Inhibition Of Akt Kinase Activity Research Articles

AKT Kinase Activity Is Required for Lithium to Modulate Mood-Related Behaviors in Mice

Bipolar disorder (BP) is a debilitating psychiatric disorder, affecting ∼2% of the worldwide population, for which the etiological basis, pathogenesis, and neurocircuitry remain poorly understood. Individuals with BP suffer from recurrent episodes of mania and depression, which are commonly treated with the mood stabilizer lithium. However, nearly half of BP patients do not respond adequately to lithium therapy and the clinically relevant mechanisms of lithium for mood stabilization remain elusive. Here, we modeled lithium responsiveness using cellular assays of glycogen synthase kinase 3 (GSK-3) signaling and mood-related behavioral assays in inbred strains of mice that differ in their response to lithium. We found that activating AKT through phosphosrylation of a key regulatory site (Thr308) was associated with lithium response-activation of signaling pathways downstream of GSK-3 in cells and attenuation of mood-related behaviors in mice-and this response was attenuated by selective and direct inhibition of AKT kinase activity. Conversely, the expression of constitutively active AKT1 in both the cellular and behavioral assays conferred lithium sensitivity. In contrast, selective and direct GSK-3 inhibition by the ATP-competitive inhibitor CHIR99021 bypassed the requirement for AKT activation and modulated behavior in both lithium-responsive and non-responsive mouse strains. These results distinguish the mechanism of action of lithium from direct GSK-3 inhibition both in vivo and in vitro, and highlight the therapeutic potential for selective GSK-3 inhibitors in BP treatment.

Lycopene Enhances Docetaxel's Effect in Castration-Resistant Prostate Cancer Associated with Insulin-like Growth Factor I Receptor Levels

Docetaxel is currently the most effective drug for the treatment of castration-resistant prostate cancer (CRPC), but it only extends life by an average of 2 months. Lycopene, an antioxidant phytochemical, has antitumor activity against prostate cancer (PCa) in several models and is generally safe. We present data on the interaction between docetaxel and lycopene in CRPC models. The growth-inhibitory effect of lycopene on PCa cell lines was positively associated with insulin-like growth factor I receptor (IGF-IR) levels. In addition, lycopene treatment enhanced the growth-inhibitory effect of docetaxel more effectively on DU145 cells with IGF-IR high expression than on those PCa cell lines with IGF-IR low expression. In a DU145 xenograft tumor model, docetaxel plus lycopene caused tumor regression, with a 38% increase in antitumor efficacy (P = .047) when compared with docetaxel alone. Lycopene inhibited IGF-IR activation through inhibiting IGF-I stimulation and by increasing the expression and secretion of IGF-BP3. Downstream effects include inhibition of AKT kinase activity and survivin expression, followed by apoptosis. Together, the enhancement of docetaxel's antitumor efficacy by lycopene supplementation justifies further clinical investigation of lycopene and docetaxel combination for CRPC patients. CRPC patients with IGF-IR-overexpressing tumors may be most likely to benefit from this combination.

The N-terminal Fragment from Caspase-cleaved Serine/Arginine Protein-specific Kinase2 (SRPK2) Translocates into the Nucleus and Promotes Apoptosis

SRPK2 belongs to a family of serine/arginine (SR) protein-specific kinases (SRPKs), which phosphorylate SR domain-containing proteins in the nuclear speckles and mediate the pre-mRNA splicing. Previous studies have shown that SRPK2 plays a pivotal role in cell proliferation and apoptosis. However, how SRPK2 is regulated during the apoptosis is unclear. Here, we show that SRPK2 is cleaved by caspases at Asp-139 and -403 residues. Its N terminus cleaved product translocates into the nucleus and promotes VP16-induced apoptosis. Akt phosphorylation of SRPK2 prevents its apoptotic cleavage by caspases. 14-3-3β, the binding partner of Akt-phosphorylated SRPK2, further protects it from degradation. Hence, our results suggest that the N-terminal domain of SRPK2 cleaved by caspases translocates into the nucleus, where it promotes chromatin condensation and apoptotic cell death.

Topotecan as a molecular targeting agent which blocks the Akt and VEGF cascade in platinum-resistant ovarian cancers

(Objective) Topotecan, a novel topoisomerase-1 inhibitor, is a drug that appears to be effective against platinum-resistant ovarian cancers. However, the molecular mechanisms by which Topotecan treatment inhibits cancer cell proliferation are unclear. We investigated whether Topotecan increases the efficacy of Cisplatin in platinum-resistant ovarian cancer models in vitro and in vivo. (Methods) We used Cisplatin-resistant Caov-3 cells and Cisplatin-sensitive A2780 cells. We examined the effect of Cisplatin and Topotecan on the cell viability of Caov-3 and A2780 cells by MTS assay. We examined the Akt kinase activity, VEGF and HIF-1α expression after Cisplatin and Topotecan by a Western blot analysis. Moreover, we also evaluated the effects of Cisplatin and Topotecan on the intraabdominal dissemination of ovarian cancer in vivo. (Results) Topotecan significantly inhibited Cisplatin-induced Akt activation in Caov-3 cells, but not in A2780 cells. In the presence of Topotecan, Cisplatin-induced growth inhibition and apoptosis were significantly enhanced in Caov-3 cells. Topotecan inhibited not only Cisplatin-induced Akt activation but also VEGF and HIF-1α expression. Moreover, treatment with Topotecan increased the efficacy of Cisplatin-induced growth inhibition in the intraabdominal dissemination and production of ascites in athymic nude mice inoculated with Caov-3 cells. (Conclusion) We herein demonstrated that Topotecan inhibits Akt kinase activity and VEGF transcriptional activation after Cisplatin treatment in platinum-resistant ovarian cancers. We clarified how Topotecan enhanced the clinical activity in the platinum-resistant ovarian cancer. These results provide a rationale for using Topotecan in clinical regimens aimed at molecular targeting agents in platinum-resistant ovarian cancers.

Causal reasoning identifies mechanisms of sensitivity for a novel AKT kinase inhibitor, GSK690693

BackgroundInappropriate activation of AKT signaling is a relatively common occurrence in human tumors, and can be caused by activation of components of, or by loss or decreased activity of inhibitors of, this signaling pathway. A novel, pan AKT kinase inhibitor, GSK690693, was developed in order to interfere with the inappropriate AKT signaling seen in these human malignancies. Causal network modeling is a systematic computational analysis that identifies upstream changes in gene regulation that can serve as explanations for observed changes in gene expression. In this study, causal network modeling is employed to elucidate mechanisms of action of GSK690693 that contribute to its observed biological effects. The mechanism of action of GSK690693 was evaluated in multiple human tumor cell lines from different tissues in 2-D cultures and xenografts using RNA expression and phosphoproteomics data. Understanding the molecular mechanism of action of novel targeted agents can enhance our understanding of various biological processes regulated by the intended target and facilitate their clinical development.ResultsCausal network modeling on transcriptomic and proteomic data identified molecular networks that are comprised of activated or inhibited mechanisms that could explain observed changes in the sensitive cell lines treated with GSK690693. Four networks common to all cell lines and xenografts tested were identified linking GSK690693 inhibition of AKT kinase activity to decreased proliferation. These networks included increased RB1 activity, decreased MYC activity, decreased TFRC activity, and increased FOXO1/FOXO3 activity.ConclusionAKT is involved in regulating both cell proliferation and apoptotic pathways; however, the primary effect with GSK690693 appears to be anti-proliferative in the cell lines and xenografts evaluated. Furthermore, these results indicate that anti-proliferative responses to GSK690693 in either 2-D culture or xenograft models may share common mechanisms within and across sensitive cell lines.

Delayed growth of glioma by Scutellaria flavonoids involve inhibition of Akt, GSK-3 and NF-κB signaling

Plants of the genus Scutellaria constitute one of the common components of Eastern as well as traditional American medicine against various human diseases, including cancer. In this study, we examined the in vivo anti-glioma activity of a leaf extract of Scutellaria ocmulgee (SocL) while also exploring their potential molecular mechanisms of action. Oral administration of SocL extract delayed the growth of F98 glioma in F344 rats, both in intracranial and subcutaneous tumor models. Immunohistochemistry revealed inhibition of Akt, GSK-3α/β and NF-κB phosphorylation in the subcutaneous tumors following treatment with Scutellaria. The SocL extract as well as the constituent flavonoid wogonin also showed dose- and time-dependent inhibition of Akt, GSK-3α/β and NF-κB in F98 cell cultures in vitro, as determined by western blot analysis. Pharmacologic inhibitors of PI3K and NF-κB also significantly inhibited the in vitro proliferation of F98 glioma cells, indicating the key role of these signaling molecules in the growth of malignant gliomas. Transfection of F98 cells with constitutively active mutant of AKT (AKT/CA), however, did not significantly reverse Scutellaria-mediated inhibition of proliferation, indicating that Scutellaria flavonoids either directly inhibited Akt kinase activity or acted downstream of Akt. In vitro Akt kinase assay demonstrated that the SocL extract or wogonin could indeed bind to Akt and inhibit its kinase activity. This study provides the first in vivo evidence and mechanistic support for anti-glioma activity of Scutellaria flavonoids and has implications in potential usage of Scutellaria flavonoids in adjuvant therapy for malignant tumors, including gliomas.

Eradication of Intracellular Salmonella enterica Serovar Typhimurium with a Small-Molecule, Host Cell-Directed Agent

Eradication of intracellular pathogenic bacteria with host-directed chemical agents has been an anticipated innovation in the treatment of antibiotic-resistant bacteria. We previously synthesized and characterized a novel small-molecule agent, AR-12, that induces autophagy and inhibits the Akt kinase in cancer cells. As both autophagy and the Akt kinase have been shown recently to play roles in the intracellular survival of several intracellular bacteria, including Salmonella enterica serovar Typhimurium, we investigated the effect of AR-12 on the intracellular survival of Salmonella serovar Typhimurium in macrophages. Our results show that AR-12 induces autophagy in macrophages, as indicated by increased autophagosome formation, and potently inhibits the survival of serovar Typhimurium in macrophages in association with increased colocalization of intracellular bacteria with autophagosomes. Intracellular bacterial growth was partially rescued in the presence of AR-12 by the short hairpin RNA-mediated knockdown of Beclin-1 or Atg7 in macrophages. Moreover, AR-12 inhibits Akt kinase activity in infected macrophages, which we show to be important for its antibacterial effect as the enforced expression of constitutively activated Akt1 in these cells reverses the AR-12-induced inhibition of intracellular serovar Typhimurium survival. Finally, oral administration of AR-12 at 2.5 mg/kg/day to serovar Typhimurium-infected mice reduced hepatic and splenic bacterial burdens and significantly prolonged survival. These findings show that AR-12 represents a proof of principle that the survival of intracellular bacteria can be suppressed by small-molecule agents that target both innate immunity and host cell factors modulated by bacteria.

Akt inhibitor Akt-IV blocks virus replication through an Akt-independent mechanism.

Many viruses activate the phosphatidylinositol 3'-kinase (PI3k)/Akt intracellular signaling pathway to promote viral replication. We have analyzed whether a rapidly replicating rhabdovirus, vesicular stomatitis virus (VSV), requires the PI3k/Akt signaling pathway for its replication. Through the use of chemical inhibitors of PI3k and Akt, we show that VSV replication and cytopathic effects do not require activation of these kinases. Inhibitors that block the activating phosphorylations of Akt at threonine 308 (Thr308) and serine 473 (Ser473) did not inhibit VSV protein expression or the induction of the cytopathic effects of VSV. One compound, Akt inhibitor Akt-IV, inhibited the replication of VSV, respiratory syncytial virus, and vaccinia virus but increased the phosphorylation of Akt at positions Thr308 and Ser473 and did not inhibit Akt kinase activity in vitro. Together, our data suggest that the PI3k/Akt pathway is of limited relevance to the replication of VSV but that Akt inhibitor Akt-IV is a novel broad-spectrum antiviral compound with a mechanism differing from that of its previously reported effect on the PI3k/Akt pathway. Identification of other targets for this compound may define a new approach for blocking virus replication.

Characterization of an Akt Kinase Inhibitor with Potent Pharmacodynamic and Antitumor Activity

Akt kinases 1, 2, and 3 are important regulators of cell survival and have been shown to be constitutively active in a variety of human tumors. GSK690693 is a novel ATP-competitive, low-nanomolar pan-Akt kinase inhibitor. It is selective for the Akt isoforms versus the majority of kinases in other families; however, it does inhibit additional members of the AGC kinase family. It causes dose-dependent reductions in the phosphorylation state of multiple proteins downstream of Akt, including GSK3 beta, PRAS40, and Forkhead. GSK690693 inhibited proliferation and induced apoptosis in a subset of tumor cells with potency consistent with intracellular inhibition of Akt kinase activity. In immune-compromised mice implanted with human BT474 breast carcinoma xenografts, a single i.p. administration of GSK690693 inhibited GSK3 beta phosphorylation in a dose- and time-dependent manner. After a single dose of GSK690693, >3 micromol/L drug concentration in BT474 tumor xenografts correlated with a sustained decrease in GSK3 beta phosphorylation. Consistent with the role of Akt in insulin signaling, treatment with GSK690693 resulted in acute and transient increases in blood glucose level. Daily administration of GSK690693 produced significant antitumor activity in mice bearing established human SKOV-3 ovarian, LNCaP prostate, and BT474 and HCC-1954 breast carcinoma xenografts. Immunohistochemical analysis of tumor xenografts after repeat dosing with GSK690693 showed reductions in phosphorylated Akt substrates in vivo. These results support further evaluation of GSK690693 as an anticancer agent.

Epigallocatechin-3 gallate induces growth inhibition and apoptosis in human breast cancer cells through survivin suppression

Recent investigations have demonstrated that polyphenolic catechins inhibit cancer cell proliferation and tumor growth. However, how the major active component of tea catechins, epigallocatechin-3 gallate (EGCG), mediates anticancerous effects has not been extensively examined. We have investigated the cell growth inhibitory effects of EGCG on cell growth of the human breast cancer cell line MCF-7, and the mechanism of its action with emphasis on the regulation of tumor cell survival. A significant EGCG dose-dependent growth inhibition was observed coordinated with EGCG-induced apoptosis. Analysis of survivin expression after addition of EGCG showed that both survivin mRNA and protein were decreased. The survivin-promoter luciferase activity in EGCG-treated cells was significantly inhibited by 91+/-2.0% (P<0.001), compared with the control. Interestingly, EGCG strongly inhibited the basal activation of phospho-AKT and AKT kinase activity as early as 30 min after treatment. Furthermore, inhibition of AKT kinase activity by EGCG preceded the suppression of survivin (1 h post treatment), followed by increased caspase-9 activity (6 h post treatment). A dominant negative AKT or the phosphatidylinositol 3-kinase inhibitor, LY294002, also strongly inhibited survivin promoter activity, providing further evidence to support the hypothesis that the inhibitory effect of EGCG on survivin is mediated via the AKT pathway. Therefore, EGCG is a potent proapoptotic agent in MCF-7 breast cancer cells that targets survivin expression via suppression of the AKT pathway.

Silymarin Prevents Palmitate‐Induced Lipotoxicity in HepG2 Cells: Involvement of Maintenance of Akt Kinase Activation

Whereas adipocytes have a unique capacity to store excess free fatty acids in the form of triglyceride in lipid droplets, non-adipose tissues, such as liver, have a limited capacity for storage of lipids. Saturated long-chain fatty acids, such as palmitate, are the major contributors to lipotoxicity. Silymarin is a mixture of flavonolignans, extracted from the milk thistle (Silibum marianum). Its hepatoprotective properties have been studied both in vitro and in vivo; however, its effect on palmitate-induced lipotoxicity has not been investigated. The objective of this study was to investigate (i) whether silymarin could protect HepG2 cells from palmitate-induced cell death in an in vitro model, and (ii) possible mechanisms involved in this hepatoprotective role of silymarin. HepG2 cells were treated with palmitate in the absence or presence of silymarin and supernatants or cell lysates were collected at varying time-points. Cell death was assayed by measuring DNA fragmentation, caspase-3 activity and lactate dehydrogenase release. Lipid peroxidation was assessed by measuring malondialdehyde and 4-hydroxyalkenals. Akt kinase activity was also measured. Incubation with palmitate caused significant death in HepG2 cells. Palmitate incubation did not cause significant changes in reactive oxygen species production or intracellular glutathione content, but markedly inhibited Akt kinase activity. Pre-treatment of HepG2 cells with silymarin prevented palmitate-induced inhibition of Akt kinase activity and attenuated cell death. Our results suggest that silymarin may be an effective agent in protecting hepatocytes from saturated fatty acids-induced cell death. These data also provide a further rationale for exploration of the use of silymarin in the treatment of non-alcoholic steatohepatitis.

Antiproliferative activity of sulforaphane in Akt-overexpressing ovarian cancer cells

Epidemiologic studies show a correlation between increased consumption of fruits and vegetables with reduced risk of ovarian cancer. One major bioactive compound found in cruciferous vegetables, particularly broccoli, is sulforaphane, derived from the breakdown of glucoraphanin. We observed potent antiproliferative effects of sulforaphane on human ovarian cancer cell line SKOV3 (IC(50) 40 micromol/L) and mouse ovarian cancer cell lines C3 and T3 (IC(50) 25 micromol/L each) by cell viability assays. The loss of viability is reflected by a down-regulation of cell cycle transition regulators cyclin D1, cyclin-dependent kinase 4 (cdk4), and cdk6. The upstream mediators of sulforaphane effects on the cell cycle in ovarian cancer are still unknown. However, because the Akt signal transduction pathway is overactivated in ovarian cancer, we investigated the effects of sulforaphane on this prosurvival pathway. Both total Akt protein and active phosphorylated levels of Akt (Ser(473)) and phosphoinositide 3-kinase were significantly decreased in sulforaphane-treated SKOV3, C3, and T3 cells with a concomitant inhibition of Akt kinase activity by sulforaphane in SKOV3 and C3 cells. This inhibitory effect of sulforaphane leads to a potent induction of apoptosis in all three cell lines, along with the cleavage of poly(ADP)ribose polymerase. Our study is the first to report the antiproliferative effects of sulforaphane in ovarian cancer and identifying the Akt pathway as a target of sulforaphane, with implications for the inhibition of carcinogenesis by diet-based chemoprevention.

Downregulation of catalase by reactive oxygen species via PI 3 kinase/Akt signaling in mesangial cells

Reactive oxygen species (ROS) contribute to many glomerular diseases by targeting mesangial cells. ROS have been shown to regulate expression of many antioxidant enzymes including catalase. The mechanism by which the expression of catalase protein is regulated by ROS is not precisely known. Here we report that increased intracellular ROS level by hydrogen peroxide (H(2)O(2)) reduced the expression of catalase. H(2)O(2) increased phosphorylation of Akt kinase in a dose-dependent and sustained manner with a concomitant increase in the phosphorylation of FoxO1 transcription factor. Further analysis revealed that H(2)O(2) promoted rapid activation of phosphatidylinositol (PI) 3 kinase. The PI 3 kinase inhibitor Ly294002 and expression of tumor suppressor protein PTEN inhibited Akt kinase activity, resulting in the attenuation of FoxO1 phosphorylation and preventing the downregulating effect of H(2)O(2) on catalase protein level. Dominant negative Akt attenuated the inhibitory effect of H(2)O(2) on expression of catalase. Constitutively active FoxO1 increased the expression of catalase. However, dominant negative FoxO1 inhibited catalase protein level. Catalase transcription was reduced by H(2)O(2) treatment. Furthermore, expression of dominant negative Akt and constitutively active FoxO1 increased catalase transcription, respectively. These results demonstrate that ROS downregulate the expression of catalase in mesangial cells by PI 3 kinase/Akt signaling via FoxO1 as a target.

Protein Kinase C Negatively Regulates Akt Activity and Modifies UVC-induced Apoptosis in Mouse Keratinocytes

Skin keratinocytes are subject to frequent chemical and physical injury and have developed elaborate cell survival mechanisms to compensate. Among these, the Akt/protein kinase B (PKB) pathway protects keratinocytes from the toxic effects of ultraviolet light (UV). In contrast, the protein kinase C (PKC) family is involved in several keratinocyte death pathways. During an examination of potential interactions among these two pathways, we found that the insulin-like growth factor (IGF-1) activates both the PKC and the Akt signaling pathways in cultured primary mouse keratinocytes as indicated by increased phospho-PKC and phospho-Ser-473-Akt. IGF-1 also selectively induced translocation of PKCdelta and PKCepsilon from soluble to particulate fractions in mouse keratinocytes. Furthermore, the PKC-specific inhibitor, GF109203X, increased IGF-1-induced phospho-Ser-473-Akt and Akt kinase activity and enhanced IGF-1 protection from UVC-induced apoptosis. Selective activation of PKC by 12-O-tetradecanoylphorbol-13-acetate (TPA) reduced phospho-Ser-473-Akt, suggesting that activation of PKC inhibits Akt activity. TPA also attenuated IGF-1 and epidermal growth factor-induced phospho-Ser-473-Akt, reduced Akt kinase activity, and blocked IGF-1 protection from UVC-induced apoptosis. The inhibition of Akt activity by TPA was reduced by inhibitors of protein phosphatase 2A, and TPA stimulated the association of phosphatase 2A with Akt. Individual PKC isoforms were overexpressed in cultured keratinocytes by transduction with adenoviral vectors or inhibited with PKC-selective inhibitors. These studies indicated that PKCdelta and PKCepsilon were selectively potent at causing dephosphorylation of Akt and modifying cell survival, whereas PKCalpha enhanced phosphorylation of Akt on Ser-473. Our results suggested that activation of PKCdelta and PKCepsilon provide a negative regulation for Akt phosphorylation and kinase activity in mouse keratinocytes and serve as modulators of cell survival pathways in response to external stimuli.

A small molecule compound inhibits AKT pathway in ovarian cancer cell lines

Background and objective. Overactivation of AKT1 and gene amplification of AKT2 are frequently detected in ovarian cancer. Activated AKT kinases provide a cell survival signal that may confer resistance to apoptosis induced by conventional therapies in cancer cells. Therefore, development of potent inhibitors that block AKT pathway is an attractive therapeutic strategy for treating ovarian carcinoma. Methods. Ovarian cancer cell lines, A2780, MDAH2774, OVCAR-8, Caov-3, and normal murine fibroblasts (NIH3T3) were used. Cells were treated with different doses of a non-peptide small molecule compound, 9-methoxy-2-methylellipticinium acetate (termed API-59-OME) that potentially inhibit AKT pathway. Kinase assays and the phosphorylation of AKT, GSK-3α/β, PDK1, ERK1/2, SGK, p38, FAK, EGFR, JAK2, PKC isoforms, and the cleavage of poly (ADP-ribose) polymerase (PARP) were examined in treated and untreated cell lines. Further, cells treated with API-59-OME were analyzed for induction of apoptosis using sub-G1 profile with propidium iodide staining. Results. API-59-OME inhibited AKT kinase activity but did not inhibit ERK or JNK kinase activities in A2780, MDAH2774, and OVCAR-8 cell lines. API-59-OME did not reduce phosphorylation of other protein kinases in these cell lines. API-59-OME induced apoptosis and the cleavage of PARP in A2780, MDAH2774, and OVCAR-8 ovarian cancer cell lines that express elevated levels of phosphorylated AKT. In contrast, in Caov-3 and NIH3T3 cell lines, which lack constitutive AKT activity, API-59-OME only had minimal effect to induce apoptosis. Conclusion. These data suggest that API-59-OME may be a potent agent to target constitutively activated AKT pathway in ovarian cancer cells.

ArgBP2γ Interacts with Akt and p21-activated Kinase-1 and Promotes Cell Survival

Akt/protein kinase B is a major cell survival pathway through phosphorylation of proapoptotic proteins Bad and Bax and of additional apoptotic pathways linked to Forkhead proteins glycogen synthase kinase-3beta and ASK1. To further explore the mechanism by which Akt regulates cell survival, we identified an Akt interaction protein by yeast two-hybrid screening. It is highly homologous to ARG-binding protein 2 (ArgBP2) with splicing exon 8 of the coding region of the ArgBP2. As two splicing isoforms (ArgBP2alpha and -beta) of ArgBP2 have been identified (Wang, B., Golemis, E. A., and Kruh, G. D. (1997) J. Biol. Chem. 272, 17542-17550), it was named ArgBP2gamma. ArgBP2gamma contains four Akt phosphorylation consensus sites, a SoHo motif, and three Src homology (SH) 3 domains and binds to C-terminal proline-rich motifs of Akt through its first and second SH3 domains. It also interacts with p21-activated protein kinase (PAK1) via its first and third SH3 domains, indicating the SH3 domains of ArgBP2gamma as docking sites for Akt and PAK1. Akt phosphorylates ArgBP2gamma in vitro and in vivo. Expression of ArgBP2gamma induces PAK1 activity and overrides apoptosis induced by ectopic expression of Bad or DNA damage. Nonphosphorylatable ArgBP2gamma-4A and SH3 domain-truncated mutant ArgBP2gamma inhibit Akt-induced PAK1 activation and reduce Akt and PAK1 phosphorylation of Bad and antiapoptotic function. These data indicate that ArgBP2gamma is a physiological substrate of Akt, functions as an adaptor for Akt and PAK1, and plays a role in Akt/PAK1 cell survival pathway.

Inhibition of Nuclear Translocation of Nuclear Factor-κB Contributes to 3,3′-Diindolylmethane-Induced Apoptosis in Breast Cancer Cells

AbstractDietary indole-3-carbinol (I3C), a natural compound present in vegetables of the genus Brassica, showed clinical benefits and caused apoptosis in breast cancer cells. Our laboratory and others have shown that I3C induces apoptosis in breast cancer cells mediated by inactivation of Akt and nuclear factor-κB (NF-κB) pathway. 3,3′-Diindolylmethane (DIM), a major in vivo acid-catalyzed condensation product of I3C, also showed some benefit in breast cancer. However, the precise molecular mechanism(s) by which DIM induces apoptosis in breast cancer cells has not been fully elucidated. Hence, we investigated whether DIM-induced apoptosis of breast cancer cells could also be mediated by inactivation of Akt and NF-κB. We found that DIM induces apoptotic processes in MCF10A derived malignant (MCF10CA1a) cell lines but not in nontumorigenic parental MCF10A cells. DIM specifically inhibits Akt kinase activity and abrogates the epidermal growth factor–induced activation of Akt in breast cancer cells, similar to those observed for I3C. We also found that DIM reduces phosphorylation of IκBα, an inhibitor of NF-κB. Our confocal microscopy study clearly showed that DIM blocks the translocation of p65, a subunit of NF-κB to the nucleus. DNA binding analysis and transfection studies with IκB kinase cDNA revealed that overexpression of IκB kinase mediates IκBα phosphorylation, which activates NF-κB, and this activation was completely abrogated by DIM treatment. Taken together, these results showed for the first time that the inactivation of Akt and NF-κB activity also plays important roles in DIM-induced apoptosis in breast cancer cells, which seems to be more relevant to in vivo situations.

Inhibition of AKT survival pathway by a small molecule inhibitor in human endometrial cancer cells

The PTEN (phosphatase and tensin homolog deleted on chromosome 10) tumour suppressor is mutated in 40–50% of human endometrial cancers. PTEN exerts its effects in part via inhibition of the antiapoptotic protein AKT. We demonstrate that two endometrial cancer cell lines that harbour PTEN mutations, Ishikawa and RL95-2, have high levels of phosphorylated AKT and high AKT kinase activity. Two additional endometrial cancer cell lines that express wild-type PTEN, Hec1A and KLE, have little phosphorylated AKT and minimal demonstrable AKT kinase activity. We tested a potential inhibitor of the AKT pathway, API-59CJ-OMe, in these four cell lines. We found that API-59CJ-OMe inhibits AKT kinase activity and induces apoptosis in the Ishikawa and RL95-2 cell lines with high AKT activity, but has little effect on Hec1A and KLE cells without AKT activity. API-59CJ-OMe may therefore have therapeutic potential for those endometrial cancers that harbour PTEN mutations and AKT activation.

Apigenin Induces Apoptosis through Proteasomal Degradation of HER2/neu in HER2/neu-overexpressing Breast Cancer Cells via the Phosphatidylinositol 3-Kinase/Akt-dependent Pathway

Apigenin is a low toxicity and non-mutagenic phytopolyphenol and protein kinase inhibitor. It exhibits anti-proliferating effects on human breast cancer cells. Here we examined several human breast cancer cell lines having different levels of HER2/neu expression and found that apigenin exhibited potent growth-inhibitory activity in HER2/neu-overexpressing breast cancer cells but was much less effective for those cells expressing basal levels of HER2/neu. Induction of apoptosis was also observed in HER2/neu-overexpressing breast cancer cells in a dose- and time-dependent manner. However, the one or more molecular mechanisms of apigenin-induced apoptosis in HER2/neu-overexpressing breast cancer cells remained to be elucidated. A cell survival pathway involving phosphatidylinositol 3-kinase (PI3K), and Akt is known to play an important role in inhibiting apoptosis in response to HER2/neu-overexpressing breast cancer cells, which prompted us to investigate whether this pathway plays a role in apigenin-induced apoptosis in HER2/neu-overexpressing breast cancer cells. Our results showed that apigenin inhibits Akt function in tumor cells in a complex manner. First, apigenin directly inhibited the PI3K activity while indirectly inhibiting the Akt kinase activity. Second, inhibition of HER2/neu autophosphorylation and transphosphorylation resulting from depleting HER2/neu protein in vivo was also observed. In addition, apigenin inhibited Akt kinase activity by preventing the docking of PI3K to HER2/HER3 heterodimers. Therefore, we proposed that apigenin-induced cellular effects result from loss of HER2/neu and HER3 expression with subsequent inactivation of PI3K and AKT in cells that are dependent on this pathway for cell proliferation and inhibition of apoptosis. This implies that the inhibition of the HER2/HER3 heterodimer function provided an especially effective strategy for blocking the HER2/neu-mediated transformation of breast cancer cells. Our results also demonstrated that apigenin dissociated the complex of HER2/neu and GRP94 that preceded the depletion of HER2/neu. Apigenin-induced degradation of mature HER2/neu involves polyubiquitination of HER2/neu and subsequent hydrolysis by the proteasome.

Inactivation of Akt and NF-κB Play Important RolesDuring Indole-3-Carbinol-Induced Apoptosis in Breast Cancer Cells

Despite significant advances in treatment, breast cancer is still the second leading cause of cancer-related deaths in women in the United States. Therefore, significant efforts are being given to develop novel strategies for the prevention of breast cancer in recent years. Our laboratory and others have been studying the effects of a potential chemopreventive agent, indole-3-carbinol (I3C), in breast cancer cells. We have previously shown that I3C induces apoptosis in breast cancer cells and found that the induction of apoptotic processes was partly mediated by dysregulation of anti- and pro-apoptotic molecules. However, the precise molecular mechanism(s) by which I3C induces apoptosis in breast cancer cells has not been fully elucidated. For the present study, we focused our investigation on important cell signaling molecules such as Akt and NF-κB during I3C-induced apoptosis in breast cancer cells. We found that I3C induces apoptotic processes in MCF10A-derived cell lines with premalignant (DCIS.com) and malignant (MCF10CA1a) phenotypes but not in nontumorigenic parental MCF10A cells. Immunoprecipitation, kinase assays, and Western blot analysis showed that I3C specifically inhibits Akt kinase activity and abrogates the EGF-induced activation of Akt in breast cancer cells. NF-κB DNA-binding analysis and transfection studies with Akt cDNA and NF-κB-Luc reporter constructs revealed that Akt gene transfection directly activates NF-κB, and this activation was completely abrogated by I3C treatment. In addition, I3C also abrogated the EGF-induced activation of NF-κB, which was mediated via the Akt signaling pathway. From these results, we conclude that there is a direct cross-talk between Akt and NF-κB pathways and that the inactivation of Akt and NF-κB activity plays important roles in mediating I3C-induced apoptosis in breast cancer cells. These results also suggest that I3C may be a potential chemopreventive agent by virtue of its selective apoptosis-inducing ability in premalignant and malignant breast epithelial cells.