Selective AKT Inhibitor Research Articles

Effects of a novel HSP90 inhibitor on estrogen receptor α signaling pathways in breast cancer cells

Introduction. Heat shock proteins (HSP), also known as molecular chaperones, are a large family of proteins that play crucial roles in histogenesis, homeostasis, and the folding and functional regulation of numerous client proteins. Among them, HSP90 is a key player, particularly in supporting the growth of tumor cells. HSP90 impacts multiple carcinogenic signaling pathways, including BCR-ABL, Raf-1, AKT, human epidermal growth factor receptor 2 (ERBB2/HER2), hypoxia-inducible factor 1-α (HIF-1α), janus kinase 2 (JAK2), STAT3, p53, and estrogen receptor α (ERα). As a result, the search for new, selective inhibitors of this chaperone is a high priority in medicinal chemistry and oncology.Aim. To evaluate the antiproliferative activity of a novel HSP90 inhibitor, THB5T-1, on ERα-positive breast cancer cell lines and assess its anti-estrogenic potential and selectivity. Materials and methods. The study was conducted on hormone-dependent breast cancer cell lines MCF7 and T47D, along with the normal fibroblast line hFB-hTERT. The antiproliferative activity of THB5T-1 was measured using the MTT assay, while immunoblotting was employed to analyze the effects of HSP90 inhibition on cell signaling pathways. Anti-estrogenic activity was assessed in MCF7 cells via a reporter assay, and molecular modeling was used to construct a model of THB5T-1 interaction with the ligand-binding domain of ERα.Results. The half-maximal inhibitory concentration (IC50) of THB5T-1 was determined to be 4.3 μM for MCF7 cells and 5.6 μM for T47D cells. At a concentration of 25 μM, cell survival decreased to 20%. The selectivity index for THB5T-1 varied from 3.7 to 5.0 in different breast cancer cell lines. The compound’s effects on hormonal pathways in MCF7 cells, as observed via reporter assay and immunoblotting, were dose-dependent. These findings were further supported by molecular docking studies, showing THB5T-1 interaction with the ligand-binding domain of ERα. Additionally, the antiproliferative activity of THB5T-1 in MCF7 cells was associated with reduced expression of cell cycle regulators cyclin D1 and cyclin-dependent kinase 4 (CDK4). Significant efficacy of compound THB5T-1 in combination with a selective AKT inhibitor was revealed.Conclusion. Compound THB5T-1 demonstrated significant antiproliferative effects on ERα-positive breast cancer cells and exhibited high selectivity. Its anti-estrogenic effects highlight its potential as a selective inhibitor of the HSP90/ ERα/GREB1 pathway, effectively blocking ERα-mediated cell proliferation.

New therapies for hormone-positive HER2-negative metastatic breast cancer with AKT signaling alterations: the Expert Panel Decision

On May 20, 2024, the Expert Panel discussed key issues of the diagnosis of alterations of the AKT signaling pathway, the effectiveness and safety of targeted therapy for hormone-positive (HR+) HER2-negative metastatic breast cancer (BC) concerning the emergence of a new selective AKT inhibitor capivasertib. The results of the CAPItello-291 study are presented. The experts concluded that a new effective treatment line has become available for patients with hormone-resistant advanced hormone-positive HER2-negative BC with genetic alterations in the AKT signaling pathway and relapse on previous adjuvant endocrine therapy with aromatase inhibitors or within the first year after the therapy or with progression during previous therapy with aromatase inhibitors for metastatic BC. Modern genetic testing methods for the alterations in the AKT signaling pathway can confirm the presence of this key biological target in the tumor for tailoring effective targeted therapy.

Increased in vitro production of bovine embryos resulting from oocyte maturation in the presence of triciribine, a specific inhibitor of AKT

The aim of this study was to evaluate the effect of different concentrations of triciribine, a selective Akt inhibitor, on various aspects of oocyte maturation and on the IVF of bovine embryos. Cumulus-oocyte complexes (COCs) were matured in vitro in medium supplemented with: 0 (control), 1, 5, 10, and 20 μM of triciribine. The nuclear maturation was assessed by staining with acetic orcein, while the cytoplasmic maturation was evaluated by mitochondrial (MitoTracker® Red CMXRos) and lipid droplets distribution (LipidTOX). COCs were fertilized in vitro and cultured for nine days. Cleavage rates, blastocyst production, and hatching rates were determined on days three, seven, and nine of in vitro culture, respectively. Oocytes from COCs treated with 1 μM of triciribine were stained at 3, 6, and 9 h of IVM to determine the inhibitor's involvement in germinal vesicle breakdown. Analysis of variance (ANOVA) of the data was performed and the means were compared using the SNK test at a 5 % significance level. Exposure of COCs to 1, 5, and 10 μM of triciribine did not alter the number of matured oocytes (P < 0.05), a concentration of 20 μM reduced the number of oocytes in MII with a consequent increase in oocytes in MI (P < 0.05). This concentration markedly reduced the number of oocytes with peripheral cortical granules and the rates of cleavage and blastocysts (P < 0.05). On the other hand, when COCs were matured in the presence of 1 μM, there was an increase in the blastocyst rate (P < 0.05), but without altering the timing of meiosis resumption (P < 0.05). It is concluded that the Akt pathway participates in the nuclear and cytoplasmic events of in vitro maturation of bovine oocytes, but through mechanisms that do not interfere with germinal vesicle breakdown. Modulation of Akt activity in bovine COCs during IVM with 1 μM of triciribine increases the in vitro production of bovine embryos.

MFGE8 promotes adult hippocampal neurogenesis in rats following experimental subarachnoid hemorrhage via modifying the integrin β3/Akt signaling pathway

Subarachnoid hemorrhage (SAH) is one of the most severe type of cerebral strokes, which can cause multiple cellular changes in the brain leading to neuronal injury and neurological deficits. Specifically, SAH can impair adult neurogenesis in the hippocampal dentate gyrus, thus may affecting poststroke neurological and cognitive recovery. Here, we identified a non-canonical role of milk fat globule epidermal growth factor 8 (MFGE8) in rat brain after experimental SAH, involving a stimulation on adult hippocampal neurogenesis(AHN). Experimental SAH was induced in Sprague-Dawley rats via endovascular perforation, with the in vivo effect of MFGE8 evaluated via the application of recombinant human MFGE8 (rhMFGE8) along with pharmacological interventions, as determined by hemorrhagic grading, neurobehavioral test, and histological and biochemical analyses of neurogenesis related markers. Results: Levels of the endogenous hippocampal MFGE8 protein, integrin-β3 and protein kinase B (p-Akt) were elevated in the SAH relative to control groups, while that of hippocalcin (HPCA) and cyclin D1 showed the opposite change. Intraventricular rhMGFE8 infusion reversed the decrease in doublecortin (DCX) immature neurons in the DG after SAH, along with improved the short/long term neurobehavioral scores. rhMGFE8 treatment elevated the levels of phosphatidylinositol 3-kinase (PI3K), p-Akt, mammalian target of rapamycin (mTOR), CyclinD1, HPCA and DCX in hippocampal lysates, but not that of integrin β3 and Akt, at 24 hr after SAH. Treatment of integrin β3 siRNA, the PI3K selective inhibitor ly294002 or Akt selective inhibitor MK2206 abolished the effects of rhMGFE8 after SAH. In conclusion, MFGE8 is upregulated in the hippocampus in adult rats with reduced granule cell genesis. rhMFGE8 administration can rescue this impaired adult neurogenesis and improve neurobehavioral recovery. Mechanistically, the effect of MFGE8 on hippocampal adult neurogenesis is mediated by the activation of integrin β3/Akt pathway. These findings suggest that exogenous MFGE8 may be of potential therapeutic value in SAH management.Graphical abstract and proposed pathway of rhMFGE8 administration attenuate hippocampal injury by improving neurogenesis in SAH models. SAH caused hippocampal injury and neurogenesis interruption. Administered exogenous MFGE8, recombinant human MFGE8(rhMFGE8), could ameliorate hippocampal injury and improve neurological functions after SAH. Mechanistically, MFGE8 bind to the receptor integrin β3, which activated the PI3K/Akt pathway to increase the mTOR expression, and further promote the expression of cyclin D1, HPCA and DCX. rhMFGE8 could attenuated hippocampal injury by improving neurogenesis after SAH, however, know down integrin β3 or pharmacological inhibited PI3K/Akt by ly294002 or MK2206 reversed the neuro-protective effect of rhMFGE8.

PFKFB3 facilitates cell proliferation and migration in anaplastic thyroid carcinoma via the WNT/β-catenin signaling pathway.

Despite the involvement of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase3 (PFKFB3) in the proliferation and metastasis of diverse tumor types, its biological functions and related molecular mechanisms in anaplastic thyroid carcinoma (ATC) remain largely unclear. Datasets from the Gene Expression Omnibus, the Cancer Genome Atlas and immunohistochemistry (IHC) analyses were employed to measure the expression level of PFKFB3 in ATC. A series of assays were performed to analyze the role of PFKFB3 and its inhibitor KAN0438757 in ATC cell proliferation and migration. Furthermore, Western blotting (WB), IHC and luciferase reporter assay were conducted to investigate the potential mechanisms underlying the involvement of PFKFB3 and KAN0438757 in ATC. Additionally, we established a subcutaneous xenograft tumor model in nude mice to evaluate the in vivo tumor growth. PFKFB3 exhibited a significant increase in its expression level in ATC tissues. The overexpression of PFKFB3 resulted in the stimulation of ATC cell proliferation and migration. Furthermore, this overexpression was associated with the elevated expression levels of p-AKT (ser473), p-GSK3α/β (ser21/9), nuclear β-catenin, fibronectin1 (FN1), matrix metallopeptidase 9 (MMP-9) and cyclin D1. It also promoted the nuclear translocation of β-catenin and the transcription of downstream molecules. Conversely, contrasting results were observed with the downregulation or KAN0438757-mediated inhibition of PFKFB3 in ATC cells. The selective AKT inhibitor MK2206 was noted to reverse the increased expression of p-AKT (ser473) and p-GSK3α/β (ser21/9) induced by PFKFB3 overexpression. The level of lactate was increased in PFKFB3-overexpressing ATC cells, while the presence of KAN0438757 inhibited lactate production. Moreover, the simultaneous use of PFKFB3 downregulation and KAN0438757 was found to suppress subcutaneous tumor growth in vivo. PFKFB3 can enhance ATC cell proliferation and migration via the WNT/β-catenin signaling pathway and plays a crucial role in the regulation of aerobic glycolysis in ATC cells.

Targeted delivery of AZD5363 to T-cell acute lymphocytic leukemia by mSiO2-Au nanovehicles.

T-cell acute lymphocytic leukemia (T-ALL) is the most common cancer in children, with a low survival rate because of drug resistance and a high recurrence rate. Targeted delivery of chemotherapy drugs can reduce their side effects and improve their efficacy. The abnormality of phosphatidylinositol-3-kinase/protein kinase B/ mammalian target of rapamycin (PI3K/Akt/mTOR) pathway plays a key role in T-ALL occurrence. AZD5363 is a selective Akt inhibitor with promising therapeutic potential for tumors encoded by the PI3K/Akt/mTOR pathway. However, the toxicity and side effects have limited its application in treating T-ALL. This study aimed to design a delivery system for targeting AZD5363 to T-ALL by sgc8c aptamer designed as mesoporous silica (mSiO2) decorated with Au nanoparticles. The cell-specific targeting and cytotoxicity of mSiO2-Au-AZD5363-Apt were investigated. The mSiO2-Au nanovehicles were found feasible for AZD5363 delivery, with high loading efficiency and pH-responsive release in the acidic lysosome. More importantly, mSiO2-Au-AZD5363-Apt nanovehicles could specifically recognize and enter T-ALL cells in vitro and in vivo, effectively inhibiting the proliferation of CCRF-CEM cells. In conclusion, mSiO2-Au-AZD5363-Apt provided an effective therapeutic method for the targeted treatment of T-ALL.

Everolimus prevents doxorubicin-induced apoptosis in H9c2 cardiomyocytes but not in MCF-7 cancer cells: Cardioprotective roles of autophagy, mitophagy, and AKT

Cardiotoxicity is a severe side effect of the chemotherapeutic agent doxorubicin (DOX). We recently showed that DOX-induced cardiomyocyte apoptosis and death were attenuated through autophagy pre-induction. Herein, we assessed how the autophagy/mitophagy-inducing antitumor drug everolimus (EVL) affected DOX-induced cytotoxicity in the rat cardiomyocyte cell line H9c2 and human breast cancer cell line MCF-7. Apoptosis was assessed using annexin V assay. Autophagy and mitophagy were assessed using fluorescence assays. Cellular protein levels were determined using western blotting. Pretreatment with EVL (1 nM) before DOX exposure inhibited mammalian target of rapamycin (mTOR) activity, induced autophagy and mitophagy, and activated protein kinase B (AKT) in H9c2 cells. In mitochondria, DOX (1 μM) induced structural damage (decreased membrane potential and release of cytochrome c), increased superoxide levels, decreased apoptosis inhibitor Bcl-2, and increased apoptosis inducer Bax, leading to apoptosis and reduced viability in H9c2 cells. EVL pretreatment suppressed DOX-induced changes. EVL anti-apoptotic effects were inhibited by treatment with MK-2206, a selective AKT inhibitor. Furthermore, EVL suppressed DOX-induced cardiotoxicity through autophagy/mitophagy and AKT activation but did not attenuate DOX-induced apoptosis or reduction in viability in MCF-7 cells. Altogether, EVL can protect cardiomyocytes from DOX-induced apoptosis and toxicity without reducing DOX antitumor effects, allowing safer chemotherapy.

Insulin regulates gap junction intercellular communication in porcine granulosa cells through modulation of connexin43 protein expression

Gap junction intercellular communication (GJIC) among granulosa cells plays an important role in folliculogenesis, and it is temporal-spatially regulated during follicular development. Connexin (Cx) proteins predominantly form the basal structure of gap junctions in granulosa cells. In our study, immunohistochemical analysis revealed that Cx43 is the most widely expressed connexin in porcine follicles, especially among the large antral follicles. With application of insulin on porcine granulosa cells, we found that insulin significantly facilitated the protein level of Cx43, not mRNA level. This process is dependent on the phosphorylated activities of AKT and Erk since selective AKT and Erk inhibitors, LY294002 and U0126, respectively, hampered the potential of insulin to up-regulate Cx43 protein expression. As a consequence, the insulin-enhanced Cx43-couple GJIC activity in porcine granulosa cells was corresponding attenuated by the administration of LY294002 and U0126. Our findings provide a new insight into the molecular mechanisms by which insulin mediates cell-cell communication in porcine granulosa cells and sheds light on nutrition-reproduction interactions.

Combining the AKT inhibitor capivasertib and SERD fulvestrant is effective in palbociclib-resistant ER+ breast cancer preclinical models

Combining the selective AKT inhibitor, capivasertib, and SERD, fulvestrant improved PFS in a Phase III clinical trial (CAPItello-291), treating HR+ breast cancer patients following aromatase inhibitors, with or without CDK4/6 inhibitors. However, clinical data suggests CDK4/6 treatment may reduce response to subsequent monotherapy endocrine treatment. To support understanding of trials such as CAPItello-291 and gain insight into this emerging population of patients, we explored how CDK4/6 inhibitor treatment influences ER+ breast tumour cell function and response to fulvestrant and capivasertib after CDK4/6 inhibitor treatment. In RB+, RB− T47D and MCF7 palbociclib-resistant cells ER pathway ER and Greb-1 expression were reduced versus naïve cells. PI3K-AKT pathway activation was also modified in RB+ cells, with capivasertib less effective at reducing pS6 in RB+ cells compared to parental cells. Expression profiling of parental versus palbociclib-resistant cells confirmed capivasertib, fulvestrant and the combination differentially impacted gene expression modulation in resistant cells, with different responses seen in T47D and MCF7 cells. Fulvestrant inhibition of ER-dependent genes was reduced. In resistant cells, the combination was less effective at reducing cell cycle genes, but a consistent reduction in cell fraction in S-phase was observed in naïve and resistant cells. Despite modified signalling responses, both RB+ and RB− resistant cells responded to combination treatment despite some reduction in relative efficacy and was effective in vivo in palbociclib-resistant PDX models. Collectively these findings demonstrate that simultaneous inhibition of AKT and ER signalling can be effective in models representing palbociclib resistance despite changes in pathway dependency.

Abstract B065: Combining the androgen receptor inhibitor darolutamide with PI3K/AKT/mTOR pathway inhibitors has superior efficacy in preclinical models of prostate cancer

Abstract The phosphoinositide 3 kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway is dysregulated in the majority of advanced prostate cancer patients, often linked to phosphate and tensin homolog (PTEN) inactivation. An increasing number of studies demonstrates the reciprocal crosstalk between the PI3K/AKT/mTOR and the androgen receptor (AR) pathways with the inhibition of one of them leading to the compensatory activation of the other one. We compared the effects of different PI3K/AKT/mTOR pathway inhibitors as single agents or in combination with the AR inhibitor darolutamide.In vitro impact of the compounds was evaluated in prostate cancer cell lines by proliferation and apoptosis assays, and by RNA-seq analysis. In vivo effects were determined in a patient-derived (PDX) prostate cancer model in an efficacy study followed by immunohistochemistry analysis. Analysis of VCaP cells treated with the androgen R1881 showed that beside androgen response, the PI3K/AKT/mTOR pathway was an essential hallmark characteristic of androgen action. Additional darolutamide treatment opposed androgen effects on both pathways. We therefore evaluated the impact of selective inhibitors of PI3K or AKT and saw strong in vitro antiproliferative effects in the prostate cancer cell lines tested. We then focused on the pan-PI3K inhibitor copanlisib and observed pronounced synergistic effects with darolutamide in the VCaP model. This was accompanied by induction of PARP cleavage and activation of caspase 3/7. A detailed transcriptomic analysis revealed that the combination of both inhibitors resulted in more pronounced transcript changes, compared to individual treatments. Principal component analysis furthermore showed the combination to be closer to the DMSO control along the axis that represents androgen regulation. We observed a pronounced downregulation of cell proliferation and cell division genes following combination treatment compared to individual treatments, a prominent example being the proliferation marker KI67. In vivo studies performed with the PDX LuCaP 35 model revealed a superior inhibitory effect of the combination treatment with darolutamide and copanlisib when compared to single agents. Immunohistochemistry analysis revealed a significant upregulation of a pro-apoptotic pathway in tumors treated with darolutamide and copanlisib which was not observed in single treatment arms, when compared to vehicle control. In summary, we found that different PI3K and AKT inhibitors impaired the in vitro proliferation of prostate cancer cell lines. Combining the AR inhibitor darolutamide with the pan-PI3K inhibitor copanlisib led to increased apoptosis and down-regulation of cell division and proliferation genes. Importantly, the improved tumor inhibition by the combination was also observed in vivo. These results warrant further analysis of the impact of combined AR and PI3K pathway inhibition in prostate cancer, especially when aberrations in the PI3K/AKT/mTOR pathway or PTEN loss are observed. Citation Format: Simon Heller, Tatsuo Sugawara, Ekaterina Nevedomskaya, Simon J. Baumgart, Holly Nguyen, Eva Corey, Annika Böhme, Oliver von Ahsen, Oliver Politz, Bernard Haendler. Combining the androgen receptor inhibitor darolutamide with PI3K/AKT/mTOR pathway inhibitors has superior efficacy in preclinical models of prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B065.

CircZNF215 promotes tumor growth and metastasis through inactivation of the PTEN/AKT pathway in intrahepatic cholangiocarcinoma

BackgroundIncreasing evidence shows that circular RNAs (circRNAs), a novel class of noncoding RNAs, play a crucial role in the development of cancers, including intrahepatic cholangiocarcinoma (iCCA). Nevertheless, their functions and exact mechanisms in iCCA progression and metastasis are still unclear. Ipatasertib is a highly selective inhibitor of AKT that inhibits tumor growth by blocking the PI3K/AKT pathway. In addition, phosphatase and tensin homolog (PTEN) can also inhibit the activation of the PI3K/AKT pathway, but it is not clear whether the cZNF215-PRDX-PTEN axis plays a role in the antitumor activity of ipatasertib.MethodsWe identified a new circRNA (circZNF215, termed cZNF215) through high-throughput circRNA sequencing (circRNA-seq). In addition, RT‒qPCR, immunoblot assay, RNA pull-down assay, RNA immunoprecipitation (RIP) assay, and fluorescence in situ hybridization assay (FISH) were used to investigate the interaction of cZNF215 with peroxiredoxin 1 (PRDX1). Coimmunoprecipitation (Co-IP) assays and duolink in situ proximity ligation assays (PLAs) were conducted to analyze the effects of cZNF215 on the interaction between PRDX1 and PTEN. Finally, we tested the potential effects of cZNF215 on the antitumor activity of ipatasertib with in vivo experiments.ResultsWe found that cZNF215 expression was obviously upregulated in iCCA tissues with postoperative metastases and was correlated with iCCA metastasis and poor outcome in patients with iCCA. We further revealed that overexpression of cZNF215 promoted iCCA cell growth and metastasis in vitro and in vivo, while cZNF215 knockdown had the opposite effect. Mechanistic studies suggested that cZNF215 competitively interacted with PRDX1, which blocked the association between PRDX1 and PTEN, subsequently leading to oxidation-induced inactivation of the PTEN/AKT pathway and finally contributing to iCCA progression and metastasis. Additionally, we also revealed that silencing cZNF215 in iCCA cells had the potential to enhance the antitumor effect of ipatasertib.ConclusionsOur study demonstrates that cZNF215 facilitates iCCA progression and metastasis by regulating the PTEN/AKT pathway and may serve as a novel prognostic predictor in patients with iCCA.

MicroRNA‐17‐3p protects against excessive posthypoxic autophagy in H9C2 cardiomyocytes via PTEN–Akt–mTOR signaling pathway

The activity of phosphatase and tensin homolog (PTEN) can be inhibited by miR-17-3p, which results in attenuating myocardial ischemia/reperfusion injury (IRI), however, the mechanism behind this phenomenon is still elusive. Suppression of PTEN leads to augmented protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling strength and constrained autophagy activation, which might be the one mechanism for the ameliorated myocardial IRI. Thus, we tested the hypothesis that miR-17-3p attenuated hypoxia/reoxygenation (H/R)-mediated damage in cardiomyocytes by downregulating excessive autophagy via the PTEN-Akt-mTOR axis. The expression of miR-17-3p was remarkably increased after H/R treatment (6-h hypoxia followed by 6-h reoxygenation; H6/R6), which was concomitant with the increase of the release of lactic acid dehydrogenase (cell injury marker) and the enhancement LC3II/I ratio (autophagy markers) in H9C2 cardiomyocytes. Ectoexpression of miR-17-3p agomir led to remarkable augmentation of miR-17-3p expression and evidently attenuated H/R-mediated cell damage and excessive autophagy. Furthermore, an increase in miR-17-3p expression elicited constrained phosphorylation of PTEN (Ser380 ) while enhanced the phosphorylation of Akt (Thr308 , Ser473 ) and mTOR (Ser536 ) after H/R stimulation. In addition, pretreatment with LY-294002 (an Akt selective inhibitor) and rapamycin (an mTOR selective inhibitor) significantly abrogated the protective function of miR-17-3p on H/R-mediated cell damage and autophagy in H9C2 cardiomyocytes. Taken together, these observations indicated that the enhancement of the PTEN/Akt/mTOR axis and the consequent suppression of autophagy overactivation might represent an underlying mechanism by which miR-17-3p attenuated H/R-mediated damage in H9C2 cells.

Ipatasertib, an oral AKT inhibitor, in combination with carboplatin exhibits anti-proliferative effects in uterine serous carcinoma

Purpose Uterine serous carcinoma (USC) exhibits worse survival rates compared to the endometrioid subtype, and there is currently no effective treatment options for recurrence of this disease after platinum-based chemotherapy. Activation of PIK3CA/AKT/mTOR signaling pathway is a common biological feature in USC. Materials and Methods Ipatasertib (IPAT) is an investigational, orally administered, ATP-competitive, highly selective inhibitor of pan AKT that has demonstrated anti-proliferative activity in a variety of tumor cells and tumor models. In this study, we used IPAT, carboplatin and their combination to investigate the anti-tumor activity in SPEC-2 and ARK-1 cells. Results Our results indicate that IPAT combined with carboplatin at low doses was more effective at reducing proliferation, inducing apoptosis and causing cellular stress than IPAT or carboplatin alone. In particular, inhibition of the PIK3CA/AKT/mTOR pathway and induction of DNA damage were involved in the synergistic inhibition by combination treatment of cell viability in USC cells treated with the combination. Furthermore, IPAT in combination with carboplatin significantly reduced cell adhesion and inhibited cell invasion. Conclusions These findings suggest that the combination of IPAT and carboplatin has potential clinical implications for developing new USC treatment strategies.

Vasohibin-1 promotes osteoclast differentiation in periodontal disease by stimulating the expression of RANKL in gingival fibroblasts

Vasohibin-1 (VASH1) is a key inhibitor of vascular endothelial growth factor-induced angiogenesis. Although the involvement of VASH1 in various pathological processes has been extensively studied, its role in periodontal disease (PD) remains unclear. We aimed to investigate the role of VASH1 in PD by focusing on osteoclastogenesis regulation. We investigated VASH1 expression in PD by analyzing data from the online Gene Expression Omnibus (GEO) database and using a mouse ligature-induced periodontitis model. The effects of VASH1 on osteoclast differentiation and osteoclastogenesis-supporting cells were assessed in mouse bone marrow-derived macrophages (BMMs) and human gingival fibroblasts (GFs). To identify the stimulant of VASH1, we used culture broth from Porphyromonas gingivalis (Pg), a periopathogen. The GEO database and mouse periodontitis model revealed that VASH1 expression was upregulated in periodontitis-affected gingival tissues, which was further supported by immunohistochemistry and qRT-PCR analyses. VASH1 expression was significantly stimulated in GFs after treatment with the Pg broth. Direct treatment with recombinant VASH1 protein did not stimulate osteoclast differentiation in BMMs but did contribute to osteoclast differentiation by inducing RANKL expression in GFs through a paracrine mechanism. Small interfering RNA-mediated silencing of VASH1 in GFs abrogated RANKL-mediated osteoclast differentiation in BMMs. Additionally, VASH1-activated RANKL expression in GFs was significantly suppressed by MK-2206, a selective inhibitor of AKT. These results suggest that Pg-induced VASH1 may be associated with RANKL expression in GFs in a paracrine manner, contributing to osteoclastogenesis via an AKT-dependent mechanism during PD progression.

Synthesis of dihydrofuran-3-one and 9,10-phenanthrenequinone hybrid molecules and biological evaluation against colon cancer cells as selective Akt kinase inhibitors.

A series of dihydrofuran-3-one and 9,10-phenanthrenequinone hybrid compounds were synthetized through a one-pot gold-catalyzed oxidative cyclization and Aldol-type addition cascade reaction of homopropargylic alcohols with 9,10-phenanthrenequinone. The cytotoxicity of newly synthesized compounds was evaluated in CCK8 assay against different human cancer cells, showing significantly antiproliferative activity against tested tumor cell lines with a lowest IC50 value of 0.92μM over HCT-116. Further investigation revealed that the treatment of HCT-116 cell line with the promising compound 4c induced cell death as a selective Akt inhibitor. In addition, controlled experiments and molecular docking study suggested that the significant antitumor activity might be attributed to the unique hybrid structure, which implied the promising potential of this dual heterocycle hybrid method in the discovery of novel bioactive molecules with structural diversity.

Abstract 156: Dynamic live-cell visualization and quantification of Akt activity using a genetically-encoded, fluorescent kinase translocation reporter

Abstract Dysregulation of signal transduction pathways is associated with cancer initiation, progression, and recurrence. The PI3K/Akt signaling pathway has been extensively investigated as a therapeutic target due to its mechanistic association with several hallmarks of cancer. Studying dynamic changes in kinase activity can be difficult, and assays to measure these changes in live cells in a physiologically relevant environment are lacking. Standard approaches to monitoring Akt kinase activity are limited to end point assays which lack the ability to monitor the effects of treatments over time. Here we demonstrate the utility of the Incucyte® Kinase Akt Lentivirus Reagent, encoding a kinase translocation reporter based on a green fluorescent protein-tagged Akt substrate whose subcellular localization is phosphorylation-dependent, and a red fluorescent nuclear protein to denote the nuclear/cytoplasmic boundary. To demonstrate inhibition of Akt, A549 cells stably expressing the reporter were treated with compounds targeting the PI3K/Akt kinase pathway, including allosteric Akt inhibitors MK2206 and API-1, competitive Akt inhibitors AZD5363 and Ipatasertib, and upstream PI3K kinase inhibitors LY294002 and PI-103. Quantification of treatment responses using the Incucyte® Live Cell Analysis System showed concentration-dependent inhibition of Akt activity for all compounds with varying kinetic profiles over 24 hours. To study activation of Akt, HeLa cells were first cultured in the absence of serum to reduce Akt activity. After 4 hours of incubation in serum-free conditions, the cells were treated with either epidermal growth factor (EGF) or recombinant insulin-like growth factor (R3-IGF-1) to activate Akt. An increase in Akt activity was observed for both treatments. While R3-IGF-1-induced activation was sustained over the 12-hour time course, activation by EGF diminished over time. Cell lines with mutations in the tumor suppressor PTEN showed no response to serum starvation or activation with EGF or R3-IGF-1. In addition to monitoring Akt activity over time, the integrated red nuclear restricted protein of the reporter enables concurrent measurements of proliferation. The selective Akt inhibitor MK2206 decreased Akt activity in a similar concentration-dependent manner in both T-47D and MDA-MB-231 cell lines. In contrast, measurements of red object count from the same cells reveal differential effects of MK2206 on proliferation between the two cell lines, with concentration-dependent inhibition of T-47D cell growth but little effect on MDA-MB-231 cells. Overall, these data highlight the utility of the Incucyte® Kinase Akt Lentivirus Reagent to provide valuable kinetic measurements of Akt activity using live cells within a physiologically relevant environment. Citation Format: John N. Rauch, Susan K. Foltin, Libuse Oupicka, Matthew Dilsaver, Grigory S. Filonov, Gillian Lovell, Jasmine Trigg, Cicely L. Schramm. Dynamic live-cell visualization and quantification of Akt activity using a genetically-encoded, fluorescent kinase translocation reporter [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 156.

Vasorelaxant effect of curcubisabolanin A isolated from Curcuma longa through the PI3K/Akt/eNOS signaling pathway

Ethnopharmacological relevanceCurcuma longa L. (Zingiberaceae) is a known blood-activating and stasis-removing traditional Chinese medicine and has relevant pharmacological properties. The rhizomes of C. longa have been used for the treatment of cardiovascular disease (CVD) in China. Previous studies have shown that sesquiterpenoids from C. longa have significant vasorelaxant effects, which are closely associated with the prevention and treatment of CVD. Aim of the studyTo explore the sesquiterpenoids with vasorelaxant effects from C. longa and investigate the underlying mechanisms. Materials and methodsThe compound was isolated from C. longa by multiple chromatography technologies. Its structure was determined by extensive spectroscopic analyses, nuclear magnetic resonance (NMR) data calculations, electronic circular dichroism (ECD) data calculations, and optical rotation (OR) data calculations. The vasorelaxant effect of the isolated compound was evaluated by KCl- or phenylephrine (PHE)-inducing contraction of the rat thoracic aortic rings. Endothelial removal and L-NAME pretreatment experiments were used to verify the endothelium-dependent vasorelaxant effect of the isolated compound in rat thoracic aortic rings. NO production was monitored in human umbilical vein endothelial cells (HUVECs). Western blot was carried out in HUVECs to elucidate the potential mechanisms. ResultsA new bisabolane-type sesquiterpenoid, curcubisabolanin A [(+)-(1S,7S,9E)-bisabola-2(3),4(15),9(10)-trien-11-ol], was isolated from the rhizomes of C. longa. curcubisabolanin A exhibited endothelium-dependent relaxation on rat thoracic aortic rings, while pre-treatment of intact aortic rings with an eNOS inhibitor (L-NAME) attenuated the vasorelaxant response of curcubisabolanin A. In addition, curcubisabolanin A induced intracellular NO production and significantly increased the levels of phosphorylated PI3K (p-PI3K), phosphorylated Akt (p-Akt), and phosphorylated eNOS (p-eNOS) in HUVECs. LY294002 (a blocker of PI3K) and MK-2206 (a highly selective inhibitor of Akt) significantly decreased these effects of curcubisabolanin A. ConclusionsThese findings demonstrated that the vasorelaxant effect of curcubisabolanin A was partially endothelium-dependent and was related to regulation of NO production in vascular endothelial cells through the PI3K/Akt/eNOS signaling pathway.

Assessment of cytochrome P450 3A4-mediated drug\u2013drug interactions for ipatasertib using a fit-for-purpose physiologically based pharmacokinetic model

PurposeIpatasertib, a potent and highly selective small-molecule inhibitor of AKT, is currently under investigation for treatment of cancer. Ipatasertib is a substrate and a time-dependent inhibitor of CYP3A4. It exhibits non-linear pharmacokinetics at subclinical doses in the clinical dose escalation study. To assess the DDI risk of ipatasertib at the intended clinical dose of 400 mg with CYP3A4 inhibitors, inducers, and substrates, a fit-for-purpose physiologically based pharmacokinetic (PBPK) model of ipatasertib was developed.MethodsThe PBPK model was constructed in Simcyp using in silico, in vitro, and clinical data and was optimized and verified using clinical data.ResultsThe PBPK model described non-linear pharmacokinetics of ipatasertib and captured the magnitude of the observed clinical DDIs. Following repeated doses of 400 mg ipatasertib once daily (QD), the PBPK model predicted a 3.3-fold increase of ipatasertib exposure with itraconazole; a 2–2.5-fold increase with moderate CYP3A4 inhibitors, erythromycin and diltiazem; and no change with a weak CYP3A4 inhibitor, fluvoxamine. Additionally, in the presence of strong or moderate CYP3A4 inducers, rifampicin and efavirenz, ipatasertib exposures were predicted to decrease by 86% and 74%, respectively. As a perpetrator, the model predicted that ipatasertib (400 mg) caused a 1.7-fold increase in midazolam exposure.ConclusionThis study demonstrates the value of using a fit-for-purpose PBPK model to assess the clinical DDIs for ipatasertib and to provide dosing strategies for the concurrent use of other CYP3A4 perpetrators or victims.

10-DEBC Hydrochloride as a Promising New Agent against Infection of Mycobacterium abscessus.

Mycobacterium abscessus (M. abscessus) causes chronic pulmonary infections. Its resistance to current antimicrobial drugs makes it the most difficult non-tuberculous mycobacteria (NTM) to treat with a treatment success rate of 45.6%. Therefore, there is a need for new therapeutic agents against M. abscessus. We identified 10-DEBC hydrochloride (10-DEBC), a selective AKT inhibitor that exhibits inhibitory activity against M. abscessus. To evaluate the potential of 10-DEBC as a treatment for lung disease caused by M. abscessus, we measured its effectiveness in vitro. We established the intracellular activity of 10-DEBC against M. abscessus in human macrophages and human embryonic cell-derived macrophages (iMACs). 10-DEBC significantly inhibited the growth of wild-type M. abscessus and clinical isolates and clarithromycin (CLR)-resistant M. abscessus strains. 10-DEBC’s drug efficacy did not have cytotoxicity in the infected macrophages. In addition, 10-DEBC operates under anaerobic conditions without replication as well as in the presence of biofilms. The alternative caseum binding assay is a unique tool for evaluating drug efficacy against slow and nonreplicating bacilli in their native caseum media. In the surrogate caseum, the mean undiluted fraction unbound (fu) for 10-DEBC is 5.696. The results of an in vitro study on the activity of M. abscessus suggest that 10-DEBC is a potential new drug for treating M. abscessus infections.

Chronic lithium treatment ameliorates ketamine-induced mania-like behavior via the PI3K-AKT signaling pathway.

Ketamine, a rapid-acting antidepressant drug, has been used to treat major depressive disorder and bipolar disorder (BD). Recent studies have shown that ketamine may increase the potential risk of treatment-induced mania in patients. Ketamine has also been applied to establish animal models of mania. At present, however, the underlying mechanism is still unclear. In the current study, we found that chronic lithium exposure attenuated ketamine-induced mania-like behavior and c-Fos expression in the medial prefrontal cortex (mPFC) of adult male mice. Transcriptome sequencing was performed to determine the effect of lithium administration on the transcriptome of the PFC in ketamine-treated mice, showing inactivation of the phosphoinositide 3-kinase (PI3K)-protein kinase B (AKT) signaling pathway. Pharmacological inhibition of AKT signaling by MK2206 (40 mg/kg), a selective AKT inhibitor, reversed ketamine-induced mania. Furthermore, selective knockdown of AKT via AAV-AKT-shRNA-EGFP in the mPFC also reversed ketamine-induced mania-like behavior. Importantly, pharmacological activation of AKT signaling by SC79 (40 mg/kg), an AKT activator, contributed to mania in low-dose ketamine-treated mice. Inhibition of PI3K signaling by LY294002 (25 mg/kg), a specific PI3K inhibitor, reversed the mania-like behavior in ketamine-treated mice. However, pharmacological inhibition of mammalian target of rapamycin (mTOR) signaling with rapamycin (10 mg/kg), a specific mTOR inhibitor, had no effect on ketamine-induced mania-like behavior. These results suggest that chronic lithium treatment ameliorates ketamine-induced mania-like behavior via the PI3K-AKT signaling pathway, which may be a novel target for the development of BD treatment.