Pemetrexed Induces S-Phase Arrest and Apoptosis via a Deregulated Activation of Akt Signaling Pathway

Pemetrexed is approved for first-line and maintenance treatment of patients with advanced or metastatic non-small-cell lung cancer (NSCLC). The protein kinase Akt/protein kinase B is a well-known regulator of cell survival which is activated by pemetrexed, but its role in pemetrexed-mediated cell death and its molecular mechanisms are unclear. This study showed that stimulation with pemetrexed induced S-phase arrest and cell apoptosis and a parallel increase in sustained Akt phosphorylation and nuclear accumulation in the NSCLC A549 cell line. Inhibition of Akt expression by Akt specific siRNA blocked S-phase arrest and protected cells from apoptosis, indicating an unexpected proapoptotic role of Akt in the pemetrexed-mediated toxicity. Treatment of A549 cells with pharmacological inhibitors of phosphatidylinositol 3-kinase (PI₃K), wortmannin and {"type":"entrez-nucleotide","attrs":{"text":"Ly294002","term_id":"1257998346","term_text":"LY294002"}}Ly294002, similarly inhibited pemetrexed-induced S-phase arrest and apoptosis and Akt phosphorylation, indicating that PI₃K is an upstream mediator of Akt and is involved in pemetrexed-mediated cell death. Previously, we identified cyclin A-associated cyclin-dependent kinase 2 (Cdk2) as the principal kinase that was required for pemetrexed-induced S-phase arrest and apoptosis. The current study showed that inhibition of Akt function and expression by pharmacological inhibitors as well as Akt siRNA drastically inhibited cyclin A/Cdk2 activation. These pemetrexed-mediated biological and molecular events were also observed in a H1299 cell line. Overall, our results indicate that, in contrast to its normal prosurvival role, the activated Akt plays a proapoptotic role in pemetrexed-mediated S-phase arrest and cell death through a mechanism that involves Cdk2/cyclin A activation.     

Synergy between PI3K/Akt and Raf/MEK/ERK pathways in IGF-1R mediated cell cycle progression and prevention of apoptosis in hematopoietic cells

The insulin like growth factor-1 (IGF-1) receptor (R) induced PI3K/Akt signal transduction cascade has critical roles in prevention of apoptosis and regulation of cell cycle progression. Here, we discuss the effects of IGF-1R-mediated signal transduction on hematopoietic cells which normally require interleukin-3 (IL-3) for growth and prevention of apoptosis. Cytokine-dependent FDC-P1 hematopoietic cells were conditionally transformed to grow in response to overexpression of IGF-1R in the presence of IGF-1. When these cells were deprived of IL-3 or IGF-1 for 24 hrs, they exited the cell cycle, activated caspase 3 and underwent apoptosis. The effects of inhibitors which targeted the PI3K/Akt and Raf/MEK/ERK pathways were determined. When the cells were cultured with IGF-1 and either PI3K or MEK inhibitors, cell cycle progression and DNA synthesis were inhibited and caspase 3 activity and apoptosis were induced. Coinhibition of both pathways synergized to prevent cell cycle progression, inhibit DNA synthesis and induce apoptosis. These inhibitors had more apoptotic inducing effects when the cells were grown in response to IGF-1 than IL-3, indicating that IL-3 can induce additional anti-apoptotic pathways. These results demonstrate that the PI3K/Akt and Raf/MEK/ERK pathways are intimately involved in IGF-1R-mediated cell cycle progression and prevention of apoptosis in hematopoietic cells.     

Inhibition of cell proliferation by CD44: Akt is inactivated and EGR‐1 is down‐regulated

Objective: CD44 is a transmembrane glycoprotein and can facilitate signal transduction by serving as a platform for molecular recruitment and assembly. A number of studies have suggested that CD44 can either positively or negatively regulate cell proliferation. The purpose of this study was to investigate how CD44 can inhibit cell proliferation. Materials and methods: We engineered E6.1 Jurkat cells to express CD44. Importantly, these cells lack endogenous CD44 expression. Molecular pathways involved with cell proliferation were studied using RT²‐PCR array, siRNA, Western blotting and by employing pharmacological inhibitors of ERK1/2, p38 and the PI3K/Akt pathways. Results: We found that CD44 expression significantly inhibited cell proliferation and down‐regulated EGR‐1 expression and EGR‐1 targets cyclin D1 and cyclin D2. Transfection of control E6.1 Jurkat cells with EGR‐1 siRNA also inhibited cell proliferation, confirming its role. Disruption of the PI3K/Akt pathway with pharmacological inhibitors reduced both EGR‐1 expression and cell proliferation, recapitulating the properties of CD44 expressing cells. Akt was hypophosphorylated in cells expressing CD44 showing its potential role in negatively regulating Akt activation. Strikingly, constitutively active Akt rescued the proliferation defect showing requirement for active Akt, in our system. Conclusion: Our results suggest a novel pathway by which CD44 inactivates Akt, down‐regulates EGR‐1 expression and inhibits cell proliferation.     

Anti-Myeloma Activity of Akt Inhibition Is Linked to the Activation Status of PI3K/Akt and MEK/ERK Pathway

The PI3K/Akt/mTOR signal transduction pathway plays a central role in multiple myeloma (MM) disease progression and development of therapeutic resistance. mTORC1 inhibitors have shown limited efficacy in the clinic, largely attributed to the reactivation of Akt due to rapamycin induced mTORC2 activity. Here, we present promising anti-myeloma activity of MK-2206, a novel allosteric pan-Akt inhibitor, in MM cell lines and patient cells. MK-2206 was able to induce cytotoxicity and inhibit proliferation in all MM cell lines tested, albeit with significant heterogeneity that was highly dependent on basal pAkt levels. MK-2206 was able to inhibit proliferation of MM cells even when cultured with marrow stromal cells or tumor promoting cytokines. The induction of cytotoxicity was due to apoptosis, which at least partially was mediated by caspases. MK-2206 inhibited pAkt and its down-stream targets and up-regulated pErk in MM cells. Using MK-2206 in combination with rapamycin (mTORC1 inhibitor), {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (PI3K inhibitor), or U0126 (MEK1/2 inhibitor), we show that Erk- mediated downstream activation of PI3K/Akt pathway results in resistance to Akt inhibition. These provide the basis for clinical evaluation of MK-2206 alone or in combination in MM and potential use of baseline pAkt and pErk as biomarkers for patient selection.     

NF-kappaB inhibition enhances caspase-3 degradation of Akt1 and apoptosis in response to camptothecin

DNA damaging agents, such as camptothecin, and ionizing radiation (IR), can induce both NF-kappaB activation and apoptosis, however, the mechanism of their inter-regulation is not yet clear. In the present study, we discovered that Akt1 is degraded when cells deficient in Ataxia Telangiectasia mutated (ATM) were treated to CPT for apoptosis induction. While CPT-induced NF-kappaB activation could not be detected in ATM-deficient AT5BIVA cells, caspase-3 activation occurred and was even further enhanced by pretreatment with proteasome inhibitor-1 (Pro1), a NF-kappaB inhibitor. In contrast, activation of NF-kappaB but not of caspase-3 by CPT could be found in normal MRC5CV1 cells. NF-kappaB inhibition by Pro1, dominant negative mutant IkappaBalpha (S32/36) or p65 (N250), however, induced the caspase-3 activation in the normal cells, indicating the role of ATM-mediated NF-kappaB activation against cell apoptosis. On the other hand, interestingly, CPT significantly reduced the level of Akt1, this effect further enhanced by Pro1 pretreatment in AT5BIVA cells. In MRC5CV1 cells, however, Akt1 level could be reduced only when CPT and NF-kappaB inhibitors were co-treated to the cells, and this reversed by DEVD-cho treatment, demonstrating the caspase-3-mediated Akt1 degradation. Moreover, although MRC5CV1 cells were much more resistant to CPT compared with AT5BIVA, wortmannin and LY294002 significantly increased the chemosensitivity of MRC5CV1 cells to CPT. Given the accumulating evidences demonstrating Akt as a promising anticancer therapeutic target, all these results suggest that DNA damage induced apoptosis could be regulated by ATM-mediated NF-kappaB activation, and that Akt1 degradation be necessarily required for this apoptotic process.     

Guggulsterone targets smokeless tobacco induced PI3K/Akt pathway in head and neck cancer cells

Background

Epidemiological association of head and neck cancer with smokeless tobacco (ST) emphasizes the need to unravel the molecular mechanisms implicated in cancer development, and identify pharmacologically safe agents for early intervention and prevention of disease recurrence. Guggulsterone (GS), a biosafe nutraceutical, inhibits the PI3K/Akt pathway that plays a critical role in HNSCC development. However, the potential of GS to suppress ST and nicotine (major component of ST) induced HNSCC remains unexplored. We hypothesized GS can abrogate the effects of ST and nicotine on apoptosis in HNSCC cells, in part by activation of PI3K/Akt pathway and its downstream targets, Bax and Bad.

Methods and Results

Our results showed ST and nicotine treatment resulted in activation of PI3K, PDK1, Akt, and its downstream proteins - Raf, GSK3β and pS6 while GS induced a time dependent decrease in activation of PI3K/Akt pathway. ST and nicotine treatment also resulted in induction of Bad and Bax phosphorylation, increased the association of Bad with 14-3-3ζresulting in its sequestration in the cytoplasm of head and neck cancer cells, thus blocking its pro-apoptotic function. Notably, GS pre-treatment inhibited ST/nicotine induced activation of PI3K/Akt pathway, and inhibited the Akt mediated phosphorylation of Bax and Bad.

Conclusions

In conclusion, GS treatment not only inhibited proliferation, but also induced apoptosis by abrogating the effects of ST / nicotine on PI3K/Akt pathway in head and neck cancer cells. These findings provide a rationale for designing future studies to evaluate the chemopreventive potential of GS in ST / nicotine associated head and neck cancer.     

PI-3K/Akt signal pathway plays a crucial role in arsenite-induced cell proliferation of human keratinocytes through induction of cyclin D1

Exposure of arsenite can induce hyperproliferation of skin cells, which is believed to play important roles in arsenite-induced carcinogenesis by affecting both promotion and progression stages. However, the signal pathways and target genes activated by arsenite exposure responsible for the proliferation remain to be defined. In the present study, we found that: (1) exposure of human keratinocytic HaCat cells to arsenite caused an increase in cell proliferation, which was significantly inhibited by pretreatment of wortmannin, a specific chemical inhibitor of PI-3K/Akt signal pathway; (2) arsenite exposure was also able to activate PI-3K/Akt signal pathway, which thereby induced the elevation of cyclin D1 expression level in both HaCat cells and human primary keratinocytes based on that inhibition of PI-3K/Akt pathway by either pretreatment of wortmannin or the transfection of their dominant mutants, significantly inhibited cyclin D1 expression upon arsenite exposure; (3) PI-3K/Akt pathway is implicated in arsenite-induced proliferation of HaCat cells through the induction of cyclin D1 because either knockdown of cyclin D1 by its siRNA or inhibition of PI-3K/Akt signal pathway by their dominant mutants markedly impaired the proliferation of HaCat cells induced by arsenite exposure. Taken together, we provide the direct evidence that PI-3K/Akt pathway plays a role in the regulation of cell proliferation through the induction of cyclin D1 in human keratinocytes upon arsenite treatment. Given the importance of aberrant cell proliferation in cell transformation, we propose that the activation of PI-3K/Akt pathway and cyclin D1 induction may be the important mediators of human skin carcinogenic effect of arsenite.     

Inhibition of Protein Kinase Akt1 by Apoptosis Signal-regulating Kinase-1 (ASK1) Is Involved in Apoptotic Inhibition of Regulatory Volume Increase

Most animal cell types regulate their cell volume after an osmotic volume change. The regulatory volume increase (RVI) occurs through uptake of NaCl and osmotically obliged water after osmotic shrinkage. However, apoptotic cells undergo persistent cell shrinkage without showing signs of RVI. Persistence of the apoptotic volume decrease is a prerequisite to apoptosis induction. We previously demonstrated that volume regulation is inhibited in human epithelial HeLa cells stimulated with the apoptosis inducer. Here, we studied signaling mechanisms underlying the apoptotic inhibition of RVI in HeLa cells. Hypertonic stimulation was found to induce phosphorylation of a Ser/Thr protein kinase Akt (protein kinase B). Shrinkage-induced Akt activation was essential for RVI induction because RVI was suppressed by an Akt inhibitor, expression of a dominant negative form of Akt, or small interfering RNA-mediated knockdown of Akt1 (but not Akt2). Staurosporine, tumor necrosis factor-α, or a Fas ligand inhibited both RVI and hypertonicity-induced Akt activation in a manner sensitive to a scavenger for reactive oxygen species (ROS). Any of apoptosis inducers also induced phosphorylation of apoptosis signal-regulating kinase 1 (ASK1) in a ROS-dependent manner. Suppression of (ASK1) expression blocked the effects of apoptosis, in hypertonic conditions, on both RVI induction and Akt activation. Thus, it is concluded that in human epithelial cells, shrinkage-induced activation of Akt1 is involved in the RVI process and that apoptotic inhibition of RVI is caused by inhibition of Akt activation, which results from ROS-mediated activation of ASK1.     

Epidermal growth factor inhibition of c-Myc-mediated apoptosis through Akt and Erk involves Bcl-xL upregulation in mammary epithelial cells

In earlier studies, we and others have established that activation of EGFR can promote survival in association with upregulation of Bcl-x(L). However, the mechanism responsible for upregulation of Bcl-x(L) is unknown. For the current studies we have chosen pro-apoptotic, c-Myc-overexpressing murine mammary epithelial cells (MMECs) derived from MMTV-c-Myc transgenic mouse tumors. We now demonstrate that EGFR activation promotes survival through Akt and Erk1/2. Blockade of EGFR kinase activity and the PI3-K/Akt and MEK/Erk pathways with pharmacological inhibitors resulted in a significant induction of cellular apoptosis, paralleled by a downregulation of both Akt and Erk1/2 proteins. Consistent with a survival-promoting role of Akt, we observed that constitutively activated Akt (Myr-Akt) inhibited apoptosis of pro-apoptotic, c-Myc-overexpressing cells following the inhibition of EGFR tyrosine kinase activity. In addressing possible downstream effectors of EGFR through activated Akt, we detected significant upregulation of Bcl-x(L) protein, suggesting this pro-survival protein is a target of Akt in MMECs. By using pharmacological inhibitors of PI3-K/Akt and MEK/Erk together with dominant-negative Akt and Erk1 we observed the decrease in Bcl-x(L) protein. Our findings may be of importance for understanding the emerging role of Bcl-x(L) as a potential marker of poor prognosis in breast cancer.     

Akt phosphorylation and stabilization of X-linked inhibitor of apoptosis protein (XIAP)

Akt negatively regulates apoptotic pathways at a premitochondrial level through phosphorylation and modulation of proteins such as Bad, Forkhead proteins, and GSK-3beta. Akt has also been shown to protect cell death at a post-mitochondrial level, although its downstream targets have not been well documented. Here, we demonstrate that Akt, including AKT1 and AKT2, interacts with and phosphorylates X-linked inhibitor of apoptosis protein (XIAP) at residue serine-87 in vitro and in vivo. Phosphorylation of XIAP by Akt protects XIAP from ubiquitination and degradation in response to cisplatin. Moreover, autoubiquitination of XIAP is also inhibited by Akt. Consistent with this, an XIAP mutant introduced into cells which mimics the Akt-phosphorylated form (i.e. XIAP-S87D) displays reduced ubiquitination and degradation as compared with wild type XIAP. The greater stability of XIAP-S87D in cells translated to increased cell survival after cisplatin treatment. Conversely, a mutant that could not be phosphorylated by Akt (XIAP-S87A) was more rapidly degraded and showed increased cisplatin-induced apoptosis. Furthermore, suppression of XIAP by either siRNA or adenovirus of antisense of XIAP induced programmed cell death and inhibited Akt-stimulated cell survival in ovarian cancer cells. These data identify XIAP as a new downstream target of Akt and a potentially important mediator of the effect of Akt on cell survival.     

Pivotal role of Akt activation in mitochondrial protection and cell survival by poly(ADP-ribose)polymerase-1 inhibition in oxidative stress

According to the classical view, the cytoprotective effect of inhibitors of poly(ADP-ribose)polymerase (PARP) in oxidative stress was based on the prevention of NAD+ and ATP depletion, thus the attenuation of necrosis. Our previous data on PARP inhibitors in an inflammatory model suggested that PARP-catalyzed ADP-ribosylations may affect signaling pathways, which can play a significant role in cell survival. To clarify the molecular mechanism of cytoprotection, PARP activity was inhibited pharmacologically by suppressing PARP-1 expression by a small interfering RNA (siRNA) technique or by transdominantly expressing the N-terminal DNA-binding domain of PARP-1 (PARP-DBD) in cultured cells. Cell survival, activation of the phosphatidylinositol 3-kinase (PI3-kinase)/Akt system, and the preservation of mitochondrial membrane potential were studied in hydrogen peroxide-treated WRL-68 cells. Our data showed that suppression of the single-stranded DNA break-induced PARP-1 activation by pharmacological inhibitor, siRNA, or by the transdominant expression of PARP-DBD protected cells from oxidative stress and induced the phosphorylation and activation of Akt. Furthermore, prevention of Akt activation by inhibiting PI3-kinase counteracted the cytoprotective effect of PARP inhibition. Microscopy data showed that PARP inhibition-induced Akt activation was responsible for protection of mitochondria in oxidative stress because PI3-kinase inhibitors diminished the protective effect of PARP inhibition. Similarly, Src kinase inhibitors, which decrease Akt phosphorylation, also counteracted the protection of mitochondrial membrane potential supporting the pivotal role of Akt in cytoprotection. These data together with the finding that PARP inhibition in the absence of oxidative stress induced the phosphorylation and activation of Akt indicate that PARP inhibition-induced Akt activation is dominantly responsible for the cytoprotection in oxidative stress.     

Role of laminin-111 in neurotrophin-3 production of canine adipose-derived stem cells: involvement of Akt, mTOR, and p70S6K

Extracellular matrix (ECM) components play an important role in the regulation and maintenance of neural stem cells (NSCs). Laminin, an ECM component, is a key factor in promoting axonal regeneration and differentiation of NSCs. Since NSCs cannot be easily harvested with low morbidity, adipose-derived stem cells have been suggested for therapeutic applications of neural tissue damage. Therefore, the potential of laminin-111 to enhance the production of neurotrophin-3 (NT3) and its related signal pathways from canine adipose tissue-derived stem cells (cADSCs) was investigated. Laminin-111 enhanced NT3 production in neural induction medium (NIM). Treatment of NIM or laminin-111 on cADSCs distinctively changed integrin β1 mRNA and protein expression levels. In addition, laminin-111-induced Akt phosphorylation was inhibited by integrin β1 small interfering RNA (siRNA) and PI3K inhibitors (LY294002 and wortmannin). Furthermore, increased phosphorylations of mTOR and p70S6K by laminin-111 were blocked by inhibitors or specific siRNA, respectively. Moreover, laminin-111-induced NT3 production was blocked by these inhibitors. In experiments to induce the differentiation of cADSCs, laminin-111 increased the expression of neuronal markers β 3 tubulin, MAP2, and NeuN, and decreased the expression of the NSC markers nestin and vimentin. In conclusion, laminin-111 increases NT3 production through Akt, mTOR, and p70S6K pathways via integrin β1 in cADSCs cultured in NIM.     

Heme oxygenase-1 inhibits apoptosis in Caco-2 cells via activation of Akt pathway

Heme oxygenase-1 can play a protective role against cellular stress. In colon cancer cells, these effects would be relevant to oncogenesis and resistance to chemotherapy. The aim of the study was to examine the effects of heme oxygenase-1 induction on cell survival in a human colon cancer cell line, Caco-2. Serum deprivation induced apoptosis, reduced Akt and p38 phosphorylation, and increased p21(Cip/WAF1) levels. Heme oxygenase-1 induction by treatment with cobalt protoporphyrin IX resulted in resistance to apoptosis, activation of Akt, reduction in p21(Cip/WAF1) levels and modification of bcl2/bax ratio towards survival. Indomethacin reduced apoptosis but in contrast to heme oxygenase-1, arrested cells in G0/G1. Apoptosis was also inhibited by the heme oxygenase metabolites bilirubin and biliverdin but the CO donor tricarbonyldichlororuthenium(II) dimer did not exert significant effects. Protection against apoptosis in cells treated with cobalt protoporphyrin IX was reverted by incubation with heme oxygenase-1 small interfering RNA. This study shows an antiapoptotic effect of heme oxygenase-1 in colon cancer cells which could be mediated by the formation of bilirubin and biliverdin. Our results support an antiapoptotic role for HO-1 in these cells and provide a mechanism by which overexpression of HO-1 may promote tumor resistance to stress in conditions of limited nutrient supply. We have extended these observations by demonstrating that these effects are independent of p38 but are mediated via Akt pathway.     

Andrographolide Analogue Induces Apoptosis and Autophagy Mediated Cell Death in U937 Cells by Inhibition of PI3K/Akt/mTOR Pathway

Background

Current chemotherapeutic agents based on apoptosis induction are lacking in desired efficacy. Therefore, there is continuous effort to bring about new dimension in control and gradual eradication of cancer by means of ever evolving therapeutic strategies. Various forms of PCD are being increasingly implicated in anti-cancer therapy and the complex interplay among them is vital for the ultimate fate of proliferating cells. We elaborated and illustrated the underlying mechanism of the most potent Andrographolide analogue (AG–4) mediated action that involved the induction of dual modes of cell death—apoptosis and autophagy in human leukemic U937 cells.

Principal Findings

AG–4 induced cytotoxicity was associated with redox imbalance and apoptosis which involved mitochondrial depolarisation, altered apoptotic protein expressions, activation of the caspase cascade leading to cell cycle arrest. Incubation with caspase inhibitor Z-VAD-fmk or Bax siRNA decreased cytotoxic efficacy of AG–4 emphasising critical roles of caspase and Bax. In addition, AG–4 induced autophagy as evident from LC3-II accumulation, increased Atg protein expressions and autophagosome formation. Pre-treatment with 3-MA or Atg 5 siRNA suppressed the cytotoxic effect of AG–4 implying the pro-death role of autophagy. Furthermore, incubation with Z-VAD-fmk or Bax siRNA subdued AG–4 induced autophagy and pre-treatment with 3-MA or Atg 5 siRNA curbed AG–4 induced apoptosis—implying that apoptosis and autophagy acted as partners in the context of AG–4 mediated action. AG–4 also inhibited PI3K/Akt/mTOR pathway. Inhibition of mTOR or Akt augmented AG–4 induced apoptosis and autophagy signifying its crucial role in its mechanism of action.

Conclusions

Thus, these findings prove the dual ability of AG–4 to induce apoptosis and autophagy which provide a new perspective to it as a potential molecule targeting PCD for future cancer therapeutics.     

Akt and XIAP regulate the sensitivity of human uterine cancer cells to cisplatin,doxorubicin and taxol

We have investigated the interrelationship between two anti-apoptotic factors, XIAP and Akt, and their role in chemoresistance of uterine cancer cells. We used one cervical cancer cell line (HeLa) and two endometrial cancer cell lines (KLE and Ishikawa) as a model. The three drugs decreased Akt and XIAP content and induced apoptosis in P-Akt-negative HeLa cells. In P-Akt1/3-positive Ishikawa cells apoptosis induction correlated with XIAP decrease. P-Akt1/2/3-positive KLE cells showed maximum chemoresistance as XIAP and Akt levels/phosphorylation remained stable in response to the three drugs, and only cisplatin could significantly induce apoptosis. We found that XIAP and Akt were functionally linked in uterine cancer cells, as downregulation of XIAP with RNAi decreased P-Akt levels, and inhibition of PI3-K/Akt activity using LY294002 decreased XIAP content. Overexpression of constitutively active Akt isoforms in HeLa cells induced isoform-specific sensitivity to doxorubicin and taxol but not cisplatin. XIAP RNAi increased the cell-specific sensitivity to cisplatin and doxorubicin but not taxol. Finally, we found P-Akt immunoreactivity in epithelial cells from multiple human endometrial carcinoma tumors, suggesting that Akt may also regulate chemosensitivity in uterine cancers in vivo. Altogether these results highlight an intertwined role for specific Akt isoforms and XIAP in chemoresistance of uterine cancer cells.     

XB130 Mediates Cancer Cell Proliferation and Survival through Multiple Signaling Events Downstream of Akt

XB130, a novel adaptor protein, mediates RET/PTC chromosome rearrangement-related thyroid cancer cell proliferation and survival through phosphatidyl-inositol-3-kinase (PI3K)/Akt pathway. Recently, XB130 was found in different cancer cells in the absence of RET/PTC. To determine whether RET/PTC is required of XB130-related cancer cell proliferation and survival, WRO thyroid cancer cells (with RET/PTC mutation) and A549 lung cancer cells (without RET/PTC) were treated with XB130 siRNA, and multiple Akt down-stream signals were examined. Knocking-down of XB130 inhibited G(1)-S phase progression, and induced spontaneous apoptosis and enhanced intrinsic and extrinsic apoptotic stimulus-induced cell death. Knocking-down of XB130 reduced phosphorylation of p21Cip1/WAF1, p27Kip1, FOXO3a and GSK3β, increased p21Cip1/WAF1protein levels and cleavages of caspase-8 and-9. However, the phosphorylation of FOXO1 and the protein levels of p53 were not affected by XB130 siRNA. We also found XB130 can be phosphorylated by multiple protein tyrosine kinases. These results indicate that XB130 is a substrate of multiple protein tyrosine kinases, and it can regulate cell proliferation and survival through modulating selected down-stream signals of PI3K/Akt pathway. XB130 could be involved in growth and survival of different cancer cells.     

Apelin suppresses apoptosis of human vascular smooth muscle cells via APJ/PI3-K/Akt signaling pathways

Apoptosis of vascular smooth muscle cells (VSMCs) plays an important role in regulating vascular remodeling during cardiovascular diseases. Apelin is the endogenous ligand for the G-protein-coupled receptor APJ and plays an important role in the cardiovascular system. However, the mechanisms of apelin on apoptosis of VSMCs have not been elucidated. Using a culture of human VSMCs as a model for the study of apoptosis, the relationship between apelin and apoptosis of human VSMCs and the signal pathway involved were investigated. Using western blotting, we confirmed that VSMCs could express APJ. To evaluate the possible role of apelin in VSMC apoptosis, we assessed its effect on apoptosis of human VSMCs. The results showed that apelin inhibited human VSMCs apoptosis induced by serum deprivation. Suppression of APJ with small-interfering RNA (siRNA) abolished the anti-apoptotic activity of apelin. Apelin increased Bcl-2 protein expression, but decreased Bax protein expression. An increase in activation of extracellular signal-regulated protein kinase (ERK) and Akt (a downstream effector of phosphatidylinositol 3-kinase) was shown after apelin stimulation. Suppression of APJ with siRNA abolished the apelin-induced activation of ERK and Akt. LY294002 (a PI3-K inhibitor) blocked apelin-induced activation of Akt and abolished the apelin-induced antiapoptotic activity. Our study suggests that apelin suppresses serum deprivation-induced apoptosis of human VSMCs, and that the anti-apoptotic action is mediated through the APJ/PI3-K/Akt signaling pathways.     

Suppression of apoptosis by UVB irradiation: survival signaling via PI3-kinase/Akt pathway

UVB irradiation induces apoptosis in several cell types. However, we report here that UVB irradiation prevents induction of apoptosis in cells detached from the extracellular matrix under serum-free conditions. NIH3T3 cells cultured in bovine serum albumin-coated dishes (detached from the extracellular matrix) underwent apoptosis under serum-free conditions, which was inhibited by UVB (<0.1 J/cm(2)) irradiation, keeping suspension conditions, as determined by chromatin condensation and the appearance of a subG1 DNA fraction. Furthermore, UVB irradiation decreased caspase-3/7, -8/6, and -9 activation and eliminated loss of mitochondrial inner transmembrane potential, suggesting suppression upstream of the caspase cascade. Treatment with PI3-kinase inhibitors, wortmannin, and LY294002 partly eliminated the UV-mediated inhibition of cell death and recovered the inhibited caspase-3/7 activity. Phosphorylation of Akt was observed from 15 min after UVB irradiation. These results suggested that UVB irradiation transduced a survival signal via PI3 kinase activation and phosphorylation of Akt, and induced some apoptosis inhibition factors upstream of the caspase cascade.