Targeting Phosphatidylinositol 3-kinase Research Articles

Hirsutenone Directly Targets PI3K and ERK to Inhibit Adipogenesis in 3T3-L1 Preadipocytes.

Adipogenesis is a key driver of the expansion of adipose tissue mass that causes obesity. Hirsutenone (HST) is an active botanical diarylheptanoid present in Alnus species. In this study, we evaluated the effects of HST on adipogenesis, its mechanisms of action and the molecular targets involved. Using Oil Red O staining, we observed that HST dose-dependently suppresses lipid accumulation during adipogenesis in 3T3-L1 preadipocytes, concomitant with a decrease in peroxisome proliferator-activated receptor-γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα) and fatty acid synthase (FAS) protein expression. This inhibitory effect was largely limited to the early stage of adipogenesis, which includes mitotic clonal expansion (MCE), as evidenced by delayed cell cycle entry of preadipocytes from G1 to S phase. Furthermore, the regulation of MCE was accompanied by suppression of phosphatidylinositol 3-kinase (PI3K) and extracellular-regulated kinase (ERK) activity. HST was also shown to bind directly to PI3K and ERK1 in a non-ATP competitive manner. Our results suggest that HST attenuates adipogenesis by directly targeting PI3K and ERK during MCE in 3T3-L1 preadipocytes, underscoring the potential therapeutic application of HST in preventing obesity.

Management of chronic lymphocytic leukemia.

Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL) is usually diagnosed in asymptomatic patients with early-stage disease. The standard management approach is careful observation, irrespective of risk factors unless patients meet the International Workshop on CLL (IWCLL) criteria for "active disease," which requires treatment. The initial standard therapy for most patients combines an anti-CD20 antibody (such as rituximab, ofatumumab, or obinutuzumab) with chemotherapy (fludarabine/cyclophosphamide [FC], bendamustine, or chlorambucil) depending on multiple factors including the physical fitness of the patient. However, patients with very high-risk CLL because of a 17p13 deletion (17p-) with or without mutation of TP53 (17p-/TP53mut) have poor responses to chemoimmunotherapy and require alternative treatment regimens containing B-cell receptor (BCR) signaling pathway inhibitors. The BCR signaling pathway inhibitors (ibrutinib targeting Bruton's tyrosine kinase [BTK] and idelalisib targeting phosphatidyl-inositol 3-kinase delta [PI3K-delta], respectively) are currently approved for the treatment of relapsed/refractory CLL and all patients with 17p- (ibrutinib), and in combination with rituximab for relapsed/refractory patients (idelalisib). These agents offer great efficacy, even in chemotherapy refractory CLL, with increased tolerability, safety, and survival. Ongoing studies aim to determine the best therapy combinations with the goal of achieving long-term disease control and the possibility of developing a curative regimen for some patients. CLL is associated with a wide range of infectious, autoimmune, and malignant complications. These complications result in considerable morbidity and mortality that can be minimized by early detection and aggressive management. This active monitoring requires ongoing patient education, provider vigilance, and a team approach to patient care.

Comparison between endothelial and tumor cells in the response to verteporfin-photodynamic therapy and a PI3K pathway inhibitor

BackgroundPhotodynamic therapy (PDT) is an established cancer treatment. Molecular-targeted agents targeting phosphatidylinositol 3-kinase (PI3K) pathway is showing great promise as anticancer drugs. This study compared SVEC mouse endothelial and PC-3 human prostate tumor cells in the response to verteporfin-mediated photodynamic therapy (PDT) and a pan-PI3K pathway inhibitor LY294002. MethodsVerteporfin cellular uptake and intracellular localization was determined by spectrofluorometry and confocal fluorescence microscopy, respectively, in the SVEC and PC-3 cells. Cytotoxicity induced by LY294002 and verteporfin-PDT was assessed by the MTS assay. Effects of treatments on cell survival and death signaling were examined by Western blot analysis. ResultsPC-3 cells had a higher cellular uptake of verteporfin than SVEC cells at 15min after incubation with verteporfin. Verteporfin was mainly localized in mitochondria in both SVEC and PC-3 cells. Verteporfin-PDT alone as well as PDT in combination with LY294002 induced more apoptosis and caused more reduction in cell viability in SVEC cells than in PC-3 cells. PC-3 cells exhibited a higher level of anti-apoptotic Bcl-2 family proteins than SVEC cells. ConclusionsSVEC cells were more responsive to verteporfin-PDT and PI3K pathway inhibitor LY294002 than PC-3 cells. Such differences in response were likely due to differences in Bcl-2 family protein level. These results support tumor vascular targeting by verteporfin-PDT and its therapeutic enhancement by PI3K pathway inhibition.

Abstract C96: Development of combination therapy with an IGF1R inhibitor and a PI3K inhibitor ZSTK474 in cancer cells exhibiting the intrinsic resistance to PI3K inhibitors.

Abstract Background: Tumor cells can vary markedly in their response to anti-cancer drugs. Therefore predicting sensitivity or resistance of tumor cells to the drug and developing therapeutic strategies for overcoming the resistance is required to realize personalized medicine for cancer treatment. Although multiple drugs targeting phosphatidylinositol 3-kinase (PI3K) including ZSTK474 are undergoing clinical trials, little is known about the resistance. We previously reported that overexpression of insulin-like growth factor 1 receptor (IGF1R) contributed to acquired resistance to PI3K inhibitors (PI3Kis). In this study, we examined whether IGF1R contributes to intrinsic resistance to PI3Kis and whether combination of IGF1R tyrosine kinase inhibitor (IGF1R-TKI) and PI3Ki is an effective strategy for cancer cells exhibiting the intrinsic resistance to PI3Kis. Materials and Methods: Correlation analyses between the expression level of IGF1R with the efficacy of PI3Ki across an in vitro panel of 39 human cancer cell lines (termed JFCR39) and a panel of 24 human tumor xenografts implanted in nude mice (termed JFCR24 xenografts) were performed. Specific siRNAs against IGF1R were used to examine the functional involvement of IGF1R in intrinsic resistance. Combination effect of ZSTK474 and an IGF1R-TKI OSI-906 was examined in vitro using isobologram analysis and in vivo using human tumor xenografts implanted in nude mice. Results: Correlation analysis revealed that PI3Ki-naïve cancer cells expressing high levels of IGF1R exhibited resistance to ZSTK474 in vitro and in vivo. Targeted knockdown of IGF1R expression using specific siRNAs enhanced the efficacy of ZSTK474 in intrinsic resistant cell lines overexpressing IGF1R. In these cell lines, higher concentration of ZSTK474 was needed to dephosphorylate Akt compared to IGF1R-negative cell lines, and specific knockdown of IGF1R made it easier to dephosphorylate Akt after ZSTK474 treatment. Finally, we found a synergistic therapeutic effect of ZSTK474 in combination with OSI-906 in vivo, as well as in vitro. Conclusion: PI3Ki-naïve cancer cells overexpressing IGF1R exhibited resistance to ZSTK474, but combination of ZSTK474 with an IGF1R-TKI exerted a remarkable therapeutic response in such cancer cells. We hope that our findings would contribute to personalized medicine using PI3Kis in future clinical application. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C96. Citation Format: Sho Isoyama, Gensei Kajiwara, Naomi Tamaki, Hisashi Yoshimi, Naoki Nakamura, Mutsumi Okamura, Yumiko Nishimura, Takao Yamori, Shingo Dan. Development of combination therapy with an IGF1R inhibitor and a PI3K inhibitor ZSTK474 in cancer cells exhibiting the intrinsic resistance to PI3K inhibitors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C96.

Abstract 72: Targeting PI-3K/Akt signaling pathway by a PI-3K inhibitor BKM120 for radiosensitization in murine hepatocellular carcinoma.

Abstract Purpose: Tumor control of hepatocellular carcinoma (HCC) by radiotherapy remains unsatisfactory and the sub-lethal effect associates with secondary spread. Phosphatidylinositol 3-kinase (PI-3K)/Akt pathway plays a critical role in promoting cancer cell proliferation and inhibiting cell death. Elevated PI-3K/Akt activity is associated with increased cellular resistance to irradiation. Our aim was to assess if the inhibition of PI-3K/Akt activity by a PI-3K inhibitor, BKM120, contributes to the increasing sensitivity of murine liver cancer cells to irradiation. Methods and Materials: Murine HCC cell line (BNL) was used to evaluate the in vitro synergism of combining BKM120 with irradiation. Flow cytometry analyzed the cell cycle changes, whereas Western blot investigated the protein expressions after the combined treatment. Balb/c mice bearing ectopic BNL xenografts were treated with BKM120 and/or radiotherapy for the in vivo response. Results: Clonogenic cell survival decreased dose-dependently either with irradiation or BKM120 treatment. Synergisms were achieved in cells treated together with greater or equal than 0.5μM BKM120 and 2 Gy radiation. BKM120 pretreatment significantly inhibited radiation-induced Akt phosphorylation. Western blots of active caspase-3 and cleaved PARP revealed that the combined BKM120 and radiation treatment strongly increased the expression of these apoptotic markers in response to irradiation compared with radiation alone. In ectopic xenografts, pretreatment with BKM120 significantly enhanced the tumor-suppressive effect by radiotherapy. Conclusion: The apparent synergism between BKM120 and irradiation or effect of pre-treatment with BKM120 suggests an option to mitigate the activation of Akt by radiation, leading to increased cell apoptosis in murine liver cancer. These data suggest that the BKM120/radiation combination be a strategy worthy of further clinical trials. Citation Format: Wei-Lin Liu, Melissa Gao, Ann-Lii Cheng, Jason Chia-Hsien Cheng. Targeting PI-3K/Akt signaling pathway by a PI-3K inhibitor BKM120 for radiosensitization in murine hepatocellular carcinoma. [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 72. doi:10.1158/1538-7445.AM2013-72

Abstract 2589: The P110 subunit of PI3-K is a therapeutic target of acacetin in skin cancer.

Abstract Verifying potential target proteins of phytochemicals is important for explaining their health benefits. In the present study, we examined the chemopreventive effect and molecular target of acacetin, which is a flavonoid from Robinia Psuedoacacia also known as Black Locust. Treatment with acacetin significantly inhibits EGF-induced transformation of JB6 P+ cells. Western blot assay results revealed that acacetin suppresses EGF-induced phosphorylation of Akt/p70S6K, which is a well-known downstream signaling molecule of phosphatidylinositol 3-kinase (PI3-K). In vitro kinase and pull-down assay results showed that acacetin significantly inhibits PI3-K activity by direct binding. Additionally, acacetin also attenuated EGF-induced PI3-K activity and binds with PI3-K in JB6 P+ cells. Acacetin showed significant effects against anchorage-dependent and -independent cell growth in cells displaying higher PI3-K activity compared with those exhibiting lower PI3-K activity. Binding assay data and computational modeling showed that acacetin binds in an ATP-competitive manner with the p110 kinase domain of PI3-K and interacts with Val828, Glu826 Asp911 Trp760, Ile777, Ile825, Tyr813, Ile910 and Met900. Acacetin reduced SK-MEL-28 xenograft tumor growth and phosphorylation of Akt in vivo. Overall, these results indicate that acacetin is an ATP competitive PI3-K inhibitor and might be a promising chemopreventive agent targeting PI3-K activity. Citation Format: Jon Eun Kim, Sung Keun Jung, Sanguine Byun, Sung-Young Lee, Myoung Ok Kim, Tae Gyu Lim, Ann M. Bode, Hyong Joo Lee, Ki Won Lee, Zigang Dong. The P110 subunit of PI3-K is a therapeutic target of acacetin in skin cancer. [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 2589. doi:10.1158/1538-7445.AM2013-2589

Targeting PI3K in Cancer: Any Good News?

The phosphatidylinositol 3-kinase (PI3K) signaling pathway regulates several cellular processes and it’s one of the most frequently deregulated pathway in human tumors. Given its prominent role in cancer, there is great interest in the development of inhibitors able to target several members of PI3K signaling pathway in clinical trials. These drug candidates include PI3K inhibitors, both pan- and isoform-specific inhibitors, AKT, mTOR, and dual PI3K/mTOR inhibitors. As novel compounds progress into clinical trials, it’s becoming urgent to identify and select patient population that most likely benefit from PI3K inhibition. In this review we will discuss individual PIK3CA mutations as predictors of sensitivity and resistance to targeted therapies, leading to use of novel PI3K/mTOR/AKT inhibitors to a more “personalized” treatment.

Abstract P5-07-03: Gallotannins and Gallic acid From Mango Fruit (Mangifera Indica L) Suppress Breast Cancer Tumor Growth by Targeting Phosphatidylinositol3-kinase (PI3K)-Akt-NF-kB Pathway and Associated microRNAs

Abstract The cytotoxic and anti-inflammatory properties of polyphenolics have been demonstrated in numerous types of cancer and the objective of this research was to understand the post-transcriptional targets involved in anti-inflammatory and pro-apoptotic pathways in the breast cancer cell line BT474, treated with a polyphenolic extract (2.5–40µg/ml) rich in gallotannins and gallic acid from mango fruit in vitro and in athymic nude mice bearing BT474 cells as xenografts. Results show that cell proliferation as well as tumor size and weight in vivo was decreased by polyphenolics 60% and 70% in vitro and vivo, respectively. NF-kB protein and mRNA expression as well as activation were decreased in a dose-dependent manner (0.6 fold). The extract also inhibited PI3K-dependent phosphorylation of AKT and expression and this was accompanied by down-regulation of miRNA 126 of BT474 (2.25 fold) and in vivo. NFκB-dependent genes involved in apoptosis-inhibition, metastasis and angiogenesis, such as survivin, VCAM-1, and VEGF were also decreased, in vitro and in vivo. A microRNA-array was also used to investigate whether microRNAs with target regions within affected genes were affected by the polyphenolic extract and results show that in addition to miR-126, miR-let-7a and miR-let-7b among others were significantly increased by the extract. In summary the anti-carcinogenic and anti-inflammatory activity of mango polyphenolics in breast cancer cells were at least in part due to targeting miR-126 -Phosphatidylinositol 3-kinase (PI3K)-Akt-NF-kB which plays an important role in the proliferative/ inflammatory phenotype exhibited in this breast cancer cell line. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-07-03.

Abstract IA6: Targeting PI3K in cancer, mechanistic insights from genetic mouse models

Abstract The class 1a phosphatidylinositol 3-kinase (PI3K) signaling pathway is frequently activated in human cancer by a number of mechanisms, including amplification and/or activation of RTKs (receptor tyrosine kinases), loss of PTEN (phosphatase and tensin homologue) function and somatic mutation in the PIK3CA gene that encodes the p110α catalytic subunit of PI3K. Thus PI3K represents an attractive target for cancer therapy and tremendous efforts are being devoted to developing effective PI3K inhibitors for cancer treatment. Despite the great promise of targeted therapy, drug resistance is likely to develop, causing treatment failure. To elucidate resistance mechanisms to PI3K-targeted therapy, we constructed mouse models of breast cancer and brain tumors conditionally expressing an oncogenic allele of PIK3CA. Interestingly, we found that oncogenic PIK3CA-driven tumors in both mammary gland and brain tissues frequently recur in PI3K pathway-dependent and -independent manners. Genomic and functional analyses of recurrent tumors revealed multiple lesions, providing mechanistic insights for tumor recurrence and resistance to PI3K-targeted therapy.

Cyanidin suppresses neoplastic cell transformation by directly targeting phosphatidylinositol 3-kinase

Cyanidin, an anthocyanidin found in fruits and vegetables, has been reported to possess anti-cancer effects. However, there is no study on the chemopreventive effect of cyanidin against neoplastic cell transformation and its molecular mechanisms. In the present study, we compare the anti-carcinogenic effects of cyanidin and cyanidin-3-glucoside (C3G) and investigate their underlying mechanisms. The inhibitory effect of cyanidin on EGF-induced cell transformation was higher than those of C3G in JB6 P+ mouse epidermal (JB6 P+) cells. Both cyanidin and C3G showed dose-dependent radical scavenging activities. It is indicated that the divergent inhibitory effects of cyanidin and C3G are not due to their antioxidant activities. We found that cyanidin, but not C3G, inhibited the EGF-induced Akt/p70S6K phosphorylation. Moreover, cyanidin directly suppressed the activity of PI3K by binding to PI3K directly in an ATP-competitive manner, which indicates that PI3K is one of the molecular targets of cyanidin.

Proteomic and transcriptomic profiling reveals a link between the PI3K pathway and lower estrogen-receptor (ER) levels and activity in ER+ breast cancer.

IntroductionAccumulating evidence suggests that both levels and activity of the estrogen receptor (ER) and the progesterone receptor (PR) are dramatically influenced by growth-factor receptor (GFR) signaling pathways, and that this crosstalk is a major determinant of both breast cancer progression and response to therapy. The phosphatidylinositol 3-kinase (PI3K) pathway, a key mediator of GFR signaling, is one of the most altered pathways in breast cancer. We thus examined whether deregulated PI3K signaling in luminal ER+ breast tumors is associated with ER level and activity and intrinsic molecular subtype.MethodsWe defined two independent molecular signatures of the PI3K pathway: a proteomic (reverse-phase proteomic array) PI3K signature, based on protein measurement for PI3K signaling intermediates, and a PI3K transcriptional (mRNA) signature based on the set of genes either induced or repressed by PI3K inhibitors. By using these signatures, we scored each ER+ breast tumor represented in multiple independent expression-profiling datasets (four mRNA, n = 915; one protein, n = 429) for activation of the PI3K pathway. Effects of PI3K inhibitor BEZ-235 on ER expression and activity levels and cell growth were tested by quantitative real-time PCR and cell proliferation assays.ResultsWithin ER+ tumors, ER levels were negatively correlated with the PI3K activation scores, both at the proteomic and transcriptional levels, in all datasets examined. PI3K signature scores were also higher in ER+ tumors and cell lines of the more aggressive luminal B molecular subtype versus those of the less aggressive luminal A subtype. Notably, BEZ-235 treatment in four different ER+ cell lines increased expression of ER and ER target genes including PR, and treatment with IGF-I (which signals via PI3K) decreased expression of ER and target genes, thus further establishing an inverse functional relation between ER and PI3K. BEZ-235 had an additional effect on tamoxifen in inhibiting the growth of a number of ER+ cell lines.ConclusionsOur data suggest that luminal B tumors have hyperactive GFR/PI3K signaling associated with lower ER levels, which has been correlated with resistance to endocrine therapy. Targeting PI3K in these tumors might reverse loss of ER expression and signaling and restore hormonal sensitivity.

Gene Expression Signatures of PI3K Signaling Are Associated with Low ER Levels and the Luminal B Subtype in Breast Cancer Cell Lines and Human Tumors, and in Patients Predicts Poor Outcome.

Abstract Background: Accumulating evidence suggests that both levels and activity of ER and progesterone receptor (PR) are dramatically influenced by growth factor receptor (GFR) signaling pathways, and that this crosstalk is a major determinant of both breast cancer (BC) progression and response to therapy. The phosphatidylinositol 3-kinase (PI3K) pathway, a key mediator of GFR signaling, is one of the most altered pathways in BC. We thus examined whether deregulated PI3K signaling in luminal ER+ breast tumors is associated with ER level and activity, molecular subtype, and outcome.Materials and Methods: To directly test for an association between ER levels and PI3K activation in BC, we defined a PI3K transcriptional signature as the set of genes either induced or repressed by PI3K inhibitors in the public Connectivity Map (CMap) expression profile dataset (www.broad.mit.edu/cmap). Using this signature, we assigned a PI3K activation score to each ER+ human breast tumor from two independent expression profile datasets (van de Vijver et al., n=226; Loi et al., n=348). Furthermore, within a broad panel of luminal BC cell lines, we compared the gene signature-based CMap PI3K score with a proteomic (Reversed Phase Proteomic Array, RPPA)-based PI3K score (which relied on protein measurement for PI3K signaling intermediates). We also compared the CMap-derived PI3K activation scores with scores based on a signature of PTEN loss in human breast tumors (Saal et al.), and on a signature of Akt activation in a transgenic mouse model (Majumder et al.).Results: Within ER+ tumors, ER mRNA level was negatively associated with the CMap PI3K activation score (van de Vijver: Pearson's rs=-0.32, p<1E-06; Loi: rs=-0.18, p<0.002). PI3K activation scores based on CMap, Saal, or Majumder gene signatures were highly correlated with each other (van de Vijver rs=0.65-0.71). When scoring for luminal B versus luminal A molecular subtypes, CMap PI3K scores were associated with the more aggressive luminal B subtype (van de Vijver rs=0.75, p<1E-40). High PI3K activation scores were associated with poorer outcome in ER+ tumors treated or untreated with tamoxifen (tamoxifen treated: Loi p=0.02, n=263; van de Vijver p=0.05, n=34, log-rank statistic). Analysis of 18 previously identified luminal BC cell lines showed a significant association between PI3K activation at the genomic and proteomic levels (rs=0.47, p<0.02) and, as in the clinical tumors, there was a strong and significant association between PI3K activation and the luminal B subtype.Discussion: Our data suggest that luminal B tumors have hyperactive GFR/PI3K signaling associated with lower ER levels, which has been correlated with resistance to endocrine therapy. Targeting PI3K in these tumors might reverse loss of ER expression and signaling and restore hormonal sensitivity. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 31.

Inhibition of phosphatidylinositol 3-kinase signaling in hepatic stellate cells blocks the progression of hepatic fibrosis #

The hepatic stellate cell (HSC) is the primary cell type in the liver responsible for excess collagen deposition during fibrosis. Following a fibrogenic stimulus the cell changes from a quiescent vitamin A-storing cell to an activated cell type associated with increased extracellular matrix synthesis and increased cell proliferation. The phosphatidylinositol 3-kinase (PI3K) signaling pathway has been shown to regulate several aspects of HSC activation in vitro, including collagen synthesis and cell proliferation. Using a targeted approach to inhibit PI3K signaling specifically in HSCs, we investigated the role of PI3K in HSCs using a rodent model of hepatic fibrosis. An adenovirus expressing a dominant negative form of PI3K under control of the smooth muscle alpha-actin (alphaSMA) promoter was generated (Ad-SMAdnPI3K). Transducing HSCs with Ad-SMAdnPI3K resulted in decreased proliferation, migration, collagen expression, and several additional profibrogenic genes, while also promoting cell death. Inhibition of PI3K signaling was also associated with reduced activation of Akt, p70 S6 kinase, and extracellular regulated kinase signaling as well as reduced cyclin D1 expression. Administering Ad-SMAdnPI3K to mice following bile duct ligation resulted in reduced HSC activation and decreased extracellular matrix deposition, including collagen expression. A reduction in profibrogenic mediators, including transforming growth factor beta, tissue inhibitor of metalloproteinase 1, and connective tissue growth factor was also noted. However, liver damage, assessed by alanine aminotransferase levels, was not reduced. Inhibition of PI3K signaling in HSCs during active fibrogenesis inhibits extracellular matrix deposition, including synthesis of type I collagen, and reduces expression of profibrogenic factors. These data suggest that targeting PI3K signaling in HSCs may represent an effective therapeutic target for hepatic fibrosis.

Effect of ZSTK474, a Novel Phosphatidylinositol 3-Kinase Inhibitor, on DNA-Dependent Protein Kinase

Phosphatidylinositol 3-kinase (PI3K) has been implicated in a variety of diseases including cancer. A number of PI3K inhibitors have recently been developed for use in cancer therapy. ZSTK474 is a highly promising antitumor agent targeting PI3K. We previously reported that ZSTK474 showed potent inhibition against four class I PI3K isoforms but not against 140 protein kinases. However, whether ZSTK474 inhibits DNA-dependent protein kinase (DNA-PK), which is structurally similar to PI3K, remains unknown. To investigate the inhibition of DNA-PK, we developed a new DNA-PK assay method using Kinase-Glo. The inhibition activity of ZSTK474 against DNA-PK was determined, and shown to be far weaker compared with that observed against PI3K. The inhibition selectivity of ZSTK474 for PI3K over DNA-PK was significantly higher than other PI3K inhibitors, namely NVP-BEZ235, PI-103 and LY294002. These results indicated that ZSTK474 was the most specific agent among these PI3K inhibitors.

Combined inhibition of the phosphatidylinositol 3-kinase/Akt and Ras/mitogen-activated protein kinase pathways results in synergistic effects in glioblastoma cells

The present study uses cell-based screening assays to assess the anticancer effects of targeting phosphatidylinositol 3-kinase-regulated integrin-linked kinase (ILK) in combination with small-molecule inhibitors of Raf-1 or mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase kinase (MEK). The objective was to determine if synergistic interactions are achievable through the use of agents targeting two key cell signaling pathways involved in regulating glioblastoma cancer. The phosphatidylinositol 3-kinase/protein kinase B (PKB)/Akt and the Ras/MAPK pathway were targeted for their involvement in cell survival and cell proliferation, respectively. The glioblastoma cell lines U87MG, SF-188, and U251MG were transiently transfected with an antisense oligonucleotide targeting ILK (ILKAS) alone or in combination with the Raf-1 inhibitor GW5074 or with the MEK inhibitor U0126. Dose and combination effects were analyzed by the Chou and Talalay median-effect method and indicated that combinations targeting ILK with either Raf-1 or MEK resulted in a synergistic interaction. Glioblastoma cells transfected with ILKAS exhibited reduced levels of ILK and phosphorylated PKB/Akt on Ser473 but not PKB/Akt on Thr308 as shown by immunoblot analysis. These results were confirmed using glioblastoma cells transfected with ILK small interfering RNA, which also suggested enhanced gene silencing when used in combination with U0126. U87MG glioblastoma cells showed a 90% (P < 0.05) reduction in colony formation in soft agar with exposure to ILKAS in combination with GW5074 compared with control colonies. A substantial increase in Annexin V-positive cells as determined by using fluorescence-activated cell sorting methods were seen in combinations that included ILKAS. Combinations targeting ILK and components of the Ras/MAPK pathway result in synergy and could potentially be more effective against glioblastoma cancer than monotherapy.

The Targeting of Phosphoinositide-3 Kinase Attenuates Pulmonary Metastatic Tumor Growth Following Laparotomy

We aimed to characterize a potential role for phosphatidylinositol 3-kinase (PI3k) in leading to accelerated postoperative metastatic tumor growth. PI3k enhances tumor cell survival in part by phosphorylating Akt and reducing apoptosis. Postoperatively, apoptosis is reduced within local recurrences and distant metastases. This reduction is associated with the phenomenon of accelerated postoperative tumor growth. Balb/c mice underwent a tail vein injection of 1x10 metastatic murine mammary adenocarcinoma 4T1 cells. Animals were divided into the following treatment groups (n=10/group): group A, controls; group B, DMSO intraperitoneally (IP) daily from days 14 to 21; group C, IP LY294002 daily from days 14 to 21; group D, laparotomy only; group E, laparotomy followed by IP DMSO for 7 days; and group F, laparotomy followed by LY294002 IP for 7 days. All laparotomies were performed on day 14. Animals were killed at day 28. Metastatic tumor burden was assessed using the lung/body weight ratio and a histologic metastatic index. Mitotic counts and apoptotic indices were established using a combination of hematoxylin and eosin histology and TUNEL immunohistochemistry. A parallel survival study was performed, and PI3k activity was assessed using western blots for phospho-Akt. Laparotomy was associated with increased systemic tumor burden (P=0.001). Postoperatively, LY294002 significantly attenuated metastatic tumor growth (P<0.001). Effective PI3k inhibition was confirmed by demonstrating a reduced Akt phosphorylation. Moreover, PI3k inhibition led to reduced proliferation, increased apoptosis (P<0.001), and enhanced postoperative survival (P<0.001). Targeting PI3k with postoperative LY294002 significantly attenuates the acceleration in postoperative metastatic tumor growth seen following laparotomy.

Epidermal growth factor receptor agonists increase expression of glutamate transporter GLT-1 in astrocytes through pathways dependent on phosphatidylinositol 3-kinase and transcription factor NF-kappaB.

The glial glutamate transporter GLT-1 may be the predominant Na(+)-dependent glutamate transporter in forebrain. Expression of GLT-1 correlates with astrocyte maturation in vivo and increases during synaptogenesis. In astrocyte cultures, GLT-1 expression parallels differentiation induced by cAMP analogs or by coculturing with neurons. Molecule(s) secreted by neuronal cultures contribute to this induction of GLT-1, but little is known about the signaling pathways mediating this regulation. In the present study, we determined whether growth factors previously implicated in astrocyte differentiation regulate GLT-1 expression. Of the six growth factors tested, two [epidermal growth factor (EGF) and transforming growth factor-alpha] induced expression of GLT-1 protein in cultured astrocytes. Induction of GLT-1 protein was accompanied by an increase in mRNA and in the V(max) for Na(+)-dependent glutamate transport activity. The effects of dibutyryl-cAMP and EGF were additive but were independently blocked by inhibitors of protein kinase A or protein tyrosine kinases, respectively. The induction of GLT-1 in both EGF- and dibutyryl-cAMP-treated astrocytes was blocked by inhibitors targeting phosphatidylinositol 3-kinase (PI3K) or the nuclear transcription factor-kappaB. Furthermore, transient transfection of astrocyte cultures with a constitutively active PI3K construct was sufficient to induce expression of GLT-1. These data suggest that independent but converging pathways mediate expression of GLT-1. Although an EGF receptor-specific antagonist did not block the effects of neuron-conditioned medium, the induction of GLT-1 by neuron-conditioned medium was completely abolished by inhibition of PI3K or nuclear factor-kappaB. EGF also increased expression of GLT-1 in spinal cord organotypic cultures. Together, these data suggest that activation of specific signaling pathways with EGF-like molecules may provide a novel approach for limiting excitotoxic brain injury.