Downregulation Of Akt Research Articles

Neosetophomone B induces apoptosis in multiple myeloma cells via targeting of AKT/SKP2 signaling pathway.

Multiple myeloma (MM) is a hematologic malignancy associated with malignant plasma cell proliferation in the bone marrow. Despite the available treatments, drug resistance and adverse side effects pose significant challenges, underscoring the need for alternative therapeutic strategies. Natural products, like the fungal metabolite neosetophomone B (NSP-B), have emerged as potential therapeutic agents due to their bioactive properties. Our study investigated NSP-B's antitumor effects on MM cell lines (U266 and RPMI8226) and the involved molecular mechanisms. NSP-B demonstrated significant growth inhibition and apoptotic induction, triggered by reduced AKT activation and downregulation of the inhibitors of apoptotic proteins and S-phase kinase protein. This was accompanied by an upregulation of p21Kip1 and p27Cip1 and an elevated Bax/BCL2 ratio, culminating in caspase-dependent apoptosis. Interestingly, NSP-B also enhanced the cytotoxicity of bortezomib (BTZ), an existing MM treatment. Overall, our findings demonstrated that NSP-B induces caspase-dependent apoptosis, increases cell damage, and suppresses MM cell proliferation while improving the cytotoxic impact of BTZ. These findings suggest that NSP-B can be used alone or in combination with other medicines to treat MM, highlighting its importance as a promising phytoconstituent in cancer therapy.

Activation of AHR by ITE improves cardiac remodelling and function in rats after myocardial infarction.

Left ventricular remodelling subsequent to myocardial infarction (MI) constitutes a pivotal underlying cause of heart failure. Intervention with the nontoxic endogenous aryl hydrocarbon receptor (AHR) agonist 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) in the acute phase of MI has been shown to ameliorate cardiac function, but its role in the chronic phase remains obscured. This study explores the beneficial role of ITE in delaying the progression of heart failure in the chronic phase of MI. MI rats established by ligating the left anterior descending coronary artery were treated with the indicated concentration of the AHR agonist ITE or vehicle alone. Echocardiography was performed to determine cardiac structure and function; myocardial morphology and fibrosis were observed by haematoxylin and eosin and Masson's trichrome staining; serum biochemical indices, BNP, and inflammatory cytokine levels were detected by enzyme-linked immunosorbent assay; F4/80+ iNOS+ M1 macrophages and F4/80+ CD206+ M2 macrophages were detected by immunofluorescence; the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assay was used to detect the apoptosis of cardiomyocytes; ultrastructural changes in myocardial tissue were observed by transmission electron microscopy; and Cyp1a1, Akt, P-Akt, p70S6K, P-p70S6K, Bcl-2, Bax, caspase-3, and cleaved caspase-3 protein levels were determined via Western blotting. We found that therapy with the AHR agonist ITE rescued cardiac remodelling and dysfunction in rats with MI and attenuated myocardial fibrosis, inflammation, and mitochondrial damage. Further studies confirmed that ITE dose-dependently improved myocardial cell apoptosis after MI, as demonstrated by reduced levels of the apoptosis-related proteins cleaved caspase-3 and Bax but increased expression levels of Bcl-2. These effects were attributed to ITE-induced activation of AHR receptors, leading to the down-regulation of Akt and p70S6K phosphorylation. The AHR agonist ITE alleviates cardiomyocyte apoptosis through the Akt/p70S6K signalling pathway, thereby rescuing left ventricular adverse remodelling and cardiac dysfunction after MI.

Violet phosphorene nanosheets and cold atmospheric plasma for synergetic cancer therapy

Violet phosphorene nanosheets (VPNS), another type of two-dimensional nonmetal nanostructure, is a promising nanomaterial for biomedical applications. Cold atmospheric plasma (CAP) is an emerging anti-cancer strategy. Here, for the first time, a synergistic combination of VPNS and CAP exhibited remarkable efficacy in cancer treatment. Our findings revealed that VPNS and CAP effectively suppress cancer cell viability by inhibiting cell proliferation and inducing cell apoptosis in vitro. Moreover, we demonstrated the synergistic anti-cancer effect of VPNS and CAP in vivo, with low or no side effects. By metabolism analysis in vivo, we first reported that VPNS-1 inhibited tumor growth by down-regulating Delta-12-Prostaglandin J2, 8,9-DiHETrE, and Hepoxilin B, thereby suppressing arachidonic acid metabolism. Additionally, the down-regulation of AKT and anti-apoptotic proteins (Bcl-2, Bcl-xL and Mcl-1) of Bcl-2 family contributed to the inhibition of cell growth and the initiation of cell apoptosis. Our results unveiled a novel and promising anti-cancer approach involving the combined treatment of VPNS and CAP.

In vitro siRNA-mediated GPX4 and AKT1 silencing in oxaliplatin resistance cancer cells induces ferroptosis and apoptosis.

Oxaliplatin is a member of platinum-based chemotherapy drugs frequently used in colorectal cancer (CRC). However, resistance to oxaliplatin causes tumor progression and metastasis. Akt1 and Gpx4 are essential regulator genes of apoptosis and ferroptosis pathways. Inhibition of these genes might eradicate oxaliplatin resistance in resistant CRC cells. We compared two cell death strategies to reverse drug resistance in Caco-2 and HT-29 oxaliplatin-resistant cell lines. We used the AKT1-specific siRNA to induce apoptosis. Also, GPX4-specific siRNA and FIN56 were utilized to generate ferroptosis. The effect of these treatments was assessed by reactive oxygen species (ROS) formation, cell viability, and protein expression level assays. Besides, the expression of GPX4, CoQ10, and NRF2 was assessed in both cell lines after treatments. Correctly measuring the expression of these responsible genes and proteins confirms the occurrence of different types of cell death. In addition, the ability of Akt1/ GPX4 siRNA in resensitizing HT-29 and Caco-2 oxaliplatin resistance cells was evaluated. Our finding showed that the upregulation of GPX4/siRNA caused a reduction in GPX4 and CoQ10 expressions in both cell lines. However, the expression level of NRF2 showed the same level in our cell lines, so we observed a downregulation of NRF2 in resistant CRC cell lines. Cell viability assay indicated that induction of ferroptosis by GPX4/siRNA or FIN56 and apoptosis by Akt1/siRNA in resistant cell lines could reverse the oxaliplatin resistance. We concluded that downregulation of Akt1 or Gpx4 could increase the efficacy of oxaliplatin to overcome the resistance compared to FIN56.

Mogroside ⅡE, an in vivo metabolite of sweet agent, alleviates acute lung injury via Pla2g2a-EGFR inhibition

In the face of increasingly serious environmental pollution, the health of human lung tissues is also facing serious threats. Mogroside ⅡE (M2E) is the main metabolite of sweetening agents mogrosides from the anti-tussive Chinese herbal Siraitia grosvenori. The study elucidated the anti-inflammatory action and molecular mechanism of M2E against acute lung injury (ALI). A lipopolysaccharide (LPS)-induced ALI model was established in mice and MH-S cells were employed to explore the protective mechanism of M2E through the western blotting, co-immunoprecipitation, and quantitative real time-PCR analysis. The results indicated that M2E alleviated LPS-induced lung injury through restraining the activation of secreted phospholipase A2 type IIA (Pla2g2a)-epidermal growth factor receptor (EGFR). The interaction of Pla2g2a and EGFR was identified by co-immunoprecipitation. In addition, M2E protected ALI induced with LPS against inflammatory and damage which were significantly dependent upon the downregulation of AKT and mTOR via the inhibition of Pla2g2a-EGFR. Pla2g2a may represent a potential target for M2E in the improvement of LPS-induced lung injury, which may represent a promising strategy to treat ALI.

Down regulation of Akt/mTOR signaling pathway proteins in hippocampus of Alzheimer’s disease mouse model

AbstractBackgroundAkt and mechanistic target of rapamycin (mTOR) signaling pathways are implicated in AD pathology. Activation of these pathways by external cues facilitates new protein synthesis at the synapse which leads to the synaptic plasticity. We have demonstrated that activity dependent Akt1 mediated new protein synthesis is abolished in primary cortical neurons of APP/PS1 mice. However, the molecular underpinnings for the impairment of new protein synthesis at the synapse are still elusive. Our aim is to understand the molecular mechanisms prior to manifestation of pathological symptoms by examining the Akt1 and mTOR signaling cascades and new protein synthesis in hippocampus of WT and APP/PS1 male mice.MethodsWe isolated post nuclear supernatant (PNS) and synaptosomes from brain cortices of WT and APP/PS1 mice at different ages. Synaptosomes and PNS were resolved on SDS‐PAGE and immunoblotted against respective antibodies. Further, synaptoneurosomes were also prepared from hippocampus and performed 35S methionine incorporation assay without or with KCl stimulation. Statistical comparisons were performed using two‐way ANOVA followed by Tukey’s post‐hoc test or two‐tailed Mann‐Whitney U test.ResultsWe observed that pAkt1, pGSK3β, pmTOR, pS6 ribosomal protein, p4E‐BP1 levels are significantly decreased in both post nuclear supernatant (PNS) and synaptosomal fractions isolated from hippocampus of one month old (presymptomatic) APP/PS1 compared to WT male mice. Further, activity dependent new protein synthesis at the synapse was significantly impaired in synaptoneurosomes isolated from hippocampus of presymptomatic APP/PS1 mice, and this deficit is sustained at young adults (three months). However, no impairment was observed in basal protein synthesis at both ages in hippocampus of APP/PS1 compared to WT male mice. Down regulation of Akt1 precludes synaptic activity dependent protein translation at the dendrites but not in the soma in hippocampal neurons from APP/PS1 mice compared to WT mice.ConclusionsOur finding demonstrate that the dysregulation of Akt1/mTOR and its downstream signaling molecules in hippocampus contributes to synaptic dysfunction in AD through loss of activity dependent new protein synthesis at the synapse in hippocampus which is essential for synaptic plasticity and maintenance.

Akt2 deficiency alleviates oxidative stress in the heart and liver via up-regulating SIRT6 during high-fat diet-induced obesity.

The present study aims to investigate the role of AKT2 in the pathogenesis of hepatic and cardiac lipotoxicity induced by lipid overload-induced obesity and identify its downstream targets. WT and Akt2 KO mice were fed either normal diet, or high-fat diet (HFD) to induce obesity model in vivo. Human hepatic cell line (L02 cells) and neonatal rat cardiomyocytes (NRCMs) were used as in vitro models. We observed that during HFD-induced obesity, Akt2 loss-of-function mitigated lipid accumulation and oxidative stress in the liver and heart tissue. Mechanistically, down-regulation of Akt2 promotes SIRT6 expression in L02 cells and NRCMs, the latter deacetylates SOD2, which promotes SOD2 activity and therefore alleviates oxidative stress-induced injury of hepatocytes and cardiomyocytes. Furthermore, we also proved that AKT2 inhibitor protects hepatocytes and cardiomyocytes from HFD-induced oxidative stress. Therefore, our work prove that AKT2 plays an important role in the regulation of obesity-induced lipid metabolic disorder in the liver and heart. Our study also indicates AKT2 inhibitor as a potential therapy for obesity-induced hepatic and cardiac injury.

Inhibition of stemness and EMT by taxifolin ruthenium-p-cymene complex via downregulating the SOX2 and OCT4 expression on lung cancer

Lung carcinoma is perhaps the most often reported cancer incidence throughout the world. The flavonoid metal complexes have exhibited a potential chemotherapeutic effect. This study investigated the chemotherapeutic effect of taxifolin ruthenium-p-cymene complex against lung carcinoma through MTT assay, transwell migration assay, sphere formation assay, western blot, histopathology, immunohistochemistry, and TUNEL assay. The in silico study determined the target proteins of the complex. The synthesized organometallic complex was characterized via various spectroscopical techniques. The complex exhibited strong binding affinity for PI3K, EGFR, and β-catenin. The complex promoted apoptosis and inhibited the colony formation of the cancer cells. Moreover, the cancer cell migration and cancer stemness were reduced with decreased SOX2 and OCT4 expression. The complex induced cell cycle arrest at sub G0/G1 phase, S phase and G2/M phase and promoted caspase-3 mediated apoptosis. The in vivo study demonstrated successful restoration of lung tissue due to the treatment with the complex in benzo-α-pyrene induced lung cancer model. Additionally, the expression of p53 and caspase-3 was increased with significant downregulation of Akt, mTOR and β-catenin. In conclusion, the complex inhibited cancer cell viability, stemness, and EMT in both in vitro and in vivo model systems through the alteration of PI3K/Akt/mTOR/EGFR pathway and expression of stem cell markers including SOX2 and OCT4 that eventually abrogated the EMT mediated metastasis of cancer cells.

Knockdown of Stromal Interaction Molecule 1 (STIM1) Suppresses Acute Myeloblastic Leukemia-M5 Cell Line Survival Through Inhibition of Reactive Oxygen Species Activities

This study aimed to investigate the role of the stromal interaction molecule 1 (STIM1) gene in the survival of the acute myeloblastic leukemia (AML)-M5 cell line (THP-1). The STIM1 effect was assessed via dicersubstrate siRNA-mediated STIM1 knockdown. The effect of STIM1 knockdown on the expression of AKT and MAPK pathway-related genes and reactive oxygen species (ROS) generation-related genes was tested using real-time polymerase chain reaction. Cellular functions, including ROS generation, cell proliferation, and colony formation, were also evaluated following STIM1 knockdown. The findings revealed that STIM1 knockdown reduced intracellular ROS levels via downregulation of NOX2 and PKC. These findings were associated with the downregulation of AKT, KRAS, MAPK, and CMYC. BCL2 was also downregulated, while BAX was upregulated following STIM1 knockdown. Furthermore, STIM1 knockdown reduced THP-1 cell proliferation and colony formation. This study has demonstrated the role of STIM1 in promoting AML cell proliferation and survival through enhanced ROS generation and regulation of AKT/MAPK-related pathways. These findings may help establish STIM1 as a potential therapeutic target for AML treatment.

Salidroside ameliorates severe acute pancreatitis-induced cell injury and pyroptosis by inactivating Akt/NF-κB and caspase-3/GSDME pathways

Our previous studies showed that Salidroside (Sal), a glucoside of the phenylpropanoid tyrosol isolated from Rhodiola rosea L, alleviated severe acute pancreatitis (SAP) by inhibiting inflammation. However, the detailed mechanism remains unclear. Recent evidence has indicated a critical role of Sal in ameliorating inflammatory disorders by regulating pyroptosis. The present study aimed to explore the involvement of Sal and pyroptosis in the pathogenesis of SAP and investigate the potential mechanism. The effects of Sal on pyroptosis were first evaluated using SAP rat and cell model. Our results revealed that Sal treatment significantly decreased SAP-induced pancreatic cell damage and pyroptosis in vivo and in vitro, as well as reduced the release of lactate dehydrogenase (LDH), IL-1β and IL-18. Search Tool for Interacting Chemicals (STITCH) online tool identified 4 genes (CASP3, AKT1, HIF1A and IL10) as candidate targets of Sal in both rattus norvegicus and homo sapiens. Western blot and immunohistochemistry staining validated that Sal treatment decreased the phosphorylation levels of Akt and NF-κB p65, as well as cleaved caspase-3 and N-terminal fragments of GSDME (GSDME-N), suggesting that Sal might suppress pyroptosis through inactivating Akt/NF-κB and Caspase-3/GSDME pathways. Furthermore, overexpression of AKT1 or CASP3 could partially reverse the inhibitory effects of Sal on cell injury and pyroptosis, while downregulation of AKT1 or CASP3 promoted the inhibitory effects of Sal. Taken together, our data indicate that Sal suppresses SAP-induced pyroptosis through inactivating Akt/NF-κB and Caspase-3/GSDME pathways.

E-cigarettes Induce Dysregulation of Autophagy Leading to Endothelial Dysfunction in Pulmonary Arterial Hypertension.

Given the increasing popularity of electronic cigarettes (e-cigs), it is imperative to evaluate the potential health risks of e-cigs, especially in users with preexisting health concerns such as pulmonary arterial hypertension (PAH). The aim of the present study was to investigate whether differential susceptibility exists between healthy and patients with PAH to e-cig exposure and the molecular mechanisms contributing to it. Patient-specific induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) from healthy individuals and patients with PAH were used to investigate whether e-cig contributes to the pathophysiology of PAH and affects EC homeostasis in PAH. Our results showed that PAH iPSC-ECs showed a greater amount of damage than healthy iPSC-ECs upon e-cig exposure. Transcriptomic analyses revealed that differential expression of Akt3 may be responsible for increased autophagic flux impairment in PAH iPSC-ECs, which underlies increased susceptibility upon e-cig exposure. Moreover, knockdown of Akt3 in healthy iPSC-ECs significantly induced autophagic flux impairment and endothelial dysfunction, which further increased with e-cig treatment, thus mimicking the PAH cell phenotype after e-cig exposure. In addition, functional disruption of mTORC2 by knocking down Rictor in PAH iPSC-ECs caused autophagic flux impairment, which was mediated by downregulation of Akt3. Finally, pharmacological induction of autophagy via direct inhibition of mTORC1 and indirect activation of mTORC2 with rapamycin reverses e-cig-induced decreased Akt3 expression, endothelial dysfunction, autophagic flux impairment, and decreased cell viability, and migration in PAH iPSC-ECs. Taken together, these data suggest a potential link between autophagy and Akt3-mediated increased susceptibility to e-cig in PAH.

Alpha-B-Crystallin overexpression is sufficient to promote tumorigenesis and metastasis in mice

BackgroundαB-Crystallin is a heat shock chaperone protein which binds to misfolded proteins to prevent their aggregation. It is overexpressed in a wide-variety of cancers. Previous studies using human cancer cell lines and human xenograft models have suggested potential tumor promoter (oncogene) roles for αB-Crystallin in a wide-spectrum of cancers.MethodsTo determine the causal relationship between CRYAB overexpression and cancer, we generated a Cryab overexpression knock-in mouse model and monitor them for development of spontaneous and carcinogen (DMBA)-induced tumorigenesis. In order to investigate the mechanism of malignancies observed in this model multiple techniques were used such as immunohistochemical characterizations of tumors, bioinformatics analysis of publically available human tumor datasets, and generation of mouse embryonic fibroblasts (MEFs) for in vitro assays (clonogenic survival and migration assays and proteome analysis by mass-spectrometry).ResultsThis model revealed that constitutive overexpression of Cryab results in the formation of a variety of lethal spontaneous primary and metastatic tumors in mice. In vivo, the overexpression of Cryab correlated with the upregulation of epithelial-to-mesenchymal (EMT) markers, angiogenesis and some oncogenic proteins including Basigin. In vitro, using E1A/Ras transformed MEFs, we observed that the overexpression of Cryab led to the promotion of cell survival via upregulation of Akt signaling and downregulation of pro-apoptotic pathway mediator JNK, with subsequent attenuation of apoptosis as assessed by cleaved caspase-3 and Annexin V staining.ConclusionsOverall, through the generation and characterization of Cryab overexpression model, we provide evidence supporting the role of αB-Crystallin as an oncogene, where its upregulation is sufficient to induce tumors, promote cell survival and inhibit apoptosis.

Synergistic role of resveratrol and exercise training in management of diabetic neuropathy and myopathy via SIRT1/NGF/GAP43 linkage

AimsOxidative stress also plays an important role in the pathogenesis of diabetic neuropathy (DN). Both resveratrol (RES) and exercise (EX) have potent anti-oxidative benefits. Low levels of nerve growth factor (NGF) and SIRT1 (a member of sirtuin family) have been reported in patients with DN. The current study has been designed to investigate the role of serum NGF and SIRT1 on DN-induced hyperalgesia and motor incoordination and to evaluate the possible protective role of RES and/or EX. Main methodsA total of 40 male adult albino rats divided into five groups; control, DN, DN + RES, DN + EX, and DN + RES and EX. DN was confirmed by sensorimotor disturbance and diminished nerve conduction velocity (NCV). NGF and SIRT1 levels were measured by western blot. Calcitonin gene-related peptide (CGRP) was measured by PCR. Myofibrillar degeneration and inflammation scores were revealed via H&E microscopic analysis of the gastrocnemius muscle. Immunohistochemical evaluation of caspase3 and TNF-α was performed in the lumber segment of spinal cord and gastrocnemius muscle sections. Ultrastructural evaluation of sciatic nerve axonal degeneration has also been assessed. Key findingsDN group showed decreased SIRT1 level, decreased NGF level and correlated with CGRP level and Na+/K+ ATPase. Treatment with RES and/or EX resulted in improvement of sensorimotor disturbance. DN characterized by reduced SOD level, whereas RES and/or EX could limit oxidative damage by up-regulation Bcl2, Akt and GAP-43 and down-regulation of caspase3 and TNF-α. In conclusion, increased level of SIRT1and NGF by incorporation of RES (natural supplementation) and EX (life style modification) could improve the neuroinflammatory state in DN.

Inhibition of sPLA2 enzyme activity by cell-permeable antioxidant EUK-8 and downregulation of p38, Akt, and p65 signals induced by sPLA2 in inflammatory mouse paw edema model.

The arachidonic acid (AA) metabolic pathway, plays a vital role in the production of eicosanoids by the action of pro-inflammatory secretory phospholipase A2 (PLA2 ). Release of eicosanoids is known to be involved in many inflammatory diseases. Identification of the inhibitory molecules of this AA pathway enzyme along with the regulation of intracellular signaling cascades may be a finer choice to develop as a powerful anti-inflammatory drug. In this regard, we have screened few cell-permeable antioxidant molecules Tempo, Mito-TEMPO, N,N'-Bis(salicylideneamino)ethane-manganese(II) (EUK)-134, and EUK-8 against pro-inflammatory sPLA2 s. Among these, we found EUK-8 is a potent inhibitor with its IC50 value ranges 0.7-2.0 µM for sPLA2 s isolated from different sources. Furthermore, docking studies confirm the strong binding of EUK-8 towards sPLA2 . In vivo effect of EUK-8 was studied in HSF-sPLA2 -induced edema in mouse paw model. In addition to neutralizing the edema, EUK-8 significantly reduces the phosphorylation level of inflammatory proteins such as p38 member of MAPK pathway, Akt, and p65 along with the suppression of pro-inflammatory cytokine (interleukin-6) and chemokine (CXCL1) in edematous tissue. This shows that EUK-8 not only inhibits the sPLA2 activity, it also plays an important role in the regulation of sPLA2 -inducedcell signaling cascades. Apart from the sPLA2 inhibition, we also examine the regulatory actions of EUK-8 with other downstream enzymes of AA pathway such as 5-LOX assay in human polymorphonuclear leukocytes (PMNs) and COX-2 expression in carrageenan-λ induced paw edema. Here EUK-8 significantly inhibits 5-LOX enzyme activity and downregulates COX-2 expression. These data indicate that EUK-8 found to be a promising multitargeted inhibitory molecule toward inflammatory pathway. In conclusion, mitochondrial targeted antioxidant EUK-8 is not only the powerful antioxidant, also a potent anti-inflammatory molecule and may be a choice of molecule for pharmacological applications.

Isobavachalcone Induces Multiple Cell Death in Human Triple-Negative Breast Cancer MDA-MB-231 Cells.

Standardized treatment guidelines and effective drugs are not available for human triple-negative breast cancer (TNBC). Many efforts have recently been exerted to investigate the efficacy of natural compounds as anticancer agents owing to their low toxicity. However, no study has examined the effects of isobavachalcone (IBC) on the programmed cell death (PCD) of human triple-negative breast MDA-MB-231 cancer cells. In this study, IBC substantially inhibited the proliferation of MDA-MB-231 cells in concentration- and time-dependent manners. In addition, we found that IBC induced multiple cell death processes, such as apoptosis, necroptosis, and autophagy in MDA-MB-231 cells. The initial mechanism of IBC-mediated cell death in MDA-MB-231 cells involves the downregulation of Akt and p-Akt-473, an increase in the Bax/Bcl-2 ratio, and cleaved caspases-3 induced apoptosis; the upregulation of RIP3, p-RIP3 and MLKL induced necroptosis; as well as a simultaneous increase in LC3-II/I ratio induced autophagy. In addition, we observed that IBC induced mitochondrial dysfunction, thereby decreasing cellular ATP levels and increasing reactive oxygen species accumulation to induce PCD. These results suggest that IBC is a promising lead compound with anti-TNBC activity.

EP08.02-164 The Effect of Metformin on Survival Outcomes of Advanced EGFR-mutant NSCLC Patients Treated with EGFR TKIs in Thailand

EGFR tyrosine kinase inhibitor (TKI) is mainstay treatment of advanced mutant NSCLC. Mechanism of action of EGFR TKI is inhibitory effects on cell proliferation via through down regulation of AKT and MAPK signaling pathways. Metformin may have direct effect on cancer cells by the similar pathway. The synergistic effect of metformin and EGFR TKI on survival outcomes has been explored. This is the first study in Thailand aim to evaluate the progression free survival (PFS) and overall survival (OS) in advanced EGFR-mutant non-small cell lung cancer (NSCLC) patients with or without type 2 diabetes (T2DM) who were treated with EGFR TKI and metformin/ or other oral hypoglycemic agent, and EKFR TKI alone, respectively.

Apoptotic and Anti-metastatic Effects of Atractylodes lancea (Thunb.) DC. in a Hamster Model of Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a highly aggressive tumor with a greater risk of distant metastasis. The promising anti-CCA activity and safety profile of Atractylodes lancea (AL) have previously been reported in a series of in vitro, in vivo and clinical studies. The present study investigated the effect of AL extract on apoptosis and metastasis signaling pathways in the Opisthorchis viverrini/dimethylnitrosamine (OV/DMN)-induced CCA hamster model. Hamster liver tissues were obtained from the four groups (n = 5 per group), i.e., (i) 5-FU treated CCA (40 µg/mL); (ii) CCA; (iii) AL-treated CCA (5,000 mg/kg), and (iv) normal hamsters. Total RNA was isolated, and the expression levels of apoptosis-related and metastasis-related genes were determined by qRT-PCR analysis. The expression levels of p16, caspase-3, caspase-8, caspase-9, Apaf-1, p53 and Eef1a1 were downregulated, while that of the remaining genes were upregulated in CCA hamsters compared with normal hamsters. AL treatment increased the expression of p16, caspase-9, caspase-3, Apaf-1, p53 and E-cadherin and decreased the expression of cyclin D1, cdk4, Bax, Akt/PKB, Bcl-2, Mfge-8, Lass4, S100A6, TGF-β, Smad-2, Smad-3, Smad-4, MMP-9, and N-cadherin. The expression of Eef1a1 was unchanged. The anti-CCA activity of AL in OV/DMN-induced CCA hamsters could be due to the induction of cell cycle arrest at the G1 phase and activation of the apoptosis pathway, resulting in cancer cell death. The activation of the apoptosis pathway mainly involved the intrinsic pathway (activation of caspase-3 and caspase-9 through p53 and Mfge-8 modulation and downregulation of anti-apoptotic genes Akt and Bcl-2). In addition, AL could also inhibit the canonical TGF-β signaling pathway, MMP-9 and N-cadherin to suppress tumor metastasis.

Brusatol Inhibits Proliferation, Migration, and Invasion of Nonsmall Cell Lung Cancer PC-9 Cells

Objective: The purpose of this study was to investigate the inhibitory effect of brusatol, a nigakilactone extracted from Brucea javanica, on lung cancer for development of therapeutic drugs. We explored the effects of brusatol on the proliferation, migration, and invasion of lung cancer PC-9 cells in vitro and analyzed the mechanisms involved. Materials and Methods: MTT assay was used to determine the effect of brusatol on the proliferative capacity of PC-9 and H1975 cells in vitro. The half-maximal inhibitory concentrations (IC50) were calculated and used as a reference for subsequent experiments. Variations in the number and size of tumor cell clusters were monitored by the colony formation assay as evidence for the effect of brusatol on cell proliferation. The effect of brusatol on the migration and invasion of PC-9 cells was evaluated using wound healing and transwell assays, respectively. Apoptosis in lung cancer cells was detected using the Annexin V-FITC/propidium iodide assay. The correlated anticancer mechanism was detected using Western blotting. Results: The IC50 values of brusatol acting on PC-9 and H1975 cells were 1.58 ± 0.30 mM and 31.34 ± 2.72 mM, respectively, according to the MTT experiment. In addition, brusatol suppressed PC-9 cell proliferation, migration, and invasion, as well as induced apoptosis, which may be related to the downregulation of epidermal growth factor receptor (EGFR), β-catenin, Akt, and STAT3 expression. Conclusions: Brusatol showed potent anticancer activity against lung cancer PC-9 cells, inhibiting the proliferative capacity and metastatic potential of PC-9 cells. Its anticancer effect may be related to the downregulation of EGFR, β-catenin, Akt, and STAT3.

Bacillus subtilis Produces Amino Acids to Stimulate Protein Synthesis in Ruminal Tissue Explants via the Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Beta-Serine/Threonine Kinase-Mammalian Target of Rapamycin Complex 1 Pathway.

BackgroundBacillus subtilis is a probiotic strain that is widely used as a feed supplement for ruminants. In this study, one B. subtilis strain isolated from the ruminal fluid of Holstein dairy cows was used for an ex vivo study with ruminal tissue explants. The main goal was to assess the potential endosymbiotic links between B. subtilis and the ruminal epithelium using molecular analyses and amino acid profiling. The explant culture protocol was first optimized to determine the ideal conditions in terms of tissue viability before performing the actual experiments involving active and inactive bacteria with or without protein synthesis inhibitors, such as LY294002 (phosphatidylinositol 3-kinase inhibitor) or rapamycin [mammalian target of rapamycin (mTOR) inhibitor].ResultsThe mRNA levels of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit beta (PIK3CB), serine/threonine kinase (AKT), mTOR, P70S6K1, and eukaryotic translation initiation factor 4E binding protein 1 were the highest (p < 0.01), while those of programmed cell death 4 were the lowest when the tissue was incubated with 107 of B. subtilis. Compared with the inactivated bacteria, the expression levels of PIK3CB and AKT, and overall changes in mTOR and P70S6K1 were greater in rumen explants with living bacteria (p < 0.05). With an increase in B. subtilis concentration, the trends of protein and corresponding gene changes were consistent. There were differences in the concentrations of individual amino acids in the supernatants of living and inactivated bacterial culture groups, with most amino acids enriched in pathways, such as aminoacyl tRNA biosynthesis, cyanoamino acid metabolism, monobactam biosynthesis, or glycine, serine, and threonine metabolism. The addition of psilocybin upregulated the expression levels of PIK3CB and AKT. A significant decrease (p < 0.05) in PIK3CB and mTOR protein expression levels was detected after the addition of LY294002 and rapamycin. In addition, These responses were associated with the downregulation (p < 0.05) of AKT and P70S6K protein expression levels.ConclusionsWe confirmed that the in vivo ruminal tissue culture system is a suitable model for studying probiotic-induced alterations in tissue function. As such, this study provides a means for future mechanistic studies related to microbial regulation and the dietary supply of proteins. In addition, living and inactivated B. subtilis can promote protein synthesis in ruminal tissue explants by altering the expression levels of related factors in the PIK3CB–AKT–mTORC1 pathway, which could further aid in optimizing the feed efficiency and increasing the use of inactivated bacteria as additives in dairy cow farming.

Abstract 150: Prevalent PKC-ι/ζ signaling is crucial for apoptosis inhibition and invasiveness of glioblastoma through upregulation of Pdk1/Akt1/14-3-3 cascade and Smad2/3

Abstract Glioblastoma (GB) is an aggressive form of cancer derived from astrocytes and often found in brain or spinal cord. Poor prognosis of GB has limitations in current therapeutics mainly due to the development of resistance. Our results demonstrated that atypical protein kinase C-iota and zeta (aPKC-ι/ζ) levels were over-expressed in GB cells (U-87 and T-98) and tissue samples compared to normal brain cells/tissues which is correlated with increased invasiveness. The present study shows the downstream effects of siRNA knockdown of PKC-ι/ζ on the expression of aPKCs, E-cadherin, Vimentin, p-Vimentin, 14-3-3 and p-14-3-3, BAD, p-BAD, Pdk1, Akt1, c-Raf, Tak1, Smad2/3 and p-Smad2/3. Downregulation of Vimentin, p-Vimentin (S33 and S56) and increased E-cadherin indicated the downregulation of epithelial-mesenchymal transition (EMT). Proteolytic E-cadherin fragment of 80 kDa indicates the reduction of cancer progression. Result also indicated that SNAIL1, SLUG and PRRX1 transcriptional activities were downregulated as a result of aPKC diminution. These transcription factors are known to induce and promote EMT. 14-3-3 is known to bind Pdk1, BAD, Akt1 and c-Raf and perform various anti-apoptotic tasks. Our results indicated that total 14-3-3 levels were significantly increased as a result of aPKC attenuation along with dephosphorylated BAD. In addition, p-14-3-3, p-BAD, pAkt1, Pdk1 levels were significantly decreased. Unphosphorylated 14-3-3 does not bind with their partners: phosphorylation on conserved S259 and S621 through Tak1 which is essential for its activation. Our results indicated that Tak1 levels significantly decreased as a result of aPKC diminution. In-vitro migration/invasion assays, immunoprecipitation (IP), immunofluorescence and immuno-gold transmission electron microscopic techniques, real time qPCR and Western blot techniques are being used in this study to further analyze role of aPKCs in GB malignancy. Our IP and kinase activity assay data confirmed that PKC-ι heavily interacts with Pdk1 and PKC-ζ interacts with Akt1, thereby activating those two kinases via phosphorylation. Data confirmed that aPKC attenuation triggers multiple pro apoptotic signaling cascades through downregulation of Akt1, Pdk1, 14-3-3 and Smad2/3.We have used a novel PKC-ι specific inhibitor 5-amino-1-(2,3-dihydroxy-4-(hydroxymethyl)cyclopentyl)-1H-imidazole-4-carboxamide (ICA-1S) and a PKC-ζ specific inhibitor 8-hydroxy-1,3,6-naphthalenetrisulfonic acid (ζ-Stat) to conduct in-vivo experiments on murine models. Excised tumors will be analyzed for pathways observed in in-vitro experiments. Overall results suggest that aPKCs are crucial for increased invasiveness in GB. Results suggested that aPKC can be targeted to develop customized, tailored therapies for Glioblastoma which merit further research. Citation Format: Wishrawana Sarathi Ratnayake, Khandker M. Khalid, Sloan Breedy, Luke Lajmi, Tracess Smalley, Christopher A. Apostolatos, Robert Hill, James Fackrell, Mildred Acevedo-Duncan. Prevalent PKC-ι/ζ signaling is crucial for apoptosis inhibition and invasiveness of glioblastoma through upregulation of Pdk1/Akt1/14-3-3 cascade and Smad2/3 [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 150.