LY294002 In Combination Research Articles

Autophagy activation in breast cancer cells in vitro after the treatment with PI3K/AKT/mTOR inhibitors

Introduction. Current chemotherapy of breast cancer has a wide range of disadvantages, in particular, the development of therapy-related infections and hormonal imbalance. Combination of main cytostatic with glucocorticoids allows to broaden its therapeutic interval and to decrease the total toxicity of the treatment. However, long-term treatment with glucocorticoids leads to the development of severe side effects via activation of multiple molecular mechanisms. Thus, glucocorticoids activate prosurvival mTOR-dependent autophagy. Therefore, the evaluation of PI3K (phosphoinositide 3-kinases) / Akt (protein kinase B) / mTOR (mammalian target of rapamycin) inhibitors as adjuvants for breast cancer therapy is important for optimization of treatment protocol.Aim. Analysis of the effects of PI3K/Akt/mTOR inhibitors, rapamycin, wortmannin and LY-294002 in combination with glucocorticoids in breast cancer cell lines of different subtypes.Materials and methods. We demonstrated the inhibition of PI3K/Akt/mTOR signaling and the autophagy induction after the treatment of breast cancer cells with rapamycin, wortmannin and LY-294002 by Western blotting analysis of Beclin-1, phospho-Beclin-1 (Ser93 and Ser30).Conclusion. PI3K/Akt/mTOR inhibitors in combination with Dexamethasone cooperatively inhibited mTOR signaling and activated autophagy in breast cancer cells in vitro.

Effect of Oxaliplatin, Olaparib and LY294002 in Combination on Triple-Negative Breast Cancer Cells.

Triple-negative breast cancer (TNBC) has a poor prognosis as the therapy has several limitations, most importantly, treatment resistance. In this study we examined the different responses of triple-negative breast cancer line MDA-MB-231 and hormone receptor-positive breast cancer line MCF7 to a combined treatment including olaparib, a poly-(ADP ribose) polymerase (PARP) inhibitor, oxaliplatin, a third-generation platinum compound and LY294002, an Akt pathway inhibitor. We applied the drugs in a single, therapeutically relevant concentration individually and in all possible combinations, and we assessed the viability, type of cell death, reactive oxygen species production, cell-cycle phases, colony formation and invasive growth. In agreement with the literature, the MDA-MB-231 cells were more treatment resistant than the MCF7 cells. However, and in contrast with the findings of others, we detected no synergistic effect between olaparib and oxaliplatin, and we found that the Akt pathway inhibitor augmented the cytostatic properties of the platinum compound and/or prevented the cytoprotective effects of PARP inhibition. Our results suggest that, at therapeutically relevant concentrations, the cytotoxicity of the platinum compound dominated over that of the PARP inhibitor and the PI3K inhibitor, even though a regression-based model could have indicated an overall synergy at lower and/or higher concentrations.

Autophagy Inhibition Enhances CDK4/6 Inhibitor-Induced Apoptosis in t(8;21) Acute Myeloid Leukemia Cells

Acute myeloid leukemia (AML) is a genetically and clinically heterogeneous disease. Although t(8;21) AML patients have a more favorable prognosis than other cytogenetic subgroups, nearly 40% of t(8;21) AML patients experience relapse. Therefore, novel therapeutic approaches based on a better understanding of the biology of t(8;21) AML need to be developed. In this study, at first, we re-analyzed the sequencing data of 149 pediatric t(8;21) AML patients from St. Jude Children's Research Hospital tissue resource core facility and the JPLSG AML-05 study, and 134 adult t(8;21) AML patients from CALGB/Alliance trials and the University Hospital of Ulm. In pediatric patients, 13 CCND2 mutations were detected in 11 patients (11/149, 7.4%), and in adult patients, 14 CCND2 mutations were detected in 12 patients (12/134, 9.0%). In both cohorts, CCND2 mutations were located on the PEST domain, suggesting that the mutations stabilize the cyclin D2 protein. Next, we compared CCND2 mRNA expression between t(8;21) AML patients (n=24) and non-t(8;21) AML patients (n=163) using the TARGET AML cohort. In non-t(8;21) AML patients, CCND2 expression varied from low to high levels, whereas in t(8;21) AML patients, CCND2 expression was restricted to higher levels. Consistently, CCND2 expression was higher in t(8;21) AML cell lines (n=2: Kasumi-1 and SKNO-1), compared with non-t(8;21) AML cell lines (n=32). Kasumi-1 cells transfected with shCCND2 showed cell cycle arrest at G1 phase and impaired cell proliferation. These results suggest that the frequency of CCND2 mutations and CCND2 expression are increased in t(8;21) AML, and high CCND2 expression plays an important role in t(8;21) AML cell proliferation. Because CCND2 is not a druggable target, we examined the effect of CDK4/6 inhibitors (palbociclib and abemaciclib) on t(8;21) AML cells. Analysis of 19 AML cell lines showed that t(8;21) AML cells had lower IC50 values for CDK4/6 inhibitors than non-t(8;21) AML cells. CDK4/6 inhibitors caused cell cycle arrest at G1 phase and impaired cell proliferation in t(8;21) AML cells. To identify potential therapeutic approaches in combination with CDK4/6 inhibitors in t(8;21) AML, we performed microarray analysis and examined the effects of CDK4/6 inhibition. In addition to the pathways associated with the cell cycle (regulation of sister chromatid separation, retinoblastoma gene, and cell cycle), the MAP-ERK and PI3K-AKT-mTOR signaling pathways were downregulated by CDK4/6 inhibition. Because these pathways are involved in autophagy regulation via mTOR, we focused on examining autophagy in subsequent experiments. Assessment of the effect of CDK4/6 inhibition on autophagy in t(8;21) AML cells showed that the CDK4/6 inhibitor (abemaciclib) treatment induced LC3B-I to LC3B-II conversion in both Kasumi-1 and SKNO-1 cells. Transmission electron microscopic examination of autophagosome formation detected a large number of autophagosomes in the cytoplasm of Kasumi-1 and SKNO-1 cells treated with abemaciclib, whereas few autophagosomes were detected in control samples. These results suggest that autophagy is induced by CDK4/6 inhibition in t(8;21) AML cells. Autophagy is involved in the resistance to chemotherapy in cancer cells, therefore, we hypothesized that autophagy inhibition may be a promising therapeutic approach. Treatment of t(8;21) AML cells with the autophagy inhibitors chloroquine (CQ) or LY294002 in combination with abemaciclib significantly increased the frequency of apoptotic (Annexin V positive) cells compared with that in untreated cells, whereas CQ or LY294002 single treatment had no significant effect on apoptosis. Consistently, combinatorial inhibition of CDK4/6 and autophagy upregulated cleaved caspase 3 expression. The combinatorial effect was confirmed by silencing the autophagy-related protein ATG7 using small interfering RNA in abemaciclib-treated t(8;21) AML cells. These results suggest that autophagy inhibition enhances CDK4/6 inhibitor-induced apoptosis in t(8;21) AML cells. In conclusion, the present results indicate that inhibition of CDK4/6 and autophagy may be a novel and promising biomarker-driven therapeutic strategy for the treatment of t(8;21) AML. Disclosures: No relevant conflicts of interest to declare.

Inhibition of CDK4/6 and autophagy synergistically induces apoptosis in t(8;21) acute myeloid leukemia cells.

The t(8;21) translocation is the most common cytogenetic abnormality in acute myeloid leukemia (AML). Although t(8;21) AML patients have a relatively favorable prognosis, relapse is a frequent occurrence, underscoring the need to develop novel therapeutic approaches. Here, we showed that t(8;21) AML is characterized by frequent mutation and overexpression of CCND2. Analysis of 19 AML cell lines showed that t(8;21) AML cells had lower IC50 values for the selective CDK4/6 inhibitors palbociclib and abemaciclib than non-t(8;21) AML cells. CDK4/6 inhibitors caused cell cycle arrest at G1 phase and impaired cell proliferation in t(8;21) AML cells. CDK4/6 inhibition decreased MAP-ERK and PI3K-AKT-mTOR signaling pathway activity, induced LC3B-I to LC3B-II conversion, and enhanced autophagosome formation, suggesting autophagy induction. Treatment of t(8;21) AML cells with the autophagy inhibitors chloroquine (CQ) or LY294002 in combination with the CDK4/6 inhibitor abemaciclib significantly increased the percentage of apoptotic (Annexin V positive) cells, whereas CQ or LY294002 single treatment had no significant effects. The effectiveness of co-inhibiting CDK4/6 and autophagy was confirmed in primary t(8;21) AML cells. The results suggest that the combination of CDK4/6 and autophagy inhibitors had a synergistic effect on inducing apoptosis, suggesting a novel therapeutic approach for the treatment of t(8;21) AML.

Heat shock protein 90 inhibitor AUY922 attenuates platelet-derived growth factor-BB-induced migration and proliferation of vascular smooth muscle cells.

Luminespib (AUY922), a heat shock proteins 90 inhibitor, has anti-neoplastic and antitumor effects. However, it is not clear whether AUY922 affects events in vascular diseases. We investigated the effects of AUY922 on the platelet-derived growth factor (PDGF)-BB-stimulated proliferation and migration of vascular smooth muscle cells (VSMC). VSMC viability was detected using the XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) reagent. To detect the attenuating effects of AUY922 on PDGF-BB-induced VSMCs migration in vitro, we performed the Boyden chamber and scratch wound healing assays. To identify AUY922-mediated changes in the signaling pathway, the phosphorylation of protein kinase B (Akt) and extracellular signal-regulated kinase (ERK) 1/2 was analyzed by immunoblotting. The inhibitory effects of AUY922 on migration and proliferation ex vivo were tested using an aortic ring assay. AUY922 was not cytotoxic at concentrations up to 5 nM. PDGF-BB-induced VSMC proliferation, migration, and sprout outgrowth were significantly decreased by AUY922 in a dose-dependent manner. AUY922 significantly reduced the PDGF-BB-stimulated phosphorylation of Akt and ERK1/2. Furthermore, PD98059 (a selective ERK1/2 inhibitor) and LY294002 (a selective Akt inhibitor) decreased VSMC migration and proliferation by inhibiting phosphorylation of Akt and ERK1/2. Greater attenuation of PDGF-BB-induced cell viability and migration was observed upon treatment with PD98059 or LY294002 in combination with AUY922. AUY922 showed anti-proliferation and anti-migration effects towards PDGF-BB-induced VSMCs by regulating the phosphorylation of ERK1/2 and Akt. Thus, AUY922 is a candidate for the treatment of atherosclerosis and restenosis.

SC79, the AKT Activator Protects Cerebral Ischemia in a Rat Model of Ischemia/Reperfusion Injury

BackgroundActivation of AKT pathway attenuates brain damage and neuronal apoptosis during cerebral ischemia/reperfusion (I/R) injury. SC79 is a novel, selective and highly-efficient Akt activator. This study aimed to investigate the neuroprotective effect of SC79 against cerebral I/R injury in a rat model, and to explore the possible underlying mechanisms.Material/MethodsMale Sprague-Dawley rats received cerebral ischemia for 1 hour, followed by brain reperfusion for 0.5–24 hours. The cerebral I/R injury animal model were treated with SC79 alone or SC79 in combination with LY294002. Western blots were used to detect the levels of expression of phosphatidylinositol AKT (p-Akt), Bax, and bcl-2. Twenty-four hours after cerebral I/R, the degree of brain injury was evaluated by detecting the neurological deficit score (NDS). The infarct rate of brain tissue was observed by TTC (2, 3, 5-triphenyltetrazolium chloride) staining. TUNEL (terminal deoxynucleotidyl transferase-mediated UTP nick end labeling) staining was used to detect cell apoptosis.Resultsp-Akt was activated during early cerebral I/R at 0.5 hours, and reached the highest levels at 4 hours, then gradually decreased from 6 hours, and reached and maintained the lowest levels at 12–24 hours. Bax expression was gradually increased from 6 hours and reached the highest level at 24 hours. However, bcl-2 expression was gradually increased and reached the highest levels at 4 hours, then gradually decreased from 6 hours, and reached the lowest levels at 24 hours. Administration of SC79 decreased infarct volumes and improved neurological function significantly. LY294002 in combination with SC79 lost the capability of SC79 to resist the cerebral I/R injury. SC79 treatment alone activated p-Akt and promoted anti-apoptotic bcl-2 and inhibited anti-apoptotic Bax expression in middle cerebral artery occlusion (MCAO) mice. However, combined SC79 and LY294002 treatment abolished SC79-induced p-Akt activity, inhibited anti-apoptotic bcl-2 and promoted anti-apoptotic Bax expression in MCAO mice. Furthermore, SC79 treatment alone attenuated apoptotic neuronal cell death, but abolished this effect in SC79 in combination with LY294002 treated groups.ConclusionsSC79 significantly increased Akt activation and reduced infarct volume and subsequently improved neurological function in ischemic brain after cerebral I/R injury in rats. These findings suggested that SC79 may be as a neuroprotective drug to be potentially used in the clinic.

Abstract 4618: mTOR signaling complex can be targeted using Torin2 in colorectal cancer to induce efficient apoptosis

Abstract Colorectal cancer (CRC) is the third most common cancer in the world and the 4th most common cause of cancer related death. Despite the considerable advances in awareness, diagnosis and modern therapeutic strategies, the incidence of CRC is increasing in the Middle Eastern population. In order to explore the molecular cause of increasing incidence of CRC in this region, we sought to evaluate the role of mammalian target of rapamycin (mTOR) survival pathway in CRC of this region. mTOR; a component of the phosphatidylinositol 3-kinase (PI3K) cell survival pathway, is known to play an important role in the regulation of many cellular activities leading to cell growth and proliferation through the formation of its two complexes mTORC1 and mTORC2. We therefore examined the expression of mTOR in 700 Saudi CRC cases by immunohistochemistry in a tissue microarray format and found that mTORC1 and mTORC2 were activated in 43.4% (294/677) and 46.2% (313/677) of cases respectively. mTORC1 activation was found to be significantly associated with Ki67 and mTORC2. In addition, mTORC2 activation was also found to be directly associated with their downstream target; p-AKT. In vitro, we found that treatment with a second generation mTOR inhibitor, Torin2 led to inhibition of cell viability and induction of apoptosis via inactivation of mTORC1 and mTORC2 in majority of CRC cell lines. Inactivation of mTORC1 and mTORC2 following Torin2 treatment also led to dephosphorylation of mTOR downstream targets; P70S6, 4E-BP1, AKT and BAD. Interestingly, one CRC cell line; HT29 that had over-activation of mTORC2 was found to be resistant to Torin2 treatment suggesting that increased activation of mTORC2 confers resistance to Torin2 treatment. As our clinical data demonstrated a significant association between mTORC2 over-expression and activation of AKT in clinical CRC samples, we synergistically targeted HT29 cells with combination of sub-optimal doses of PI3-kinase/AKT inhibitor; LY294002 in combination with Torin2. Our data showed that combination of Torin2 and LY294002 synergistically induced apoptosis in HT29 cells via inactivation of mTORC2 and AKT. These data highlight the utility of targeting mTOR signaling complex using second generation mTOR inhibitors such as Torin2 alone or in combination with other inhibitors such as LY294002 or chemotherapeutic agents to effectively treat these aggressive subgroups of CRC with over-activation of mTOR and AKT simultaneously. Citation Format: Saeeda O. Ahmed, Maqbool Ahmed, Abdul K. Siraj, Shaham Beg, Saravanan Thangavel, Nasser Al-Sanea, Fouad Al-Dayel, Azhar R. Hussain, Khawla S. Al-Kuraya. mTOR signaling complex can be targeted using Torin2 in colorectal cancer to induce efficient apoptosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4618.

Involvement of PI3K/Akt pathway in rat condylar chondrocytes regulated by PTHrP treatment

ObjectiveThe aim of this study was to explore the mutual communication of the parathyroid hormone-related peptide (PTHrP) and phosphatidylinositol 3-kinase/threonine protein kinase (PI3K/Akt) pathway on the proliferation and differentiation of condylar chondrocytes from Sprague-Dawley (SD) rats. MethodsCondylar chondrocytes from the condylar cartilage were cultured and an organ culture system of mandibular condyles was employed. The distribution of PI3K, phospho-Akt (p-Akt), and PTHrP in condylar cartilage was detected by either immunohistochemistry or immunofluorescence. The second passage chondrocytes and condyle specimens in the organ culture system were treated with PTHrP, LY294002, PTHrP and LY294002 in combination, or dimethyl sulfoxide (DMSO), separately. The mRNA and protein levels of type II (Col II) and type X collagen (Col X) were investigated by real-time polymerase chain reaction (PCR) and Western blot analysis. The condyle growth in organ culture system was analysed by haematoxylin–eosin staining. ResultsPTHrP, PI3K, and p-Akt were mainly located in the proliferative and hypertrophic zones. PTHrP promoted the proliferation of condylar chondrocytes, while LY294002 limited this effect. The mRNA and protein levels of Col II and Col X in these cells were reduced by PTHrP and enhanced by LY294002. Organ culture showed a significant enhancement of condyle elongation with PTHrP treatment or a combination of PTHrP and LY294002 treatment. After treatment with LY294002, the length of condyles was reduced compared with the samples treated with DMSO. ConclusionsWe conclude that the PI3K/Akt pathway plays an essential role in the proliferation and differentiation of condylar chondrocytes and is a potential target for PTHrP in regulating chondrocyte differentiation at condylar cartilage.

Inhibitory effect of soluble CD40 ligand combined with LY294002 on growth of gastric cancer cells

Objective To investigate the effects of soluble CD40 ligand combined with LY294002,a specific inhibitor of phosphatidylinositol 3 kinase (PI3K),on the proliferation and apoptosis of gastric cancer cells (AGS),and its potential mechanisms.Methods The effect of gradient concentration of soluble CD40 ligand (sCD40L) and its combination with LY294002 on cellular proliferation was determined by using methyl thiazol tetrazolium (MTT) assay.The cellular cycle and apoptosis were evaluated by flow cytometry (FCM).The cellular morphologic change was detected by election microscope.The protein expression of PI3K and p-protein kinase B (p-Akt) was tested by using Western blotting.Results The inhibitory rate of cellular proliferation in LY294002,sCD40L and LY294002 + sCD40L groups was (65.72 ±3.19)％,(70.14 ±2.98)％ and (41.25 ±2.74)％ respectively (P ＜0.05).The cellular cycle arrest was increased in cells treated with sCD40L plus LY294002 when compared with those treated with sCD40L or LY294002 alone (P ＜ 0.05).LY294002 could increase cellular apoptosis with the apoptosis rate being (8.97 ± 1.23) ％,and sCD40L did not obviously increase cellular apoptosis with the apoptosis rate bing (4.23 ± 0.74)％.As compared with LY294002 alone,the rate of cellular apoptosis was increased obviously after being treated by LY294002 in combination with sCD40L with the apoptosis rate being (29.32 ±2.37)％ (P ＜0.05).The protein expression of PI3K and p-Akt was increased in cells treated with sCD40L,but reduced drastically in those treated with sCD40L plus LY294002 (P ＜ 0.05).Conclusion Inhibiting PI3K/Akt signaling pathway can enhance the inhibitory effect of proliferation by sCD40L on AGS,which may provide a new way for biochemical therapy of gastric cancer. Key words: Gastric cancer; Soluble CD40 ligand; Phosphatidylinositol 3 kinase/protein kinase B pathway; Tumor treatment

Abstract 2385: Identification of a molecular marker for cetuximab sensitivity and development of a novel combination therapy for non-small cell lung cancers.

Abstract The epidermal growth factor receptor (EGFR) is an important molecular target for non-small-cell lung cancers (NSCLCs). A recent phase III study (the FLEX study) revealed that the combination of the anti-EGFR antibody cetuximab with conventional chemotherapy improved the survival of patients with NSCLCs. However, the survival benefit was only about 5 weeks. Therefore, the identification of a molecular marker for cetuximab sensitivity is urgently needed. In this study, we investigated a new molecular marker for cetuximab sensitivity and developed a combination therapy to improve the effectiveness of cetuximab. First, we established a NSCLC cell line that showed resistance to cetuximab by culturing a parental sensitive NSCLC cell line in the presence of increasing concentrations of cetuximab. Next, we performed microarray analysis to compare the gene expression patterns of cetuximab-sensitive and -resistant cell lines. Using pathway analysis, we found that the Akt and Wnt pathways were significantly activated in the resistant cell line as compared to their activation in the sensitive cell line. We confirmed this result by performing western blot analysis, which showed Akt phosphorylation and beta-catenin dephosphorylation in the resistant cell line. Therefore, the activation status of either the Akt or Wnt pathway is the candidate pathway that influences cetuximab sensitivity. Finally, we focused on the Akt pathway and treated both resistant and parental sensitive cell lines with the PI3-K inhibitor LY294002 in combination with cetuximab. This combination therapy induced a synergistic growth-inhibiting effect in both cell lines. The data suggest that the activation status of the Akt pathway is one of the markers for cetuximab sensitivity and the combination of an Akt pathway inhibitor and cetuximab would be a promising anti-EGFR therapy for NSCLCs. Citation Format: Miyako Takata, Hiroki Chikumi, Kosuke Yamaguchi, Jun Kurai, Masaki Nakamoto, Naoto Burioka, Tadashi Igishi, Eiji Shimizu. Identification of a molecular marker for cetuximab sensitivity and development of a novel combination therapy for non-small cell lung cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2385. doi:10.1158/1538-7445.AM2013-2385 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

LY294002 enhances inhibitory effect of gemcitabine on proliferation of human pancreatic carcinoma PANC-1 cells

Phosphatidylinositide 3-kinase (PI3K)/protein kinase B (PKB, Akt) pathway plays a major role in proliferation and survival of many types of cells. The inhibitory effect of LY294002, widely applied as an inhibitor of PI3K, in combination with gemcitabine on proliferation of PANC-1 cells was investigated. The expression of PI3K, phosphorylated Akt (p-Akt) and multidrug-resistance like protein (MRP) in normal pancreas tissues, chronic pancreatitis tissues and pancreatic carcinoma tissues was detected. The effects of LY294002 combined with gemcitabine on proliferation of PANC-1 cells and protein levels of p-Akt and MRP were detected. The results showed that the positive expression rate of PI3K, p-Akt and MRP in pancreatic carcinoma tissues was significantly higher than that in normal pancreas tissues and chronic pancreatitis tissues (P<0.01 and P<0.05 respectively). LY294002 could effectively enhance the inhibitory effect of gemcitabine on proliferation of PANC-1 cells. Furthermore, Western blotting revealed that LY294002 combined with gemcitabine reduced the protein levels of p-Akt and MRP, which contributed to the inhibition of proliferation. It is concluded that LY294002 in combination with gemcitabine may represent an alternative therapy for pancreatic carcinoma.

Targeting PI3K/Akt represses Hypoxia inducible factor-1α activation and sensitizes Rhabdomyosarcoma and Ewing’s sarcoma cells for apoptosis

BackgroundHypoxia inducible factor-1 α (HIF-1α) has been identified as an important novel target in apoptosis resistance of pediatric tumors such as Rhabdomyosarcoma (RMS) and Ewing’s sarcoma (ES). Evidence suggests that PI3K/Akt signaling plays a role in regulation of HIF-1α activation as well as apoptosis resistance in various adult tumors. However the relevance of PI3K/Akt signaling in HIF-1bα activation and apoptosis resistance in childhood tumors has not been addressed yet. Thus, this study was to investigate whether PI3K/Akt signaling is involved in hypoxia induced activation of HIF-1α as well as in resistance to hypoxia-induced apoptosis in childhood tumors such as RMS and ES.MethodsConstitutive activation of PI3K/Akt signaling was analyzed by Western blotting. Hypoxic activation of HIF-1α was determined by Western Blot analysis and electrophoretic mobility shift assay. Apoptosis was determined by flow cytometric analysis of the propidium iodine stained nuclei of cells treated with PI3K inhibitor LY294002 in combination with either TNF-related apoptosis-inducing ligand (TRAIL) or doxorubicin.ResultsThis study demonstrated that PI3K/Akt signaling was constitutively activated in RMS and ES cell lines, A204 and A673, respectively. Targeting PI3K/Akt signaling by the inhibitor LY294002 (30 μM) significantly decreased the protein expression as well as DNA binding activity of HIF-1α and restored the apoptosis-inducing ability of cells in hypoxia Additionally, pretreatment with LY294002 sensitized A204 and A673 cells to TRAIL or doxorubicin induced apoptosis under hypoxia.ConclusionThese results suggest that the constitutively active PI3K/Akt signaling contributes to hypoxic activation of HIF-1α as well as HIF1α-mediated apoptosis resistance in RMS and ES cells under hypoxia.

Inhibition of phosphoinositide 3-kinase enhances the cytotoxicity of AG1478, an epidermal growth factor receptor inhibitor, in breast cancer cells

Aberrant activation and dysfunction of the EGFR/PI3K/Akt signaling pathways are commonly reported in breast cancer. Constitutive activation of the PI3K/Akt pathway by the lack of PTEN regulation is associated with resistance to novel targeted therapies including EGFR inhibitors. We aimed to study whether Ly294002, an inhibitor of PI3K, could enhance the cytotoxicity of AG1478, an inhibitor of EGFR, on breast cancer cells. We tested these agents in the MDA-MB-468 and MCF-7 breast cancer cell lines with different EGFR and PTEN profiles (MDA-MB-468: high expression of EGFR and PTEN mutation; MCF-7: low expression of EGFR and PTEN wild type). Simultaneous inhibition of EGFR and PI3K in MDA-MB-468 cells with combined Ly294002 and AG1478 treatment had a greater anti-proliferative effect and increased mitotic death than either treatment alone. In addition, more apoptosis and increased induction of cell arrest at G0/G1 phase were observed in MDA-MB-468 cells with the combined treatment. Phosphor-EGFR and its downstream signal transducer, phosphor-Akt, were fully attenuated only by simultaneous treatment with Ly294002 and AG1478. These data suggest that the inhibition of PI3K could enhance the cytotoxicity of EGFR inhibitors on breast cancer cells and tumors which overexpress EGFR and demonstrate mutated PTEN. This dual inhibition treatment protocol may have important therapeutic implication in the treatment of a subset of breast cancer patients with high expression of EGFR and deficient function of PTEN.

Abstract 822: Drug resistance towards vincristine in acute lymphoblastic leukemia is mediated by the PI3K-Akt pathway

Abstract Contemporary acute lymphoblastic leukemia (ALL) therapy involves the use of powerful anti-mitotic agents such as vincristine (VCR). VCR targets the microtubule cytoskeleton and disrupts important cellular processes, leading to cell death. Nonetheless, around 20% of patients relapse and become resistant to most drugs. At present, mechanisms of resistance toward VCR remain elusive despite its extensive clinical use, warranting a more in-depth study with respect to ALL. Five VCR-resistant (VCR-R) subclones were established by gradually exposing five ALL VCR-sensitive (VCR-S) cell lines (Jurkat, REH, SEM, RS4;11, 697) to incremental doses of VCR. Comparing resistant to sensitive cells, the IC50 at 48h increased 3-6 logs. Both VCR-S and VCR-R cell lines were subjected to cell cycle analysis by flow cytometry using propidium iodide staining. After a 48h exposure to VCR, cell death for VCR-S cells was the result of a typical G2/M phase arrest. In contrast, a G2/M phase arrest did not precede the VCR-induced cell death of VCR-R cells. VCR-R subclones were also observed to be more resistant to vinblastine, an alternative tubulin binding agent. In addition, there was increased resistance towards other chemotherapeutic drugs such as prednisolone, dexamethasone, daunorubicin and doxorubicin, indicating that resistance to VCR may contribute to cross-resistance after a prolonged incubation. The PI3K/Akt/mTOR signaling pathway is known to play important roles in cancer cell proliferation and survival. The combination of inhibitors with other chemotherapeutic agents also has the potential to enhance leukemic cell death. To further investigate if the PI3K/Akt/mTOR signaling pathway is involved in VCR resistance, we screened a panel of twelve kinase inhibitors (Merck) which selectively targets this pathway. Interestingly, PDK1/Akt/Flt Dual Pathway Inhibitor KP372-1, was highly potent toward all cell lines with IC50 less than 100nM. Our results also showed that the administration of PI3Kα Inhibitor VIII, Akt Inhibitor IV and PI-103, as well as pan-selective PI3K inhibitors Wortmannin and LY294002 in combination with VCR was able to promote increased levels of cell death. Cell cycle analysis also revealed that the combination treatment elicits different methods of cell death between VCR-S and VCR-R cells. This is especially evident upon the co-treatment with pan-selective inhibitors. Optimising the sensitivity of ALL cells and/or reversing resistance to VCR promise to maximise the therapeutic value of this important chemotherapy. This study provides evidence that VCR resistance potentially contributes to cross-resistance. Our data also shows that the PI3K-Akt pathway may be involved in VCR resistance in ALL, providing a valid rationale for further studies and the future application of inhibitors in clinical settings. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 822. doi:1538-7445.AM2012-822

Bufalin Induces Lung Cancer Cell Apoptosis via the Inhibition of PI3K/Akt Pathway

Bufalin is a class of toxic steroids which could induce the differentiation and apoptosis of leukemia cells, and induce the apoptosis of gastric, colon and breast cancer cells. However, the anti-tumor effects of bufalin have not been demonstrated in lung cancer. In this study we used A549 human lung adenocarcinoma epithelial cell line as the experimental model to evaluate the potential of bufalin in lung cancer chemotherapy. A549 cells were treated with bufalin, then the proliferation was detected by MTT assay and apoptosis was detected by flow cytometry analysis and Giemsa staining. In addition, A549 cells were treated by Akt inhibitor LY294002 in combination with bufalin and the activation of Akt and Caspase-3 as well as the expression levels of Bax, Bcl-2 and livin were examined by Western blot analysis. The results showed that Bufalin inhibited the proliferation of A549 cells and induced the apoptosis of A549 cells in a dose and time dependent manner. Mechanistically, we found that bufalin inhibited the activation of Akt. Moreover, bufalin synergized with Akt inhibitor to induce the apoptosis of A549 cells and this was associated with the upregulation of Bax expression, the downregulation of Bcl-2 and livin expression, and the activation of Caspase-3. In conclusion, our findings demonstrate that bufalin induces lung cancer cell apoptosis via the inhibition of PI3K/Akt pathway and suggest that bufalin is a potential regimen for combined chemotherapy to overcome the resistance of lung cancer cells to chemotherapeutics induced apoptosis.

Abstract B58: OPNc and OPNb osteopontin splicing isofoms activate prostate cancer prosurvival features through PI3K/AKT/BIM signaling pathway

Abstract Alternative splicing of the osteopontin (OPN) gene generates three protein splicing isoforms (OPN-SI), designated as OPNa, OPNb and OPNc. We previously demonstrated that both OPNb e OPNc are able to activate typical features of prostate cancer (PCa) progression. These data suggested that some of these pro-tumorigenic roles are mediated by the activation of pro-survival pathways in PCa cells. The aim of this study was to delineate molecular and signaling pathways by which these two isoforms are able to mediate PCa cell survival and growth. PC3 cells treated with docetaxel were used as an in vitro cell model to induce cell death. We then investigated the involvement of PI3K/Akt signaling and anti e pro-apoptotic proteins on inducing PC3 cell survival on cells overexpressing each OPN-SI. PC3 cells overexpressing the three OPN-SI were treated with 1×10-6μg/mL of docetaxel and then analysed for cell proliferation and viability using crystal violet and 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) MMT assays, respectively. PC3 whole protein cell extracts were prepared to evaluate protein expression of members of PI3K/Akt and cell death signaling. PI3K/Akt signaling was analysed by immunoblot tests using specific antibodies against total and phosphorylated (ser473) Akt. The expression of apoptotic proteins was investigated by using Bcl-2, Mcl-1 e Bim specific antibodies. LY294002 was used as an specific inhibitor of PI3K signaling. PC3 cells were incubated with LY294002 in combination or not with docetaxel treatment in order to evaluate cell survival pathways. Cells overexpressing OPNb and OPNc isoforms presented a significant increase of Akt (Ser473) phosphorylation, as compared to cells overexpressing OPNa and empty vector control. An additional increase in Akt (Ser473) phosphorylation was also observed after docetaxel treatment. PC3 cells overexpressing OPNb and OPNc isoforms also promoted additional resistance to cell death induced by docetaxel treatment, as compared to cells overexpressing OPNa. Combined treatment with LY294002 and docetaxel significantly inhibited PC3 cell survival mediated by the overexpression of OPNb and OPNc isoforms. The expression of Bcl-2 protein was similar among cells overexpressing the three OPN-SI, before and after docetaxel treatment. On the other hand, cells overexpressing OPNb e OPNc presented significant reduction on the expression of Mcl-1. Cells overexpressing OPNc, but not OPNb, presented a significant inhibition of pro-apoptotic protein Bim. As a whole, our data indicate an important role of PI3K/Akt signaling activation and inhibition of pro-apoptotic proteins on the survival of PC3 cells as a result of OPNb e OPNc overexpression. Citation Format: Kivvi Duarte Mello, Tatiana Martins Tilli, Ana Carolina S. Ferreira, Claudete Esteves Klumb, Etel Rodrigues Pereira Gimba. OPNc and OPNb osteopontin splicing isofoms activate prostate cancer prosurvival features through PI3K/AKT/BIM signaling pathway [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr B58.

LY294002 enhances cytotoxicity of temozolomide in glioma by down-regulation of the PI3K/Akt pathway

The introduction of temozolomide (TMZ) has advanced chemotherapy for malignant gliomas. However, a considerable number of glioblastoma (GBM) cases are refractory to TMZ. Previous studies have revealed that the PI3K/Akt pathway is activated in an ataxia telangiectasia and Rad3 related-dependent manner in response to TMZ. Thus, we hypothesized that PI3K inhibitors may act as antitumor agents against gliomas and potentiate the cytotoxicity of TMZ. The cytotoxicity of a PI3K inhibitor, LY294002, was examined both alone and in combination with TMZ in human glioma cell lines. Proliferation of tumor cells treated with LY294002 in combination with TMZ was significantly suppressed compared to treatment with either drug used alone. The combination treatment induced a higher apoptosis rate, while reducing the invasive capability of U87 cells. The apoptosis-associated proteins, cleaved-caspase-3 and Bax, were more significantly up-regulated by the combined treatment than by TMZ used alone. In addition, p-Akt and Bcl-2, which can promote TMZ resistance, were markedly decreased by LY294002. These findings suggest that LY294002 enhances the cytotoxicity of TMZ by down‑regulation of the PI3K/Akt pathway.

Abstract 2928: Regulation of MST1 and MST2 by mTOR and androgen receptor signaling in prostate cancer

Abstract The serine/threonine kinases MST1 and MST2 (hippo in Drosophila) were originally identified as pro-apoptotic proteins. In addition to their pro-apoptotic functions, evidence suggests that deficiency in MST1 and MST2 and their downstream effectors has been linked to developmental defects, tissue overgrowth, and cancer, including prostate cancer (PCa). However, the molecular mechanism that may modulate MST1 or MST2 signaling in PCa cells remains unknown. Here we investigate the impact of androgen receptor (AR) and PI3K-AKT-mTOR signaling on MST1 or MST2. We used biochemical, mutagenesis, histochemical, and pharmacological approaches to carry out this investigation. First, we demonstrate that AR and MST1 co-localize and form protein complexes in cell nuclei. Androgen further stimulates the interaction between the two proteins and reduces MST1 kinase activity in LNCaP PCa cells, suggesting that AR may sequester MST1 in an inactive form. Second, we showed that MST1 at Threonine120 (T120) was exclusively phosphorylated in cell nuclei, whereas MST2 at Threonine117 (T117), corresponding to MST1-T120, was phosphorylated only in the cytoplasm, although MST1 and MST2 were found in both locations. Similar phosphorylation patterns were also observed in prostate cancer tissue samples. Third, we have found that the T120 is not a physiologic target of AKT kinase in vivo, because the inhibition of AKT activity by a specific PI3K inhibitor, LY294002, had no effect on the phosphorylation of MST1 at the T120 site, but it did inhibit MST1-T117 phosphorylation. Furthermore, attenuation of mTOR activity by Ku0063794, a specific mTOR1 and mTOR2 inhibitor, abolished MST2-T117 phosphorylation, but without having an effect on the levels of phospho-MST1-Thr120. Lastly, the use of combined LY294002 and Ku0063794 compounds synergistically inhibited PCa cell growth, relative to a single compound. Our data suggest that AR and mTOR pathway signaling may oppose MST1 and MST2 in discrete cell locations. Thus, understanding the functional relationship between these signal networks in the context of cell proliferation and cell death may have important therapeutic implications in prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2928. doi:10.1158/1538-7445.AM2011-2928

Phosphoinositide-3-Kinase Inhibition Enhances Radiosensitization of Cervical Cancer In Vivo

Phosphoinositide-3-kinase (PI3K)/Akt pathway is downregulated in several human cancers, and PI3K inhibition can sensitize these cancer cells to radiation. However, no research on cervical cancer in vivo has been reported. The present study further investigated whether PI3K inhibition could sensitize cervical cancer to radiation in vivo. HeLa cells with sustained PI3K activity and Akt phosphorylation were injected subcutaneously into BALB/C nude mice to establish tumor cell xenograft, which were randomly assigned to control, PI3K inhibitor LY294002 alone, radiation alone, or combined LY294002 and radiation group. Akt phosphorylation was detected by Western blotting to evaluate the blocking efficiency on PI3K activity. The radiosensitization of PI3K inhibition was measured by clonogenic assays, apoptosis analysis, and tumor regrowth assays. The combination of LY294002 and radiation resulted in significant and synergistic suppression of cervical cancer cells in a dose-dependent manner in clonogenic assays (P < 0.05), higher ratio of apoptosis cells, and more remarkable reduction of Akt phosphorylation. Tumor regrowth delay curve showed the lowest increase of tumor volume in the combined group (37 days in average) (P = 0.003). Besides, LY294002 (100 mg/kg) alone decreased cell survival and produced xenograft regrowth delay. Phosphoinositide-3-kinase inhibition by LY294002 can synergistically enhance radiation efficacy via dephosphorylation of Akt in cervical cancer, and PI3K inhibition alone can also suppress tumor regrowth. This may provide novel therapeutic opportunities to enhance the effect of radiotherapy against cervical cancer.

Inhibition of phosphatidylinositol 3-kinase sensitizes ovarian cancer cells to carboplatin and allows adjunct chemotherapy treatment

Signal transduction pathways associated with cancer progression and chemotherapeutic resistance are being investigated as molecular targets for chemotherapy. The phosphatidylinositol 3-kinase (PI3K) pathway has been found to be frequently amplified and have increased activity in ovarian cancer. The current study investigates the efficacy of an antagonist of PI3K, LY294002, in inhibiting ovarian cancer cell growth and survival both in vitro and in vivo. The hypothesis tested is that inhibition of PI3K signaling makes ovarian cancer cells susceptible to the effects of platinum-based chemotherapy. Observations show that LY294002 is an effective inhibitor of ovarian cancer cell growth and survival in vitro. Inhibition of PI3K/Akt signaling increased the sensitivity of ovarian cell cultures to the cytotoxic effects of carboplatin. The combined treatment of LY294002 and carboplatin was needed to optimally promote cellular apoptosis and decrease ovarian cancer cell survival in vitro. To extend these observations, a model involving in vivo i.p. growth of human ovarian tumors in a nude mouse was used. LY294002 in combination with carboplatin was more effective in inhibiting ovarian cancer cell xenograft growth than either agent alone. The results of this study suggest that the combined treatment of carboplatin and LY294002 can effectively decrease ovarian tumor progression and support the use of a PI3K inhibitor (e.g., LY294002) as an adjunct platinum-based drug therapy for treatment of ovarian cancer.