Sulforaphane sensitizes tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis through downregulation of ERK and Akt in lung adenocarcinoma A549 cells

The cytotoxic effect of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is limited in some cancer cells, including A549 lung adenocarcinoma cells. However, treatment with TRAIL in combination with subtoxic concentrations of sulforaphane (SFN) sensitizes TRAIL-resistant A549 cells to TRAIL-mediated apoptosis. Combined treatment with SFN and TRAIL induced chromatin condensation, DNA fragmentation, annexin V staining and sub-G(1) phase DNA content. These indicators of apoptosis correlate with the induction of caspase-3 activity that results in the cleavage of poly(ADP-ribose) polymerase and the release of lactate dehydrogenase. Both the cytotoxic effect and apoptotic characteristics induced by combined treatment were significantly inhibited by z-DEVD-fmk, a caspase-3 inhibitor, demonstrating the important role of caspase-3 in the observed cytotoxic effect. Combined treatment also triggered the activation of p38 MAPK and JNK, and downregulation of ERK and Akt. Inhibitors of ERK (PD98059) or Akt (LY294002), but not p38 MAPK, resulted in significantly decreased cell viability. Although the activation of JNK was increased in response to combined treatment, inhibition of the JNK pathway significantly attenuated cell viability. These results indicate that caspase-3 is a key regulator of apoptosis in response to combined SFN and TRAIL in human lung adenocarcinoma A549 cells through downregulation of ERK and Akt.     

力达霉素联合硼替佐米的抗骨髓瘤作用及对MAPKs表达的影响

目的 研究力达霉素(lidamycin, LDM)联合硼替佐米(bortezomib, BZM)的抗骨髓瘤作用及对丝裂原活化蛋白激酶(Mitogen-activated protein kinases, MAPKs)的影响,并探讨MAPKs在两药联合抗骨髓瘤中的作用。方法 选取适当的药物浓度和通路抑制剂浓度,MTS法检测细胞增殖情况;Westernblot 检测相关蛋白及蛋白磷酸化水平。结果 BZM能增强LDM对骨髓瘤细胞的增殖抑制作用,LDM激活c-Jun氨基末端激酶(c-Jun NH2-terminal kinase, JNK)、p38 MAPK的表达和细胞外信号调节激酶(Extracellular signal regulated rotein kinase, ERK),两药联合后可使JNK和p38 MAPK的激活显著增强,而ERK的激活显著下降。JNK抑制剂(SP600125)、p38抑制剂(SB203580)和MEK抑制剂(U0126) 3种抑制剂单独作用对细胞的增殖抑制作用均不明显,但SP600125或SB203580分别与LDM联合BZM合用后均降低了两药联合对细胞的增殖抑制作用,而U0126与LDM联合BZM合用后提高了两药联合对细胞的增殖抑制作用。结论 LDM通过进一步激活JNK、p38 MAPK和降低ERK的激活来增强BZM抗骨髓瘤敏感度。     

N-(4-hydroxyphenyl)retinamide-induced apoptosis triggered by reactive oxygen species is mediated by activation of MAPKs in head and neck squamous carcinoma cells

N-(4-hydroxyphenyl)retinamide (4HPR), a synthetic retinoid effective in cancer chemoprevention and therapy, is thought to act via apoptosis induction resulting from increased reactive oxygen species (ROS) generation. As ROS can activate MAP kinases and protein kinase C (PKC), we examined the role of such enzymes in 4HPR-induced apoptosis in HNSCC UMSCC22B cells. 4HPR increased ROS level within 1 h and induced activation of caspase 3 and PARP cleavage within 24 h. Activation of MKK3/6 and MKK4, JNK, p38 and ERK was detected between 6 and 12 h, increased up to 24 h and preceded apoptosis. 4HPR-induced activation of these kinases was abrogated by the antioxidants BHA and vitamin C. SP600125, a JNK inhibitor, suppressed 4HPR-induced c-Jun phosphorylation, cytochrome c release from mitochondria and apoptosis. Suppression of JNK1 and JNK2 using siRNA decreased, whereas overexpression of wild type-JNK1 enhanced 4HPR-induced apoptosis. PD169316, a p38, inhibitor suppressed phosphorylation of Hsp27 and apoptosis. PD98059, an MEK1/2 inhibitor, also suppressed ERK1/2 activation and apoptosis induced by 4HPR. Likewise, PKC inhibitor GF109203X suppressed ERK and p38 phosphorylation and PARP cleavage. These data indicate that 4HPR-induced apoptosis is triggered by ROS increase, leading to the activation of the mitogen-activated protein serine/threonine kinases JNK, p38, PKC and ERK, and subsequent apoptosis.     

Rotenone induces apoptosis in MCF‐7 human breast cancer cell‐mediated ROS through JNK and p38 signaling

Rotenone is an inhibitor of the mitochondrial electron transport chain complex I, resulting in the generation of reactive oxygen species (ROS). Rotenone has been shown to display anticancer activity through the induction of apoptosis in various cancer cells. However, the underlying mechanism is still not fully understood. Here, rotenone showed a strong growth inhibitory effect against human breast cancer MCF‐7 cells. DNA flow cytometric analysis, chromatin condensation, and poly (ADP‐ribose) polymerase (PARP) cleavage indicated rotenone actively induced apoptosis in MCF‐7 cells. The antiapoptotic protein, Bcl‐2, was decreased, whereas the apoptotic protein, Bax, was increased in a time‐dependent manner in rotenone‐induced apoptosis. Moreover, the treatment of rotenone in MCF‐7 cells caused the activation of c‐jun N‐terminal kinase (JNK) and p38 mitogen‐activated protein kinases (MAPKs), and the inactivation of extracellular signal‐regulated protein kinase 1/2 (ERK1/2). The pharmacological inhibition of JNK and p38 MAPK revealed significant protection against rotenone‐induced apoptosis. Taken together, these results indicate rotenone may induce apoptosis through ROS and JNK/p38 MAPKs activation in MCF‐7 cells. © 2009 Wiley‐Liss, Inc.     

The inhibition of ERK/MAPK not the activation of JNK/SAPK is primarily required to induce apoptosis in chronic myelogenous leukemic K562 cells

In this study, the downstream signaling of Bcr-Abl tyrosine kinase responsible for apoptosis resistance was investigated. DNA fragmentation, a hallmark of apoptosis, was observed after 2 days of herbimycin A treatment with a peak on 3 day. During the apoptosis induced by the treatment of herbimycin A, stress-activated protein kinase (SAPK) and p38 kinase were activated time- and dose-dependently, while extracellular signal-regulated kinase (ERK) was inhibited. However, apoptosis was induced by the treatment of PD98059, a specific inhibitor of MEK (MAPK or ERK kinase), not by the treatment of sorbitol, a strong activator of SAPK and p38 kinase. Although K562 cells were very resistant to sorbitol-induced apoptosis, DNA fragmentation was induced rapidly in Jurkat, HL-60 and U937 cells after exposure to sorbitol, despite that these apoptosis-sensitive cells have similar or lower activities of JNK/SAPK and p38 kinase compared with K562 cells after treatment of sorbitol. K562 cells had a much higher basal activity of ERK/MAPK than other apoptosis-sensitive cell lines, which were very susceptible to apoptosis induced by low dose of PD98059 compared with K562 cells. In HL-60 cells, sorbitol-induced apoptosis was prevented by the treatment of phorbol myristate 13-acetate (PMA), which activates the ERK/MAPK pathway, and this was blocked by PD98059. From these results, it could be suggested that the inhibition of ERK/MAPK not the activation of JNK/SAPK is primarily required to induce apoptosis in K562 cells.     

Ajoene-induced cell death in human promyeloleukemic cells does not require JNK but is amplified by the inhibition of ERK

Treatment of human promyeloleukemic HL-60 cells with the experimental antileukemic drug ajoene induces the activation of the mitogen-activated protein kinases (MAPKs) c-Jun NH(2)-terminal kinase (JNK), p38 and extracellular signal-regulated kinases (ERK) 1/2 as well as the survival kinase Akt. JNK activation occurred in HL-60/neo, HL-60/bcl-x(L), and in HL-60 cells pretreated with the pan-caspase inhibitor zVAD-fmk, indicating that JNK activation is not dependent on ajoene-induced mitochondria perturbation and subsequent caspase activation. Cells overexpressing a dominant-negative JNK showed no altered sensitivity towards ajoene suggesting that the activation of JNK is not necessary for ajoene-induced cell death. Inhibition of p38 MAPK by SB 203580 had no influence on ajoene-mediated apoptosis. In contrast, inhibition of ERK1/2 vastly enhanced ajoene-induced cell death. The survival kinase Akt, in contrast, did not participate in ajoene-induced death signaling as shown by the use of the phosphatidylinositol-3-kinase inhibitor wortmannin. Thus in contrast to the previous findings regarding stress-induced cell death, ajoene-mediated activation of JNK and p38 has no impact on ajoene-induced apoptosis in HL-60 cells. Blockade of ERK1/2 but not Akt pathways leads to sensitization of cells against ajoene-mediated apoptosis supporting the view that inhibition of ERK1/2 is a valuable strategy to increase the sensitivity of promyeloleukemic cells towards ajoene.     

Induction of apoptosis in human leukemia cells by the tyrosine kinase inhibitor adaphostin proceeds through a RAF-1/MEK/ERK- and AKT-dependent process

Effects of the tyrphostin tyrosine kinase inhibitor adaphostin (NSC 680410) have been examined in human leukemia cells (Jurkat, U937) in relation to mitochondrial events, apoptosis, and perturbations in signaling and cell cycle regulatory events. Exposure of cells to adaphostin concentrations > or =0.75 microM for intervals > or =6 h resulted in a pronounced release of cytochrome c and AIF, activation of caspase-9, -8, and -3, and apoptosis. These events were accompanied by the caspase-independent downregulation of Raf-1, inactivation of MEK1/2, ERK, Akt, p70S6K, dephosphorylation of GSK-3, and activation of c-Jun-N-terminal kinase (JNK) and p38 MAPK. Adaphostin also induced cleavage and dephosphorylation of pRb on CDK2- and CDK4-specific sites, as well as the caspase-dependent downregulation of cyclin D1. Inducible expression of a constitutively active MEK1 construct markedly diminished adaphostin-induced cytochrome c and AIF release, JNK activation, and apoptosis in Jurkat cells. Ectopic expression of Raf-1 or constitutively activated (myristolated) Akt also significantly attenuated adaphostin-induced apoptosis, but protection was less than that conferred by enforced activation of MEK. Lastly, antioxidants (e.g., L-N-acetylcysteine; L-NAC) opposed adaphostin-mediated mitochondrial dysfunction, Raf-1/MEK/ERK downregulation, JNK activation, and apoptosis. However, in contrast to L-NAC, enforced activation of MEK failed to block adaphostin-mediated ROS generation. Together, these findings demonstrate that the tyrphostin adaphostin induces multiple perturbations in signal transduction pathways in human leukemia cells, particularly inactivation of the cytoprotective Raf-1/MEK/ERK and Akt cascades, that culminate in mitochondrial injury, caspase activation, and apoptosis. They also suggest that adaphostin-related oxidative stress acts upstream of perturbations in these signaling pathways to trigger the cell death process.     

X-ray irradiation increases production of IL-8 in lung cancer cell line A549

Objective To observe the effect of irradiation on the production of IL-8 in lung cancer cell line A549 and explore its possible mechanism. Methods A549 cells irradiated with different doses of X-rays were used to collect cell supernatant, cellular RNA and protein at different time points after irradiation. The expression level of IL-8 mRNA in A549 cells after irradiation was detected by RT-PCR, which was further validated by real-time quantitative PCR. The expression level of IL-8 in the cell supernatant was quantitatively measured by ELISA. The expression levels of cellular signaling pathway molecules in A549 cells after irradiation were detected byWestern Blot. The A549 cells were pretreated with p38 MAPK inhibitor, NF-κB inhibitor and ROS scavenger. The effect of these inhibitors on the expression of IL-8 in A549 cells induced by irradiation was evaluated by ELISA. Results Irradiation up-regulated the expression of IL-8 in A549 cells in a dose-and time-dependent manner. Irradiation activated the p38 MAPK and NF-κB signaling pathway in A549 cells. p38 MAPK and NF-κB inhibitors blocked the induction of IL-8 of A549 cells by irradiation. Inhibition of ROS failed to inhibit the induction of IL-8 of A549 cells by irradiation. Conclusion Irradiation can increase the production of IL-8 in lung cancer cells A549, possibly through the activation of p38 MAPK and NF-κB signaling pathways in a ROS-independent pattern.     

X线照射增加肺癌细胞A549产生IL-8的研究

目的 观察照射对肺癌细胞A549产生IL-8的影响及探索其可能机制。方法 采用不同剂量X线照射A549细胞,于照射后不同时间收集细胞上清,细胞RNA以及蛋白质,采用RT-PCR检测照射后A549细胞IL-8 mRNA表达水平,并进一步行实时定量PCR验证照射后A549细胞IL-8 mRNA表达水平,行ELISA检测照射后上清中IL-8表达水平,Western Blot检测照射后A549细胞信号通路分子表达情况,采用p38 MAPK抑制剂、NF-κB抑制剂及ROS清除剂预处理细胞,ELISA验证抑制剂对照射诱导A549细胞产生IL-8表达水平的影响。结果 照射增加A549细胞的IL-8的表达水平,并具有剂量、时间效应。照射激活A549细胞中p38 MAPK和NF-κB信号通路分子。抑制p38 MAPK和抑制NF-κB能够阻断照射诱导A549细胞产生的IL-8。抑制ROS后并不能抑制照射诱导A549细胞产生的IL-8。结论 X线照射能增加A549细胞IL-8的产生,可能与通过激活p38 MAPK和NF-κB信号通路相关,并呈ROS非依赖性模式。     

Role of mitogen-activated protein kinases in phenethyl isothiocyanate-induced apoptosis in human prostate cancer cells

The present study was undertaken to examine the role of mitogen-activated protein kinases (MAPKs) in apoptosis induction by phenethyl isothiocyanate (PEITC), a cruciferous vegetable-derived cancer chemopreventive agent, with DU145 and LNCaP human prostate cancer cells as a model. The MAPK family of serine/threonine kinases, including extracellular signal-regulated kinase1/2 (ERK1/2), c-jun N-terminal kinase1/2/3 (JNK1/2/3), and p38 MAPK play an important role in cell proliferation and apoptosis in response to different stimuli. Exposure of DU145 and LNCaP cells to growth suppressive concentrations of PEITC resulted in activation of ERK1/2 and JNKs, but not p38 MAPK, in both cell lines. In DU145 cells, the apoptosis induction by PEITC was statistically significantly attenuated by pharmacological inhibition of JNKs with SP600125. Adenovirus-mediated overexpression of Flag-tagged JNK binding domain (JBD) of JNK-interacting protein-1 (JIP-1), an inhibitor of JNK, also inhibited PEITC-induced apoptosis in DU145 cells. On the other hand, inhibition of ERK1/2 activation with MEK1 inhibitor PD98059 failed to offer protection against PEITC-induced apoptosis in DU145 cells. In LNCaP cells, the PEITC-induced cell death was not affected by either pretreatment with PD98059 or SP600125 or overexpression of JBD of JIP-1. These results indicate that involvement of MAPKs in apoptosis induction by PEITC in human prostate cancer cells is cell line-specific.     

Rhein lysinate inhibits cell growth by modulating various mitogen-activated protein kinases in cervical cancer cells

In previous studies, we found that rhein lysinate (RHL; the salt of rhein and lysine, easily dissolved in water) inhibited the growth of tumor cells in breast and ovarian cancer and hepatocellular carcinoma. This study aimed to investigate the effect of RHL on the growth of human cervical carcinoma HeLa cells and any underlying mechanisms. RHL inhibited the growth of HeLa cells in a dose- and time-dependent manner. It was also noted that RHL induced apoptosis in HeLa cells in a dose-dependent manner. Mechanistically, RHL triggered HeLa cell apoptosis by increasing the levels of cleaved poly ADP-ribose polymerase (PARP) and caspase-3/7. In addition, the activation of p38 mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK) was a critical mediator in RHL-induced growth inhibition. Inhibition of the expression of p38 MAPK and JNK by pharmacological inhibitors reversed RHL-induced growth inhibition by decreasing the level of cleaved PARP and caspase-3/7. Phosphorylation of the extracellular signal-related kinase (ERK) was increased by RHL; conversely, the MEK inhibitor which inhibits ERK activity, synergistically enhanced RHL-induced growth inhibition in HeLa cells. The results showed that RHL inhibits Hela cell growth through the activation of p38 MAPK and JNK, and is a potential chemotherapeutic agent for cervical cancer.     

Synergistic anti‐tumor effect of paclitaxel with CRM197, an inhibitor of HB‐EGF,in ovarian cancer

Heparin‐binding EGF‐like growth factor (HB‐EGF) plays a pivotal role in tumor growth and clinical outcomes in patients with ovarian cancer, leading to the validation of HB‐EGF as a target for ovarian cancer therapy. In this study, we investigated the anti‐tumor effects of paclitaxel, as an anti‐cancer agent, and CRM197, as a specific inhibitor off HB‐EGF, in ovarian cancer. Paclitaxel induced transient ERK activation and sustained activation of JNK and p38 MAPK through the ectodomain shedding of HB‐EGF in SKOV3 cells. In addition, the overexpression of HB‐EGF in paclitaxel‐treated SKOV3 cells resulted in modulation of paclitaxel‐evoked MAPK signaling, including marked activation of ERK and Akt, and minimized activation of JNK and p38 MAPK, indicating that HB‐EGF is involved in drug sensitivity through the balance of anti‐apoptotic and pro‐apoptotic signals induced by paclitaxel. The combination of paclitaxel with CRM197 had an inhibitory effect on cell proliferation and enhanced apoptosis via the inhibition of ERK and Akt activation and the stimulation of p38 and JNK activation. More prominently, the administration of paclitaxel with CRM197 resulted in synergistic anti‐tumor effects in SKOV3 cells and in SKOV3 cells overexpressing HB‐EGF in xenografted mice. Accordingly, inhibitory agents against HB‐EGF, such as CRM197, represent possible chemotherapeutic and chemosensitizing agents for ovarian cancer. © 2008 Wiley‐Liss, Inc.     

Inhibition of extracellular signal-regulated kinase (ERK) mediates cell cycle phase independent apoptosis in vinblastine-treated ML-1 cells

Chemotherapeutic agents induce alterations in intracellular signal transduction cascades that culminate in the initiation of the apoptotic program. Here, the relationship between the mitogen-activated protein kinase (MAPK) response and apoptosis in ML-1 cells treated with vinblastine and paclitaxel was investigated. We show that these compounds elicit different effects on MAPKs with vinblastine, but not paclitaxel, increasing both c-Jun-NH2-terminal kinase (JNK) and p38 activity. However, vinblastine and paclitaxel both induced apoptosis with similar kinetics, suggesting that increased JNK and p38 activity is not required for apoptosis that is induced by microtubule interfering agents. Strikingly, the abrogation of extracellular signal-regulated kinase (ERK)-signaling by the MAPK/ERK kinase (MEK)1/2 inhibitor PD098059 in combination with vinblastine robustly induced apoptosis in ML-1 cells at a rate much faster than treatment with vinblastine alone and occurred at all phases of the cell cycle. This apoptotic induction was attributed to JNK activation because: (a) non-JNK-activating concentrations of vinblastine failed to increase apoptosis in the presence of PD098059; (b) apoptosis induced by paclitaxel, which did not activate JNK, was not potentiated by PD098059; and (c) transduction of an inhibitor of JNK activity partially suppressed both JNK activity and apoptosis induced by vinblastine plus PD098059. Additionally, we found that the activation of JNK by vinblastine occurred upstream of effector caspase activation because treatment with a pan-specific caspase inhibitor (valine-alanine-aspartate-fluoromethylketone) resulted in complete abrogation of apoptosis with no effect on MAPK signaling. Taken together, these data suggest that inhibition of the MEK-->ERK signal transduction cascade alleviates cell cycle dependence for vinblastine-induced apoptosis by a mechanism that requires JNK activation.     

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

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

Dihydroartemisinin induces apoptosis in HL-60 leukemia cells dependent of iron and p38 mitogen-activated protein kinase activation but independent of reactive oxygen species

Dihydroartemisinin (DHA), the main active metabolite of artemisinin derivatives, is one of the most effective anti-malarial analogs of artemisinin. In the current study, we found that DHA inhibited the proliferation of a panel of tumor cells originated from different tissue types. DHA effectively induced apoptosis in human promyelocytic leukemia HL-60 cells, which was accompanied with mitochondrial dysfunction and caspases activation. Further studies indicated that DHA-induced apoptosis was iron-dependent. Though DHA slightly elicited superoxide anion, these reactive oxygen species (ROS) contribute little to DHA-induced apoptosis in HL-60 cells. Moreover, DHA time-dependently activated mitogen-activeted protein kinases (MAPKs) and specific inhibition of p38 MAPK, but not c-Jun-NH2-terminal kinase (JNK) or extracellular signal-regulated kinase (ERK), abolished DHA-induced apoptosis, indicating that activation of p38 MAPK is required for DHA-induced apoptosis in HL-60 cells. Altogether, our data uncover that DHA induces apoptosis is dependent of iron and p38 MAPK activation but not ROS in HL-60 cells.     

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.     

Mechanism of 2-methoxyestradiol-induced apoptosis and growth arrest in human breast cancer cells

2-Methoxyestradiol, a well-known nonpolar endogenous metabolite of 17beta-estradiol, has been shown to selectively induce apoptosis in a number of cancer cell lines, but not in normal cells. The mechanism of 2-methoxyestradiol-induced apoptosis appears to vary considerably in different cell lines examined. In the present study, we systematically analyzed the mechanisms of 2-methoxyestradiol-induced apoptosis in the estrogen receptor-negative MDA-MB-435s human breast cancer cells. We found that 2-methoxyestradiol induced the activation of JNK, ERK, and p38 MAPKs. 2-methoxyestradiol-induced JNK activation was associated with the induction of apoptosis through the mitochondrial pathways as a result of increased phosphorylation (inactivation) of the anti-apoptotic Bcl-2 and Bcl-xL proteins. In comparison, 2-methoxyestradiol-induced activation of ERK and p38 in these cells was found to have a protective effect against 2-MeO-E(2)-induced apoptosis. Consistent with this observation, the presence of pharmacological inhibitor of ERK or p38 enhanced 2-methoxyestradiol-induced apoptosis. Mechanistically, inhibition of ERK and p38 activity was associated with activation of various caspases and PARP cleavage, and it also stabilized the pro-apoptotic proteins Bax and Bim, thereby preventing them from degradation during 2-methoxyestradiol treatment. These results suggest that ERK and p38 MAPKs may serve as viable targets for the sensitization of human breast cancer cells to 2-methoxyestradiol-induced apoptosis.     

Requirement of p38 MAPK for a cell-death pathway triggered by vorinostat in MDA-MB-231 human breast cancer cells

Vorinostat is a histone deacetylase inhibitor that effectively suppresses cancer-cell proliferation by inducing cell-cycle arrest and/or apoptosis. We now show the involvement of p38 mitogen-activated protein kinase (MAPK) in the regulation of vorinostat-induced apoptosis in MDA-MB-231 human breast cancer cells. Vorinostat induced the hyperacetylation of histone H3, which correlated to apoptosis induction. Vorinostat-induced apoptosis occurred in parallel with the phosphorylation of p38 MAPK and the dephosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). Knockdown of p38 MAPK prominently abrogated apoptosis induction and was accompanied by decreased caspase-3 cleavage. These findings support the notion that the activation of the p38 MAPK pathway followed by caspase-3 cleavage is responsible for vorinostat-induced apoptosis in MDA-MB-231 cells.