor p21 impaired apoptosis induction by p14 ARF as evidenced by nuclear DNA fragmentation, phosphatidyl serine exposure, and caspase activation, which included caspase-3/7- and caspase-9-like activities. However, lack of functional p21 resulted in the accumulation of cells in G2/M phase of the cell cycle and markedly enhanced p14 ARF-induced apoptosis that was, nevertheless, efficiently inhibited by the cell permeable broad-spectrum caspase inhibitor zVAD-fmk (valyl-alanyl-aspartyl-(O)-methyl)-fluoromethylketone). Thus, loss of cell cycle restriction point control in the absence of p21 may interfere with p14 ARF-induced apoptosis. Finally, these data indicate that the signaling events required for G1 cell cycle arrest and apoptosis induction by p14 ARF dissociate upstream of p53.     

Curcumin inhibits protein phosphatases 2A and 5, leading to activation of mitogen-activated protein kinases and death in tumor cells

Curcumin can induce p53-independent apoptosis. However, the underlying mechanism remains to be defined. Here, we show that curcumin-induced apoptosis in a panel of tumor cells with mutant p53. Curcumin rapidly induced activation of the mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated kinase 1/2 (Erk1/2) and c-Jun N-terminal kinase (JNK). Inhibition of JNK (with SP600125) or Erk1/2 (with U0126) partially prevented curcumin-induced cell death in the cells. Similarly, expression of dominant negative c-Jun or downregulation of Erk1/2 in part attenuated curcumin-induced cell death. It appears that curcumin-induced activation of MAPKs and apoptosis was due to induction of reactive oxygen species (ROS), as pretreatment with N-acetyl-L-cysteine, a ROS scavenger, blocked these events. Furthermore, we found that curcumin-induced activation of MAPK pathways was related to inhibition of the serine/threonine protein phosphatases 2A (PP2A) and 5 (PP5). Overexpression of PP2A or PP5 partially prevented curcumin-induced activation of JNK and Erk1/2 phosphorylation as well as cell death. The results suggest that curcumin induction of ROS activates MAPKs, at least partially by inhibiting PP2A and PP5, thereby leading to p53-independent apoptosis in tumor cells.     

The prosurvival activity of p53 protects cells from UV-induced apoptosis by inhibiting c-Jun NH2-terminal kinase activity and mitochondrial death signaling

The cytoprotective function of p53 recently has been exploited as a therapeutic advantage for cancer prevention; agents activating the prosurvival activity of p53 are shown to prevent UV-induced damages. To explore the mechanisms of p53-mediated protection from UV-induced apoptosis, we have established stable clones of H1299 lung carcinoma cells expressing a temperature-sensitive p53 mutant, tsp53(V143A). At the permissive temperature of 32 degrees C, the tsp53(V143A)-expressing cells were arrested in G(1) phase without the occurrence of apoptosis; consistent with this is the preferential induction of genes related to growth arrest and DNA damage repair. Previous expression of functional tsp53(V143A) for > or =18 hours inhibited the release of proapoptotic molecules from mitochondria and protected the cells from UV-induced apoptosis; moreover, it suppressed the activation of c-Jun NH(2)-terminal kinase (JNK) signaling and relieved the effect of UV on p53 target gene activation. p53 associated with JNK and inhibited its kinase activity. Using the p53-null H1299 cells, we showed that inhibition of JNK blocked the UV-elicited mitochondrial death signaling and caspase activation. Our results suggest that the ability of p53 to bind and inactivate JNK, together with the activation of the p53 target genes related to cell cycle arrest and DNA damage repair, is responsible for its protection of cells against UV-induced apoptosis.