Mesenchymal progenitor cells derived from traumatized muscle enhance neurite growth

The success of peripheral nerve regeneration is governed by the rate and quality of axon bridging and myelination that occurs across the damaged region. Neurite growth and the migration of Schwann cells is regulated by neurotrophic factors produced as the nerve regenerates, and these processes can be enhanced by mesenchymal stem cells (MSCs), which also produce neurotrophic factors and other factors that improve functional tissue regeneration. Our laboratory has recently identified a population of mesenchymal progenitor cells (MPCs) that can be harvested from traumatized muscle tissue debrided and collected during orthopaedic reconstructive surgery. The objective of this study was to determine whether the traumatized muscle‐derived MPCs exhibit neurotrophic function equivalent to that of bone marrow‐derived MSCs. Similar gene‐ and protein‐level expression of specific neurotrophic factors was observed for both cell types, and we localized neurogenic intracellular cell markers (brain‐derived neurotrophic factor and nestin) to a subpopulation of both MPCs and MSCs. Furthermore, we demonstrated that the MPC‐secreted factors were sufficient to enhance in vitro axon growth and cell migration in a chick embryonic dorsal root ganglia (DRG) model. Finally, DRGs in co‐culture with the MPCs appeared to increase their neurotrophic function via soluble factor communication. Our findings suggest that the neurotrophic function of traumatized muscle‐derived MPCs is substantially equivalent to that of the well‐characterized population of bone marrow‐derived MPCs, and suggest that the MPCs may be further developed as a cellular therapy to promote peripheral nerve regeneration. Copyright © 2012 John Wiley & Sons, Ltd.     

Traumatized muscle‐derived multipotent progenitor cells recruit endothelial cells through vascular endothelial growth factor‐A action

Traumatized muscle, such as that debrided from blast injury sites, is considered a promising and convenient tissue source for multipotent progenitor cells (MPCs), a population of adult mesenchymal stem cell (MSC)‐like cells. The present study aimed to assess the regenerative therapeutic potential of human traumatized muscle‐derived MPCs, e.g., for injury repair in the blast‐traumatized extremity, by comparing their pro‐angiogenic potential in vitro and capillary recruitment activity in vivo to those of MSCs isolated from human bone marrow, a widely‐used tissue source. MPCs were tested for their direct and indirect effects on human microvascular endothelial cells (ECs) in vitro. The findings reported here showed that MPC‐conditioned culture medium (MPC‐CM), like MSC‐CM, promoted EC‐cord network branching. Silent (si)RNA‐mediated silencing of vascular endothelial growth factor‐A (VEGF‐A) expression in MPCs attenuated this effect. In a chick embryonic chorioallantoic membrane in vivo angiogenesis assay, MPCs encapsulated in photocrosslinked gelatin scaffold recruited blood vessels more efficiently than either MSCs or human foreskin fibroblasts. Together, these findings support the potential application of traumatized muscle‐derived MPCs in cell‐based regenerative medicine therapies as a result of their influence on EC organization. Copyright © 2017 John Wiley & Sons, Ltd.     

A Gi‐independent mechanism mediating Akt phosphorylation in platelets

Summary. Background: The serine‐threonine kinase Akt plays an important role in regulating platelet activation. Stimulation of platelets with various agonists results in Akt activation as indicated by Akt phosphorylation. However, the mechanisms of Akt phosphorylation in platelets are not completely understood. Objectives and Methods: We used P2Y₁₂ knockout mice to address the role of P2Y₁₂ in Akt phosphorylation in response to thrombin receptors in platelets. Results: Thrombin or the PAR4 thrombin receptor peptide AYPGKF at high concentrations stimulated substantial phosphorylation of Akt residues Thr³⁰⁸ and Ser⁴⁷³ in P2Y₁₂‐deficient platelets. AYPGKF‐induced Akt phosphorylation is enhanced by expression of recombinant human PAR4 cDNA in Chinese hamster ovary (CHO) cells. P2Y₁₂‐independent Akt phosphorylation was not inhibited by integrin inhibitor peptide RGDS or integrin β₃ deficiency. Akt phosphorylation induced by thrombin or AYPGKF in P2Y₁₂‐deficient platelets was inhibited by the calcium chelator dimethyl‐BAPTA, the Src family kinase inhibitor PP2, and PI3K inhibitors, respectively. Conclusions: Our results reveal a novel P2Y₁₂‐independent signaling pathway mediating Akt phosphorylation in response to thrombin receptors.     

Sustained Delivery of VEGF Maintains Innervation and Promotes Reperfusion in Ischemic Skeletal Muscles Via NGF/GDNF Signaling

Tissue reinnervation following trauma, disease, or transplantation often presents a significant challenge. Here, we show that the delivery of vascular endothelial growth factor (VEGF) from alginate hydrogels ameliorates loss of skeletal muscle innervation after ischemic injury by promoting both maintenance and regrowth of damaged axons in mice. Nerve growth factor (NGF) and glial-derived neurotrophic factor (GDNF) mediated VEGF-induced axonal regeneration, and the expression of both is induced by VEGF presentation. Using both in vitro and in vivo modeling approaches, we demonstrate that the activity of NGF and GDNF regulates VEGF-driven angiogenesis, controlling endothelial cell sprouting and blood vessel maturation. Altogether, these studies produce evidence of new mechanisms of VEGF action, further broaden the understanding of the roles of NGF and GDNF in angiogenesis and axonal regeneration, and suggest approaches to improve axonal and ischemic tissue repair therapies.     

Contribution of the P2Y12 receptor‐mediated pathway to platelet hyperreactivity in hypercholesterolemia

Summary. Background: In hypercholesterolemia, platelets demonstrate increased reactivity and promote the development of cardiovascular disease. Objective: This study was carried out to investigate the contribution of the ADP receptor P2Y12‐mediated pathway to platelet hyperreactivity due to hypercholesterolemia. Methods: Low‐density lipoprotein receptor‐deficient mice and C57Bl/6 wild‐type mice were fed on normal chow and high‐fat (Western or Paigen) diets for 8 weeks to generate differently elevated cholesterol levels. P2Y12 receptor‐induced functional responses via G_i signaling were studied ex vivo when washed murine platelets were activated by 2MeSADP and PAR4 agonist AYPGKF in the presence and absence of indomethacin. Platelet aggregation and secretion, α_IIbβ₃ receptor activation and the phosphorylation of extracellular signal‐regulated protein kinase (ERK) and Akt were analyzed. Results: Plasma cholesterol levels ranged from 69 ± 10 to 1011 ± 185 mg dL^?1 depending on diet in mice with different genotypes. Agonist‐dependent aggregation, dense and α‐granule secretion and JON/A binding were gradually and significantly (P < 0.05) augmented at low agonist concentration in correlation with the increasing plasma cholesterol levels, even if elevated thromboxane generation was blocked. These functional responses were induced via increased levels of G_i‐mediated ERK and Akt phosphorylation in hypercholesterolemic mice vs. normocholesterolemic animals. In addition, blocking of the P2Y12 receptor by AR‐C69931MX (Cangrelor) resulted in strongly reduced platelet aggregation in mice with elevated cholesterol levels compared with normocholesterolemic controls. Conclusions: These data revealed that the P2Y12 receptor pathway was substantially involved in platelet hyperreactivity associated with mild and severe hypercholesterolemia.     

Glycogen synthase kinase‐3 negatively regulates tissue factor expression in monocytes interacting with activated platelets

Summary. Background: At the site of vascular injury, monocytes (MN) interacting with activated platelets (PLT) synthesize tissue factor (TF) and promote thrombus formation. Intracellular signals necessary for the expression of TF in MN, in the context of a developing thrombus, remain unknown. Objective: The study was designed to investigate the role of the glycogen synthase kinase 3 (GSK3, a serine‐threonine kinase) downstream insulin receptor pathway, in PLT‐induced TF expression in MN. Methods: To this purpose we used a well‐characterized in vitro model of human MN‐PLT interactions that allows detailed analysis of TF activity, TF protein and gene expression.Results: The results demonstrated that, in MN interacting with activated PLT: (i) TF activity, antigen and mRNA were low until 8–10 h and dramatically increased thereafter, up to 24 h; (ii) according to the kinetics of TF expression in MN, GSK3β undergoes phosphorylation on serine 9, a process associated with down‐regulation of enzyme activity; (iii) pharmacological blockade of GSK3 further increased TF expression and was accompanied by increased accumulation of NF‐kB, in the nucleus; (iv) blockade of phosphoinositide‐3 kinase (PI(3)K) by wortmannin inhibited PLT‐induced TF expression; and (v) according to the established role of the GSK3 downstream insulin receptor, insulin increased PLT‐induced TF expression in a PI(3)K‐dependent manner. Conclusion: GSK3 acts as a molecular brake on the signaling pathway, leading to TF expression in MN interacting with activated PLT. PI(3)K, through Akt‐dependent phosphorylation of GSK3, relieves this brake and allows TF gene expression. This study identifies a novel molecular link between thrombotic risk and metabolic disorders.     

Functional association of phosphoinositide‐3‐kinase with platelet glycoprotein Ibα, the major ligand‐binding subunit of the glycoprotein Ib–IX–V complex

Summary. Background: The adhesion receptor glycoprotein (GP)Ib–IX–V, which binds von Willebrand factor (VWF) and other ligands, initiates platelet activation and thrombus formation at arterial shear rates, and may control other vascular processes, such as coagulation, inflammation, and platelet‐mediated tumor metastasis. The cytoplasmic C‐terminal domain of the ligand‐binding GPIbα subunit contains binding sites for filamin (residues 561–572, critically Phe568/Trp570), 14‐3‐3ζ (involving phosphorylation sites Ser587/590 and Ser609), and the phosphoinositide‐3‐kinase (PI3‐kinase) regulatory subunit, p85. Objectives: We previously showed that, as compared with wild‐type receptor, deleting the contiguous sequence 580–590 or 591–610, but not upstream sequences, of GPIbα expressed as a GPIb–IX complex in Chinese hamster ovary cells inhibited VWF‐dependent Akt phosphorylation, which is used as a read‐out for PI3‐kinase activity. Pulldown experiments using glutathione‐S‐transferase (GST)–p85 or GST–14‐3‐3ζ constructs, and competitive inhibitors of 14‐3‐3ζ binding, suggested an independent association of 14‐3‐3ζ and PI3‐kinase with GPIbα. The objective of this study was to analyze a further panel of GPIbα deletion mutations within residues 580–610. Results: We identified a novel deletion mutant, Δ591–595, that uniquely disrupts 14‐3‐3ζ binding but retains the functional p85/PI3‐kinase association. Deletion of other sequences within the 580–610 region were less discriminatory, and either partially affected p85/PI3‐kinase and 14‐3‐3ζ binding (Δ580–585, Δ586–590, Δ596–600, Δ601–605), or strongly inhibited binding of both proteins (Δ606–610). Conclusions: Together, these findings have significant implications for interpreting the functional role of p85 and/or 14‐3‐3ζ in GPIb‐dependent signaling or platelet functional studies involving truncation of the C‐terminal residues in cell‐based assays and mouse models. The Δ591–595 mutation provides another strategy for determining the function of GPIbα‐associated 14‐3‐3ζ by selective disruption of 14‐3‐3ζ but not p85/PI3‐kinase binding.     

Salvianolic acid A inhibits platelet activation and arterial thrombosis via inhibition of phosphoinositide 3‐kinase

Summary. Background and objective: Salvianolic acid A (SAA) is a water‐soluble component from the root of Salvia miltiorrhiza Bunge, a herb that is widely used for atherothrombotic disease treatment in Asian medicine. As platelets play pivotal roles in atherothrombogenesis, we studied the effect of SAA on platelet activation and its underlying mechanisms. Methods and Results: SAA dose‐dependently inhibited platelet aggregation induced by ADP, thrombin, collagen and U46619. It reduced ADP‐enhanced platelet P‐selectin expression and fibrinogen binding, which consequently hampered ADP‐induced platelet–leukocyte aggregation. SAA also inhibited platelet spreading on fibrinogen, a process mediated by outside‐in signaling. Under an arterial shear rate of 1000 s^?1, SAA decreased platelet adhesion on collagen surfaces by ～ 40%. Western blot analysis showed that SAA, like the phosphoinositide 3‐kinase (PI3K) inhibitors LY294002 and TGX‐221, potently inhibited PI3K, as shown by reduced Akt phosphorylation. The in vitro findings were further evaluated in the mouse model of arterial thrombosis, in which SAA prolonged the mesenteric arterial occlusion time in wild‐type mice (35 ± 2 min without SAA and 56 ± 4 min with SAA; P < 0.01). Interestingly, SAA could even counteract the shortened arterial occlusion time in Ldlr^tm1Her mutant mice (21 ± 2 min without SAA and 45 ± 4 min with SAA; P < 0.01). Conclusions: SAA inhibits platelet activation via the inhibition of PI3K, and attenuates arterial thrombus formation in vivo. Our data suggest that SAA may be developed as a novel therapeutic agent for the prevention of thrombotic disorders.     

Advanced maturation by electrical stimulation: Differences in response between C2C12 and primary muscle progenitor cells

Skeletal muscle tissue engineering still does not result in the desired functional properties and texture as preferred for regenerative medicine and meat production applications. Electrical stimulation has been appropriately used as a tool to advance muscle cell maturation in vitro, thereby simulating nerve stimulation, as part of the muscle cell niche in vivo. We first investigated the effects of electrical stimulation protocols in two‐dimensional (2D) monolayers of C2C12 and translated these protocols to a three‐dimensional (3D) model system, based on a collagen type I/Matrigel^? hydrogel. More importantly, we addressed the ongoing debate of the translation of results found in cell lines (C2C12) to a primary cell source [muscle progenitor cells (MPCs)] in our 3D system. Striking differences in maturation level were found between the different cell sources. Constructs with MPCs were much more mature than C2C12 constructs, based on developed cross‐striations and expression levels of mature myosin heavy chain (MHC) isoforms. Overall, electrical stimulation, when optimally timed, accelerated sarcomere assembly in both 2D and 3D. In addition, MPC constructs were more susceptible to the electrical stimulus, resulting in a shift of MHC expression to slower isoforms. Copyright © 2010 John Wiley & Sons, Ltd.     

Intraperitoneal administration of adipose tissue‐derived stem cells for the rescue of retinal degeneration in a mouse model via indigenous CNTF up‐regulation by IL‐6

As the world's population begins to age, retinal degeneration is an increasing problem, and various treatment modalities are being developed. However, there have been no therapies for degenerative retinal conditions that are not characterized by neovascularization. We investigated whether transplantation of mouse adipose tissue‐derived stem cells (mADSC) into the intraperitoneal space has a rescue effect on NaIO₃‐induced retinal degeneration in mice. In this study, mADSC transplantation recovered visual function and preserved the retinal outer layer structure compared to the control group without any integration of mADSC into the retina. Moreover, endogenous ciliary neurotrophic factor (CNTF) was elevated in the retinas of mADSC‐treated mice. We found that lipopolysaccharide (LPS) or LPS‐stimulated monocyte supernatant induced the secretion of granulocyte colony stimulating factor (GCSF), CD54, CXCL10, interleukin‐6 (IL‐6), and CCL5 from the mADSC by cytokine array. Network inference was conducted to investigate signaling networks related to CNTF regulation. Based on bioinformatics data, the expression of IL‐6 was related to the expression of CNTF. Additionally, intravitreal injection of IL‐6 in rats produced up‐regulation of endogenous CNTF in the retina. mADSC had a rescue effect on retinal degeneration through the up‐regulation of endogenous CNTF by IL‐6. Thus, transplantation of mADSC could be a potential treatment option for retinal degeneration.     

Three‐dimensional porous scaffold allows long‐term wild‐type cell delivery in dystrophic muscle

Duchenne muscular dystrophy (DMD) is caused by the lack of dystrophin; affected muscles are characterized by continuous bouts of muscle degeneration, eventually leading to the exhaustion of the endogenous satellite cell pool. At present, only palliative treatments are available, although several gene and cell therapy‐based approaches are being studied. In this study we proposed to overcome the limitations hampering intramuscular cell injection by using a biomaterial‐based strategy. In particular, we used a three‐dimensional (3D) collagen porous scaffold to deliver myogenic precursor cells (MPCs) in vivo in the mdx murine model of DMD. MPCs, derived from single fibres of wild‐type donors, were expanded in vitro, seeded onto collagen scaffolds and implanted into the tibialis anterior muscles of normal and mdx mice. As a control, cells were delivered via direct intramuscular cell injection in the contralateral muscles. Scaffold‐delivered MPCs displayed lower apoptosis and higher proliferation than injected cells; in terms of dystrophin restoration, collagen scaffolds yielded better results than direct injections. Importantly, time‐course experiments indicated that the scaffolds acted as a cell reservoir, although cell migration was mostly contained within 400 µm from the scaffold–host tissue interface. Copyright © 2010 John Wiley & Sons, Ltd.     

Erythropoietin attenuates hypertrophy of neonatal rat cardiac myocytes induced by angiotensin‐II in vitro

Objective: Erythropoietin (EPO) is a haematopoietic hormone that has been confirmed as a novel cardioprotective agent. In this study, we test the hypothesis that EPO inhibits angiotensin‐II (Ang‐II)‐induced hypertrophy in cultured neonatal rat cardiomyocytes. Material and methods: Cultured neonatal rat cardiomyocytes were used to evaluate the effects of EPO on Ang‐II‐induced hypertrophy in vitro. The surface area and mRNA expression of atrial natriuretic (ANF) myocytes were employed to detect cardiac hypertrophy. A phosphatidylinositol 3′‐kinase (PI3K) inhibitor LY294002 and an endothelial nitric oxide synthase (eNOS) inhibitor l‐NAME were also employed to detect the underlying mechanism of EPO. Intracellular signal molecules, such as Akt (PKB), phosphorylated Akt, eNOS and transforming growth factor‐β1 (TGF‐β1) protein expression were determined by Western blot. Nitric oxide (NO) levels in the supernatant of cultured cardiomyocytes were assayed using an NO assay kit. Results: The results indicate that EPO significantly attenuates Ang‐II‐induced hypertrophy shown as inhibition of increases in cell surface area and ANF mRNA levels. NO production was also increased proportionally in the EPO‐treated group. EPO enhanced Akt activation and eNOS protein expression, whereas LY294002 or l‐NAME partially abolished the anti‐hypertrophic effect of EPO, accompanied by a decrease in Akt activation, eNOS protein expression and/or a reduction of NO production. EPO also down‐regulated the protein expression of TGF‐β1. Conclusion: We conclude that EPO attenuates cardiac hypertrophy via activation of the PI3K‐Akt‐eNOS‐NO pathway and the down‐regulation of TGF‐β1.     

Platelet endothelial cell adhesion molecule‐1 regulates collagen‐stimulated platelet function by modulating the association of phosphatidylinositol 3‐kinase with Grb‐2‐associated binding protein‐1 and linker for activation of T cells

Summary. Background: Platelet activation by collagen depends on signals transduced by the glycoprotein (GP)VI–Fc receptor (FcR)γ‐chain collagen receptor complex, which involves recruitment of phosphatidylinositol 3‐kinase (PI3K) to phosphorylated tyrosines in the linker for activation of T cells (LAT). An interaction between the p85 regulatory subunit of PI3K and the scaffolding molecule Grb‐2‐associated binding protein‐1 (Gab1), which is regulated by binding of the Src homology 2 domain‐containing protein tyrosine phosphatase‐2 (SHP‐2) to Gab1, has been shown in other cell types to sustain PI3K activity to elicit cellular responses. Platelet endothelial cell adhesion molecule‐1 (PECAM‐1) functions as a negative regulator of platelet reactivity and thrombosis, at least in part by inhibiting GPVI–FcRγ‐chain signaling via recruitment of SHP‐2 to phosphorylated immunoreceptor tyrosine‐based inhibitory motifs in PECAM‐1. Objective: To investigate the possibility that PECAM‐1 regulates the formation of the Gab1–p85 signaling complexes, and the potential effect of such interactions on GPVI‐mediated platelet activation in platelets. Methods: The ability of PECAM‐1 signaling to modulate the LAT signalosome was investigated with immunoblotting assays on human platelets and knockout mouse platelets. Results: PECAM‐1‐associated SHP‐2 in collagen‐stimulated platelets binds to p85, which results in diminished levels of association with both Gab1 and LAT and reduced collagen‐stimulated PI3K signaling. We therefore propose that PECAM‐1‐mediated inhibition of GPVI‐dependent platelet responses result, at least in part, from recruitment of SHP‐2–p85 complexes to tyrosine‐phosphorylated PECAM‐1, which diminishes the association of PI3K with activatory signaling molecules, such as Gab1 and LAT.     

A brief primer on the mediational role of BDNF in the exercise‐memory link

One of the most amazing aspects of the human brain is its ability to learn information and use it to change behaviour. A key neurotrophin that influences memory function is brain‐derived neurotrophic factor (BDNF). This review briefly discusses the mechanistic role that BDNF may play in facilitating learning and memory. We also describe the role of exercise on this relationship. As discussed herein, BDNF may influence memory via BDNF‐induced alterations in membrane receptor expression and translocation, as well as activating several pathways (PLC‐y, PI3K, ERK) that act together to facilitate cellular effects that influence synaptic plasticity. Exercise may help to facilitate BDNF expression and its downstream cellular pathways from both direct and indirect mechanisms.     

Regulatory mechanisms of C4b‐binding protein (C4BP)α and β expression in rat hepatocytes by lipopolysaccharide and interleukin‐6

Summary. Background: C4b‐binding protein (C4BP), a multimeric protein structurally composed of α chains (C4BPα) and a β chain (C4BPβ), regulates the anticoagulant activity of protein S (PS). Patients with sepsis have increased levels of plasma C4BP, which appears to be induced by interleukin (IL)‐6. However, it is not fully understood how lipopolysaccharide (LPS) and IL‐6 affect the plasma C4BP antigen level and C4BPα and C4BPβ expression in hepatocytes. Objectives: To assess the effect of LPS and IL‐6 on plasma C4BP, PS–C4BP complex levels, PS activity, and C4BP expression by rat liver in vivo and on C4BP expression by isolated rat hepatocytes in vitro. Results: Plasma C4BP antigen level transiently decreased from 2 to 12 h after LPS (2 mg kg^?1) injection, and then it abruptly increased up to 24 h after LPS injection. Plasma C4BP antigen level increased until 8 h after IL‐6 (10 μg kg^?1) injection, and then gradually decreased up to 24 h after IL‐6 injection. LPS significantly decreased the protein and mRNA expression of both C4BPα and C4BPβ in rat hepatocytes, and this effect was inhibited by NFκB and MEK/ERK inhibitors. IL‐6 mediated increase in C4BPβ expression in rat hepatocytes, which leads to increased plasma PS–C4BP complex level and to decreased plasma PS activity, was inhibited by inhibition of STAT‐3. Conclusion: LPS decreases both C4BPα and C4BPβ expression via the NFκB and MEK/ERK pathways, whereas IL‐6 specifically increases C4BPβ expression via the STAT‐3 pathway, causing an increase in plasma PS–C4BP complex, and thus decreasing the anticoagulant activity of PS.     

Rapid onset of perfused blood vessels after implantation of ECFCs and MPCs in collagen,PuraMatrix and fibrin provisional matrices

We developed an in vivo vascularization model in which human endothelial colony‐forming cells (ECFCs) and human mesenchymal progenitor cells (MPCs) form blood vessel networks when co‐injected (ECFC + MPC) into nude mice in rat tail type I collagen, bovine fibrin or synthetic peptide PuraMatrix matrices. We used three approaches to determine the onset of functional vascularization when ECFC + MPC suspended in these matrices were implanted in vivo. The first was immunohistochemistry to detect vessels lined by human endothelial cells and filled with red blood cells. The second was in vivo vascular staining by tail vein injection of a mixture of Ulex europaeus agglutinin I (UEA‐I), a lectin specific for human endothelium, and Griffonia simplicifolia isolectin B₄ (GS‐IB₄), a lectin specific for rodent endothelium. The third approach employed contrast‐enhanced ultrasound to measure the perfusion volumes of implants in individual animals over time. Human endothelial‐lined tubular structures were detected in vivo on days 1 and 2 after implantation, with perfused human vessels detected on days 3 and 4. Contrast‐enhanced ultrasound revealed significant perfusion of ECFC + MPC/collagen implants on days 1–4, at up to 14% perfused vascular volume. ECFC + MPC implanted in fibrin and PuraMatrix matrices also supported perfusion at day 1, as assessed by ultrasound (at 12% and 23% perfused vascular volume, respectively). This model demonstrates that ECFC + MPC suspended in any of the three matrices initiated a rapid onset of vascularization. We propose that ECFC + MPC delivered in vivo provide a means to achieve rapid perfusion of tissue‐engineered organs or for in situ tissue repair. Copyright © 2013 John Wiley & Sons, Ltd.     

Phosphatidylinositol 3‐kinases inhibitor LY294002 potentiates the cytotoxic effects of doxorubicin,vincristine, and etoposide in a panel of cancer cell lines

Many novel therapeutic approaches to overcome chemoresistance have involved targeting specific signaling pathways such as the phosphatidylinositol 3‐kinase (PI3K) pathway. PI3K is a known stress response pathway which is involved in the regulation of cell survival, apoptosis, and growth. Inhibition of this pathway may possibly restore or augment the effectiveness of chemotherapy. Using three human malignant cell lines, we examined the effects of LY294002 (PI3K inhibitor) on chemotherapeutic agent‐induced apoptosis and cytotoxicity. An antimicrotubule agent vincristine, a topoisomerase II inhibitor etoposide, and a DNA cross‐linking agent doxorubicin were used accompanied with LY294002. Cell viability was determined by MTT assay, and the induction of apoptosis was assessed by immunoblotting of caspase‐3. Blocking the PI3K/Akt cascade with a PI3K inhibitor LY294002 (10 μM) increased the cytotoxic effect of vincristine and doxorubicin on SK‐OV‐3 cell line. Furthermore, LY294002 showed a greater promoting effect in etoposide‐ and doxorubicin‐induced cytotoxicity on MDA‐MB‐468 and A549 cells. The quantity of cleaved caspase‐3 in cancer cells that had combination therapy was increased compared with that in the cells treated with each drug alone. We suggest that inhibitors of the PI3K/Akt pathway in combination with chemotherapeutic agents may induce cell death effectively and be a potent modality to treat various types of cancer. The effectiveness of such combination therapy is depending to the used cell line and class of anticancer drug.