The development of phosphatidylinositol ether lipid analogues as inhibitors of the serine/threonine kinase, Akt

The serine/threonine kinase Akt is a component of the phosphatidylinositol 3'-kinase/Akt signal transduction pathway that is activated by receptor tyrosine kinases, activated Ras and integrins. As Akt regulates many processes crucial to carcinogenesis, and Akt activation has been observed in human cancers, intense efforts are underway to develop Akt inhibitors as cancer therapeutics. Towards this aim, phosphatidylinositol ether lipid analogues (PIAs), which are structurally similar to the products of phosphatidylinositol 3'-kinase, have been synthesised. PIAs inhibit Akt translocation, phosphorylation and kinase activity. Furthermore, they selectively induce apoptosis in cancer cell lines that depend on Akt for survival. This review will trace the development of PIAs, cover the biological activities of PIAs and discuss future steps and challenges in their development.     

Phosphatidylinositol analogues that block the activation of Akt for the treatment of cancer

D-myo-phosphatidylinositol analogues with Akt antagonist activity are claimed in a patent from Georgetown university. The compounds contain an inositol ring with different hydroxyl and/or ether substitution patterns together with a C1 linker and a lipophillic bis-ether side chain. The compounds are indicated to be useful in the treatment of cancer and other human diseases.     

Investigational serine/threonine kinase inhibitors against prostate cancer metastases

Introduction: Androgen deprivation therapy (ADT) is used as first therapeutic approach in prostate cancer (PCa) although castration resistant disease (CRPC) develops with high frequency. CRPC is the consequence of lack of apoptotic responses to ADT. Alternative targeting of the androgen axis with abiraterone and enzalutamide, as well as taxane-based chemotherapy were used in CRPC. Serine/threonine protein kinases (STKs) regulate different molecular pathways of normal and neoplastic cells and participate to development of CRPC as well as to the progression towards a bone metastatic disease (mCRPC).

Areas covered: The present review provide data on STK expression and activity in the development of CRPC as well as summarize recent reports of different strategies to block STK activity for the control of PCa progression.

Expert Opinion: Inhibitors for different STKs have been developed but clinical trials in PCa are comparatively rare and few exhibit satisfactory ‘drug-like’ properties. It is, however, necessary to intensify, when possible, the number of clinical trials with these drugs in order to insert new therapies or combinations with standard hormone- and chemo-therapies in the treatment guidelines of the mPCA.     

Oxidized low-density lipoprotein-induced apoptosis is attenuated by insulin-activated phosphatidylinositol 3-kinase/Akt through p38 mitogen-activated protein kinase

1. The aim of the present study was to investigate whether p38 mitogen-activated protein kinase (p38 MAPK) is involved in oxidized low-density lipoprotein (oxLDL)-induced apoptosis of human umbilical vein endothelial cells (HUVECs). We also sought to determine whether this apoptosis is regulated by the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. 2. Low-density lipoprotein was oxidized with CuSO4 and used as oxLDL. Using HUVEC, we determined whether LDL/oxLDL induces apoptosis by DNA fragmentation and the cell cycle distribution (SubG1 method). The mechanism and activation of p38 MAPK and Akt were determined by western blot analysis. 3. The results showed that oxLDL induced DNA fragmentation, whereas cell cycle distribution showed that it also significantly increased the rate of cell death compared with the LDL group. SB203580 significantly inhibited cell death induced by oxLDL, as did the administration of insulin. Western blot analysis showed the activation of p38 MAPK by oxLDL, but not with LDL. It was found that Akt was activated in the presence of insulin. In the presence of either SB203580 or insulin, activation of p38 MAPK was significantly inhibited compared with stimulation by oxLDL alone. However, application of both insulin and wortmannin resulted in no significant difference compared with HUVEC stimulated by oxLDL only. 4. The results showed that apoptosis in HUVEC can be induced by oxLDL and involves p38 MAPK. It was also demonstrated that insulin inhibited oxLDL-induced apoptosis and may inhibit the activation of p38 MAPK through the PI3K/Akt pathway.     

阿魏酸通过磷酸肌醇 3激酶 /丝氨酸 /苏氨酸蛋白激酶途径保护大鼠免受心肌缺血 /再灌注损伤

目的研究阿魏酸对大鼠心肌缺血 /再灌注(I/R)损伤的保护作用及其可能的机制。方法将雄性 SD大鼠采用随机数字表法分为四组( n=12):假手术组( Sham组), I/R模型组( I/R组);阿魏酸 +I/R组( Fer+I/R组);阿魏酸 +蛋白激酶抑制剂(LY294002)+I/R组( Fer+LY+I/R)。通过结扎左冠状动脉前降支 30 min,然后再灌注 2h,建立 I/R损伤大鼠模型。通过苏木精 -伊红( HE)染色分析大鼠的左心室病理学变化,结合透射电子显微镜观察其超微结构;通过 TUNEL/DAPI双重染色检测大鼠心肌细胞凋亡率。通过蛋白质印迹法( Western blotting)检测 PI3K/Akt信号通路关键调控因子的蛋白表达水平［包括 PI3K、p-Akt(Ser 473)、 Akt、eNOS、p-eNOS(Ser1177)和 p-mTOR(Ser2448)］以及血清超氧化物歧化酶( SOD)、丙二醛( MDA)、肿瘤坏死因子-α(TNF-α)、白细胞介素 6(IL-6)和 IL-1β水平。结果 HE染色,结果显示,相较于假手术组, I/R组大鼠心肌中表现出明显的炎性细胞浸润、细胞膜损伤、细胞坏死和水肿;而 Fer+I/R组大鼠表现心肌细胞坏死、炎性细胞浸润,细胞水肿均明显低于 I/R组,并且心肌纤维结构清晰完整。 TUNEL/DAPI双重染色结果显示, Fer+I/R组大鼠［(18.73±1.01)%］心肌细胞凋亡率明显低于 I/R组［(55.45±1.14)%］(P<0.001)。 Western blotting检测结果表明,与 I/R组相比, Fer+I/R组大鼠 Cleaved caspase-3蛋白表达水平显著降低( P<0.05)PI3K、p-Akt、Akt、p-eNOS(Ser1177)和 p-mTOR(Ser2448)蛋白表达明显上调( P<0.05)。相较于 I/R组,阿魏酸显著抑制 MDA5、T,NF-α、IL-6和 IL-1β的表达( P<0.05)并上调 SOD1蛋白表达水平( P<0.05);而 LY294002处理逆转了阿魏酸在 I/R模型大鼠中对上述细胞因子的调控作用。结论阿,魏酸通过激活 PI3K/Akt信号通路抑制心肌细胞凋亡,减轻炎症反应和氧化应激水平,保护大鼠免受缺血 /再灌注诱导的心肌损伤。     

Hyperinsulinaemia increases the gene expression of endothelial nitric oxide synthase and the phosphatidylinositol 3-kinase/Akt pathway in rat aorta

1. Hyperinsulinaemia has been reported to be an independent risk factor for cardiovascular diseases. Insulin stimulates both the phosphatidylinositol 3-kinase (PI3-K)/Akt and mitogen-activated protein kinase (MAPK) pathways. To investigate the direct effects of insulin on vascular tissues, we examined the gene and protein expression of insulin signalling molecules, endothelial nitric oxide synthase (eNOS) and MAPK in aortas obtained from established hyperinsulinaemic rats under deep urethane anaesthesia (1.2 g/kg, i.p.). 2. High plasma insulin levels significantly enhanced the gene and protein expression of eNOS in aortas. This was accompanied not only by increased mRNA levels of insulin receptor substrate (IRS)-1, IRS-2, PI3-K and Akt, but also by a high protein content of Akt and phospho-Akt (Ser473). 3. In contrast, MAPK mRNA levels were decreased in hyperinsulinaemic rats compared with normoinsulinaemic rats. 4. Insulin receptor mRNA levels were also lower in insulin-treated rats rather than controls. The overexpression of mRNA for vascular endothelial growth factor (VEGF) and insulin-like growth factor (IGF)-I receptor was also observed in aortas from hyperinsulinaemic rats. 5. To our knowledge, these data provide the first direct measurements of the mRNA of insulin signalling molecules and the downstream eNOS and MAPK. We conclude that hyperinsulinaemia itself can lead to the upregulation of eNOS and the PI3-K/Akt pathway in the vasculature and may also induce the overexpression of VEGF and IGF-I receptor genes.     

Akt inhibitors in clinical development for the treatment of cancer

Importance of the field: The evolution of targeted therapies is dependent upon identification of cellular moieties that can be pharmacologically modulated. As one such example, the serine-threonine kinase Akt was identified nearly two decades ago. Since then, its role in mediating multiple signaling cascades (ultimately leading to cell growth and proliferation) has since been identified. More recently, several agents have been developed that antagonize Akt – these agents are in various stages of clinical testing.

Areas covered in this review: Herein, we outline development of several promising Akt inhibitors, including perifosine, MK-2206, RX-0201, PBI-05204, GSK2141795 and others.

What the reader will gain: The reader will gain insight into the current pipeline of Akt inhibitors, and the degree to which these agents have been examined both clinically and preclinically.

Take home message: With an emerging pipeline of agents targeting Akt, it will be critical to decipher which amongst them holds the greatest promise. Herein, we explore this drug pipeline and provide strategies for determining the future clinical application of these agents.     

Leucanone A and naamine J,glycerol ether lipid and imidazole alkaloid from the marine sponge Leucandra sp.

Chemical investigation on CH₂Cl₂ extract of the marine sponge Leucandra sp. afforded two new compounds named leucanone A (1) and naamine J (2), together with eight known compounds (3–10). Their structures were elucidated on the basis of NMR spectroscopic analyses, and comparing with the literature. The cytotoxic activities of the compounds were evaluated against four cancer cell lines, and compound 2 showed mild cytotoxic activities against MCF-7, A549, HeLa, and PC9 cancer cell lines with IC₅₀ values in the range of 20.1–45.3 μM.     

The PI3K/Akt pathway: recent progress in the development of ATP-competitive and allosteric Akt kinase inhibitors

This article describes recent advances in the development and biological evaluation of allosteric and ATP-competitive small molecule inhibitors for the serine/threonine kinase Akt (protein kinase B, PKB). Unregulated activation of the PI3K/Akt/PTEN pathway is a prominent feature of many human cancers and Akt is over-expressed or activated in all major cancers making Akt an exciting new target for cancer therapy. The development of Akt inhibitors has been complicated and hampered by the presence of three Akt isozymes, (Akt1, Akt2 and Akt3) which differ in function and tissue distribution, as well as a lack of Akt specific inhibitors. In the past 18 months, a large number of reports have appeared describing the discovery and development of allosteric Akt kinase inhibitors and classical ATP-competitive Akt kinase inhibitors. This review will discuss the PI3K/Akt/PTEN pathway, allosteric and ATP-competitive Akt kinase inhibitors, their biological evaluation and progress towards target validation.     

Targeting Akt to increase the sensitivity of neuroblastoma to chemotherapy: lessons learned from the brain-derived neurotrophic factor/TrkB signal transduction pathway

Neuroblastoma (NB) is a neural crest precursor cell-derived extracranial solid tumor in children. Patients with a poor prognosis are often resistant to chemotherapy and have tumors that express the neuronal growth/survival factor brain-derived neurotrophic factor and its tyrosine kinase receptor, TrkB. In this article, the authors discuss a growth/survival factor-stimulated mechanism leading to chemoresistance in NB that is mediated by the PI3K/Akt signaling pathway. Besides brain-derived neurotrophic factor/TrkB, other growth/survival factors and their receptors also activate the PI3K/Akt pathway and have the potential to mediate chemoresistance in NB. These findings raise the possibility of a new therapeutic approach in NB that would target Akt, the common downstream mediator of multiple growth/survival factor signaling pathways, to enhance the efficacy of chemotherapeutics. Several classes of Akt inhibitors, including phosphatidylinositol ether lipid analogs, alkylphospholipid analogs, allosteric Akt kinase inhibitors, HSP90 inhibitor and HIV protease inhibitors are discussed.     

Induction of apoptosis by the ginsenoside Rh2 by internalization of lipid rafts and caveolae and inactivation of Akt

Background and purpose:

Lipid rafts and caveolae are membrane microdomains with important roles in cell survival signalling involving the Akt pathway. Cholesterol is important for the structure and function of these microdomains. The ginsenoside Rh2 exhibits anti-tumour activity. Because Rh2 is structurally similar to cholesterol, we investigated the possibility that Rh2 exerted its anti-tumour effect by modulating rafts and caveolae.

Experimental approach:

A431 cells (human epidermoid carcinoma cell line) were treated with Rh2 and the effects on cell apoptosis, raft localization and Akt activation measured. We also examined the effects of over-expression of Akt and active-Akt on Rh2-induced cell death.

Key results:

Rh2 induced apoptosis concentration- and time-dependently. Rh2 reduced the levels of rafts and caveolae in the plasma membrane and increased their internalization. Furthermore, Akt activity was decreased and consequently, Akt-dependent phosphorylation of Bad, a pro-survival protein, was decreased whereas the pro-apoptotic proteins, Bim and Bax, were increased upon Rh2 treatment. Unlike microdomain internalization induce by cholesterol depletion, Rh2-mediated internalization of rafts and caveolae was not reversed by cholesterol addition. Also, cholesterol addition did not restore Akt activation or rescue cells from Rh2-induced cell death. Rh2-induced cell death was attenuated in MDA-MB-231 cells over-expressing either wild-type or dominant-active Akt.

Conclusions and implications:

Rh2 induced internalization of rafts and caveolae, leading to Akt inactivation, and ultimately apoptosis. Because elevated levels of membrane rafts and caveolae, and Akt activation have been correlated with cancer development, internalization of these microdomains by Rh2 could potentially be used as an anti-cancer therapy.     

A new lipophilic amino alcohol,chemically similar to compound FTY720, attenuates the pathogenesis of experimental autoimmune encephalomyelitis by PI3K/Akt pathway inhibition

Experimental autoimmune encephalomyelitis (EAE) is one of the main animal models used for the study of Multiple Sclerosis (MS). Long-chain lipophilic amino alcohols with immunoregulatory activities have already been studied in some models of inflammatory diseases, but the action of these compounds in EAE and MS is still unknown. In this study, we investigated whether the lipophilic amino alcohol 4b would act to improve the clinical signs of EAE and reduce the demyelination process and the neuroinflammatory parameters in the spinal cord, as well as the inflammatory process in the inguinal lymph nodes, of C57Bl/6 mice induced with EAE after stimulation with MOG_35-55 and pertussis toxin. The 4b treatment (1.0 mg/kg/day) was orally administered, starting on the day of onset of clinical signs of the disease (10th) and ending on the 20th day after immunization. This treatment was able to reduce the cell count on the inguinal lymph nodes, the migration of inflammatory cells into the central nervous system (CNS), as well as the processes of microgliosis, astrogliosis, and the production of chemokines and pro-inflammatory cytokines, thus increasing the IL-10 anti-inflammatory cytokine levels in EAE mice. The inhibition of Akt phosphorylation in the CNS of EAE mice after treatment with 4b indicates that the immunoregulatory action of 4b is related to the PI3K/Akt signaling pathway. Our results indicate the immunoregulatory efficacy of the new compound 4b in the control of some inflammatory parameters and in the glial proliferation. In addition, 4b was able to reduce the demyelination of neurons and the worsening of clinical signs of EAE as effectively as the compound FTY720, the first oral drug approved by the FDA for the treatment of MS.     

Antisense oligodeoxynucleotides targeting the serine／threonine kinase Pim-2 inhibited proliferation of DU-145 cells

Aim: To investigate the effect of antisense oligodeoxynucleotides (ASODN) targeting Pim-2 on cell proliferation of DU-145 cells. Methods: Three ASODN targeting Pim-2 were designed and synthesized. After transfection with ASODN, cell proliferation was analyzed using an MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] assay. In addition, Pim-2 mRNA, protein levels, and cell cycles were examined. Results: The ASODN designed and synthesized by our laboratory significantly reduced Pim-2 mRNA level and protein content in DU-145 cells. After transfection with ASODN for 48 h, a marked reduction in cell viability was observed in DU-145 cells in a dose-dependent manner. No remarkable apoptosis occurred in cells treated with ASODN compared with control cells. However, it should be noted that G1 phase arrest was clearly observed in ASODN-treated cells. Conclusion: ASODN targeting Pim-2 resulted in a marked reduction in DU-145 cell proliferation, and induction of G1 phase cell cycle arrest is one of the important mechanisms for ASODN to reduce cell growth. Moreover, antisense inhibition of Pim-2 expression provides a new promising therapy target for prostate cancer.     

SZ-685C,a marine anthraquinone,is a potent inducer of apoptosis with anticancer activity by suppression of the Akt/FOXO pathway

Background and purpose:

The aims of this study were to investigate the anti-cancer activity of SZ-685C, an anthracycline analogue isolated from marine-derived mangrove endophytic fungi, and to explore the molecular mechanisms underlying such activity.

Experimental approach:

The effect of SZ-685C on the viability of cancer cell lines was investigated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. SZ-685C-induced apoptosis was assessed by Annexin V-fluorescein isothiocyanate/propidium iodide staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assay and analysis of caspase activation. The effect of SZ-685C on the Akt/FOXO pathway was studied using Western blotting analysis, and the in vivo anti-tumour efficacy was examined in an MDA-MB-435 breast cancer xenograft model.

Key results:

SZ-685C suppressed the proliferation of six cancer cell lines derived from human breast cancer, prostate cancer, glioma and hepatoma (IC₅₀ values ranged from 3.0 to 9.6 µM) and the growth of breast cancer xenografts in mice. SZ-685C had a direct apoptosis-inducing effect through both the extrinsic and intrinsic apoptotic pathways, as shown by activation of caspase-8 and 9 as well as effector caspase-3 and poly (ADP-ribose) polymerase. Phosphorylation of Akt and its downstream effectors, forkhead box protein O1 and forkhead box protein O3a, was down-regulated in SZ-685C-treated cancer cells. Furthermore, the pro-apoptotic protein Bim was up-regulated by SZ-685C treatment consistent with FOXO dephosphorylation.

Conclusions and implications:

SZ-685C could induce apoptosis through the Akt/FOXO pathway, which consequently leads to the observed anti-tumour effect both in vitro and in vivo. Our data suggest that SZ-685C may be a potentially promising Akt inhibitor and anti-cancer drug candidate.     

Vanadate is a potent activator of endothelial nitric-oxide synthase: evidence for the role of the serine/threonine kinase Akt and the 90-kDa heat shock protein

We investigated the molecular mechanisms of sodium vanadate (vanadate)-induced nitric oxide (NO) production. Exposure of bovine lung microvascular cells (BLMVEC) to vanadate increased the release of biologically active NO in endothelium/smooth muscle cocultures, as measured by the accumulation of its surrogate marker, cGMP. This release was sensitive to NO synthase (NOS) inhibition and was greater than that observed with ionomycin. Although calcium chelators (BAPTA, EGTA) inhibited basal and ionomycin-induced NO production, they failed to inhibit vanadate-induced NO release. Moreover, in the absence of calcium/calmodulin, cell lysates from vanadate-treated cells exhibited greater NOS activity compared with control cells. Vanadate activates the phosphoinositide3-kinase (PI3-K)/Akt pathway, which is known to increase endothelial NOS (eNOS) activity by direct phosphorylation of Ser-1179. Treatment of BLMVEC with vanadate resulted in phosphorylation of both Akt and endothelial NOS. In addition, wortmannin, a PI3-K inhibitor, blocked both the vanadate-induced phosphorylation of eNOS and the increase in cGMP accumulation. Similarly, adenovirus-mediated gene transfer of an activation deficient form of Akt (AA-Akt) blocked the release of NO brought about by vanadate. To further investigate the mechanism of action of vanadate, eNOS was immunoprecipitated and its association with proteins that alter eNOS activity was tested. Immunoblots demonstrated that the eNOS-caveolin interaction remained unaffected by vanadate, whereas vanadate promoted recruitment of the 90-kDa heat shock protein (hsp90) to eNOS. We conclude that vanadate causes NO release via a mechanism that involves Akt-induced eNOS phosphorylation and increased binding of the activator protein hsp90 to eNOS.     

Regulation of endothelial nitric oxide synthase: involvement of protein kinase G 1 beta, serine 116 phosphorylation and lipid structures

1. Endothelial nitric oxide synthase (NOS3) is important for vascular homeostasis. The role of protein kinase G (PKG) in regulation of NOS3 activity was studied in primary cultures of newborn lamb lung microvascular endothelial cells (LMVEC). 2. We determined the presence of PKG in fetal and neonatal LMVEC as well as subcellular localization of PKG isoforms in the neonatal cells by fluorescence immunohistochemistry. We used diaminofluorescein (DAF) fluorophore to measure nitric oxide (NO) production from neonatal LMVEC. We confirmed that NO measured was from constitutive NOS3 by inhibiting it with NOS inhibitors. 3. To identify a role for PKG in basal NO production, we measured NO release from LMVEC cells using 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM; 0.5-0.8 micromol/L) with and without prior stimulation with the PKG activator 8-bromo-cGMP (8-Br-cGMP; 0.3 and 3 micromol/L) or prior PKG inhibition with beta-phenyl-1,N2-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothionate (BPC; 0.3 and 3 micromol/L). With the same drugs, we determined the role of PKG on cellular expression of NOS3 and serine 116 phosphorylated NOS (pSer116-NOS) by qualitative and quantitative immunofluorescence assays, as well as western blotting. 4. Because PKG 1 beta was distributed throughout the cytosol in a punctate expression, we used 2 mmol/L cyclodextrin, a cholesterol extractor, to determine a role for lipid vesicles in PKG regulation of NO production. 5. Protein kinase G 1 beta gave a punctate appearance, indicating its presence in intracellular vesicles. Nitric oxide production decreased by approximately 20% with 300 nmol/L and 3 micromol/L 8-Br cGMP (P < 0.05) and increased by 20.8 +/- 3.7% with 3 micromol/L BPC (P < 0.001), indicating that both stimulated and basal PKG activity has inhibitory effects on basal NOS3 function. Nitric oxide synthase immunofluorescence and immunoblot expression were decreased and pSer116-NOS immunofluorescence was increased by 800 nmol/L 8-Br-cGMP and 170 micromol/L (Z)-1-[2-(2-aminoethyl)-N-(2-ammonio-ethyl)amino]diazen-1-ium-1, 2-diolate (DETANONOate). The effect of cyclodextrin indicated that cholesterol extraction interfered with PKG inhibition of NOS. Further examination of pSer116-NOS by immunohistochemistry showed it abundant in the endoplasmic reticulum and colocalized with PKG 1 beta, especially in nuclear vesicles. 6. We conclude that endothelial PKG is involved in endogenous regulation of basal NOS3 activity with the involvement of lipid structures, the endoplasmic reticulum and the nucleus. Protein kinase G 1 beta is colocalized with pSer116-NOS, indicating that PKG action may involve serine 116 phosphorylation on NOS.     

The expression of the imprinted gene pleckstrin homology‐like domain family A member 2 in placental tissues of preeclampsia and its effects on the proliferation,migration and invasion of trophoblast cells JEG‐3

Preeclampsia (PE) is one of the most common hypertensive disorders and is a leading cause of morbidity and mortality for pregnant women and perinatal babies. Additionally, pleckstrin homology‐like domain family A member 2 (PHLDA2) is associated with placental dysfunction. However, the effect of PHLDA2 on trophoblast cell proliferation, migration and invasion has not been investigated. In this study, 15 PE patients and 15 normal pregnant women were recruited and clinical characteristics were summarized. Pleckstrin homology‐like domain family A member 2 levels in placental tissues were examined using real‐time PCR and western blot. Overexpression plasmid and PHLDA2 siRNA was introduced into JEG‐3 cells, respectively. Cell proliferation was measured using MTT assay and flow cytometry. Cell migration and invasion capacities were assessed by wound healing and Transwell assays. It was found that PE patients collectively presented proteinuria, elevated systolic blood pressure (SBP) and diastolic blood pressure (DBP), and lower gestational ages and birth weights. Pleckstrin homology‐like domain family A member 2 levels in the preeclamptic placenta were significantly upregulated. Pleckstrin homology‐like domain family A member 2 overexpression significantly arrested cells in the G0/G1 phase, inhibited cell proliferation and suppressed the migration and invasion of JEG‐3 cells. Pleckstrin homology‐like domain family A member 2 knockdown significantly blocked the cells in the S phase of the cell cycle. Knockdown of PHLDA2 alleviated the inhibition on the migration and invasion of trophoblast cells JEG‐3. These findings illustrate that PHLDA2 may participate in PE pathogenesis and indicate its potential application in the early diagnosis of PE.     

DHA2, a synthesized derivative of bisbibenzyl,exerts antitumor activity against ovarian cancer through inhibition of XIAP and Akt/mTOR pathway

The analysis of dihydroptychantol (DHA) and its chemically synthesized macrocyclic bisbibenzyl derivatives (DHA1, 2 and 3) led to the selection of DHA2 as a potential drug candidate for ovarian cancer. The exposure of ovarian cancer SKOV3 cells to DHA2 resulted in the downregulation of the anti-apoptotic X-linked inhibitor of apoptosis protein (XIAP) and Bcl-2 and led to caspase-independent cell death. The overexpression of XIAP reversed DHA2-induced cell death, and the depletion of XIAP had the opposite effect. DHA2 could induce autophagy, as evidenced by increases in the formation of acidic vesicular organelles and the processing of LC3B-I to LC3B-II. Pretreatment with autophagy inhibitors potentiated DHA2-mediated cell death. We showed that typical PI3K/Akt signaling was involved in DHA2-mediated cell death. The overexpression of Akt almost completely reversed DHA2-induced cell death, and the inactivation of Akt significantly facilitated DHA2-induced cell death. The administration of DHA2 to xenograft mice reduced tumor growth by causing Akt inactivation, the conversion of LC3B and proliferation inhibition. Our study demonstrates that the inhibition of XIAP and the inactivation of Akt/mTOR facilitate the efficacy of DHA2 in ovarian cancer cells.     

LY294,002, a specific inhibitor of PI3K/Akt kinase pathway, antagonizes P-glycoprotein-mediated multidrug resistance

The transmembrane transport pump P-glycoprotein (P-gp) causes the efflux of chemotherapeutic agents from cells and is an important system that secures multidrug resistance (MDR) of neoplastic cells. In the present study drug sensitive L1210 and multidrug resistant L1210/VCR mouse leukemic cell lines were used as an experimental model. We found that LY 294,002, a specific inhibitor of PI3K/Akt kinase pathway, reduced the degree of vincristine resistance in L1210/VCR cells significantly and in a concentration-dependent manner. This was accompanied by decrease in IC(50) value to vincristine from 3.195+/-0.447 to 1.898+/-0.676 micromol/l for 2 micromol/l, to 0.947+/-0.419 micromol/l for 4 micromol/l, and to 0.478+/-0.202 micromol/l for 8 micromol/l LY294,002. The IC(50) value of sensitive cells for vincristine was about 0.010 micromol/l. FACS analysis of the proportion of cells in apoptosis or necrosis by annexin-V apoptosis kit showed the following: (i) vincristine-induced apoptosis in resistant cell to a much lower extent than in sensitive cells; (ii) LY294,002 alone did not induce apoptosis or necrosis in both sensitive and resistant cells; (iii) LY294,002 applied together with vincristine significantly increased the number of apoptotic cells. Transport activity of P-gp in resistant cells was monitored using calcein/AM as substrate and was depressed by LY294,002 in a concentration dependent manner. Significant differences in calcein retention were not observed when cells were preincubated with LY294,002 at different times from 0.5 to 24h. Sensitive and resistant cells contain similar amounts of uncleaved (i.e., unactivated) caspase-3 but in latter cells the activation of caspase-3 by proteolytic cleavage was decreased. The reversal of vincristine resistance by LY294,002 was associated with marked activation of caspase-3. Western blot analysis revealed that the development of MDR phenotype in L1210/VCR cells was also associated with increased level of Bcl-2 protein. All the above findings point to the possible involvement of PI3K/Akt kinase pathway in modulation of P-gp mediated multidrug resistance in L1210/VCR mouse leukemic cell line. MDR reversal effect of LY294,002 is accompanied with this compound's influence on vincristine-induced apoptosis.