Blocked Akt Phosphorylation Research Articles

Trisarcglaboids A and B, two cytotoxic lindenane sesquiterpenoid trimers with a unique polymerization mode isolated from Sarcandra glabra

Trisarcglaboids A and B (1 and 2), representing the first example of lindenane sesquiterpenoid trimers repolymerized based on the classical [4 + 2] type dimer, together with known biogenic precursors chlorahololide D (3) and sarcandrolide A (4), were identified as chemical components of the root of Sarcandra glabra. The novel trimeric lindenane sesquiterpenoid skeletons, including their absolute configurations, were characterized using MS, NMR, ECD, and X-ray single crystal diffraction. The proposed Diels-Alder cycloaddition between Δ2(3) of the tiglic acyl group of the classical [4 + 2] type dimer and Δ15(4),5(6) of the third lindenane may serve as the key biogenic step. In addition, compound 1 exerted significant cytotoxicity against five human cancer cell lines with IC50 values ranging from 1 to 7 μM, potentially through blocking Akt phosphorylation and activating the endogenous apoptosis pathway.

Structurally diverse amides from Chloranthus henryi var. hupehensis and their anti-inflammatory activities by blocking Akt phosphorylation

Eleven new amides, four racemic pairs of (±)-chlorahupetamides A, B, D, E (1, 2, 4, 5) and chlorahupetamides C, F, G (3, 6, 7), have been isolated from Chloranthus henryi var. hupehensis. Compounds 1–3 are the first naturally occurring dimers via an unprecedented [2 + 2] cycloaddition derived from two dissimilar cinnamic acid amides, while compounds 4 and 5 represent the first examples of lignanamides in Chloranthus; together with two new hydroxycinnamic acid amide monomers (6–7), these compounds were obtained. Their structures were characterized by nuclear magnetic resonance (NMR), electronic circular dichroism (ECD), and X-ray diffraction analysis. Meanwhile, an LPS-induced BV-2 cell inflammatory model was used to determine the potential anti-inflammatory activity of all the isolated compounds. Intriguingly, compound −1 treatment showed a much greater inhibition of TNF-α expression with an EC50 value of 1.80 µM, while compound + 1 had more advantages in reducing IL-1β expression with an EC50 value of 19.93 µM. Moreover, compounds + 1 and −1 could significantly suppress inflammation and inhibit the Akt signaling pathway by decreasing the phosphorylated protein levels of Akt.

NAD (P)H Quinone Dehydrogenase 1-Targeting Triptolide Analogue Causes Tumor Regression and Sensitizes Cisplatin-Resistant Lung Cancer to Chemotherapy.

Cisplatin (DDP) is a first-line chemotherapeutic drug against lung cancer. Nonetheless, the effectiveness of this drug is hampered by drug resistance. Overcoming drug resistance is crucial for improving the outcomes of lung cancer treatment. Here, we reported the effect of CX-23, an activated triptolide analogue that targets NAD (P)H quinone dehydrogenase 1 (NQO1), on DDP-resistant lung cancer and sensitizes DDP-resistant lung cancer to chemotherapy. Our findings unveiled the antiproliferative activity of CX-23 against both A549- and DDP-resistant A549 (A549/DDP) cells while enhancing the chemosensitivity of these cells to DDP. Notably, CX-23 demonstrated no toxicity toward normal lung cells. Further investigations revealed that CX-23 exerts its effects by blocking AKT phosphorylation, leading to reduced expression of Mcl-1 and Bcl-2, and upregulating cleaved-caspase-3 levels, ultimately inducing apoptosis in cancer cells. CX-23 can be rapidly transformed in both A549 and A549/DDP cell lysates while remaining stable in mouse plasma and normal lung tissues. Pharmacokinetic analysis showed that CX-23 can distribute to lung tissues. Moreover, in vivo studies showed that CX-23 can prevent DDP-resistant lung cancer progression without causing any toxicity in the liver, kidneys, or lungs after 6 weeks of treatment. The combination of CX-23 and DDP not only significantly inhibited tumor progression compared to DDP alone but also attenuated DDP-induced kidney toxicity. These findings suggest that CX-23 alone or in combination with DDP may provide an alternative therapeutic option for DDP-resistant lung cancer.

Metformin promotes cGAS/STING signaling pathway activation by blocking AKT phosphorylation in gastric cancer

The cGAS/STING signaling pathway plays a pivotal role in regulating innate immunity. Emerging novel drugs aim to regulate the anti-tumor immune response by activating innate immunity. The anti-diabetic drug metformin has been reported to exhibit anti-cancer effect against various types of cancer. However, the role of metformin in regulating the cGAS/STING signaling pathway in gastric cancer remains unknown. In our study, we first used bioinformatic analysis to detect that metformin is closely related to tumor immunity in multiple tumors. Next, we validated the function of metformin in activating the cGAS/STING signaling pathway in gastric cancer cell lines. In addition, KEGG pathway enrichment analysis showed that metformin is negatively correlated with the PI3K/AKT signaling pathway in gastric cancer. We further verified that metformin activates the cGAS/STING signaling pathway by blocking AKT phosphorylation. Moreover, we found that metformin regulates the AKT signaling pathway by mediating the transcription factor SOX2. Thus, our study indicates that metformin activates the cGAS/STING signaling pathway by suppressing SOX2/AKT and has promising potential in gastric cancer immunotherapy.

MiR-155 Is a Positive Regulator of FcεRI-Induced Cyclooxygenase-2 Expression and Cytokine Production in Mast Cells.

MicroRNA-155 (miR-155) has been implicated in IgE-dependent allergic disease including asthma and atopic dermatitis. A few roles for miR-155 have been described in mast cells and some specifically related to IgE receptor signaling, but it is not completely understood. Here, we demonstrate by miRNA seq profiling and quantitative RT-PCR that miR-155 expression is significantly increased in human skin-derived mast cells (SMCs) and mouse bone marrow-derived mast cells (BMMCs) following FcεRI crosslinking with antigen. We demonstrate that FcεRI-induced expression of cyclooxygenase-2 (COX-2) was significantly inhibited in miR-155 knockout (KO) BMMCs whereas arachidonate-5-lipoxygenase (ALOX-5) expression and leukotriene C4 (LTC4) biosynthesis, and degranulation were unaffected. FcεRI-induced cytokine production (TNF, IL-6, and IL-13) from miR-155 KO BMMCs was also significantly diminished. Correspondingly, Akt phosphorylation, but not protein expression, was inhibited in the absence of miR-155 whereas p38 and p42/44 were unaffected. Interesting, lipopolysaccharide (LPS)-induced cytokine production was increased in miR-155 KO BMMCs. Together, these data demonstrate that miR-155 specifically targets the FcεRI-induced prostaglandin and cytokine pathways, but not the leukotriene or degranulation pathways, in mast cells. The data further suggest that miR-155 acts indirectly by targeting a repressor of COX-2 expression and a phosphatase that normally blocks Akt phosphorylation. Overall, this study reveals the role of miR-155 as a positive regulator of mast cell function.

Vitamin C sensitizes triple negative breast cancer to PI3K inhibition therapy.

Rationale: The clinical use of PI3K inhibitors, such as buparlisib, has been plagued with toxicity at effective doses. The aim of this study is to determine if vitamin C, a potent epigenetic regulator, can improve the therapeutic outcome and reduce the dose of buparlisib in treating PIK3CA-mutated triple negative breast cancer (TNBC).Methods: The response of TNBC cells to buparlisib was assessed by EC50 measurements, apoptosis assay, clonogenic assay, and xenograft assay in mice. Molecular approaches including Western blot, immunofluorescence, RNA sequencing, and gene silencing were utilized as experimental tools.Results: Treatment with buparlisib at lower doses, along with vitamin C, induced apoptosis and inhibited the growth of TNBC cells in vitro. Vitamin C via oral delivery rendered a sub-therapeutic dose of buparlisib able to inhibit TNBC xenograft growth and to markedly block metastasis in mice. We discovered that buparlisib and vitamin C coordinately reduced histone H3K4 methylation by enhancing the nuclear translocation of demethylase, KDM5, and by serving as a cofactor to promote KDM5-mediated H3K4 demethylation. The expression of genes in the PI3K pathway, such as AKT2 and mTOR, was suppressed by vitamin C in a KDM5-dependent manner. Vitamin C and buparlisib cooperatively blocked AKT phosphorylation. Inhibition of KDM5 largely abolished the effect of vitamin C on the response of TNBC cells to buparlisib. Additionally, vitamin C and buparlisib co-treatment changed the expression of genes, including PCNA and FILIP1L, which are critical to cancer growth and metastasis.Conclusion: Vitamin C can be used to reduce the dosage of buparlisib needed to produce a therapeutic effect, which could potentially ease the dose-dependent side effects in patients.

Abstract B06: Simultaneous inhibition of PI3Kα and CDK4/6 synergistically suppresses Kras-mutated non-small cell lung cancer

Abstract Activating KRAS mutation is the most common driver mutation in the initiation and promotion of non-small cell lung cancer (NSCLC). Direct inhibition of the enzymatic activity of mutated Kras has been proved unsuccessful. Deregulation of PI3K-Akt-mTOR signaling, one of the most important downstream effectors of Kras, plays important roles in tumorigenesis and drug resistance in NSCLC. In this study, we found that a novel PI3Kα-specific inhibitor, CYH33, possessed variable activity against a panel of Kras-mutated NSCLC cell lines. Though CYH33 blocked Akt phosphorylation in all tested cells, phosphorylated Rb decreased in CYH33-sensitive but not resistant cells, which was consistent with G1 phase arrest in CYH33-sensitive cells. Consequently, combination of a CDK4/6 inhibitor PD0332991 with CYH33 displayed synergistic activity against the proliferation of both CYH33-sensitive and -resistant cells, which was accompanied with enhanced cell population in G1 phase compared to single-agent treatment. Moreover, downregulation of cyclin D1 with siRNA sensitized A549 and H23 cells to CYH33. Reciprocally, CYH33 abrogated PD0332991-induced upregulation of phosphorylated Akt and cyclin D1 in A549 cells. Cotreatment of these two drugs demonstrated synergistic activity against A549 and H23 xenografts with enhanced inhibition on the phosphorylation of Akt and Rb compared to administration of CYH33 or PD03332991 alone. Thus, simultaneous inhibition of PI3Kα and CDK4/6 displayed synergistic activity against Kras-mutated NSCLC and provided a mechanistic rationale for a combination approach using PI3Kα inhibitor and CDK4/6 inhibitor CYH33 for the therapy of Kras-mutated NSCLC. Citation Format: Yu-xiang Wang, Xian Li, Xue-ling Liu, Bo-bo Wang, Yi Chen, Chun-hao Yang, Jian Ding, Ling-hua Meng. Simultaneous inhibition of PI3Kα and CDK4/6 synergistically suppresses Kras-mutated non-small cell lung cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B06.

Serum Ceramide Reduction by Blueberry Anthocyanin-Rich Extract Alleviates Insulin Resistance in Hyperlipidemia Mice

Blueberry anthocyanin-rich extract (BAE) was supplemented to high-fat diet (HFD)-fed mice to investigate sphingolipid metabolism modulating factors involved in the attenuated hyperinsulinemia and hyperlipidemia. A BAE-containing diet effectively controlled food intake and liver weight and significantly attenuated insulin resistance triggered by a HFD. Higher BAE (200 mg/kg of body weight) administration performed more efficiently in the improvement of hepatic steatosis and adipocyte hypertrophy, together with distinct suppressions in serum triacylglycerol and cholesterol in total and species. Serum lipid compositions revealed 200 mg/kg of BAE supplementation remarkably suppressed ceramide accumulation. Consistently, genes encoding enzymes associated with sphingomyelin conversion and ceramide de novo synthesis were modulated toward a healthy direction for restrained sphingolipid accumulation. Further, the inhibited mRNA expressions of protein phosphatase 2A and protein kinase Cζ involved in blocking Akt phosphorylation connected the controlled ceramides with the restored insulin sensitivity.

Simultaneous inhibition of PI3Kα and CDK4/6 synergistically suppresses KRAS-mutated non-small cell lung cancer.

ObjectiveActivating KRAS mutations are the most common drivers in the development of non-small cell lung cancer (NSCLC). However, unsuccess of treatment by direct inhibition of KRAS has been proven. Deregulation of PI3K signaling plays an important role in tumorigenesis and drug resistance in NSCLC. The activity of PI3Kα-selective inhibition against KRAS-mutated NSCLC remains largely unknown. MethodsCell proliferation was detected by sulforhodamine B assay. Cell cycle distribution and apoptosis were measured by flow cytometry. Cell signaling was assessed by Western blot and immunohistochemistry. RNA interference was used to down-regulate the expression of cyclin D1. Human NSCLC xenografts were employed to detect therapeutic efficacy in vivo. ResultsCYH33 possessed variable activity against a panel of KRAS-mutated NSCLC cell lines. Although CYH33 blocked AKT phosphorylation in all tested cells, Rb phosphorylation decreased in CYH33-sensitive, but not in CYH33-resistant cells, which was consistent with G1 phase arrest in sensitive cells. Combined treatment with the CDK4/6 inhibitor, PD0332991, and CYH33 displayed synergistic activity against the proliferation of both CYH33-sensitive and CYH33-resistant cells, which was accompanied by enhanced G1-phase arrest. Moreover, down-regulation of cyclin D1 sensitized NSCLC cells to CYH33. Reciprocally, CYH33 abrogated the PD0332991-induced up-regulation of cyclin D1 and phosphorylation of AKT in A549 cells. Co-treatment with these two drugs demonstrated synergistic activity against A549 and H23 xenografts, with enhanced inhibition of Rb phosphorylation.ConclusionsSimultaneous inhibition of PI3Kα and CDK4/6 displayed synergistic activity against KRAS-mutated NSCLC. These data provide a mechanistic rationale for the combination of a PI3Kα inhibitor and a CDK4/6 inhibitor for the treatment of KRAS-mutated NSCLC.

MDA-19 suppresses progression of melanoma via inhibiting the PI3K/Akt pathway

ObjectiveTo investigate the effect of MDA-19 on progression of melanoma, and explore the relevant mechanism.MethodsThe melanoma cell lines, M14 and UACC257, were treated with different concentrations of MDA-19, then CCK8, clone formation assay, Transwell and flow cytometry assays were performed to examine cell proliferation, migration, invasion and apoptosis, respectively. The expression of apoptosis-related proteins (Bcl-2, Bax and caspase 3 P17), EMT and signaling pathway-related proteins were also detected by Western blot.ResultsMDA-19 inhibited melanoma cells in a dose-dependent manner. Compared to the NC group, MDA-19 significantly inhibited cell growth capacity, migration and invasion of M14 and UACC257 cells, and accelerated cell apoptosis in a mitochondrial pathway through regulating Bcl-2/Bax and Caspase 3 in M14 and UACC257 cells. Moreover, MDA-19 was observed to up-regulate the expression of E-cad and down-regulate the expression of N-cad, Vimentin and Slug in melanoma cells in vitro. Furthermore, MDA-19 could inhibit the PI3K/Akt pathway by blocking Akt phosphorylation (p-Akt) and downstream proteins, P70 and Cyclin D1 in M14 and UACC257 cells.ConclusionOur data demonstrate that MDA-19 could inhibit progression of melanoma by suppressing the PI3K/Akt pathway, suggesting that MDA-19 is a potential anti-cancer agent for therapy of melanoma.

Neolymphostin A Is a Covalent Phosphoinositide 3-Kinase (PI3K)/Mammalian Target of Rapamycin (mTOR) Dual Inhibitor That Employs an Unusual Electrophilic Vinylogous Ester.

Using a novel chemistry-based assay for identifying electrophilic natural products in unprocessed extracts, we identified the PI3-kinase/mTOR dual inhibitor neolymphostin A from Salinispora arenicola CNY-486. The method further showed that the vinylogous ester substituent on the neolymphostin core was the exact site for enzyme conjugation. Tandem MS/MS experiments on PI3Kα treated with the inhibitor revealed that neolymphostin covalently modified Lys802 with a shift in mass of +306 amu, corresponding to addition of the inhibitor and elimination of methanol. The binding pose of the inhibitor bound to PI3Kα was modeled, and hydrogen-deuterium exchange mass spectrometry experiments supported this model. Against a panel of kinases, neolymphostin showed good selectivity for PI3-kinase and mTOR. In addition, the natural product blocked AKT phosphorylation in live cells with an IC50 of ∼3 nM. Taken together, neolymphostin is the first reported example of a covalent kinase inhibitor from the bacterial domain of life.

MiR-135a-5p Functions as a Glioma Proliferation Suppressor by Targeting Tumor Necrosis Factor Receptor–Associated Factor 5 and Predicts Patients' Prognosis

miR-135a-5p has been reported as a tumor suppressor in several extracranial tumors. However, its exact roles in gliomagenesis and relevance to the patients' prognoses are largely unknown. Herein, we detected the miR-135a-5p and tumor necrosis factor receptor-associated factor 5 (TRAF5) levels in 120 human glioma specimens and 20 nontumoral brain tissues; we found the miR-135a-5p level decreased, whereas the TRAF5 level increased, with the elevation of glioma grade. Their labeling indexes were inversely correlated with each other and showed strong negative (miR-135a-5p) and positive (TRAF5) correlation with the Ki-67 index. Cox regression demonstrated that both of their expression levels were independent survival predictors, whereas Kaplan-Meier analysis showed that subgrouping the glioma patients according to their levels could perfectly reflect the patients' prognoses regardless of the similarities in pathologic, molecular, and clinical features. In the following invitro and invivo studies, it was demonstrated that miR-135a-5p induced G1 arrest and inhibited the proliferation of glioma cells by targeting TRAF5 and subsequently blocking AKT phosphorylation as well as c-Myc and cyclin D1 expression. These effects could be reversed by TRAF5 overexpression and simulated by specific TRAF5 silencing. This study highlights the importance of miR-135a-5p and TRAF5 in gliomagenesis and progression and implies their potential prognostic and therapeutic values in malignant glioma.

Luteolin Regulates Macrophage Polarization via the PI3K/Akt Pathway to Inhibit the Apoptosis Stimulated by Angiotensin II.

The aim of this study was to investigate anti-apoptotic effects of luteolin on angiotensin II-stimulated murine peritoneal macrophages and to explore its mechanisms. The viability and cytotoxicity of murine peritoneal macrophages were assessed using the Cell Counting Kit-8 assay and measuring lactate dehydrogenase levels, respectively. Apoptotic rates were determined using Annexin V/propidium iodide staining. Protein expression was examined by western blotting, and markers of macrophage phenotypes were analyzed by flow cytometry and ELISA. Luteolin decreased the apoptotic rate of angiotensin II-stimulated macrophages. This effect was associated with increased Bcl-2 and caspase-3 levels as well as decreased Bax and cleaved caspase-3 levels. Additionally, luteolin reduced the expression of M1 macrophage phenotype markers (IL-6, TNF-α, iNOS, CD16/32) and increased the expression of M2 macrophage phenotype markers (Dectin-1, IL-10, Arg-1, CD206). Moreover, luteolin blocked Akt phosphorylation on residues 308 and 473, which were up-regulated in presence of angiotensin II. The effects of luteolin were similar to those of LY294002, a specific PI3K/Akt pathway inhibitor. These results indicated that luteolin has anti-apoptotic effects on angiotensin II-stimulated macrophages via macrophage polarization, which might be associated with PI3K/Akt signaling.

Thymosin β4 inhibits PDGF-BB induced activation, proliferation, and migration of human hepatic stellate cells via its actin-binding domain

ABSTRACTObjectives: Hepatic stellate cells (HSC) trans-differentiation is central to the development of liver fibrosis, marked by the expression of pro-fibrogenic genes and the proliferation and migration of activated HSC. Therefore, preventing and/or reverting the activation, proliferation, and migration of HSC may lead to new therapies for treating fibrosis/cirrhosis. Thymosin β4 (Tβ4) inhibits PDGF-BB-induced fibrogenesis, proliferation and migration of HSC by blocking Akt phosphorylation. Here, we utilized Tβ4-derived peptides: amino-terminal-Ac-SDKPDMAEIEKFDKS (1-15aa) and actin-binding-LKKTETQ (17-23aa) to investigate the molecular mechanisms in the anti-fibrogenic actions of Tβ4.Methods: We used RT-PCR, Western blot, and proliferation and migration assays in early passages of human HSC cultures treated with PDGF-BB and/or Tβ4 peptides.Results: We showed that 17-23aa but not 1-15aa inhibited PDGF-BB-dependent up-regulation of PDGFβ receptor, α-SMA, and collagen 1. It also blunted the phosphorylation of Akt at T 308 and S473, resulting in the inhibition of phosphorylation of PRAS40, and HSC proliferation and migration. Interestingly, 1-15aa blocked Akt phosphorylation at S473, but not T308 by inhibiting mTOR phosphorylation, thus, it did not have any effect on HSC proliferation and migration.Conclusion: These findings suggest that while 1-15aa has a minor effect on Akt phosphorylation, the anti-fibrogenic actions of Tβ4 are exerted via 17-23aa.

Efficacy of AKT Inhibitor ARQ 092 Compared with Sorafenib in a Cirrhotic Rat Model with Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the second most common cause of cancer-related mortality worldwide. The AKT pathway has been found activated in 50% of HCC cases, making it a promising target. Therefore, we assess efficacy of the allosteric AKT inhibitor ARQ 092 compared with untreated control and standard treatment, sorafenib, in vitro and in vivo ARQ 092 blocked phosphorylation of AKT in vitro and strongly inhibited cell growth with significantly higher potency than sorafenib. Similarly, apoptosis and cell migration were strongly reduced by ARQ 092 in vitro To mimic human advanced HCC, we used a diethylnitrosamine-induced cirrhotic rat model with fully developed HCC. MRI analyses showed that ARQ 092 significantly reduced overall tumor size. Furthermore, number of tumors was decreased by ARQ 092, which was associated with increased apoptosis and decreased proliferation. Tumor contrast enhancement was significantly decreased in the ARQ 092 group. Moreover, on tumor tissue sections, we observed a vascular normalization and a significant decrease in fibrosis in the surrounding liver of animals treated with ARQ 092. Finally, pAKT/AKT levels in ARQ 092-treated tumors were reduced, followed by downregulation of actors of AKT downstream signaling pathway: pmTOR, pPRAS40, pPLCγ1, and pS6K1. In conclusion, we demonstrated that ARQ 092 blocks AKT phosphorylation in vitro and in vivo In the HCC-rat model, ARQ 092 was well tolerated, showed antifibrotic effect, and had stronger antitumor effect than sorafenib. Our results confirm the importance of targeting AKT in HCC. Mol Cancer Ther; 16(10); 2157-65. ©2017 AACR.

KIF7 attenuates prostate tumor growth through LKB1-mediated AKT inhibition.

This study investigated kinesin family member 7 (KIF7) expression and function in prostate cancer (PCa). Our results showed that KIF7 was significantly downregulated in PCa, compared with normal, benign prostatic hyperplasia and prostate intraepithelial neoplasia tissues, partially through promoter hypermethylation. We further investigated the effects of KIF7 coiled coil (CC) domain and motor domain (MD) on PCa development in vitro and in vivo. Our results showed that KIF7-CC but not KIF7-MD significantly attenuated proliferation and colony formation, impeded migration and invasion, induced apoptosis and sensitized PCa cells to paclitaxel. Further analysis revealed that KIF7-CC enhanced LKB1 expression and phosphorylation at Ser428, which induced PTEN phosphorylation at Ser380/Thr382/383 and consequently blocked AKT phosphorylation at Ser473. Downregulation of LKB1 significantly attenuated the suppressive effects of KIF7-CC on cell proliferation, colony formation and AKT phosphorylation. Furthermore, our in vivo studies showed that KIF7-CC reduced prostate tumorigenesis in cell-derived xenografts. Downregulation of LKB1 abrogated the anti-tumor effects of KIF7-CC in these xenografts. Taken together, these findings provide the first evidence to support the role of KIF7 as a negative regulator that inhibits PCa development partially through LKB1-mediated AKT inhibition.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces hepatic stellate cell (HSC) activation and liver fibrosis in C57BL6 mouse via activating Akt and NF-κB signaling pathways

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a widespread environmental pollutant that could induce serious toxic effects in both humans and rodents. Some studies suggested that TCDD exposure may facilitate the activation of hepatic stellate cells (HSCs) and liver injury. However, the underlying molecular mechanism by which environmental pollutants promote liver injury remains poorly understood. In the present study, we established an animal model of TCDD exposure by intraperitoneal injection of TCDD in male C57BL/6J mice. As revealed by Sirius red staining and hematoxylin-eosin (H&E) staining evaluation, we found that TCDD-exposed mice showed extensive disruption of liver architecture, including hepatocellular necrosis, inflammatory cell infiltration, and fibrosis. Furthermore, we showed that TCDD up-regulated the expression and secretion of the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) in a dose-dependent manner in cultured HSCs. The effects of TCDD on cytokine secretion were very likely mediated by protein kinase B/Akt and Nuclear Factor kappa B (NF-κB) pathways, as indicated by the fact that TCDD markedly increased Akt phosphorylation and nuclear translocation of NF-κB p65 in HSCs. Furthermore, LY294002, an Akt inhibitor, significantly attenuated TCDD-triggered HSC activation through blocking Akt phosphorylation and NF-κB activation. These results indicate that HSCs are susceptible to the cytotoxic effects of TCDD and chronic TCDD exposure may contribute to liver fibrosis by activating HSC Akt and NF-κB signaling pathways.

Targeted inhibition of dominant PI3-kinase catalytic isoforms increase expression of stem cell genes in glioblastoma cancer stem cell models.

Cancer stem cells (CSC) exhibit therapy resistance and drive self-renewal of the tumour, making cancer stem cells an important target for therapy. The PI3K signalling pathway has been the focus of considerable research effort, including in glioblastoma (GBM), a cancer that is notoriously resistant to conventional therapy. Different isoforms of the catalytic sub-unit have been associated with proliferation, migration and differentiation in stem cells and cancer stem cells. Blocking these processes in CSC would improve patient outcome. We examined the effect of isoform specific PI3K inhibitors in two models of GBM CSC, an established GBM stem cell line 08/04 and a neurosphere formation model. We identified the dominant catalytic PI3K isoform for each model, and inhibition of the dominant isoform blocked AKT phosphorylation, as did pan-PI3K/mTOR inhibition. Analysis of SOX2, OCT4 and MSI1 expression revealed that inhibition of the dominant p110 subunit increased expression of cancer stem cell genes, while pan-PI3K/mTOR inhibition caused a similar, though not identical, increase in cancer stem cell gene expression. This suggested that PI3K inhibition enhanced, rather than blocked, CSC activity. Careful analysis of the response to specific isoform inhibition will be necessary before specific subunit inhibitors can be successfully deployed against GBM CSC.

Combination of 5-fluorouracil and 2-morphilino-8-phenyl-4H-chromen-4-one may inhibit liver cancer stem cell activity.

This work aims to evaluate the impact of 2-morpholino-8-phenyl-4H-chromen-4-one (LY294002) combined 5-fluorouracil (5-FU) for the activity of CD90+ liver cancer cells derived from the human liver cancer cell line MHCC97H. MHCC97H sphere-forming cells (MSFCs) were amplified in serum-free medium and CD90+ cells were isolated from bulk MSFCs using flow cytometry. The phenotype of these CD90+ cells which show liver cancer stem cells (LCSCs) behavior was validated in vitro and in a xenograft model in nude mice. MSFCs, CD90+ liver cancer cells (CD90+ LCCs), and parental MHCC97H cells were treated with no drug, LY294002 alone, 5-FU alone, or both drugs together and then compared in terms of stem cell-related gene expression, proliferation, and invasion. Stem cell phenotype increased with increasing proportion of CD90+ cells, in ascending order: parental MHCC97H cells, MSFCs, and CD90+ liver cancer cells. LY294002 reduced the expression of CD90, Nanog, SALL4, and SHP2 in a concentration-dependent manner in CD90+ LCCs and MSFCs, but not in parental cells. LY294002 blocked AKT phosphorylation via the PI3K/AKT signaling pathway and inhibited CD90+ LCCs proliferation and tumorigenicity in vitro and in vivo. CD90+ liver cancer cells can express liver cancer stem cell phenotype. LY294002 inhibits the proliferation and invasion of MHCC97H-derived CD90+ LCCs and sensitized CD90+ LCCs-derived tumors to 5-FU in the current study which may provide insight into the association between the LY294002 combined 5-FU and liver cancer stem cell (LCSCs).

Knockout Serum Replacement Promotes Cell Survival by Preventing BIM from Inducing Mitochondrial Cytochrome C Release.

Knockout serum replacement (KOSR) is a nutrient supplement commonly used to replace serum for culturing stem cells. We show here that KOSR has pro-survival activity in chronic myelogenous leukemia (CML) cells transformed by the BCR-ABL oncogene. Inhibitors of BCR-ABL tyrosine kinase kill CML cells by stimulating pro-apoptotic BIM and inhibiting anti-apoptotic BCL2, BCLxL and MCL1. We found that KOSR protects CML cells from killing by BCR-ABL inhibitors—imatinib, dasatinib and nilotinib. The protective effect of KOSR is reversible and not due to the selective outgrowth of drug-resistant clones. In KOSR-protected CML cells, imatinib still inhibited the BCR-ABL tyrosine kinase, reduced the phosphorylation of STAT, ERK and AKT, down-regulated BCL2, BCLxL, MCL1 and up-regulated BIM. However, these pro-apoptotic alterations failed to cause cytochrome c release from the mitochondria. With mitochondria isolated from KOSR-cultured CML cells, we showed that addition of recombinant BIM protein also failed to cause cytochrome c release. Besides the kinase inhibitors, KOSR could protect cells from menadione, an inducer of oxidative stress, but it did not protect cells from DNA damaging agents. Switching from serum to KOSR caused a transient increase in reactive oxygen species and AKT phosphorylation in CML cells that were protected by KOSR but not in those that were not protected by this nutrient supplement. Treatment of KOSR-cultured cells with the PH-domain inhibitor MK2206 blocked AKT phosphorylation, abrogated the formation of BIM-resistant mitochondria and stimulated cell death. These results show that KOSR has cell-context dependent pro-survival activity that is linked to AKT activation and the inhibition of BIM-induced cytochrome c release from the mitochondria.