p38 MAP kinase regulates benzo(a)pyrene-induced apoptosis through the regulation of p53 activation

Polycyclic aromatic hydrocarbons, such as benzo(a)pyrene (BaP), are widespread in the environment and cause untoward effects, including carcinogenesis, in mammalian cells. However, the molecular mechanism of apoptosis by BaP is remained to be elusive. Pharmacological inhibition of p38 kinase markedly inhibited the BaP-induced cytotoxicity, which was proven as apoptosis characterized by an increase in sub-G(0)/G(1) fraction of DNA content, ladder-pattern fragmentation of genomic DNA, and catalytic activation of caspase-3 with PARP cleavage. Our data also demonstrated that activation of caspase-3 was accompanied with activation of caspase-9 and mitochondrial dysfunction, which was also apparently suppressed by pretreatment with p38 kinase inhibitors. Also, pharmacological inhibition of p38 markedly inhibited the phosphorylation, accumulated expression, and transactivation activity of p53 in BaP-treated cells. Adenoviral overexpression of human p53 (wild-type) further augmented in increase of PARP cleavage and the sub-G(0)/G(1) fraction of DNA content. Furthermore, p53 mediated apoptotic activity in BaP-treated cells was inhibited by p38 kinase inhibitor. The current data collectively indicate that BaP induces apoptosis of Hepa1c1c7 cells via activation of p53-related signaling, which was, in part, regulated by p38 kinase.     

PEDF induces apoptosis in human endothelial cells by activating p38 MAP kinase dependent cleavage of multiple caspases

We examined how pigment epithelium derived factor (PEDF), an effective endogenous antiangiogenic protein, decreases survival of primary cultures of human umbilical vein endothelial cells (HUVECs) in a low serum environment supplemented with the endothelial cell growth factor (VEGF). We provide evidence that induction of apoptosis by PEDF is associated with activation of p38 followed by cleavage of caspases 3, 8, and 9 by treatment with PEDF, and PEDF's actions are caspase dependent. A key mediator in the executioner effects of PEDF is p38 since the inhibition of p38 activity blocked apoptosis and prevented cleavage of caspases 3, 8, and 9. Although PEDF-induced phosphorylation of JNK1, the inhibition of JNK1 had no effect on apoptosis, even though it prevented phosphorylation of JNK1 by PEDF. Based on these findings, we propose that the antiangiogenic action of PEDF is dependent on activation of p38 MAPkinase which regulates cleavage of multiple caspases cascades.     

Hispolon from Phellinus linteus possesses mediate caspases activation and induces human nasopharyngeal carcinomas cells apoptosis through ERK1/2, JNK1/2 and p38 MAPK pathway

Hispolon, a phenol compound isolated from Phellinus linteus (PL), possesses anti-inflammatory, antiproliferative, and antioxidant effects. However, the effects of hispolon on human nasopharyngeal carcinomas have yet to be evaluated. Here, the molecular mechanism by which hispolon anticancer effects in human nasopharyngeal carcinomas cells was investigated. The results showed that hispolon significantly inhibited cell proliferation of HONE-1 and NP-039 cell lines. Furthermore, hispolon induced apoptosis through caspases-3, -8, and -9 activations and PARP cleavage in dose- and time-dependent manner in HONE-1 and NP-039 cells. Moreover, hispolon also showed that increase phosphorylation of ERK1/2, p38 MAPK and JNK1/2 in dose- and time-dependent manner by western blot analysis. However, hispolon-induced activation of the caspase-3, -8 and -9 significantly abolished by inhibition of p38 MAPK and JNK1/2 specific inhibitors. In this study, we determine that the effects of hispolon on the apoptosis and related regulation mechanism in HONE-1 and NPC-039 cells takes place. Our findings revealed that hispolon may be a useful candidate as a chemotherapeutic agent for NPC therapy.     

Induction of apoptosis in breast cancer cells by apomine is mediated by caspase and p38 mitogen activated protein kinase activation

The 1,1-bisphosphonate ester family member apomine (SR-45023A) is known to have anti-tumour activity in various cancer cell types. The aims of this study were to determine the effect of apomine on the growth of two breast cancer cell lines, MCF-7 and MDA-MB-231, to ascertain whether any growth inhibitory effects found were due to induction of apoptosis, and to investigate the mechanism of action of apomine. Apomine caused significant growth inhibition of both cell lines after 72h of treatment. Apomine-induced growth inhibition was associated with caspase and p38 MAPK activation and DNA fragmentation. Apomine had no effect on Ras localisation, nor did addition of mevalonate to treatment media prevent apomine-induced apoptosis. We conclude that apomine induces apoptosis in breast cancer cells, an effect that is independent of oestrogen receptor status and is not via inhibition of the mevalonate pathway. Our study suggests apomine is a potential anti-neoplastic drug in breast cancer treatment.     

Methylglyoxal induces apoptosis through activation of p38 MAPK in rat Schwann cells

The formation of glucose-derived methylglyoxal (MG), a highly reactive dicarbonyl compound, is accelerated under diabetic conditions. We examined whether MG was capable of inducing apoptosis in Schwann cells (SCs), since recent studies have suggested a potential involvement of apoptotic cell death in the development of diabetic neuropathy. MG induced apoptosis in SCs in a dose-dependent manner, accompanied by a reduction of intracellular glutathione content and activation of the p38 MAPK. Inhibiting the p38 MAPK activation by SB203580 successfully suppressed the MG-induced apoptosis in SCs. Aminoguanidine and N-acetyl-l-cysteine also inhibited the MG-induced p38 MAPK activation and apoptosis along with restoration of the intracellular glutathione content. These results suggest a potential role for MG in SC injury through oxidative stress-mediated p38 MAPK activation under diabetic conditions, and it may serve as a novel insight into therapeutic strategies for diabetic neuropathy.     

Deregulation of Cdc2 kinase induces caspase-3 activation and apoptosis

Progression of the cell cycle and control of apoptosis are tightly linked processes. It has been reported that manifestation of apoptosis requires cdc2 kinase activity yet the mechanism(s) of which is largely unclear. In an attempt to study the role of human MDM2 (HDM2) in interphase and mitosis, we employed the Xenopus cell-free system to study HDM2 protein stability. Interestingly, HDM2 is specifically cleaved in Xenopus mitotic extracts but not in the interphase extracts. We demonstrate that HDM2 cleavage is dependent on caspase-3 and that activation of cdc2 kinase results in caspase-3 activation in the Xenopus cell-free system. Furthermore, expression of cdc2 kinase in mammalian cells leads to activation of caspase-3 and apoptosis. Taken together, these data indicate that deregulation of cdc2 kinase activity can trigger apoptotic machinery that leads to caspase-3 activation and apoptosis.     

p38 MAP kinase mediates apoptosis through phosphorylation of BimEL at Ser-65

The stress-activated c-Jun N-terminal protein kinase (JNK) and p38 mitogen-activated protein (MAP) kinase (p38) regulate apoptosis induced by several forms of cellular insults. Potential targets for these kinases include members of the Bcl-2 family proteins, which mediate apoptosis generated through the mitochondria-initiated, intrinsic cell death pathway. Indeed, the activities of several Bcl-2 family proteins, both pro- and anti-apoptotic, are controlled by JNK phosphorylation. For example, the pro-apoptotic activity of Bim(EL), a member of the Bcl-2 family, is stimulated by JNK phosphorylation at Ser-65. In contrast, there is no reported evidence that p38-induced apoptosis is due to direct phosphorylation of Bcl-2 family proteins. Here we report evidence that sodium arsenite-induced apoptosis in PC12 cells may be due to direct phosphorylation of Bim(EL) at Ser-65 by p38. This conclusion is supported by data showing that ectopic expression of a wild type, but not a non-phosphorylatable S65A mutant of Bim(EL), potentiates sodium arsenite-induced apoptosis and by experiments showing direct phosphorylation of Bim(EL) at Ser-65 by p38 in vitro. Furthermore, sodium arsenite induced Bim(EL) phosphorylation at Ser-65, which was blocked by p38 inhibition. This study provides the first example whereby p38 induces apoptosis by phosphorylating a member of the Bcl-2 family and illustrates that phosphorylation of Bim(EL) on Ser-65 may be a common regulatory point for cell death induced by both JNK and p38 pathways.     

Chrysin-induced apoptosis is mediated through caspase activation and Akt inactivation in U937 leukemia cells

Chrysin is a natural, biologically active compound extracted from many plants, honey, and propolis. It possesses potent anti-inflammation, anti-cancer, and anti-oxidation properties. The mechanism by which chrysin initiates apoptosis remains poorly understood. In the present report, we investigated the effect of chrysin on the apoptotic pathway in U937 human promonocytic cells. We show that chrysin induces apoptosis in association with the activation of caspase 3 and that Akt signal pathway plays a crucial role in chrysin-induced apoptosis in U937 cells. Furthermore, we have shown that inhibition of Akt phosphorylation in U937 cells by the specific PI3K inhibitor, LY294002 significantly, enhanced apoptosis. Overexpression of a constitutively active Akt (myr-Akt) in U937 cells inhibited the induction of apoptosis, activation of caspase 3, and PLC-gamma1 cleavage by chrysin. Together, these findings suggest that the Akt pathway plays a major role in regulating the apoptotic response of human leukemia cells to chrysin and raise the possibility that combined interruption of chrysin and PI3K/Akt-related pathways may represent a novel therapeutic strategy in hematological malignancies.     

Humanin delays apoptosis in K562 cells by downregulation of P38 MAP kinase

Humanin (HN) is a newly identified neuroprotective peptide. In this study, we investigated its antiapoptotic effect and the potential mechanisms in K562 cells. Upon serum deprivation, expression of HN in K562 cells decreased and its intracellular distribution changed from cytoplasm to cell membrane. In HN stably transfected K562 cells, apoptosis was delayed compared with control vector transfected cells as measured by flow cytometry. Furthermore, analysis of different mitogen-activated protein (MAP) kinases activity revealed that extracellular signal-regulated kinase (ERK) pathway was inhibited while p38 signaling was activated following serum deprivation in K562 cells. And in HN transfected K562 cells, ERK downregulation was not affected, but p38 activation was suppressed, which may responsible for the delayed apoptosis in these cells. Activation of the ERK signaling pathway by phorbol myristate 13-acetate (PMA) and sorbitol protected K562 cells from serum deprivation induced apoptosis. Additionally, overexpression of HN reduced megakaryocytic differentiation of K562 cells. The present data outline the role of ERK and p38 MAP kinases in serum deprivation induced apoptosis in K562 cells and figure out p38 signaling pathway as molecular target for HN delaying apoptosis in K562 cells.     

Triptolide induces apoptosis in human leukemia cells through caspase-3-mediated ROCK1 activation and MLC phosphorylation

The diterpene triepoxide triptolide is a major active component of Tripterygium wilfordii Hook F, a popular Chinese herbal medicine with the potential to treat hematologic malignancies. In this study, we investigated the roles of triptolide in apoptosis and cell signaling events in human leukemia cell lines and primary human leukemia blasts. Triptolide selectively induced caspase-dependent cell death that was accompanied by the loss of mitochondrial membrane potential, cytochrome c release, and Bax translocation from the cytosol to the mitochondria. Furthermore, we found that triptolide dramatically induced ROCK1 cleavage/activation and MLC and MYPT phosphorylation. ROCK1 was cleaved and activated by caspase-3, rather than RhoA. Inhibiting MLC phosphorylation by ML-7 significantly attenuated triptolide-mediated apoptosis, caspase activation, and cytochrome c release. In addition, ROCK1 inhibition also abrogated MLC and MYPT phosphorylation. Our in vivo study showed that both ROCK1 activation and MLC phosphorylation were associated with the tumor growth inhibition caused by triptolide in mouse leukemia xenograft models. Collectively, these findings suggest that triptolide-mediated ROCK1 activation and MLC phosphorylation may be a novel therapeutic strategy for treating hematological malignancies.     

Protection of oxidative stress induced apoptosis in osteosarcoma cells by dihydromyricetin through down-regulation of caspase activation and up-regulation of BcL-2

Current study was aimed to investigate the effect of dihydromyricetin on hydrogen peroxide induced oxidative stress in the osteosarcoma cells. MTT assay showed that hydrogen peroxide treatment at a concentration of 100 μM caused a significant (p < 0.005) reduction in the viability of MG63 cells. However, reduction in cell viability caused by 100 μM concentration of hydrogen peroxide was completely prevented on incubation with 30 μM dose of dihydromyricetin. Treatment with 100 μM concentration of hydrogen peroxide for 24 h led to condensation of chromatin material, rounding of cell shape and detachment of cells. The results from flow cytometry using annexin V-FITC and PI double staining showed apoptosis induction in 47.84 ± 5.21% cells on treatment with 100 μM concentration of hydrogen peroxide compared to 2.32 ± 0.54% in controlcells. The apoptotic alterations in MG63 cell morphology were prevented significantly on pre-treatment with 30 μM doses of dihydromyricetin for 48 h. Annexin V-FITC and PI staining showed reduction of hydrogen peroxide induced apoptotic cell percentage to 3.07 ± 0.86% on pre-treatment of MG63 cells with 30 μM dose of dihydromyricetin. Western blot analysis showed a significant increase in the activation of caspase-3 and -9 on treatment of MG63 cells for 24 h with 100 μM concentration of hydrogen peroxide. The expression level of Bcl-2 was decreased significantly by 100 μM concentration of hydrogen peroxide in MG63 cells. However, pre-treatment of MG63 cells with 30 μM dose of dihydromyricetin for 48 h significantly prevented hydrogen peroxide induced increase in caspase-3 and -9 levels and reduction in Bcl-2 level. Thus dihydromyricetin prevents hydrogen peroxide induced reduction in viability and induction of apoptosis in MG63 cells through down-regulation of caspase activation and up-regulation of Bcl-2 levels.     

Arisostatins A induces apoptosis through the activation of caspase-3 and reactive oxygen species generation in AMC-HN-4 cells

A microbial secondary metabolite, arisostatins A (As-A), was originally discovered as a substance carrying the antibiotic activity against Gram-positive bacteria and shown to possess potent anti-tumor properties. The mechanism by which arisostatins A initiates apoptosis remains poorly understood. In the present report we investigated the effect of arisostatins A on activation of the apoptotic pathway in HN-4 cells. Arisostatins A was shown to be responsible for the inhibition of HN-4 cell growth by inducing apoptosis. Treatment with 4 microM arisostatins A for 24h produced morphological features of apoptosis and DNA fragmentation in HN-4 cells. Arisostatins A caused dose-dependent apoptosis and DNA fragmentation of HN-4 cells used as a model. Treatment with caspase inhibitor significantly reduced the arisostatins A-induced caspase 3 activation. In addition, arisostatins A-induced apoptosis was associated with the generation of reactive oxygen species (ROS), which was prevented by an antioxidant NAC (N-acetyl-cysteine). These data indicate that cytotoxic effect of arisostatins A on HN-4 cells is attributable to the induced apoptosis and that arisostatins A-induced apoptosis is mediated by caspase-3 activation pathway, loss of mitochondrial transmembrane potential (DeltaPsi(m)), and release of cytochrome c into cytosol.     

Genistein enhances TRAIL-induced apoptosis through inhibition of p38 MAPK signaling in human hepatocellular carcinoma Hep3B cells

The cytotoxic effect of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is limited in some carcinoma cancer cells. However, it was found that treatment with TRAIL in combination with nontoxic concentrations of genistein sensitized TRAIL-resistant human hepatocellular carcinoma Hep3B cells to TRAIL-mediated apoptosis. Combined treatment with genistein and TRAIL-induced chromatin condensation and sub-G1 phase DNA content. These indicators of apoptosis were correlated with the induction of caspase activity that resulted in the cleavage of poly(ADP-ribose) polymerase (PARP). Both cell viability and the cleavage of PARP induced by combined treatment were significantly inhibited by caspase-3, -8 and -9 inhibitors, which demonstrates the important roles of caspases in the observed cytotoxic effects. Genistein treatment also triggered the inhibition of p38-β mitogen-activated protein kinase (MAPK) activation. Pretreatment with SB203580 resulted in significantly increased sub-G1 population and loss of mitochondrial membrane potential (MMP) in TRAIL-induced apoptosis. By contrast, overexpression of p38 MAPK protected apoptosis by co-treatment with genistein and TRAIL, suggesting that the p38 MAPK act as key regulators of apoptosis in response to treatment with a combination of genistein and TRAIL in human hepatocellular carcinoma Hep3B cells.     

Oleanolic Acid Induces Apoptosis in Human Leukemia Cells through Caspase Activation and Poly（ADP-ribose） Polymerase Cleavage

It has been shown that Fructus Ligustri Lucidi （FLL）, a promising traditional Chinese medicine, can inhibit the growth of tumors. However, the effective component and molecular mechanism of FLL act to inhibit tumor proliferation are unclear. In this study, we demonstrated that oleanolic acid （OA）, a principal chemical component of FLL, inhibited the proliferation of human leukemia HL60 cells in culture. MTT assay showed that treatment of HL60 cells with FLL crude extracts or OA dramatically blocked the growth of target tumor cell in a time- and dose-dependent manner. Morphological changes of the nuclei and DNA fragmentation showed that apoptotic cell death occurred in the HL60 cells after treating with FLL extracts （20 mg/ml） or OA （3.65×10^-2 mg/ml）. Furthermore, flow cytometry assay showed that treatment of HL60 cells with FLL or OA caused an increased accumulation of G1 and sub-G1 subpopulations. Western blot analysis showed that caspase-9 and caspase-3 were activated, accompanied by the cleavage of poly （ADP-ribose） polymerase （PARP） in the target cells during FLL- or OA-induced apoptosis, These results suggest that OA acts as the effective component of FLL by exerting its cytotoxicity towards target tumor cells through activation of caspases and cleavage of PARP.     

Galangin induces B16F10 melanoma cell apoptosis via mitochondrial pathway and sustained activation of p38 MAPK

Galangin, an active flavonoid present at high concentration in Alpinia officinarum Hance and propolis, shows cytotoxicity towards several cancer cell lines, including melanoma. However, the specific cellular targets of galangin-induced cytotoxicity in melanoma are still unknown. Here, we investigated the effects of galangin in B16F10 melanoma cells and explored the possible molecular mechanisms. Galangin significantly decreased cell viability of B16F10 cells, and also induced cell apoptosis shown by Hoechst 33342 staining and Annexin V-PI double staining flow cytometric assay. Furthermore, upon galangin treatment, disruption of mitochondrial membrane potential was observed by JC-1 staining. Western blotting analysis indicated that galangin activated apoptosis signaling cascades by cleavage of procaspase-9, procaspase-3 and PARP in B16F10 cells. Moreover, galangin significantly induced activation of phosphor-p38 MAPK in a time and dose dependent manner. SB203580, an inhibitor of p38, partially attenuated galangin-induced apoptosis in B16F10 cells. Taken together, this work suggests that galangin has the potential to be a promising agent for melanoma treatment and may be further evaluated as a chemotherapeutic agent.     

Withanolide induces apoptosis in HL-60 leukemia cells via mitochondria mediated cytochrome c release and caspase activation

The present study is on the growth inhibitory effect of Withania somnifera methanolic leaf extract and its active component, withanolide on HL-60 promyelocytic leukemia cells. The decrease in survival rate of HL-60 cells was noted to be associated with a time dependent decrease in the Bcl-2/Bax ratio, leading to up regulation of Bax. Both the crude leaf extract and the active component activated the apoptotic cascade through the cytochrome c release from mitochondria. The activation of caspase 9, caspase 8 and caspase 3 revealed that caspase was a key mediator in the apoptotic pathway. DNA fragmentation analysis revealed typical ladders as early as 12h indicative of caspase 3 role in the apoptotic pathway. Flow cytometry data demonstrated an increase of sub-G1 peak upon treatment by 51% at 24h, suggesting the induction of apoptotic cell death in HL-60 cells.     

Cytotoxicity of calotropin is through caspase activation and downregulation of anti-apoptotic proteins in K562 cells

Calotropin is one of cardenolides isolated from milkweed used for medicinal purposes in many Asian countries. Whereas calotropin possesses cytotoxicity against several cancer cells, the mechanisms of action remain unclear. We set out to evaluate the cytotoxic mechanism of calotropin on human chronic myeloid leukemia K562 cells. Calotropin inhibited the growth of K562 cells in a time- and dose-dependent manner by G₂/M phase arrest. It upregulated the expression of p27 leading to this arrest by downregulating the G2/M regulatory proteins, cyclins A and B, and by upregulating the cdk inhibitor, p27. Furthermore, it downregulated anti-apoptotic signaling (XIAP and survivin) and survival pathways (p-Akt and NFκB), leading to caspase-3 activation which resulted in the induction of apoptosis. In all, calotropin exerted its anticancer activity on K562 cells by modulating the pro-survival signaling that leads to induction of apoptosis.     

Heyneanol A induces apoptosis via cytochrome c release and caspase activation in human leukemic U937 cells

Heyneanol A, a tetramer of resveratrol, is isolated from the roots of Vitis amurensis by cytotoxicity based fractionation. In this study, the mechanism of apoptosis by heyneanol A was evaluated in human leukemic U937 cells. Heyneanol A (IC(50) = 6.6 microM at 24 h) exhibited stronger cytotoxic effect than resveratrol (IC(50) = 100 microM at 24 h) by 15-fold on human leukemic U937 cells by XTT assay. Apoptotic bodies were observed in U937 cells treated with 6 microM of heyneanol A by TUNEL assay. Heyneanol A effectively increased the portion of sub-G(1) DNA content in a time- and concentration-dependent manner by flow cytometric analysis. Heyneanol A also induced cytochrome c release from mitochondria into the cytosol and subsequent caspase activation involving caspase 9 and 3 to cleave PARP. However, it did not affect the expressions of Bax and Bcl-2 by western blotting. It was confirmed that the activation of caspase 8, 9 and 3 and the cleavage of PARP by heyneanol A were completely blocked by adding Z-VAD-FMK, a caspase inhibitor. These findings suggest that heyneanol A has anti-tumor activity, which may be mediated by apoptosis caused by cytochrome c release and caspase activation in human leukemic U937 cells.