Inhibition of apoptosis by amphiregulin via an insulin-like growth factor-1 receptor-dependent pathway in non-small cell lung cancer cell lines

Several abnormalities in the insulin-like growth factor-1 (IGF1) and erbB receptors pathways stimulate the growth and survival of lung cancer cells, but their mechanisms of action and cooperation are poorly understood. In this report, we have identified a new mechanism of apoptosis inhibition by amphiregulin through an IGF1-dependent survival pathway in non-small cell lung cancer (NSCLC) cells: amphiregulin activates the IGF1 receptor that in turn induces the secretion of amphiregulin and IGF1. In the absence of serum, the NSCLC cell line H358 resists apoptosis and secretes factors protecting the NSCLC cell line H322 from serum deprivation apoptosis. IGF1 receptor inhibitor AG1024 as well as epidermal growth factor receptor inhibitors AG556 and ZD1839 restore apoptosis in H322 cells cultured in H358-conditioned medium. Accordingly, the anti-apoptotic activity of H358-conditioned medium is completely abolished after incubation with anti-amphiregulin neutralizing antibody and only partially with anti-IGF1 neutralizing antibody. H358-conditioned medium and amphiregulin induce IGF1 receptor phosphorylation in H322 cells, which is prevented by anti-amphiregulin neutralizing antibody but not by AG556 or ZD1839. H358 cells secrete a high level of amphiregulin that, in combination with IGF1, prevents serum deprivation apoptosis. Finally, IGF1 receptor inhibitor blocks amphiregulin and IGF1 release by H358 cells.     

Penta-acetyl geniposide-induced C6 glioma cell apoptosis was associated with the activation of protein kinase C-delta

Herbal medicine has been utilized to treat a variety of diseases, including cancer. On the other hand, disturbance of apoptosis is often observed in cancer cells. It has been reported that protein kinase C (PKC) isoforms are involved in the signaling of apoptosis. In the present study, we investigate the antitumor effect and possible mechanism of a herbal-originated product, (Ac)(5)GP. We demonstrate that (Ac)(5)GP treatment results in DNA fragmentation of C6 glioma cells dose-dependently. Stimulated by (Ac)(5)GP, PKCdelta and PKCzeta were activated and translocated to the cell membrane fraction. Flow cytometry analysis showed that PKCdelta, but not PKCzeta inhibition blocks the (Ac)(5)GP-induced apoptosis by decreasing the cell population of sub G1 peak. However, the mRNA levels of PKCdelta and PKCzeta were not altered by (Ac)(5)GP-induced glioma cell apoptosis. These results suggested that the treatment of (Ac)(5)GP induces apoptosis of tumor cells through the activation but not the synthesis of PKCdelta.     

Activation of the IGF1 system characterizes cholangiocyte survival during progression of primary biliary cirrhosis.

We evaluated the IGF1 system in cholangiocytes of primay biliary cirrhosis (PBC) patients and investigated the relationships with apoptosis. Biopsies of PBC patients (n=32) and normal subjects (n=5) were investigated by immunohistochemistry for expression in cholangiocytes of IGF1, IGF1-R, pAKT, terminal deoxynucleotide transferase end labeling (TUNEL), Bax (proapoptotic protein), and Bcl2 (antiapoptotic protein). Whereas normal cholangiocytes were almost negative, cholangiocytes of PBC patients showed strong IHC staining for IGF1, IGF1-R, and pAKT, which increases from stage I to stage IV, where >70% of cholangiocytes were positive. Bax/Bcl2 ratio reached the highest value (4.6) in PBC stage III when apoptosis is maximal (24% TUNEL positivity), whereas it declines in stage IV (1.4) when only 7.8% cholangiocytes were TUNEL positive. In PBC stages III and IV, expression of IGF1, IGF1-R, and pAKT in cholangiocytes was directly correlated with the antiapoptotic Bcl2 and inversely correlated with proapoptotic Bax, Bax/Bcl2 ratio, and TUNEL positivity. In conclusion, cholangiocytes of PBC patients showed a marked increase in IGF1, IGF1-R, and pAKT expression involving most cholangiocytes surviving in the terminal ductopenic stage. This was associated and correlated with a balance of pro- and antiapoptotic proteins favoring survival rather than apoptosis, suggesting a major role of IGF1 system in promoting cholangiocyte survival.     

Different protein kinase C isoforms determine growth factor specificity in neuronal cells

Although mitogenic and differentiating factors often activate a number of common signaling pathways, the mechanisms leading to their distinct cellular outcomes have not been elucidated. In a previous report, we demonstrated that mitogen-activated protein (MAP) kinase (ERK) activation by the neurogenic agents fibroblast growth factor (FGF) and nerve growth factor is dependent on protein kinase Cdelta (PKCdelta), whereas MAP kinase activation in response to the mitogen epidermal growth factor (EGF) is independent of PKCdelta in rat hippocampal (H19-7) and pheochromocytoma (PC12) cells. We now show that EGF activates MAP kinase through a PKCzeta-dependent pathway involving phosphatidylinositol 3-kinase and PDK1 in H19-7 cells. PKCzeta, like PKCdelta, acts upstream of MEK, and PKCzeta can potentiate Raf-1 activation by EGF. Inhibition of PKCzeta also blocks EGF-induced DNA synthesis as monitored by bromodeoxyuridine incorporation in H19-7 cells. Finally, in embryonic rat brain hippocampal cell cultures, inhibitors of PKCzeta or PKCdelta suppress MAP kinase activation by EGF or FGF, respectively, indicating that these factors activate distinct signaling pathways in primary as well as immortalized neural cells. Taken together, these results implicate different PKC isoforms as determinants of growth factor signaling specificity within the same cell. Furthermore, these data provide a mechanism whereby different growth factors can differentially activate a common signaling intermediate and thereby generate biological diversity.     

Protein kinase Czeta abrogates the proapoptotic function of Bax through phosphorylation

Protein kinase Czeta (PKCzeta) is an atypical PKC isoform that plays an important role in supporting cell survival but the mechanism(s) involved is not fully understood. Bax is a major member of the Bcl-2 family that is required for apoptotic cell death. Because Bax is extensively co-expressed with PKCzeta in both small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) cells, it is possible that Bax may act as the downstream target of PKCzeta in regulating survival and chemosensitivity of lung cancer cells. Here we discovered that treatment of cells with nicotine not only enhances PKCzeta activity but also results in Bax phosphorylation and prolonged cell survival, which is suppressed by a PKCzeta specific inhibitor (a myristoylated PKCzeta pseudosubstrate peptide). Purified, active PKCzeta directly phosphorylates Bax in vitro. Overexpression of wild type or the constitutively active A119D but not the dominant negative K281W PKCzeta mutant results in Bax phosphorylation at serine 184. PKCzeta co-localizes and interacts with Bax at the BH3 domain. Specific depletion of PKCzeta by RNA interference blocks nicotine-stimulated Bax phosphorylation and enhances apoptotic cell death. Intriguingly, forced expression of wild type or A119D but not K281W PKCzeta mutant results in accumulation of Bax in cytoplasm and prevents Bax from undergoing a conformational change with prolonged cell survival. Purified PKCzeta can directly dissociate Bax from isolated mitochondria of C2-ceramide-treated cells. Thus, PKCzeta may function as a physiological Bax kinase to directly phosphorylate and interact with Bax, which leads to sequestration of Bax in cytoplasm and abrogation of the proapoptotic function of Bax.     

Hypoxia-mediated down-regulation of Bid and Bax in tumors occurs via hypoxia-inducible factor 1-dependent and -independent mechanisms and contributes to drug resistance

Solid tumors with disorganized, insufficient blood supply contain hypoxic cells that are resistant to radiotherapy and chemotherapy. Drug resistance, an obstacle to curative treatment of solid tumors, can occur via suppression of apoptosis, a process controlled by pro- and antiapoptotic members of the Bcl-2 protein family. Oxygen deprivation of human colon cancer cells in vitro provoked decreased mRNA and protein levels of proapoptotic Bid and Bad. Hypoxia-inducible factor 1 (HIF-1) was dispensable for the down-regulation of Bad but required for that of Bid, consistent with the binding of HIF-1alpha to a hypoxia-responsive element (positions -8484 to -8475) in the bid promoter. Oxygen deprivation resulted in proteosome-independent decreased expression of Bax in vitro, consistent with a reduction in global translation efficiency. The physiological relevance of Bid and Bax down-regulation was confirmed in tumors in vivo. Oxygen deprivation resulted in decreased drug-induced apoptosis and clonogenic resistance to agents with different mechanisms of action. The contribution of Bid and/or Bax down-regulation to drug responsiveness was demonstrated by the relative resistance of normoxic cells that had no or reduced expression of Bid and/or Bax and by the finding that forced expression of Bid in hypoxic cells resulted in increased sensitivity to the topoisomerase II inhibitor etoposide.     

Inhibition of PKCalpha induces a PKCdelta-dependent apoptotic program in salivary epithelial cells

We have used expression of a kinase dead mutant of PKCalpha (PKCalphaKD) to explore the role of this isoform in salivary epithelial cell apoptosis. Expression of PKCalphaKD by adenovirus-mediated transduction results in a dose-dependent induction of apoptosis in salivary epithelial cells as measured by the accumulation of sub-G1 DNA, activation of caspase-3, and cleavage of PKCdelta and PKCzeta, known caspase substrates. Induction of apoptosis is accompanied by nine-fold activation of c-Jun-N-terminal kinase, and an approximately two to three-fold increase in activated mitogen-activated protein kinase (MAPK) as well as total MAPK protein. Previous studies from our laboratory have shown that PKCdelta activity is essential for the apoptotic response of salivary epithelial cells to a variety of cell toxins. To explore the contribution of PKCdelta to PKCalphaKD-induced apoptosis, salivary epithelial cells were cotransduced with PKCalphaKD and PKCdeltaKD expression vectors. Inhibition of endogenous PKCdelta blocked the ability of PKCalphaKD to induce apoptosis as indicated by cell morphology, DNA fragmentation, and caspase-3 activation, indicating that PKCdelta activity is required for the apoptotic program induced under conditions where PKCalpha is inhibited. These findings indicate that PKCalpha functions as a survival factor in salivary epithelial cells, while PKCdelta functions to regulate entry into the apoptotic pathway.     

The protein kinase C delta catalytic fragment targets Mcl-1 for degradation to trigger apoptosis

Proteolytic cleavage and subsequent activation of protein kinase C (PKC) delta is required for apoptosis induced by a variety of genotoxic agent, including UV radiation. In addition, overexpression of the constitutively active PKCdelta catalytic fragment (PKCdelta-cat) is sufficient to trigger Bax activation, cytochrome c release, and apoptosis. While PKCdelta is a key apoptotic effector, the downstream target(s) responsible for the mitochondrial apoptotic cascade are not known. We found that expression of the active PKCdelta-cat in HaCaT cells triggers a reduction in the anti-apoptotic protein Mcl-1, similar to UV radiation. The down-regulation of Mcl-1 induced by PKCdelta-cat was not at the mRNA level but was due to decreased protein half-life. Overexpression of Mcl-1 protected HaCaT cells from both UV and PKCdelta-cat-induced apoptosis and blocked the release of cytochrome c from the mitochondria, indicating that Mcl-1 down-regulation was required for apoptosis signaling. Indeed, down-regulation of Mcl-1 with siRNA slightly increased the basal apoptotic rate of HaCaT cells and dramatically sensitized them to UV or PKCdelta-cat-induced apoptosis. HaCaT cells with down-regulated Mcl-1 had higher activated Bax protein, as measured by Bax cross-linking, indicating that Mcl-1 down-regulation is sufficient for Bax activation. Finally, recombinant PKCdelta could phosphorylate Mcl-1 in vitro, identifying Mcl-1 as a direct target for PKCdelta. Overall our results identify Mcl-1 as an important target for PKCdelta-cat that can mediate its pro-apoptotic effects on mitochondria to amplify the apoptotic signaling induced by a wide range of apoptotic stimuli.     

Opposing roles of prion protein in oxidative stress- and ER stress-induced apoptotic signaling

Although the prion protein is abundantly expressed in the CNS, its biological functions remain unclear. To determine the endogenous function of the cellular prion protein (PrP(c)), we compared the effects of oxidative stress and endoplasmic reticulum (ER) stress inducers on apoptotic signaling in PrP(c)-expressing and PrP(ko) (knockout) neural cells. H(2)O(2), brefeldin A (BFA), and tunicamycin (TUN) induced increases in caspase-9 and caspase-3, PKCdelta proteolytic activation, and DNA fragmentation in PrP(c) and PrP(ko) cells. Interestingly, ER stress-induced activation of caspases, PKCdelta, and apoptosis was significantly exacerbated in PrP(c) cells, whereas H(2)O(2)-induced proapoptotic changes were suppressed in PrP(c) compared to PrP(ko) cells. Additionally, caspase-12 and caspase-8 were activated only in the BFA and TUN treatments. Inhibitors of caspase-9, caspase-3, and PKCdelta significantly blocked H(2)O(2)-, BFA-, and TUN-induced apoptosis, whereas the caspase-8 inhibitor attenuated only BFA- and TUN-induced cell death, and the antioxidant MnTBAP blocked only H(2)O(2)-induced apoptosis. Overexpression of the kinase-inactive PKCdelta(K376R) or the cleavage site-resistant PKCdelta(D327A) mutant suppressed both ER and oxidative stress-induced apoptosis. Thus, PrP(c) plays a proapoptotic role during ER stress and an antiapoptotic role during oxidative stress-induced cell death. Together, these results suggest that cellular PrP enhances the susceptibility of neural cells to impairment of protein processing and trafficking, but decreases the vulnerability to oxidative insults, and that PKCdelta is a key downstream mediator of cellular stress-induced neuronal apoptosis.     

LPA protects intestinal epithelial cells from apoptosis by inhibiting the mitochondrial pathway

We previously showed (Gastroenterology 123: 206-216, 2002) that lysophosphatidic acid (LPA) protects and rescues rat intestinal epithelial cells (IEC-6) from apoptosis. Here, we provide evidence for the LPA-elicited inhibition of the mitochondrial apoptotic pathway leading to attenuation of caspase-3 activation. Pretreatment of IEC-6 cells with LPA inhibited campothecin-induced caspase-9 and caspase-3 activation and DNA fragmentation. A caspase-9 inhibitor peptide mimicked the LPA-elicited antiapoptotic activity. LPA elicited ERK1/ERK2 and PKB/Akt phosphorylation. The LPA-elicited antiapoptotic activity and inhibition of caspase-9 activity were abrogated by pertussis toxin, PD 98059, wortmannin, and LY 294002. LPA reduced cytochrome c release from mitochondria and prevented activation of caspase-9. LPA prevented translocation of Bax from cytosol to mitochondria and increased the expression of the antiapoptotic Bcl-2 mRNA and protein. LPA had no effect on Bcl-xl, Bad, and Bak mRNA or protein expression. These data indicate that LPA protects IEC-6 cells from camptothecin-induced apoptosis through G(i)-coupled inhibition of caspase-3 activation mediated by the attenuation of caspase-9 activation due to diminished cytochrome c release, involving upregulation of Bcl-2 protein expression and prevention of Bax translocation.     

Macrophage colony-stimulating factor induces the expression of mitogen-activated protein kinase phosphatase-1 through a protein kinase C-dependent pathway.

M-CSF triggers the activation of extracellular signal-regulated protein kinases (ERK)-1/2. We show that inhibition of this pathway leads to the arrest of bone marrow macrophages at the G0/G1 phase of the cell cycle without inducing apoptosis. M-CSF induces the transient expression of mitogen-activated protein kinase phosphatase-1 (MKP-1), which correlates with the inactivation of ERK-1/2. Because the time course of ERK activation must be finely controlled to induce cell proliferation, we studied the mechanisms involved in the induction of MKP-1 by M-CSF. Activation of ERK-1/2 is not required for this event. Therefore, M-CSF activates ERK-1/2 and induces MKP-1 expression through different pathways. The use of two protein kinase C (PKC) inhibitors (GF109203X and calphostin C) revealed that M-CSF induces MKP-1 expression through a PKC-dependent pathway. We analyzed the expression of different PKC isoforms in bone marrow macrophages, and we only detected PKCbetaI, PKCepsilon, and PKCzeta. PKCzeta is not inhibited by GF109203X/calphostin C. Of the other two isoforms, PKCepsilon is the best candidate to mediate MKP-1 induction. Prolonged exposure to PMA slightly inhibits MKP-1 expression in response to M-CSF. In bone marrow macrophages, this treatment leads to a complete depletion of PKCbetaI, but only a partial down-regulation of PKCepsilon. Moreover, no translocation of PKCbetaI or PKCzeta from the cytosol to particulate fractions was detected in response to M-CSF, whereas PKCepsilon was constitutively present at the membrane and underwent significant activation in M-CSF-stimulated macrophages. In conclusion, we remark the role of PKC, probably isoform epsilon, in the negative control of ERK-1/2 through the induction of their specific phosphatase.     

Cardiomyoblast apoptosis induced by insulin-like growth factor (IGF)-I resistance is IGF-II dependent and synergistically enhanced by angiotensin II

Objective: This study explores the synergistic effect of cardiomyoblast apoptosis induced by angiotensin II (Ang II) and Insulin-like growth factor (IGF)-I resistance, and elucidates the role of IGF-II via IGF-II receptor (R) and calcineurin pathways in apoptosis induced by Ang II and IGF-I resistance. Methods: Apoptosis of cultured cardiomyoblast H9c2 cells was assessed by DNA fragmentation on agarose gel electrophoresis, nuclear condensation stained with DAPI, and Western blot analysis of pro-apoptotic Bad and cytochrome c in various combinations of control, Ang II, antisense IGF (I or II), IGF (I or II) antibody, IGF (I or II) receptor (R) antibody, or calcineurin inhibitor (Cyclosporine A, (CsA)). Results: We found the following: (1) The combination of Ang II and IGF-I deficiencies had a synergistic effect on apoptosis, confirmed by DNA fragmentation, nuclei condensation, and increases in such pro-apoptotic proteins as Bad, cytochrome c, caspase 9, and caspase 3 in H9c2 cells. (2) IGF-II and IGF-IIR protein products were increased by antisense IGF-I and IGF-I resistance, but these IGF-II protein products were not affected by sense IGF-I and non-specific antibody IgG in H9c2 cells. (3) The alteration of Bad protein level and the release of cytochrome c, both induced by treatments containing combinations of Ang II and antisense IGF-I, IGF-I antibody or IGF-IR antibody, were inhibited by IGF-II antibody. (4) DNA fragmentation, Bad, and cytochrome c which was induced by treatments combining IGF-IR antibody with Ang II or combining IGF-IR antibody with IGF-II were remarkably attenuated by CsA. Conclusion: IGF-I deficiency and/or IGF-IR resistance induced apoptosis in cardiomyoblast cells. The apoptosis, which might have been caused by the upregulation of IGF-II and IGF-IIR genes possibly activated the downstream calcineurin pathway, was synergistically augmented by Ang II. The last two authors contributed equally.     

The mechanism involved in the loss of PTEN expression in NSCLC tumor cells

Loss of PTEN expression is observed in most non-small cell lung cancers (NSCLC). However, the mechanism by which PTEN expression is regulated in NSCLC has not been fully elucidated. In this study, we investigated the role of DNA methyltransferases (Dnmts), microRNA-29b (miR-29b), and anti-miR-29b inhibitor in PTEN promoter methylation and PTEN gene expression in H358 NSCLC cells in vitro and in vivo. PTEN mRNA was measured by RT-PCR. PTEN and Dnmts protein levels were measured by Western blot. miR-29b expression was detected by Northern blot. A xenograft H358 tumor mouse model was established by subcutaneously inoculating H358 cells into the right hind limbs of nude mice. We found that radiation induced cell apoptosis and hypomethylation in PTEN promoter, PTEN and miR-29b expression, and downregulation of Dnmt1, 3a and 3b expression in H358 tumor cells. The effect of radiation on gene expression and apoptosis was blocked by anti-miR-29b inhibitor. In the xenograft H358 tumor model, anti-miR-29b inhibitor reversed radiation-induced tumor growth delay, PTEN reexpression and downregulation of Dnmts expression. Our study suggested that miR-29b is an upstream molecule of PTEN. miR-29b regulates PTEN gene expression through downregulating Dnmts expression and subsequently induces hypomethylation in PTEN promoter. Targeting therapy could be established in NSCLC by upregulating miR-29b expression.     

Rapamycin-sensitive phosphorylation of PKC on a carboxy-terminal site by an atypical PKC complex.

BACKGROUND: The protein kinase C (PKC) family has been implicated in the control of many cellular functions. Although PKC isotypes are characterized by their allosteric activation, phosphorylation also plays a key role in controlling activity. In classical PKC isotypes, one of the three critical sites is a carboxy-terminal hydrophobic site also conserved in other AGC kinase subfamily members. Although this site is crucial to the control of this class of enzymes, the upstream kinase(s) has not been identified. RESULTS: A membrane-associated kinase activity that phosphorylates the hydrophobic site in PKCalpha was detected. This activity was suppressed when cells were pretreated with the immunosuppresant drug rapamycin or the phosphoinositide (Pl) 3-kinase inhibitor LY294002. These pretreatments also blocked specifically the serum-induced phosphorylation of the hydrophobic site in PKCdelta in vivo. The most highly purified hydrophobic site kinase preparations ( approximately 10,000-fold) reacted with antibodies to PKCzeta/iota. Consistent with this, rapamycin and LY294002 reduced the recovery of PKCzeta from the membrane fraction of transfected cells. An activated mutant of PKCzeta, but not wild-type PKCzeta, induced phosphorylation of the PKCdelta hydrophobic site in a rapamycin-independent manner, whereas a kinase-dead PKCzeta mutant suppressed this serum-induced phosphorylation. The immunopurified, activated mutant of PKCzeta could phosphorylate the PKCdelta hydrophobic site in vitro, whereas wild-type PKCzeta could not. CONCLUSIONS: PKCzeta is identified as a component of the upstream kinase responsible for the phosphorylation of the PKCdelta hydrophobic site in vitro and in vivo. PKCzeta can therefore control the phosphorylation of this PKCdelta site, antagonizing a rapamycin-sensitive pathway.     

Selective protein kinase C isoforms are involved in endothelin-1-induced human uterine contraction at the end of pregnancy

The role of protein kinase C (PKC) in contraction of the human myometrium induced by endothelin-1 (ET-1) was investigated at the end of pregnancy. The expression and subcellular distribution of PKC isoforms were examined by Western blot analysis using isoform-specific antibodies. At least three conventional PKC isoforms (cPKC; alpha, beta1, and beta2), two novel PKC isoforms (epsilon and delta), and an atypical PKC isoform (zeta) were detected in pregnant myometrium. Quantitative immunoblotting revealed that all these isoforms were mainly distributed in the particulate fraction. The lack of a calcium chelator to modify the particulate sequestration of cPKC suggests an interaction with an anchoring protein such as receptor-activated C kinase-1, which is evidenced in the particulate fraction of the pregnant myometrium. Of the six isoforms, only PKCbeta1, PKCbeta2, PKCdelta, and PKCzeta were translocated to the particulate fraction, and PKCepsilon to the cytoskeletal fraction, after stimulation with ET-1. Involvement of PKC in the ET-1-induced contractile response is supported by the inhibition caused by the PKC inhibitor calphostin C. However, we demonstrated that the selective cPKC isoform inhibitor, G? 6976, as well as the substantial depletion of PKCbeta1 and PKCepsilon and the partial depletion of PKCalpha and PKCdelta by a long-term treatment with phorbol 12,13-dibutyrate did not prevent ET-1-induced contraction. Accordingly, our results suggest that PKCdelta and PKCzeta activation mediated ET-1-induced contraction, whereas cPKC isoforms were not implicated in the human pregnant myometrium.     

bFGF inhibits the activation of caspase-3 and apoptosis of P19 embryonal carcinoma cells during neuronal differentiation.

P19 embryonal carcinoma (EC) cells undergo apoptosis during neuronal differentiation induced by all-trans retinoic acid (RA). Caspase-3-like proteases are activated and involved in the apoptosis of P19 EC cells during neuronal differentiation.1 Recently it has been shown that growth factor signals protect against apoptosis by phosphorylation of Bad. Phosphorylated Bad, an apoptotic member of the Bcl-2 family, cannot bind to Bcl-xL and results in Bcl-xL homodimer formation and subsequent antiapoptotic activity. In the present study, we demonstrate that this system is used generally to protect against apoptosis during neuronal differentiation. Bcl-xL inhibited the activation of caspase-3-like proteases. Basic fibroblast growth factor (bFGF) inhibited more than 90% of the caspase-3-like activity, inhibited processing of caspase-3 into its active form, and inhibited DNA fragmentation. bFGF activated phosphatidyl-inositol-3-kinase (PI3K) and stimulated the phosphorylation of Bad. Phosphorylation was inhibited by wortmannin, an inhibitor of PI3K and its downstream target Akt. Thus, Bad is a target of the FGF receptor-mediated signals involved in the protection against activation of caspase-3.     

Protective effect of cholecystokinin octapeptide on angiotensin II-induced apoptosis in H9c2 cardiomyoblast cells

Cholecystokinin (CCK) and its receptors are expressed in mammalian cardiomyocytes and are involved in cardiovascular system regulation; however, the exact effect and underlying mechanism of CCK in cardiomyocyte apoptosis remain to be elucidated. We examined whether sulfated CCK octapeptide (CCK-8) protects H9c2 cardiomyoblast cells against angiotensin II (Ang II)-induced apoptosis. The H9c2 cardiomyoblasts were subjected to Ang II with or without CCK-8 and the viability and apoptotic rate were detected using a Cell Counting Kit-8 assay, Hoechst 33342 staining, terminal deoxyribonucleotide transferase-mediated nick-end labeling assays, and flow cytometry. In addition, specific antiapoptotic mechanisms of CCK-8 were investigated using specific CCK1 (Devazepide) or CCK2 (L365260) receptor antagonists, or the PI3K inhibitor LY294002. The expression of CCK, CCK1 receptor, CCK2 receptor, Akt, p-Akt, Bad, p-Bad, Bax, Bcl-2, and caspase-3 were detected by Western blot analysis and real-time polymerase chain reaction. We found that CCK and its receptor messenger RNA (mRNA) and protein are expressed in H9c2 cardiomyoblasts. Ang II-induced increased levels of CCK mRNA and protein expression and decreased levels of CCK1 receptor protein and mRNA. Pretreatment of CCK-8 attenuated Ang II-induced cell toxicity and apoptosis. In addition, pretreatment of H9c2 cells with CCK-8 markedly induced expression of p-Akt, p-bad, and Bcl-2 and decreased the expression levels of Bax and caspase-3. The protective effects of CCK-8 were partly abolished by Devazepide or LY294002. Our results suggest that CCK-8 protects H9c2 cardiomyoblasts from Ang II-induced apoptosis partly via activation of the CCK1 receptor and the phosphatidyqinositol-3 kinase/protein kinase B (PI3K/Akt) signaling pathway.