RUNX1对氧糖剥夺诱导PC12细胞凋亡的保护作用研究

目的:研究RUNX1在PC12细胞氧糖剥夺模型中的表达及其对PC12细胞的保护作用,并探讨其相关机制。方法:体外培养PC12细胞并构建氧糖剥夺模型,将细胞分为对照组、氧糖剥夺组、RUNX1 si RNA处理组、si RNA对照处理组(sicontrol)、pc DNA3.1-RUNX1处理组(pc RUNX1)和pc DNA3.1对照处理组(pc DNA 3.1)。q RT-PCR和western blot检测RUNX1、磷酸化Akt(p-Akt)和总Akt(t-Akt)表达水平;MTT法检测细胞存活率;Annexin V-FITC/PI双染法检测细胞凋亡。结果:与对照组比较,RUNX1在PC12细胞氧糖剥夺模型中表达水平显著升高;沉默RUNX1可下调PC12细胞的存活率,促进细胞的凋亡,有效抑制p-Akt蛋白表达,而过表达RUNX1显著提高细胞存活率,抑制细胞凋亡,并上调p-Akt蛋白表达;此外,PI3K/Akt通路抑制剂LY294002明显抑制RUNX1过表达对细胞存活率的促进作用和对细胞凋亡的抑制作用。结论:RUNX1可通过PI3K/Akt信号通路保护OGD对PC12细胞的损伤作用。     

Differential effect of ascorbic acid and n-acetyl-L-cysteine on arsenic trioxide-mediated oxidative stress in human leukemia (HL-60) cells

Arsenic trioxide (ATO) has been recommended for the treatment of refractory cases of acute promyelocytic leukemia (APL). Recent studies in our laboratory indicated that oxidative stress plays a key role in ATO-induced cytotoxicity in human leukemia (HL-60) cells. In the present investigation, we performed the MTT assay and trypan blue exclusion test for cell viability. We also performed the thiobarbituric acid test to determine the levels of malondialdehyde (MDA) production in HL-60 cells coexposed to either ascorbic acid (AA) and ATO or to n-acetyl-L-cysteine (NAC) and ATO. The results of MTT assay indicated that AA exposure potentiates the cytotoxicity of ATO in HL-60 cells, as evidenced by a gradual increase in MDA levels with increasing doses of AA. In contrary, the addition of NAC to ATO-treated HL-60 cells resulted in a dose-dependent decrease of MDA production. From these results, we conclude that the addition of the AA to ATO-treated HL-60 cells enhances the formation of reactive oxygen species (ROS), whereas the addition of NAC under the same experimental condition significantly (p < .05) decreases the level of ROS formation. On the basis of these direct in vitro findings, our studies provide evidence that AA may extend the therapeutic spectrum of ATO. The coadministration of NAC with ATO shows a potential specificity for tumor cells, indicating that it may not enhance the clinical outcome associated with ATO monotherapy in vivo.     

Rifampicin Prevents SH-SY5Y Cells from Rotenone-Induced Apoptosis via the PI3K/Akt/GSK-3β/CREB Signaling Pathway

In addition to its original application for treating tuberculosis, rifampicin has multiple potential neuroprotective effects in chronic neurodegenerative diseases including Parkinson’s disease (PD) and Alzheimer’s disease. Inflammatory reactions and the PI3K/Akt pathway are strongly implicated in dopaminergic neuronal death in PD. This study aims to investigate whether rifampicin protects rotenone-lesioned SH-SY5Y cells via regulating PI3K/Akt/GSK-3β/CREB pathway. Rotenone-treated SH-SY5Y cells were used as the cell model to investigate the neuroprotective effects of rifampicin. Cell viability and apoptosis of SH-SY5Y cells were determined by CCK-8 assay and flow cytometry, respectively. The expression of Akt, p-Akt, GSK-3β, p-GSK-3β, CREB and p-CREB were measured by Western blot. Our results showed that the cell viability and level of phospho-CREB significantly decreased in SH-SY5Y cells exposed to rotenone when compared to the control group. Both the cell viability and the expression of phospho-CREB in cells pretreated with rifampicin were higher than those of cells exposed to rotenone alone. Moreover, pretreatment of SH-SY5Y cells with rifampicin enhanced phosphorylation of Akt and suppressed activity of GSK-3β. The addition of LY294002, a PI3K inhibitor, could suppress phosphorylation of Akt and CREB and activate GSK-3β, resulting in abolishment of neuroprotective effects of rifampicin on cells exposed to rotenone. Rifampicin provides neuroprotection against dopaminergic degeneration, partially via the PI3K/Akt/GSK-3β/CREB signaling pathway. These findings suggest that rifampicin could be an effective and promising neuroprotective candidate for treating PD.     

Statin induces inhibition of triple negative breast cancer (TNBC) cells via PI3K pathway

Primary TNBCs are treated as if they were a single disease entity, yet it is clear they do not behave as a single entity in response to current therapies. Recently, we reported that statins might have a potential benefit for TNBCs associated with ets-1 overexpression. The aim of this study is to investigate the role of PTEN loss in the effects of statin on TNBC cells. In addition, we analyze the relationship between AKT downstream pathways and the effects of statin on TNBC cells. We investigated the effect of a statin on TNBC cells and analyzed the association of PI3K pathways using various TNBC cells in terms of PTEN loss and AKT pathways. Simvastatin treatments resulted in decreased cell viabilities in various TNBC cell lines. Compared with PTEN wild-type TNBC cells, PTEN mutant-type TNBC cells showed a decreased response to simvastatin. Expressions of phosphorylated Akt and total Akt showed an inverse relationship with PTEN expression. The TNBC cell lines, which showed increased expression of p-Akt, appeared to attenuate the expression of p-Akt by PTEN loss in simvastatin-treated TNBC cells. The Akt inhibitor, LY294002, augmented the effect of simvastatin on PTEN wild-type TNBC cells. Simvastatin induces inhibition of TNBC cells via PI3K pathway activation.     

Death-associated protein 5 (DAP5/p97/NAT1) contributes to retinoic acid-induced granulocytic differentiation and arsenic trioxide-induced apoptosis in acute promyelocytic leukemia

All-trans-retinoic acid (ATRA) and arsenic trioxide (ATO) induce differentiation and apoptosis in acute promyelocytic leukemia (APL) cells. Here we investigated the role and regulation of death-associated protein-5 (DAP5/p97/NAT1), a novel inhibitor of translational initiation, in APL cell differentiation and apoptosis. We found that ATRA markedly induced DAP5/p97 protein and gene expression and nuclear translocation during terminal differentiation of APL (NB4) and HL60 cells but not differentiation-resistant cells (NB4.R1 and HL60R), which express very low levels of DAP5/p97. At the differentiation inducing concentrations, ATO (<0.5 μM), dimethyl sulfoxide, 1,25-dihydroxy-vitamin-D3, and phorbol-12-myristate 13-acetate also significantly induced DAP5/p97 expression in NB4 cells. However, ATO administered at apoptotic doses (1–2 μM) induced expression of DAP5/p86, a proapoptotic derivative of DAP5/p97. ATRA and ATO-induced expression of DAP5/p97 was associated with inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Furthermore, DAP5/p97 expression was upregulated by inhibition of the PI3K/Akt/mammalian target of rapamycin (mTOR) pathway via LY294002 and via rapamycin. Finally, knockdown of DAP5/p97 expression by small interfering RNA inhibited ATRA-induced granulocytic differentiation and ATO-induced apoptosis. Together, our data reveal new roles for DAP5/p97 in ATRA-induced differentiation and ATO-induced apoptosis in APL and suggest a novel regulatory mechanism by which PI3K/Akt/mTOR pathway inhibition mediates ATRA- and ATO-induced expression of DAP5/p97. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. B. Ozpolat and U. Akar contributed equally.     

p-Aminophenol-induced cytotoxicity in Jurkat T cells: protective effect of 2(RS)-n-propylthiazolidine-4(R)-carboxylic acid

Acetaminophen (APAP) is hepatotoxic and can cause toxicity in Jurkat T cells. p-Aminophenol (PAP), an industrial chemical and APAP metabolite, is nephrotoxic and hepatotoxic. Its potential toxicity in Jurkat T cells was investigated. PAP (10-250 μM) caused toxicity (decreased survival and increased LDH activity in incubation medium) and GSH depletion. At a concentration of 100 μM but not 250 μM, PAP increased DNA fragmentation. It decreased p-Akt levels (Elisa) and at higher concentrations decreased p-Akt expression (Western blotting). It had no effect on FasL expression. The cysteine precursor 2(RS)-n-propylthiazolidine-4(R)-carboxylic acid (250 μM) attenuated the PAP (100 μM)-induced decrease in viability and prevented GSH depletion and increased DNA fragmentation. It attenuated the PAP-induced decrease in p-Akt levels and protected against the decrease in p-Akt expression. The results demonstrate PAP-induced toxicity and suggest that it is due at least in part to apoptosis and involves GSH depletion and p-Akt inactivation.     

α-Lipoic acid alleviates ferroptosis in the MPP+-induced PC12 cells via activating the PI3K/Akt/Nrf2 pathway

Parkinson's disease (PD) is a typical neurodegenerative disease. α-Lipoic acid (α-LA) can reduce the incidence of neuropathy. The present study explored the role and mechanism of α-LA in 1-methyl-4-phenylpyridinium (MPP⁺)-induced cell model of PD. The PD model was induced via treating PC12 cells with MPP⁺ at different concentrations. MPP⁺ and α-LA effects on PC12 cells were assessed from cell viability and ferroptosis. Cell viability was detected using the cell counting kit-8 assay. Malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), iron, reactive xygen species (ROS), and glutathione (GSH) concentrations, and ferroptosis-related protein SLC7A11 and GPx4 expressions were used for ferroptosis evaluation. p-PI3K, p-Akt, and nuclear factor erythroid 2-related factor 2 (Nrf2) protein levels were detected. The PI3K/Akt/Nrf2 pathway inhibitors were applied to verify the role of the PI3K/Akt/Nrf2 pathway in α-LA protection against MPP⁺-induced decreased cell viability and ferroptosis. MPP⁺-reduced cell viability and induced ferroptosis as presented by increased MDA, 4-HNE, iron, and ROS concentrations, and reduced levels of GSH and ferroptosis marker proteins (SLC7A11 and GPx4). α-LA attenuated MPP⁺-induced cell viability decline and ferroptosis. The PI3K/Akt/Nrf2 pathway was activated after α-LA treatment. Inhibiting the PI3K/Akt/Nrf2 pathway weakened the protection of α-LA against MPP⁺ treatment. We highlighted that α-LA alleviated MPP⁺-induced cell viability decrease and ferroptosis in PC12 cells via activating the PI3K/Akt/Nrf2 pathway.     

Powerful tumor cell growth-inhibiting activity of a synthetic derivative of atractyligenin: Involvement of PI3K/Akt pathway and thioredoxin system

Background

The semi-synthetic ent-kaurane 15-ketoatractyligenin methyl ester (SC2017) has been previously reported to possess high antiproliferative activity against several solid tumor-derived cell lines. Our study was aimed at investigating SC2017 tumor growth-inhibiting activity and the underlying mechanisms in Jurkat cells (T-cell leukemia) and xenograft tumor models.

Methods

Cell viability was evaluated by MTT assay. Cell cycle progression, reactive oxygen species (ROS) elevation and apoptotic hallmarks were monitored by flow cytometry. Inhibition of thioredoxin reductase (TrxR) by biochemical assays. Levels and/or activation status of signaling proteins were assessed by western blotting. Xenograft tumors were generated with HCT 116 colon carcinoma cells.

Results

SC2017 displayed cell growth-inhibiting activity against Jurkat cells (half maximal inhibitory concentration values (IC50) < 2 μM), but low cell-killing potential in human peripheral blood mononuclear cells (PBMC). The primary response of Jurkat cells to SC2017 was an arrest in G₂ phase followed by caspase-dependent apoptosis. Inhibition of PI3K/Akt pathway and TrxR activity by SC2017 was demonstrated by biochemical and pharmacological approaches. At least, SC2017 was found to inhibit xenograft tumor growth.

Conclusions

Our results demonstrate that SC2017 inhibits tumor cell growth in in vitro and in vivo models, but displays moderate toxicity against PBMC. We also demonstrate that SC2017 promotes caspase-dependent apoptosis in Jurkat cells by affecting Akt activation status and TrxR functionality.

General significance

Our observations suggest the semi-synthetic ent-kaurane SC2017 as a promising chemotherapeutic compound. SC2017 has, indeed, shown to possess tumor growth inhibiting activity and be able to counteract PI3K/Akt and Trx system survival signaling.     

Activation of the PI3K/Akt signaling pathway through P2Y2 receptors by extracellular ATP is involved in osteoblastic cell proliferation

We studied the PI3K/Akt signaling pathway modulation and its involvement in the stimulation of ROS 17/2.8 osteoblast-like cell proliferation by extracellular ATP. A dose- and time-dependent increase in Akt-Ser 473 phosphorylation (p-Akt) was observed. p-Akt was increased by ATPγS and UTP, but not by ADPβS. Akt activation was abolished by PI3K inhibitors and reduced by inhibitors of PI-PLC, Src, calmodulin (CaM) but not of CaMK. p-Akt was diminished by cell incubation in a Ca²⁺-free medium but not by the use of L-type calcium channel blockers. The rise in intracellular Ca²⁺ induced by ATP was potentiated in the presence of Ro318220, a PKC inhibitor, and attenuated by the TPA, a known activator of PKC. ATP-dependent p-Akt was diminished by TPA and augmented by Ro318220 treatment in a Ca²⁺-containing but not in a Ca²⁺-free medium. ATP stimulated the proliferation of both ROS 17/2.8 cells and rat osteoblasts through PI3K/Akt. In the primary osteoblasts, ATP induces alkaline phosphatase activity via PI3K, suggesting that the nucleotide promotes osteoblast differentiation. These results suggest that ATP stimulates osteoblast proliferation through PI-PLC linked-P2Y₂ receptors and PI3K/Akt pathway activation involving Ca²⁺, CaM and Src. PKC seems to regulate Akt activation through Src and the Ca²⁺ influx/CaM pathway.     

Cancer/testis antigen LDHC promotes proliferation and metastasis by activating the PI3K/Akt/GSK-3β-signaling pathway and the in lung adenocarcinoma

The cancer/testis antigen lactate dehydrogenase-C4 (LDHC) is a specific isoenzyme of the LDH family that regulates invasion and metastasis in some malignancies; however, little is known regarding its role in progression of lung adenocarcinoma (LUAD). Thus, we investigated LDHC expression by immunohistochemistry, and analyzed its clinical significance in 88 LUAD specimens. The role and molecular mechanisms subserving LDHC in cellular proliferation, migration, and invasion were explored both in vitro and in vivo. As a result, we found that high LDHC expression was significantly correlated with clinicopathological features of aggressive LUAD and a poor prognosis. Overexpression of LDHC induced LUAD cells to produce lactate and ATP, increased their metastatic and invasive potential—, and accelerated xenograft tumor growth. We further demonstrated that overexpression of LDHC affected the expression of cell proliferation-related proteins (cyclin D1 and c-Myc) and epithelial-mesenchymal transition (EMT)-related proteins (MMP-2, MMP-9, E-cadherin, Vimentin, Twist, Slug, and Snail) both in vitro and in vivo. Finally, excessive activation of LDHC enhanced the phosphorylation levels of AKT and GSK-3β, revealing activation of the PI3K/Akt/GSK-3β oncogenic-signaling pathways. Treatment with a PI3K inhibitor reversed the effects of LDHC overexpression by inhibiting cellular proliferation, migration, and invasion, with diminished levels of p-Akt and p-GSK3β. PI3K inhibition also reversed cell proliferation-related and EMT-related proteins in LDHC-overexpressing A549 cells. In conclusion, LDHC promotes proliferation, migration, invasion, and EMT in LUAD cells via activation of the PI3K/Akt/GSK-3β pathway.     

Grape Proanthocyanidin Inhibit Pancreatic Cancer Cell Growth In Vitro and In Vivo through Induction of Apoptosis and by Targeting the PI3K/Akt Pathway

Pancreatic cancer is an aggressive malignancy that is frequently diagnosed at an advanced stage with poor prognosis. Here, we report the chemotherapeutic effects of bioactive proanthocyanidins from grape seeds (GSPs) as assessed using In Vitro and In Vivo models. Treatment of human pancreatic cancer cells (Miapaca-2, PANC-1 and AsPC-1) with GSPs In Vitro reduced cell viability and increased G2/M phase arrest of the cell cycle leading to induction of apoptosis in a dose- and time-dependent manner. The GSPs-induced apoptosis of pancreatic cancer cells was associated with a decrease in the levels of Bcl-2 and Bcl-xl and an increase in the levels of Bax and activated caspase-3. Treatment of Miapaca-2 and PANC-1 cells with GSPs also decreased the levels of phosphatidylinositol-3-kinase (PI3K) and phosphorylation of Akt at ser(473). siRNA knockdown of PI3K from pancreatic cancer cells also reduced the phosphorylation of Akt. Further, dietary administration of GSPs (0.5%, w/w) as a supplemented AIN76A control diet significantly inhibited the growth of Miapaca-2 pancreatic tumor xenografts grown subcutaneously in athymic nude mice, which was associated with: (i) inhibition of cell proliferation, (ii) induction of apoptosis of tumor cells, (iii) increased expression of Bax, reduced expression of anti-apoptotic proteins and activation of caspase-3-positive cells, and (iv) decreased expression of PI3K and p-Akt in tumor xenograft tissues. Together, these results suggest that GSPs may have a potential chemotherapeutic effect on pancreatic cancer cell growth.     

Suppression of arsenic trioxide-induced apoptosis in HeLa cells by N-acetylcysteine

Arsenic trioxide (ATO) can affect many biological functions such as apoptosis and differentiation in various cells. We investigated the involvement of ROS and GSH in ATO-induced HeLa cell death using ROS scavengers, especially N-acetylcysteine (NAC). ATO increased intracellular O(2)(*-) levels and reduced intracellular GSH content. The ROS scavengers, Tempol, Tiron and Trimetazidine, did not significantly reduce levels of ROS or GSH depletion in ATO-treated HeLa cells. Nor did they reduce the apoptosis induced by ATO. In contrast, treatment with NAC reduced ROS levels and GSH depletion in the ATO-treated HeLa cells and prevented ATO-induced apoptosis. Treatment with exogenous SOD and catalase reduced the depletion of GSH content in ATO-treated cells. Catalase strongly protected the cells from ATO-induced apoptosis. In addition, treatment with SOD, catalase and NAC slightly inhibited the G1 phase accumulation induced by ATO. In conclusion, NAC protects HeLa cells from apoptosis induced by ATO by up-regulating intracellular GSH content and partially reducing the production of O(2)(*-).     

Isoform-specific regulation of adipocyte differentiation by Akt/protein kinase Balpha

The phosphatidylinositol 3-kinase (PI3K)/Akt pathway tightly regulates adipose cell differentiation. Here we show that loss of Akt1/PKBα in primary mouse embryo fibroblast (MEF) cells results in a defect of adipocyte differentiation. Adipocyte differentiation in vitro and ex vivo was restored in cells lacking both Akt1/PKBα and Akt2/PKBβ by ectopic expression of Akt1/PKBα but not Akt2/PKBβ. Akt1/PKBα was found to be the major regulator of phosphorylation and nuclear export of FoxO1, whose presence in the nucleus strongly attenuates adipocyte differentiation. Differentiation-induced cell division was significantly abrogated in Akt1/PKBα-deficient cells, but was restored after forced expression of Akt1/PKBα. Moreover, expression of p27^Kip1, an inhibitor of the cell cycle, was down regulated in an Akt1/PKBα-specific manner during adipocyte differentiation. Based on these data, we suggest that the Akt1/PKBα isoform plays a major role in adipocyte differentiation by regulating FoxO1 and p27^Kip1.     

Induction of apoptosis in arsenic trioxide-treated lung cancer A549 cells by buthionine sulfoximine

Arsenic trioxide (ATO) affects many biological processes such as cell proliferation, apoptosis, differentiation and angiogenesis. L-buthionine sulfoximine (BSO) is an inhibitor of GSH synthesis. We tested whether ATO reduced the viability of lung cancer A549 cells in vitro, and investigated the in vitro effect of the combination of ATO and BSO on cell viability in relation to apoptosis and the cell cycle. ATO caused a dose-dependant decrease of viability of A549 cells with an IC50 of more than 50 muM. Low doses of ATO or BSO (1~10 muM) alone did not induce cell death. However, combined treatment depleted GSH content and induced apoptosis, loss of mitochondrial transmembrane potential (DeltaPsi(m)) and cell cycle arrest in G2. Reactive oxygen species (ROS) increased or decreased depending on the concentration of ATO. In addition, BSO generally increased ROS in ATO-treated A549 cells. ROS levels were at least in part related to apoptosis in cells treated with ATO and/or BSO. In conclusion, we have demonstrated that A549 lung cells are very resistant to ATO, and that BSO synergizes with clinically achievable concentration of ATO. Our results suggest that combination treatment with ATO and BSO may be useful for treating lung cancer.     

Cardiotrophin-1 stimulates the neural differentiation of human umbilical cord blood-derived mesenchymal stem cells and survival of differentiated cells through PI3K/Akt-dependent signaling pathways

Cardiotrophin-1 (CT1) plays an important role in the differentiation, development, and survival of neural stem cells. In this study, we analyzed its effects on the stimulation of human umbilical cord blood-derived mesenchymal stem cells in terms of their potential to differentiate into neuron-like cells, their survival characteristics, and the molecular mechanisms involved. The treatment of cells with neural induction medium (NIM) and CT1 generated more cells that were neuron-like and produced stronger expression of neural-lineage markers than cells treated with NIM and without CT1. Bcl-2 and Akt phosphorylation (p-Akt) expression levels increased significantly in cells treated with both NIM and CT1. This treatment also effectively blocked cell death following neural induction and decreased Bax, Bak and cleaved-caspase 3 expression compared with cells treated with NIM without CT1. In addition, the inhibition of phosphatidylinositol 3-kinase (PI3K) abrogated p-Akt and Bcl-2 expression. Thus, PI3K/Akt contribute to CT1-stimulated neural differentiation and to the survival of differentiated cells.     

Aggressive neuroblastomas have high p110alpha but low p110delta and p55alpha/p50alpha protein levels compared to low stage neuroblastomas

Background

The phosphoinositide 3-kinase (PI3K)/Akt pathway is involved in neuroblastoma development where Akt/PKB activation is associated with poor prognosis. PI3K activity subsequently activates Akt/PKB, and as mutations of PI3K are rare in neuroblastoma and high levels of PI3K subunit p110delta is associated with favorable disease with low p-Akt/PKB, the levels of other PI3K subunits could be important for Akt activation.

Methods

Protein levels of Type IA PI3K catalytic and regulatory subunits were investigated together with levels of phosphorylated Akt/PKB and the PI3K negative regulator PTEN in primary neuroblastoma tumors. Relation between clinical markers and protein levels were evaluated through t-tests.

Results

We found high levels of p-Akt/PKB correlating to aggressive disease and p-Akt/PKB (T308) showed inverse correlation to PTEN levels. The regulatory isomers p55alpha/p50alpha showed higher levels in favorable neuroblastoma as compared with aggressive neuroblastoma. The PI3K-subunit p110alpha was found mainly in advanced tumors while p110delta showed higher levels in favorable neuroblastoma.

Conclusions

Activation of the PI3K/Akt pathway is seen in neuroblastoma tumors, however the contribution of the different PI3K isoforms is unknown. Here we show that p110alpha is preferentially expressed in aggressive neuroblastomas, with high p-Akt/PKB and p110delta is mainly detected in favorable neuroblastomas, with low p-Akt/PKB. This is an important finding as PI3K-specific inhibitors are suggested for enrollment in treatment of neuroblastoma patients.

     

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.     

A dual PI3 kinase/mTOR inhibitor BEZ235 reverses doxorubicin resistance in ABCB1 overexpressing ovarian and pancreatic cancer cell lines

BackgroundMulti-drug resistance (MDR) develops because cancer cells evade toxicity of several structurally unrelated drugs. Besides other mechanisms, MDR is linked to the overexpression of ATP Binding Cassette (ABC), transporters, among which ABCB1 is the best characterized one. Since overactivation of PI3K/Akt/mTOR plays a pivotal role in the growth of human cancers, we hypothesized whether dual PI3K and mTOR inhibitor, BEZ235 (BEZ, dactolisib) reverses resistance to doxorubicin (DOX).MethodsOvarian (A2780) and pancreatic (MiaPaca2) cancer cells were used to generate DOX-resistant clones by overexpressing ABCB1 or stepwise treatment of DOX. Intracellular accumulation of DOX was measured by flow cytometry after treatment with BEZ.ResultsBEZ treatment caused an increase in intracellular levels of DOX which was almost identical to the naïve parental cell lines. BEZ was found to be a weak substrate for ABCB1 as demonstrated by minimal increase in ATPase activity. BEZ treatment caused a dose-dependent decrease in cell viability in combination with DOX, which was associated with an increase in cleaved PARP expression in the drug resistant clones.ConclusionsThese results suggest that BEZ is a non-substrate inhibitor of ABCB1 and is able to effectively re-sensitize cells overexpressing ABCB1 to the effects of DOX.General significanceDual PI3 Kinase/mTOR inhibitor, BEZ, has the potential to reverse MDR in cancer patients.