ABT-263 enhances sorafenib-induced apoptosis associated with Akt activity and the expression of Bax and p21(CIP1/WAF1) in human cancer cells

BACKGROUND AND PURPOSE

Sorafenib, a potent inhibitor that targets several kinases associated with tumourigenesis and cell survival, has been approved for clinical treatment as a single agent. However, combining sorafenib with other agents improves its anti-tumour efficacy in various preclinical tumour models. ABT-263, a second-generation BH3 mimic, binds to the anti-apoptotic family members Bcl-2, Bcl-xL and Bcl-w, and has been demonstrated to enhance TNFSF10 (TRAIL)-induced apoptosis in human hepatocarcinoma cells. Hence, we investigated the effects of ABT-263 treatment combined with sorafenib.

EXPERIMENTAL APPROACH

The effects of ABT-263 combined with sorafenib were investigated in vitro, on cell viability, clone formation and apoptosis, and the mechanism examined using western blot and flow cytometry. This combination was also evaluated in vivo, in a mouse xenograft model; tumour growth, volume and weights were measured and a TUNEL assay performed.

KEY RESULTS

ABT-263 enhanced sorafenib-induced apoptosis while sparing non-tumourigenic cells. Although ABT-263 plus sorafenib significantly stimulated intracellular reactive oxygen species production and subsequent mitochondrial depolarization, this was not sufficient to trigger cell apoptosis. ABT-263 plus sorafenib significantly decreased Akt activity, which was, at least partly, involved in its effect on apoptosis. Bax and p21 (CIP1/WAF1) were shown to play a critical role in ABT-263 plus sorafenib-induced apoptosis. Combining sorafenib with ABT-263 dramatically increased its efficacy in vivo.

CONCLUSION AND IMPLICATIONS

The anti-tumour activity of ABT-263 plus sorafenib may involve the induction of intrinsic cell apoptosis via inhibition of Akt, and reduced Bax and p21 expression. Our findings offer a novel effective therapeutic strategy for tumour treatment.     

Disruption of cellular cholesterol transport and homeostasis as a novel mechanism of action of membrane-targeted alkylphospholipid analogues

Background and purpose:

Alkylphospholipid (APL) analogues constitute a new class of synthetic anti-tumour agents that act directly on cell membranes. We have previously demonstrated that hexadecylphosphocholine (HePC) alters intracellular cholesterol traffic and metabolism in HepG2 cells. We now extended our studies to analyse the effects of other clinically relevant APLs, such as edelfosine, erucylphosphocholine and perifosine on intracellular cholesterol homeostasis.

Experimental approach:

Using radiolabelled substrates we determined the effect of APLs on cholesterol metabolism and cholesterol traffic from the plasma membrane to the endoplasmic reticulum (ER). Protein levels and gene expression of the main proteins involved in cholesterol homeostasis were analysed by Western blot and RT-PCR respectively. Membrane raft and non-raft fractions were isolated from HepG2 cells by a detergent-free method.

Key results:

All APLs inhibited the transport of cholesterol from the plasma membrane to the ER, which induced a significant cholesterogenic response in HepG2 cells. This response involved an increased gene expression and higher levels of several proteins related to the biosynthesis and the receptor-mediated uptake of cholesterol. Cell exposure to the APL-representative HePC enhanced the content of cholesterol mainly in the membrane raft fractions, compared with the untreated cells.

Conclusions and implications:

Membrane-targeted APLs exhibited a novel and common mechanism of action, through disruption of cholesterol homeostasis, which in turn affected specific lipid microdomains of cellular membranes.     

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.     

Leukamenin F suppresses liver fibrogenesis by inhibiting both hepatic stellate cell proliferation and extracellular matrix production

Aim:

To investigate the inhibitory effect of the natural product Leukamenin F on liver fibrosis and explore its potential underlying mechanisms.

Methods:

Carbon tetrachloride (CCl₄)-treated mouse model in vivo and in hepatic stellate cells (HSC) in vitro were used. The effect on CCl₄-induced liver fibrosis was studied using histochemical and biochemical analysis, while the inhibition on HSC was assessed using cell proliferation/apoptosis assay and collagen I production using real-time PCR. The inhibitory effects of Leukamenin F on Akt/mTOR/p70S6K and TGFβ/Smad pathways was studied using Western blot and cell image analysis.

Results:

Leukamenin F (0.1–1 mg/kg, ip, q.d.×28) significantly reduced α-SMA and collagen specific Sirius red staining areas in CCl₄ -treated mouse livers. This compound at 1–2 μmol/L dose-dependently inhibited α-SMA expression, cell proliferation and type I procollagen mRNA expression in activated HSC. Furthermore it inhibited the Akt/mTOR/p70S6K pathway and suppressed TGFβ -induced Smad2/Smad3 phosphorylation and nuclear translocation in HSC.

Conclusion:

Our results demonstrated that Leukamenin F could attenuate CCl₄-induced liver fibrogenesis in mice as an efficient inhibitor against both HSC proliferation and ECM production. This natural product provides a valuable structural hint for the development of anti-liver fibrosis reagents.     

Solid lipid nanoparticles of cholesteryl butyrate inhibit the proliferation of cancer cells in vitro and in vivo models

BACKGROUND AND PURPOSE

Solid lipid nanoparticles containing cholesteryl butyrate (cholbut SLN) can be a delivery system for the anti-cancer drug butyrate. These nanoparticles inhibit adhesion of polymorphonuclear and tumour cells to endothelial cells and migration of tumour cells, suggesting that they may act as anti-inflammatory and anti-tumour agents. Here we have evaluated the effects of cholbut SLN on tumour cell growth using in vitro and in vivo models.

EXPERIMENTAL APPROACH

Cholbut SLNs were incubated with cultures of four tumour cell lines, and cell growth was analysed by assessing viability, clonogenic capacity and cell cycle. Effects on intracellular signalling was assessed by Western blot analysis of Akt expression. The in vivo anti-tumour activity was measured in two models of PC-3 cell xenografts in SCID/Beige mice.

KEY RESULTS

Cholbut SLN inhibited tumour cell line viability, clonogenic activity, Akt phosphorylation and cell cycle progression. In mice injected i.v. with PC3-Luc cells and treated with cholbut SLN, . in vivo optical imaging and histological analysis showed no metastases in the lungs of the treated mice. In another set of mice injected s.c. with PC-3 cells and treated with cholbut SLN when the tumour diameter reached 2 mm, analysis of the tumour dimensions showed that treatment with cholbut SLN substantially delayed tumour growth.

CONCLUSION AND IMPLICATIONS

Cholbut SLN were effective in inhibiting tumour growth in vitro and in vivo. These effects may involve, in part, inhibition of Akt phosphorylation, which adds another mechanism to the activity of this multipotent drug.     

Luteolin is effective in the non-small cell lung cancer model with L858R/T790M EGF receptor mutation and erlotinib resistance

Background and Purpose

Non-small cell lung cancer (NSCLC) is one of the most commonly diagnosed malignancies in the world. Small-molecule inhibitors of the EGF receptor''s tyrosine kinase domain (TKIs), including gefitinib and erlotinib, have been widely used for treating NSCLC. Unfortunately, nearly all patients after initially experiencing a marked improvement while on these drugs, eventually progress to acquire resistance to TKIs. Because there is no effective therapeutic strategy to treat TKI-resistant NSCLC, we evaluated the effects of luteolin, a naturally occurring flavanoid, on T790M mutant NSCLC cells.

Experimental Approach

The effect of luteolin on the viability of NSCLC and normal cell lines was investigated using the Cell Counting Kit-8 (CCK-8) assay. Luteolin-induced apoptosis was assessed by bivariate FITC-annexin V/PI assay, and Western blots were used to measured apoptotic proteins. Co-immunoprecipitation was used to determine the effect of luteolin on the interaction between Hsp90 and mutant EGF receptors. The effect of luteolin on the Akt/mTOR pathway was studied using Western blotting analysis. Its anti-tumour efficacy in vivo was examined in a mouse xenograft model.

Key Results

Luteolin exerted significant anti-tumourigenic effects on the EGF receptor L858R/T790M mutation and erlotinib-resistant NSCLC both at the cellular and animal levels. Mechanistically, luteolin induced degradation of the EGF receptor by inhibiting the association of Hsp90 with the mutant EGF receptor, and, therefore, prevented PI3K/Akt/mTOR signalling, which resulted in NSCLC cell apoptosis.

Conclusion and Implications

Luteolin may be a potential candidate for NSCLC therapy, especially for treatment of patients with acquired erlotinib-resistant NSCLC.     

Induction of a reversible,non-cytotoxic S-phase delay by resveratrol: implications for a mechanism of lifespan prolongation and cancer protection

Background and purpose:

Resveratrol (RES) has been shown to prolong lifespan and prevent cancer formation. At present, the precise cellular mechanisms of RES actions are still not clearly understood, and this is the focus of this study.

Experimental approach:

Using human hepatocellular carcinoma-derived HepG2 cells as a model, we studied RES-induced changes in cell growth, cell cycle progression and apoptosis.

Key results:

RES at lower concentrations induced a strong but reversible S-phase delay and mild DNA synthesis inhibition, yet without causing apoptotic or necrotic cell death. At high concentrations, RES induced apoptosis, which is mainly mediated by the mitochondrial pathway. Overall, RES was a relatively weak apoptotic agent. Mechanistically, MEK inhibition was identified as an important early signalling event for RES-induced apoptosis. In comparison, activation of CDK2 and checkpoint kinase 2, and inhibition of phosphatidylinositol 3′-kinase/Akt signalling pathway contributed to the induction by RES of a reversible, non-cytotoxic S-phase delay.

Conclusion and implications:

It is hypothesized that the induction of a non-cytotoxic S-phase delay may represent a useful mechanistic strategy for lifespan prolongation and cancer prevention. When cell cycles are selectively slowed down in the S phase, it would cumulatively increase the total lifespan of an organism if the total numbers of cell divisions of a given organism are assumed to remain basically constant. Likewise, when cells proceed through the cell cycles at a reduced pace during DNA replication, it may allow cells more time to repair the damaged DNA, and thereby reduce the chances for mutagenesis and tumour initiation.     

The N-terminal region of the dopamine D2 receptor, a rhodopsin-like GPCR, regulates correct integration into the plasma membrane and endocytic routes

BACKGROUND AND PURPOSE

Functional roles of the N-terminal region of rhodopsin-like GPCR family remain unclear. Using dopamine D₂ and D₃ receptors as a model system, we probed the roles of the N-terminal region in the signalling, intracellular trafficking of receptor proteins, and explored the critical factors that determine the functionality of the N-terminal region.

EXPERIMENTAL APPROACH

The N-terminal region of the D₂ receptor was gradually shortened or switched with that of the D₃ receptor or a non-specific sequence (FLAG), or potential N-terminal glycosylation sites were mutated. Effects of these manipulations on surface expression, internalization, post-endocytic behaviours and signalling were determined.

KEY RESULTS

Shortening the N-terminal region of the D₂ receptor enhanced receptor internalization and impaired surface expression and signalling; ligand binding, desensitization and down-regulation were not affected but their association with a particular microdomain, caveolae, was disrupted. Replacement of critical residues within the N-terminal region with the FLAG epitope failed to restore surface expression but partially restored the altered internalization and signalling. When the N-terminal regions were switched between D₂ and D₃ receptors, cell surface expression pattern of each receptor was switched. Mutations of potential N-terminal glycosylation sites inhibited surface expression but enhanced internalization of D₂ receptors.

CONCLUSIONS AND IMPLICATIONS

Shortening of N-terminus or mutation of glycosylation sites located within the N-terminus enhanced receptor internalization but impaired the surface expression of D₂ receptors. The N-terminal region of the D₂ receptor, in a sequence-specific manner, controls the receptor''s conformation and integration into the plasma membrane, which determine its subcellular localization, intracellular trafficking and signalling properties.     

Angiopoietin-1 protects myocardial endothelial cell function blunted by angiopoietin-2 and high glucose condition

Aim:

To evaluate the effects of angiopoietin-1 (Ang-1) on myocardial endothelial cell function under high glucose (HG) condition.

Methods:

Mouse heart myocardial endothelial cells (MHMECs) were cultured and incubated under HG (25 mmol/L) or normal glucose (NG, 5 mmol/L) conditions for 72 h. MTT was used to determine cellular viability, and TUNEL assay and caspase-3 enzyme linked immunosorbent assays were used to assay endothelial apoptosis induced by serum starvation. Immunoprecipitation and Western blot analysis were used to analyze protein phosphorylation and expression. Endothelial tube formation was used as an in vitro assay for angiogenesis.

Results:

Exposure of MHMECs to HG resulted in dramatic decreases in phosphorylation of the Tie-2 receptor and its downstream signaling partners, Akt/eNOS, compared to that under NG conditions. Ang-1 (250 ng/mL) increased Tie-2 activation, inhibited cell apoptosis, and promoted angiogenesis. Ang-1-mediated protection of endothelial function was blunted by Ang-2 (25 ng/mL).

Conclusion:

Ang-1 activates the Tie-2 pathway and restores hyperglycemia-induced myocardial microvascular endothelial dysfunction. This suggests a protective role of Ang-1 in the ischemic myocardium, particularly in hearts affected by hyperglycemia or diabetes.     

Salidroside exerts angiogenic and cytoprotective effects on human bone marrowderived endothelial progenitor cells via Akt/mTOR/p70S6K and MAPK signalling pathways

Background and Purpose

With the increase of age, increased susceptibility to apoptosis and senescence may contribute to proliferative and functional impairment of endothelial progenitor cells (EPCs). The aim of this study was to investigate whether salidroside (SAL) can induce angiogenic differentiation and inhibit oxidative stress-induced apoptosis in bone marrow-derived EPCs (BM-EPCs), and if so, through what mechanism.

Experimental Approach

BM-EPCs were isolated and treated with different concentrations of SAL for up to 4 days. Cell proliferation, migration and tube formation ability were detected by DNA content quantification, transwell assay and Matrigel-based angiogenesis assay. Gene and protein expression were assessed by qRT-PCR and Western blot respectively.

Key Results

Treatment with SAL promoted cellular proliferation and angiogenic differentiation of BM-EPCs, and increased VEGF and NO secretion, which in turn mediated the enhanced angiogenic differentiation of BM-EPCs. Furthermore, SAL significantly attenuated hydrogen peroxide (H₂O₂)-induced cell apoptosis, reduced the intracellular level of reactive oxygen species and restored the mitochondrial membrane potential of BM-EPCs. Moreover, SAL stimulated the phosphorylation of Akt, mammalian target of rapamycin and p70 S6 kinase, as well as ERK1/2, which is associated with cell migration and capillary tube formation. Additionally, SAL reversed the phosphorylation of JNK and p38 MAPK induced by H₂O₂ and suppressed the changes in the Bax/Bcl-xL ratio observed after stimulation with H₂O₂.

Conclusions and Implications

These findings identify novel mechanisms that regulate EPC function and suggest that SAL has therapeutic potential as a new agent to enhance vasculogenesis as well as protect against oxidative endothelial injury.     

The anti-tumour compound, RH1, causes mitochondria-mediated apoptosis by activating c-Jun N-terminal kinase

BACKGROUND AND PURPOSE

2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone (RH1) is a bioreductive agent that is activated by the two-electron reductase NAD(P)H quinone oxidoreductase 1 (NQO1). Although the cytotoxic efficacy of RH1 against tumours has been studied extensively, the molecular mechanisms underlying this anti-cancer activity have not yet been fully elucidated.

EXPERIMENTAL APPROACH

2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone-induced apoptosis and related signalling pathways in NQO1-negative and NQO1-overexpressing cells were evaluated. The role of p53 in RH1-induced cell death was investigated using parental and p53-deficient RKO human colorectal cancer cells by assaying clonogenic cell survival. Specific inhibitors and siRNAs targeting factors involved in RH1-induced apoptosis were used to clarify the roles played by such factors in RH1-activated apoptotic signalling pathways.

KEY RESULTS

2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone induced apoptosis and clonogenic death, dependent on NQO1 and p53. Treatment of NQO1-overexpressing cells with RH1 caused rapid disruption of mitochondrial membrane potential, nuclear translocation of apoptosis-inducing factor (AIF) and endonuclease G (Endo G) from mitochondria, and subsequent caspase-independent apoptotic cell death. siRNA targeting AIF and Endo G effectively attenuated RH1-induced apoptotic cell death. Moreover, RH1 induced cleavage of Bax, which targets mitochondria. RH1 significantly activated the c-Jun N-terminal kinase (JNK) pathway, and inhibition of this pathway suppressed RH1-induced mitochondria-mediated apoptosis. RH1-induced generation and mitochondrial translocation of cleaved Bax were blocked by the JNK inhibitor, SP600125. Inhibition of JNK with SP600125 attenuated the mitochondrial translocation of JNK.

CONCLUSIONS AND IMPLICATIONS

2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone activated JNK, resulting in mitochondria-mediated apoptotic cell death that was NQO1-dependent.     

Promotion of self-renewal of embryonic stem cells by midkine

Aim:

To characterize the expression and function of midkine (MK) in an in vitro embryonic stem cell (ESC) culture system.

Methods:

To investigate the potential roles of MK, the expression of MK in ESCs was evaluated by RT-PCR and immunocytochemistry. The effects of MK on the self-renewal of ESCs were measured using alkaline phosphatase assays, immunocytochemistry, RT-PCR and colony-forming assays. The mechanism of the growth-promoting effect of MK in mESCs was assessed by cell cycle analysis and Western blot analysis.

Results:

MK is expressed in mouse embryonic stem cells (mESCs), human embryonic stem cells (hESCs) and mouse embryonic fibroblasts (MEFs). MK promotes proliferation and self-renewal of mESCs both in feeder and feeder free culture systems. It also promotes self-renewal and proliferation of hESCs. Further study showed that MK promotes the growth of mESCs by inhibiting apoptosis while accelerating the progression toward the S phase, and enhances mESC self-renewal through PI3K/Akt signaling pathway.

Conclusion:

MK plays profound roles in ESCs. MK/PTPζ signaling pathway is a novel pathway in the signal network maintaining pluripotency of ESCs. The results extend our knowledge on pluripotency control of ESCs and the relationship between ESCs and cancers.     

Bupivacaine causes cytotoxicity in mouse C2C12 myoblast cells： involvement of ERK and Akt signaling pathways

Aim:

The adverse effects of local anesthetics (LAs) on wound healing at surgical sites have been suggested, and may be related to their cytotoxicity. This study was aimed to compare the cellular toxicity of bupivacaine and lidocaine (two well-known LAs), and to explore the molecular mechanism(s).

Methods:

Toxicity of bupivacaine and lidocaine was assessed in cultured mouse C2C12 myoblasts by cell viability and apoptosis assays. Effects of LAs on extracellular signal-regulated kinase (ERK) and protein kinase B (Akt) activation, which are essential for cell proliferation and survival, were evaluated by immunoblotting.

Results:

Both LAs, especially bupivacaine, prevented cell growth and caused cell death in a dose-dependent manner. The half maximal inhibitory concentrations (IC₅₀) for bupivacaine and lidocaine were 0.49±0.04 and 3.37±0.53 mmol/L, respectively. When applied at the same dilutions of commercially available preparations, the apoptotic effect induced by bupivacaine was more severe than that of lidocaine in C2C12 cells. Furthermore, bupivacaine significantly diminished the ERK activation, which may underlie its anti-proliferative actions. Both LAs suppressed Akt activation, which correlated with their effects on apoptosis.

Conclusion:

Our study demonstrated that, when used at the same dilutions from clinically relevant concentrations, bupivacaine is more cytotoxic than lidocaine in vitro. Anti-proliferation and cell death with concomitant apoptosis mediated by bupivacaine may offer an explanation for its adverse effects in vivo (eg slowing wound healing at the surgical sites). A less toxic, long-acting anesthetic may be needed.     

Down-regulation of the Notch pathway mediated by a γ-secretase inhibitor induces anti-tumour effects in mouse models of T-cell leukaemia

Background and purpose:

γ-Secretase inhibitors (GSIs) block NOTCH receptor cleavage and pathway activation and have been under clinical evaluation for the treatment of malignancies such as T-cell acute lymphoblastic leukaemia (T-ALL). The ability of GSIs to decrease T-ALL cell viability in vitro is a slow process requiring >8 days, however, such treatment durations are not well tolerated in vivo. Here we study GSI''s effect on tumour and normal cellular processes to optimize dosing regimens for anti-tumour efficacy.

Experimental approach:

Inhibition of the Notch pathway in mouse intestinal epithelium was used to evaluate the effect of GSIs and guide the design of dosing regimens for xenograft models. Serum Aβ₄₀ and Notch target gene modulation in tumours were used to evaluate the degree and duration of target inhibition. Pharmacokinetic and pharmacodynamic correlations with biochemical, immunohistochemical and profiling data were used to demonstrate GSI mechanism of action in xenograft tumours.

Key results:

Three days of >70% Notch pathway inhibition was sufficient to provide an anti-tumour effect and was well tolerated. GSI-induced conversion of mouse epithelial cells to a secretory lineage was time- and dose-dependent. Anti-tumour efficacy was associated with cell cycle arrest and apoptosis that was in part due to Notch-dependent regulation of mitochondrial homeostasis.

Conclusions and implications:

Intermittent but potent inhibition of Notch signalling is sufficient for anti-tumour efficacy in these T-ALL models. These findings provide support for the use of GSI in Notch-dependent malignancies and that clinical benefits may be derived from transient but potent inhibition of Notch.     

Telmisartan protects central neurons against nutrient deprivation-induced apoptosis in vitro through activation of PPARγ and the Akt/GSK-3β pathway

Aim： To determine whether angiotensin II receptor blockers （ARBs） could protect central neurons against nutrient deprivation-induced apoptosis in vitro and to elucidate the underlying mechanisms.
Methods： Primary rat cerebellar granule cells （CGCs） underwent B27 （a serum substitute） deprivation for 24 h to induce neurotoxicity, and cell viability was analyzed using LDH assay and WST-1 assay. DNA laddering assay and TUNEL assay were used to detect cell apoptosis. The expression of caspase-3 and Bcl-2, and the phosphorylation of Akt and GSK-3β were detected using Western blot analysis. AT1a mRNA expression was determined using RT-PCR analysis.
Results： B27 deprivation significantly increased the apoptosis of CGCs, as demonstrated by LDH release, DNA laddering, caspase-3 activation and positive TUNEL staining. Pretreatment with 10 μmol/L ARBs （telmisartan, candesartan or losartan） partially blocked B27 deprivation-induced apoptosis of CGCs with telmisartan being the most effective one. B27 deprivation markedly increased the expression of AT1a receptor in CGCs, inhibited Akt and GSK-3β activation, decreased Bcl-2 level, and activated caspase-3, which were reversed by pretreatment with 1 μmol/L telmisartan. In addition, pretreatment with 10 μmol/L PPARγ agonist pioglitazone was more effective in protecting CGCs against B27 deprivation-induced apoptosis, whereas pretreatment with 20 μmol/L PPARγ antagonist GW9662 abolished all the effects of telmisartan in CGCs deprived of B27.
Conclusion： ARBs, in particular telmisartan, can protect the nutrient deprivation-induced apoptosis of CGCs in vitro through activation of PPARγ and the Akt/GSK-3β pathway.     

Recombinant viral protein promotes apoptosis and suppresses invasion of ovarian adenocarcinoma cells by targeting α5β1 integrin to down-regulate Akt and MMP-2

BACKGROUND AND PURPOSE

As prognosis for patients with metastatic ovarian cancer is generally poor, advances in treatment are needed. Here, we studied the mechanism of action of a recombinant viral capsid protein (rVP1) and explored its effect against ovarian tumour growth and metastasis in vivo.

EXPERIMENTAL APPROACH

The human ovarian cancer cell line SKOV3 and BALB/cAnN-Foxn1 female nude mice were used. Effects of rVP1 on the viability, invasive ability, matrix metalloproteinase (MMP)-2 activity and cancer cell proliferation and metastasis were determined by cell proliferation assay, Matrigel invasion assay, gelatin zymographic analysis, as well as bioluminescence imaging and immunohistological analysis in xenograft mouse models respectively. Levels of total and phosphorylated focal adhesion kinase (FAK), PKB/Akt, phosphatase and tensin homologue (PTEN) and glycogen synthase kinase-3β (GSK-3β) were detected by Western blotting.

KEY RESULTS

rVP1 promoted apoptosis and decreased invasion of human ovarian cancer cells. This effect of rVP1 was accompanied by activation of PTEN and GSK-3β as well as down-regulation of FAK, Akt and MMP-2. Anti-integrin antibodies or overexpression of constitutively active Akt reversed the cellular effects of rVP1. Orthotopic and intraperitoneal xenograft mouse models demonstrated that rVP1 attenuated survival and metastasis of human ovarian cancer SKOV3 cell line in vivo through selective regulation of Akt and GSK-3β activity as shown by bioluminescence imaging of mice and immunohistochemical analysis.

CONCLUSION AND IMPLICATIONS

These results indicate that negative regulation of Akt signalling and MMP-2 by rVP1 may have the potential to suppress ovarian tumour growth and metastasis in vivo.