Akt Protein Research Articles

Abstract 7579: Dual inhibition of enhancer of zeste homolog 1 2 induces pyroptosis, modulates oncogenic signaling pathways & sensitizes hepatocellular carcinoma to valemetostat & docetaxel treatments

Abstract HCC is a global health concern, with chemotherapy resistance and patient recurrence posing significant challenges to liver cancer treatment. EZH2 is a crucial part of the polycomb repressive complex 2 (PRC2). It, along with its homolog EZH1, is a focus for cancer therapeutic targets. Dysregulation of EZH1/2 can cause cancer development due to significant changes in geneexpression. The current study aims to investigate the interaction between EZH1/2 and oncogenic signals responsible for HCC progression and pyroptosis induction. Two cell lines, HepG2 and SK-Hep1, were subjected to two FDA-approved drugs, Valemetostat (Val) and Docetaxel (Doc), to determine the impact of EZH1/2 on tumorigenicity and drug resistance. The WST-1 assay was utilized to gauge cell proliferation. The results indisputablydemonstrated a synergistic effect of the drugs after 48 h of treatment. The inhibition of viability was attributed to apoptosis induction in both tested cell lines, and the maximum effect was observed in the combined treatment as compared to either the control or each drug alone. Furthermore, our datashowed activation of caspase-3, upregulation of BAX, indicating apoptotic induction, and downregulation of Bcl-2, Bcl-XL, AKT, S6k, oncogenic β-catenin, and Stat3 protein expression levels, suggesting a modulation of oncogenic signaling pathways at the protein level. After 24 h of Val/Doc treatment, pyroptosis was observed, characterized by cell swelling and bubbles on themembrane and a significant release of lactate dehydrogenase, indicating pyroptotic cell cytotoxicity. The antitumor effects and pyroptosis were enhanced by using a dual inhibitor, UNC1999, for EZH1/2, resulting in a marked increase in the sensitivity of tested cells to the Val/Doc treatment invitro. The present study unequivocally demonstrated that dual inhibition of EZH1/2 acts as a potent sensitizer of HCC cells to Val/Doc treatment, which might be a promising therapeutic approach for inducing cellular pyroptosis in HCC cell lines. Citation Format: Asmaa Elkhder Harira, Adham Maher, Ahmed Sultan. Dual inhibition of enhancer of zeste homolog 1 2 induces pyroptosis, modulates oncogenic signaling pathways & sensitizes hepatocellular carcinoma to valemetostat & docetaxel treatments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7579.

Abstract 7388: Deep learning-implemented molecular signature identification in vascular malignancies

Abstract Angiosarcoma is a rare, aggressive sarcoma that forms malignant endothelium. Naturally occurring angiosarcoma in domestic dogs, also called hemangiosarcoma, is relatively common and offer a useful translational model for the rare human cancer. Distinct molecular subtypes of angiosarcoma have been identified; however, current methods are cost-prohibitive and time-consuming, posing challenges in the development of clinical applications. In this study, we hypothesize that artificial intelligence enables reliable image-based diagnostics and molecular characterization specific to angiosarcoma. The aims of our work are to develop a deep learning framework to provide diagnostic support for suspicious malignant cases, to detect morphological features associated with oncogenic mutations, and to identify immune phenotypes in this malignancy. For proof-of-concept and feasibility study, we first utilized convolutional neural network models to identify malignant features in angiosarcomas. We curated 4,079 images of hematoxylin and eosin (H&E) stained slides from seventy visceral angiosarcomas and 3,536 images of thirty splenic hematomas as benign controls obtained from client-owned dogs. ResNet-50 and AlexNet were trained on the images, and area under the receiver operating characteristic curve (AUC) was determined for disease classification. ResNet-50 and AlexNet yielded AUCs of 0.999 and 0.998, respectively. Next, we calculated the gradient of the model’s prediction and applied it to the original image using Grad-CAM software. Grad-CAM results revealed that AlexNet highlighted histological regions representing malignant cellular components for decision-making, aligning with the pathological diagnostic context. In addition, we applied ResNet-50 to determine if the model could predict morphological features in angiosarcoma tissues representing the presence of PIK3CA mutation. We employed RNA-seq data to annotate PIK3CA mutation status on the matched histological images. Among 54 cases annotated with PIK3CA mutation status, 2,732 PIK3CA wild-type and 664 mutant tumor images were trained by ResNet-50. Our model demonstrated an AUC of 0.981 in the prediction of PIK3CA mutation. We further defined immunophenotypic groups using xCell algorithms on RNA-seq data, and immune scores were applied to histological images. ResNet-50 model resulted in an AUC of 0.953 to classify immune-high and immune-low angiosarcomas. RNA-seq datasets generated from 13 human angiosarcoma cases exist in our lab, along with the respective H&E slides. We also have immunohistochemical slides available for p53, phospho-p53 (Ser15 and Ser20), AKT, and phospho-AKT (Thr308) proteins conducted in both human and canine angiosarcoma tissues. Our ongoing work involves generating whole-slide images of H&E stains and immunostained slides to enhance the deep learning models for the molecular classification of angiosarcomas. Citation Format: Donghee Lee, Heon Heo, Joanne Kim, Jong Hyuk Kim. Deep learning-implemented molecular signature identification in vascular malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7388.

Abstract 551: Investigation of the antipoliferative effect of the Xylopia Aethiopicaextract in triple-negative breast cancer cells

Abstract Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer and has very limited treatment options. Xylopia Aethiopica (XA) is a common spice that is widely used in the regions of central and western Africa. It is known to have various pharmacological properties, one of which is anti-inflammatory activity. The anti-inflammatory effect may portend antiproliferative activity. We hypothesized that the extract of XA will exert antiproliferative activity against TNBC cells. The study aimed to establish the molecular mechanism underlying the antiproliferative activity of XA extract (XAE) against TNBC. The dried fruits of XA were crushed in a mixer and macerated using diethyl ether for 72 h. The macerate was subjected to vacuum and rotary evaporation to remove the organic solvent, and a stock solution was prepared in dimethyl sulfoxide (DMSO). Assessment of the efficacy of XAE was performed using serial dilutions (100 µg/ml, 50 µg/ml, 10 µg/ml, 5 µg/ml, and 1µg/ml) of the XAE stock solution, followed by in vitro testing against two TNBC cell lines (MDA-MB-231, and MDA-MB-468 cells). The cytotoxic effect of the XAE was then measured using the resazurin dye assay. XAE resulted in a concentration-dependent cell death in both cell lines. At the highest concentration (100 µg/ml), XAE induced cytotoxic cell death of 89% and 91% in MDAMB231 and MDAMB468 cells, respectively. This was consistent with IC50 values of 32.95 ± 1.32 µg/ml (MDAMB231) and 20.83 ± 1.05 µg/ml (MDAMB468). At the molecular level, western blot analysis of mitogenic markers associated with MAPK oncogenic signaling pathway demonstrated the inhibition of EGFR and AKT protein levels after a 48-h treatment. Acridine orange-ethidium bromide (AO/EB) staining showed a concentration-dependent induction of apoptosis in both cell lines. Flow cytometric analysis was performed to quantify the apoptotic cells. A significant increase of apoptotic cells was observed in treatment groups as compared to control after 72 h. Apoptotic induction was associated with increased p53 protein levels after 48 h treatment compared to control. The study results underscore the anticancer effects of XAE in TNBC cells mediated through induction of apoptosis. Citation Format: Shailvi Soni, Shail Rakesh Modi, Terrick Andey. Investigation of the antipoliferative effect of the Xylopia Aethiopicaextract in triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 551.

Lycium barbarum polysaccharide reverses drug resistance in oxaliplatin-resistant colon cancer cells by inhibiting PI3K/AKT-dependent phosphomannose isomerase.

Objective: Here, we aimed to explore the effect of LBP in combination with Oxaliplatin (OXA) on reversing drug resistance in colon cancer cells through in vitro and in vivo experiments. We also aimed to explore the possible mechanism underlying this effect. Finally, we aimed to determine potential targets of Lycium barbarum polysaccharide (LBP) in colon cancer (CC) through network pharmacology and molecular docking. Methods: The invasion ability of colon cancer cells was assessed using the invasion assay. The migration ability of these cells was assessed using the migration assay and wound healing assay. Cell cycle analysis was carried out using flow cytometry. The expression levels of phosphomannose isomerase (PMI) and ATP-binding cassette transport protein of G2 (ABCG2) proteins were determined using immunofluorescence and western blotting. The expression levels of phosphatidylinositol3-kinase (PI3K), protein kinase B (AKT), B-cell lymphoma 2 (Bcl-2), and BCL2-Associated X (Bax) were determined using western blotting. Forty BALB/c nude mice purchased from Weitong Lihua, Beijing, for the in vivo analyses. The mice were randomly divided into eight groups. They were administered HCT116 and HCT116-OXR cells to prepare colon cancer xenograft models and then treated with PBS, LBP (50mg/kg), OXA (10mg/kg), or LBP + OXA (50mg/kg + 10mg/kg). The tumor weight and volume of treated model mice were measured, and organ toxicity was evaluated using hematoxylin and eosin staining. The expression levels of PMI, ABCG2, PI3K, and AKT proteins were then assessed using immunohistochemistry. Moreover, PMI and ABCG2 expression levels were analyzed using immunofluorescence and western blotting. The active components and possible targets of LBP in colon cancer were explored using in silico analysis. GeneCards was used to identify CC targets, and an online Venn analysis tool was used to determine intersection targets between these and LBP active components. The PPI network for intersection target protein interactions and the PPI network for interactions between the intersection target proteins and PMI was built using STRING and Cytoscape. To obtain putative targets of LBP in CC, we performed GO function enrichment and KEGG pathway enrichment analyses. Results: Compared with the HCT116-OXR blank treatment group, both invasion and migration abilities of HCT116-OXR cells were inhibited in the LBP + OXA (2.5mg/mL LBP, 10μΜ OXA) group (p < 0.05). Cells in the LBP + OXA (2.5mg/mL LBP, 10μΜ OXA) group were found to arrest in the G1 phase of the cell cycle. Knockdown of PMI was found to downregulate PI3K, AKT, and Bcl-2 (p < 0.05), while it was found to upregulate Bax (p < 0.05). After treatment with L. barbarum polysaccharide, 40 colon cancer subcutaneous tumor models showed a decrease in tumor size. There was no difference in the liver index after LBP treatment (p > 0.05). However, the spleen index decreased in the OXA and LBP + OXA groups (p < 0.05), possibly as a side effect of oxaliplatin. Immunohistochemistry, immunofluorescence, and western blotting showed that LBP + OXA treatment decreased PMI and ABCG2 expression levels (p < 0.05). Moreover, immunohistochemistry showed that LBP + OXA treatment decreased the expression levels of PI3K and AKT (p < 0.05). Network pharmacology analysis revealed 45 active LBP components, including carotenoids, phenylpropanoids, quercetin, xanthophylls, and other polyphenols. It also revealed 146 therapeutic targets of LBP, including AKT, SRC, EGFR, HRAS, STAT3, and MAPK3. KEGG pathway enrichment analysis showed that the LBP target proteins were enriched in pathways, including cancer-related signaling pathways, PI3K/AKT signaling pathway, and IL-17 signaling pathways. Finally, molecular docking experiments revealed that the active LBP components bind well with ABCG2 and PMI. conclusion: Our in vitro experiments showed that PMI knockdown downregulated PI3K, AKT, and Bcl-2 and upregulated Bax. This finding confirms that PMI plays a role in drug resistance by regulating the PI3K/AKT pathway and lays a foundation to study the mechanism underlying the reversal of colon cancer cell drug resistance by the combination of LBP and OXA. Our in vivo experiments showed that LBP combined with oxaliplatin could inhibit tumor growth. LBP showed no hepatic or splenic toxicity. LBP combined with oxaliplatin could downregulate the expression levels of PMI, ABCG2, PI3K, and AKT; it may thus have positive significance for the treatment of advanced metastatic colon cancer. Our network pharmacology analysis revealed the core targets of LBP in the treatment of CC as well as the pathways they are enriched in. It further verified the results of our in vitro and in vivo experiments, showing the involvement of multi-component, multi-target, and multi-pathway synergism in the drug-reversing effect of LBP in CC. Overall, the findings of the present study provide new avenues for the future clinical treatment of CC.

The role of TIM3+ NK and TIM3- NK cells in the immune pathogenesis of severe aplastic anemia.

Natural killer (NK) cells play important immunoregulatory roles in the immune pathogenesis of severe aplastic anemia (SAA). Our previous research showed that SAA caused a decrease in T cell immunoglobulin mucin-3 (TIM3) expression on NK cells. Here we investigated the expression of surface receptors, and the cytotoxicity of peripheral TIM3+ NK and TIM3- NK cells in patients with SAA. The expressions of surface receptors and cytoplasmic protein of TIM3+ NK and TIM3- NK cells from peripheral blood were detected by FCM. The functions of mDCs, and apoptosis rate of K562 cells after co-culture with TIM3+ NK and TIM3- NK cells were maesured by FCM. Westren-blot was used to detect the changes of TIM3+ NK and TIM3- NK signaling pathway proteins (AKT, P-AKT) and compare the functional activity of the two groups. Activating receptors NKG2D and Granzyme B were higher, while inhibiting receptors NKG2A, CD158a and CD158b were lower on TIM3- NK cells compared with TIM3+ NK cells in patients with SAA. In SAA, the expression of CD80 and CD86 on mDCs (Myeloid dendritic cells) was significantly decreased after incubation with TIM3- NK cells. The apoptosis rate (AR) of K562 cells was significantly increased after being incubated with TIM3- NK cells in SAA. The level of signal pathway protein AKT of TIM3- NK cells in SAA was similar to that of TIM3+ NK cells, and the levels of P-AKT and P-AKT/AKT ratio of TIM3- NK cells were significantly higher than those of TIM3+ NK cells. Therefore, TIM3 exerts its inhibitory effect on NK cells and participates in the immune pathogenesis of SAA. Low expression of TIM3 contributes to the enhancement of NK cell activity which in turn inhibits the immune activation state of SAA and improves the disease state. Our research may aid the development of new therapeutic strategies based on TIM3-NK cells infusion for the treatment of SAA.

Thiazolyl-isatin derivatives: Synthesis, in silico studies, in vitro biological profile against breast cancer cells, mRNA expression, P-gp modulation, and interactions of Akt2 and VIM proteins

The literature reports that thiazole and isatin nuclei present a range of biological activities, with an emphasis on anticancer activity. Therefore, our proposal was to make a series of compounds using the molecular hybridization strategy, which has been used by our research group, producing hybrid molecules containing the thiazole and isatin nuclei. After structural planning and synthesis, the compounds were characterized and evaluated in vitro against breast cancer cell lines (T-47D, MCF-7 and MDA-MB-231) and against normal cells (PBMC). The activity profile on membrane proteins involved in chemoresistance and tumorigenic signaling proteins was also evaluated. Among the compounds tested, the compounds 4c and 4a stood out with IC50 values of 1.23 and 1.39 μM, respectively, against the MDA-MB-231 cell line. Both compounds exhibited IC50 values of 0.45 μM for the MCF-7 cell line. Compounds 4a and 4c significantly decreased P-gp mRNA expression levels in MCF-7, 4 and 2 folds respectively. Regarding the impact on tumorigenic signaling proteins, compound 4a inhibited Akt2 in MDA-MB-231 and compound 4c inhibited the mRNA expression of VIM in MCF-7.

Fabrication of bimetallic Ag@ZnO nanocomposite and its anti-cancer activity on cervical cancer via impeding PI3K/AKT/mTOR pathway

IntroductionBimetallic nanoparticles, specifically Zinc oxide (ZnO) and Silver (Ag), continue to much outperform other nanoparticles investigated for a variety of biological uses in the field of cancer therapy. This study introduces biosynthesis of bimetallic silver/zinc oxide nanocomposites (Ag@ZnO NCs) using the Crocus sativus extract and evaluates their anti-cancer properties against cervical cancer. MethodsThe process of generating bimetallic nanoparticles (NPs), namely Ag@ZnO NCs, through the utilization of Crocus sativus extract proved to be uncomplicated and eco-friendly. Various methods, such as UV–vis, DLS, FTIR, EDX, and SEM analyses, were utilized to characterize the generated Ag@ZnO NCs. The MTT assay was employed to assess the cytotoxic properties of biosynthesized bimetallic Ag@ZnO NCs against the HeLa cervical cancer cell line. Moreover, the impact of Ag@ZnO NCs on HeLa cells was assessed by examining cell survival, ROS production, MMP levels, and induced apoptosis. Through western blot analysis, the expression levels of the PI3K, AKT, mTOR, Cyclin D, and CDK proteins seemed to be ascertained. Using flow cytometry, the cancer cells' progression through necrosis and apoptosis, in addition to the cell cycle analysis, were investigated. ResultsBimetallic Ag@ZnO NCs that were biosynthesized showed a high degree of stability, as demonstrated by the physicochemical assessments. The median size of the particles in these NCs was approximately 80–90 nm, and their zeta potential was –14.70 mV. AgNPs and ZnO were found, according to EDX data. Further, Ag@ZnO NCs hold promise as a potential treatment for cervical cancer. After 24 hours of treatment, a dosage of 5 µg/mL or higher resulted in a maximum inhibitory effect of 58 ± 2.9. The concurrent application of Ag/ZnO NPs to HeLa cells resulted in elevated apoptotic signals and a significant generation of reactive oxygen species (ROS). As a result, the bimettalic Ag@ZnO NCs treatment has been recognized as a chemotherapeutic intervention by inhibiting the production of PI3K, AKT, and mTOR-mediated regulation of propagation and cell cycle-regulating proteins. ConclusionsThe research yielded important insights into the cytotoxic etiology of biosynthesized bimetallic Ag@ZnO NCs against HeLa cells. The biosynthesized bimetallic Ag@ZnO NCs have a significant antitumor potential, which appears to be associated with the development of oxidative stress, which inhibits the development of the cell cycle and the proliferation of cells. Therefore, in the future, biosynthesized bimetallic Ag@ZnO NCs may be used as a powerful anticancer drug to treat cervical cancer.

Clinicopathological Evaluation of Papillary Thyroid Microcarcinoma.

Clinicians sometimes encounter papillary thyroid microcarcinoma (PMC) that is less than 10 mm, associated with lymph node metastasis. In this study, we assessed PMC clinicopathologically to clarify risk factors for poor prognosis. Fifty-one patients who underwent thyroid surgery at Aichi Medical University from September 2009 to October 2016 were included. Patients were divided into two groups, pEX-positive (23 patients) and pEX-negative (28 patients), based on the pathological finding of thyroid capsule invasion. The former indicates that the tumor infiltrated the thyroid capsule and spreadto the neighboring tissue, and the latter indicates no capsule invasion. We analyzed factors such as patient characteristics, pathological findings, and serum levels of thyroid hormones in the two groups. No statistical differences were observed between the two groups in gender distribution or age at surgery. Preoperative cancer diagnoses were established for more patients in the pEX-positive group than in the pEX-negative group (n = 21 and 14, respectively; P = 0.004). The mean (±SD) pathological tumor diameter was 5.42 ± 2.77 in the pEX-negative group and 8.32 ± 1.61 in the pEX-positive group (P < 0.001). No significant differences in preoperative serum levels of free T3, free T4, thyroid-stimulating hormone, or thyroglobulin were observed between the two groups. The odds ratio for node positivity in tumors invading thyroid capsules (pEX-positive) compared to those with no capsule invasion (pEX-negative) was 13.20 (95% confidence interval, 3.45-50.42). Immunohistological staining for phosphatase and tensin homolog deleted from chromosome 10 (PTEN) and Akt (protein kinase B) revealed the facilitation of PTEN and suppression of Akt, which might indicate downregulation of the phosphoinositide 3-kinase-Akt (PI3K-Akt) cascade. In general, the prognosis of PMC is favorable. However, the prognosis is less favorable in patients with nodal metastasis or extrathyroidal invasion. It is controversial whether resection is required for proven PMCs. For PMCs associated with extrathyroidal invasion, regional lymph node resection with lobectomy should be performed due to the high risk for lymphatic spread. There might be a possibility that the natural progression of PMC seems to be controlled by the facilitation of PTEN. However, a tumor in the lateral peripheral region of the thyroid parenchyma might be associated with capsule invasion followed by lymphatic spread.

COMP promotes pancreatic fibrosis by activating pancreatic stellate cells through CD36-ERK/AKT signaling pathways

BackgroundPancreatic fibrosis is one of the most important pathological features of chronic pancreatitis (CP) and pancreatic stellate cells (PSCs) are the key cells of fibrosis. As an extracellular matrix (ECM) glycoprotein, cartilage oligomeric matrix protein (COMP) is critical for collagen assembly and ECM stability and recent studies showed that COMP exert promoting fibrosis effect in the skin, lungs and liver. However, the role of COMP in activation of PSCs and pancreatic fibrosis remain unclear. We aimed to investigate the role and specific mechanisms of COMP in regulating the profibrotic phenotype of PSCs and pancreatic fibrosis. MethodsELISA method was used to determine serum COMP in patients with CP. Mice model of CP was established by repeated intraperitoneal injection of cerulein and pancreatic fibrosis was evaluated by Hematoxylin-Eosin staining (H&E) and Sirius red staining. Immunohistochemical staining was used to detect the expression changes of COMP and fibrosis marker such as α-SMA and Fibronectin in pancreatic tissue of mice. Cell Counting Kit-8, Wound Healing and Transwell assessed the proliferation and migration of human pancreatic stellate cells (HPSCs). Western blotting, qRT-PCR and immunofluorescence staining were performed to detect the expression of fibrosis marker, AKT and MAPK family proteins in HPSCs. RNA-seq omics analysis as well as small interfering RNA of COMP, recombinant human COMP (rCOMP), MEK inhibitors and PI3K inhibitors were used to study the effect and mechanism of COMP on activation of HPSCs. ResultsELISA showed that the expression of COMP significantly increased in the serum of CP patients. H&E and Sirius red staining analysis showed that there was a large amount of collagen deposition in the mice in the CP model group and high expression of COMP, α-SMA, Fibronectin and Vimentin were observed in fibrotic tissues. TGF-β1 stimulates the activation of HPSCs and increases the expression of COMP. Knockdown of COMP inhibited proliferation and migration of HPSCs. Further, RNA-seq omics analysis and validation experiments in vitro showed that rCOMP could significantly promote the proliferation and activation of HPSCs, which may be due to promoting the phosphorylation of ERK and AKT through membrane protein receptor CD36. rCOMP simultaneously increased the expression of α-SMA, Fibronectin and Collagen I in HPSCs. ConclusionIn conclusion, this study showed that COMP was up-regulated in CP fibrotic tissues and COMP induced the activation, proliferation and migration of PSCs through the CD36-ERK/AKT signaling pathway. COMP may be a potential therapeutic candidate for the treatment of CP. Interfering with the expression of COMP or the communication between COMP and CD36 on PSCs may be the next direction for therapeutic research.

Selectivity Studies and Free Energy Calculations of AKT Inhibitors.

Protein kinase B (PKB) or AKT protein is an important target for cancer treatment. Significant advances have been made in developing ATP-competitive inhibitors and allosteric binders targeting AKT1. However, adverse effects or toxicities have been found, and the cutaneous toxicity was found to be linked to the inhibition of AKT2. Thus, selective inhibition of AKT inhibitors is of significance. Our work, using the Schrödinger Covalent Dock (CovDock) program and the Movable Type (MT)-based free energy calculation (ΔG), yielded small mean errors for the experimentally derived binding free energy (ΔG). The docking data suggested that AKT1 binding may require residues Asn54, Trp80, Tyr272, Asp274, and Asp292, whereas AKT2 binding would expect residues Phe163 and Glu279, and AKT3 binding would favor residues Glu17, Trp79, Phe306, and Glu295. These findings may help guide AKT1-selective or AKT3-selective molecular design while sparing the inhibition of AKT2 to minimize the cutaneous toxicity.

Ultrasonic-assisted hydrothermal fabrication of selenium nanorods and their anticancer properties in prostate cancer cells and associated signaling pathway

This paper describes some useful information on the ultrasonic-assisted hydrothermal fabrication of selenium nanorods (abbreviated as SeNRs) and their potential anticancer activity. The synthesis of SeNRs was confirmed and their selective anticancer activity against DU-145 cells as a standard prostate cancer cell line and human prostate normal cell line, RWPE-1 were assessed. The result of the FTIR study revealed the bending and stretching vibration of the Se-O bond. UV–vis spectroscopy data displayed a sharp maximum absorption (λmax) peak at 298 nm and the TEM image demonstrated that Se nanostructures had a rod-shaped morphology with lengths ranging from 50 to 100 nm and a diameter of around 10 nm. DLS data displayed that SeNRs had an average size of 204.68 nm and a zeta potential value of – 37.29 mV. Then, IC50 concentration of SeNRs against DU-145 and RWPE-1 cell lines were calculated to be about 15 µg/mL and 50.06 µg/mL, respectively, at 24 h. Also, it was deduced that SeNRs triggered their anticancer activity through the induction of membrane leakage, oxidative stress, apoptosis by overexpression of Bax/Bcl-2 ratio and caspase-9,-8, -3, and downexpression of AKT, PI3K, and mTOR mRNA and proteins, which was further verified by pre-incubation of cells with PI3K inhibitor, LY294002. Thus, the synthesized SeNRs can be used and developed as an effective nanostructure for the treatment of prostate cancer in future studies.

The Mechanism of Fuzheng Xiaoji Decoction in the Treatment of Non-Small Cell Lung Cancer Based on Network Pharmacology and Molecular Docking Technology

With approximately 2 million lung cancer patients and 1.8 million deaths per year, the world ranks among the highest incidence rates in the world. Non-small cell lung cancer (NSCLC) accounts for up to 85% of lung cancer patients and has a poor prognosis. To investigate the mechanism of Fuzheng Xiaoji Decoction in the treatment of NSCLC. Fuzheng Xiaoji Decoction, consisting of ten traditional Chinese medicines, was analyzed using the TCMSP database to identify its active ingredients and their corresponding targets. Simultaneously, NSCLC-related targets were obtained from GeneCards. The study aimed to predict Fuzheng Xiaoji Decoction’s targets in treating NSCLC and establish a network diagram illustrating the relationship between traditional Chinese medicine, active ingredients, targets, and the disease. The STRING platform was used to construct a PPI network, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and Gene Ontology (GO) function enrichment analysis were performed on the intersecting targets. Based on the interaction network analysis, the core target proteins, represented by the TOP5 nodes with the highest degrees, were selected. These core targets were then subjected to molecular docking with the top five key active components of Fuzheng Xiaoji Decoction to construct the docking model. We identified 132 active components in Fuzheng Xiaoji Decoction and picked the TOP 5 proteins from the PPI network as the primary active elements in Fuzheng Xiaoji Decoction for NSCLC treatment, including kaempferol, quercetin, ingenin, luteolin, and osterol, involving a total of 209 targets. The top five core targets obtained by PPI network were AKT1, TP53, STAT3, TNF and IL6. A total of 7879 targets related to NSCLC were retrieved, and 164 drug-disease intersection targets were identified. GO functional analysis results showed that 164 gene sets were involved in 377 biological processes, mainly involved in 79 processes related to cell composition and 46 molecular function expression processes. KEGG enrichment results showed 132 significantly enriched signaling pathways. Molecular docking findings indicated favorable binding interactions between the receptor and ligand, suggesting a strong affinity between the key NSCLC treatment protein and the primary active ingredient in Fuzheng Xiaoji Decoction. The potential mechanism of Fuzheng Xiaoji Decoction in NSCLC treatment may involve flavonoids, triterpenoids and other compounds regulate lung cancer-related signaling pathways by acting on AKT1, TP53, STAT3 and other proteins to affect the expression of P53, IL6 and other cytokines. This may help regulate immune function and inhibit tumor cell proliferation.

Monotropein Alleviates Ovalbumin-Induced Asthma in Mouse Model by Inhibiting AKT/NF-κB Pathway

Introduction: Clinical management of asthma remains as a prevalent challenge. Monotropein (MON) is a naturally occurring cyclic enol ether terpene glycoside with medical application potential. This study aims to evaluate the potential therapeutic effects of MON in the mouse model of chronic asthma. Methods: An ovalbumin (OVA)-induced asthmatic mouse model was established to evaluate the therapeutic effect of MON at different doses (20, 40, and 80 mg/kg). The potential involvement of protein kinase B (AKT)/nuclear factor kappa B (NF-κB) pathway in the effect of MON was investigated by the administration of an AKT activator SC79. Histological changes in pulmonary tissues were examined by hematoxylin and eosin staining. The profiles of inflammatory cytokines (interleukin [IL]-4, IL-5, IL-13, and tumor necrosis factor [TNF]-α) in bronchoalveolar lavage fluid (BALF), and OVA-specific IgE in blood samples were analyzed by enzyme-linked immunosorbent assay (ELISA). The oxidative stress in the lung tissues was determined by measuring malondialdehyde level. The phosphorylation activation of AKT and NF-κB was examined by immunoblotting in the lung tissues. Results: MON treatment suppressed the infiltration of inflammatory cells in the airways of OVA-induced asthma mice and reduced the thickness of the bronchial wall and smooth muscle layer in a dose-dependent manner. MON treatment also reduced the levels of OVA-specific IgE in serum and cytokines in BALF in asthma-induced mice, and attenuated the oxidative stress in the lung tissues. OVA induced the phosphorylation of AKT and NF-κB proteins in the lung tissues of asthmatic mice, which was significantly suppressed by MON treatment. The co-administration of AKT activator SC79 impaired the therapeutic effect of MON on asthma-induced mice. Conclusion: Our data demonstrated the potential therapeutic effect of MON on asthmatic mouse model, suggesting that MON attenuated the inflammatory and oxidative damages in ling tissues by dampening the AKT/NF-κB signaling pathway.

Inhibition of cc chemokine receptor 10 ameliorates osteoarthritis via inhibition of the phosphoinositide-3-kinase/Akt/mammalian target of rapamycin pathway

BackgroundOsteoarthritis (OA) is a joint disease characterized by inflammation and progressive cartilage degradation. Chondrocyte apoptosis is the most common pathological feature of OA. Interleukin-1β (IL-1β), a major inflammatory cytokine that promotes cartilage degradation in OA, often stimulates primary human chondrocytes in vitro to establish an in vitro OA model. Moreover, IL-1β is involved in OA pathogenesis by stimulating the phosphoinositide-3-kinase (PI3K)/Akt and mitogen-activated protein kinases pathways. The G-protein-coupled receptor, cc chemokine receptor 10 (CCR10), plays a vital role in the occurrence and development of various malignant tumors. However, the mechanism underlying the role of CCR10 in the pathogenesis of OA remains unclear. We aimed to evaluate the protective effect of CCR10 on IL-1β-stimulated CHON-001 cells and elucidate the underlying mechanism.MethodsThe CHON-001 cells were transfected with a control small interfering RNA (siRNA) or CCR10-siRNA for 24 h, and stimulated with 10 ng/mL IL-1β for 12 h to construct an OA model in vitro. The levels of CCR10, cleaved-caspase-3, MMP-3, MMP-13, Collagen II, Aggrecan, p-PI3K, PI3K, p-Akt, Akt, phosphorylated-mammalian target of rapamycin (p-mTOR), and mTOR were detected using quantitative reverse transcription polymerase chain reaction and western blotting. Viability, cytotoxicity, and apoptosis of CHON-001 cells were assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, lactate dehydrogenase assay (LDH), and flow cytometry analysis, respectively. Inflammatory cytokines (TNF-α, IL-6, and IL-8) were assessed using enzyme-linked immunosorbent assay.ResultsLevel of CCR10 was substantially higher in the IL-1β-stimulated CHON-001 cells than that in the control group, whereas CCR10 was down-regulated in the CCR10-siRNA transfected CHON-001 cells compared to that in the control-siRNA group. Notably, CCR10 inhibition alleviated IL-1β-induced inflammatory injury in the CHON-001 cells, as verified by enhanced cell viability, inhibited LDH release, reduced apoptotic cells, and cleaved-caspase-3 expression. Meanwhile, IL-1β induced the release of tumor necrosis factor alpha, IL-6, and IL-8, increase of MMP-3 and MMP-13, and decrease of Collagen II and Aggrecan in the CHON-001 cells, which were reversed by CCR10-siRNA. However, these effects were reversed upon PI3K agonist 740Y-P treatment. Further, IL-1β-induced PI3K/Akt/mTOR signaling pathway activation was inhibited by CCR10-siRNA, which was increased by 740Y-P treatment.ConclusionInhibition of CCR10 alleviates IL-1β-induced chondrocytes injury via PI3K/Akt/mTOR pathway inhibition, suggesting that CCR10 might be a promising target for novel OA therapeutic strategies.

KIF2A Upregulates PI3K/AKT Signaling through Polo-like Kinase 1 (PLK1) to Affect the Proliferation and Apoptosis Levels of Eriocheir sinensis Spermatogenic Cells.

E. sinensis is an animal model for studying the reproduction and development of crustaceans. In this study, we knocked down the Es-Kif2a gene by injecting dsRNA into E. sinensis and inhibited Es-Plk1 gene expression by injecting PLK1 inhibitor BI6727 into E. sinensis. Then, the cell proliferation level, apoptosis level, and PI3K/AKT signaling expression level were detected. Our results showed that the proliferation level of spermatogenic cells decreased, while the apoptosis level increased after Es-Kif2a knockdown or Es-Plk1 inhibition. In order to verify whether these changes are caused by regulating the PI3K/AKT pathway, we detected the expression of PI3K and AKT proteins after Es-Kif2a knockdown or Es-Plk1 inhibition. Western Blot showed that in both the Es-Kif2a knockdown group and the Es-Plk1 inhibition group, the expression of PI3K and AKT proteins decreased. In addition, immunofluorescence showed that Es-KIF2A and Es-PLK1 proteins were co-localized during E. sinensis spermatogenesis. To further explore the upstream and downstream relationship between Es-KIF2A and Es-PLK1, we detected the expression level of Es-PLK1 after Es-Kif2a knockdown as well as the expression level of Es-KIF2A after Es-Plk1 inhibition. Western Blot showed that the expression of Es-PLK1 decreased after Es-Kif2a knockdown, while there was no significant change of Es-KIF2A after Es-Plk1 inhibition, indicating that Es-PLK1 may be a downstream factor of Es-KIF2A. Taken together, these results suggest that Es-KIF2A upregulates the PI3K/AKT signaling pathway through Es-PLK1 during the spermatogenesis of E. sinensis, thereby affecting the proliferation and apoptosis levels of spermatogenic cells.

Ginseng fermentation solution affects the gut microbiota in zebrafish with alcoholic liver disease via PI3K/Akt pathway

BackgroundGinsenosides have received increased amounts of attention due to their ability to modulate the intestinal flora, which may subsequently alleviate alcoholic liver disease (ALD). The effects of ginseng fermentation solution (GFS) on the gut microbiota and metabolism in ALD patients have not been explored. PurposeThis research aimed to explore the regulatory effect of GFS on ALD both in vitro and in vivo. MethodThis study assessed the anti-ALD efficacy of GFS using an LO2 cell model and a zebrafish model. Untargeted metabolomics was used for differentially abundant metabolite analysis, and high-throughput 16S rRNA sequencing was used to examine the effect of GFS on ALD. ResultsThe LO2 cell line experiments demonstrated that GFS effectively mitigated alcohol-induced oxidative stress and reduced apoptosis by upregulating PI3K and Bcl-2 expression and decreasing the levels of malondialdehyde, total cholesterol, and triglycerides. In zebrafish, GFS improved morphological and physiological parameters and diminished oxidative stress-induced ALD. Meanwhile, the results from Western blotting indicated that GFS enhanced the expression of PI3K, Akt, and Bcl-2 proteins while reducing Bax protein expression, thereby ameliorating the ALD model in zebrafish. Metabolomics data revealed significant changes in a total of 46 potential biomarkers. Among them, metabolites such as prostaglandin F2 alpha belong to arachidonic acid metabolism. In addition, GFS also partly reversed the imbalance of gut microbiota composition caused by alcohol. At the genus level, alcohol consumption elevated the presence of Flectobacillus, Curvibacter, among others, and diminished Elizabethkingia within the intestinal microbes of zebrafish. Conversely, GFS reversed these effects, notably enhancing the abundance of Proteobacteria and Archaea. Correlation analyses further indicated a significant negative correlation between prostaglandin F2 alpha, 11,14,15-THETA, Taurocholic acid and Curvibacter. ConclusionThis study highlights a novel mechanism by which GFS modulates anti-ALD activity through the PI3K/Akt signalling pathway by influencing the intestinal flora-metabolite axis. These results indicate the potential of GFS as a functional food for ALD treatment via modulation of the gut flora.

Sex differences in insulin regulation of skeletal muscle glycogen synthase and changes during weight loss and exercise in adults.

The authors sought to understand sex differences in muscle metabolism in 73 older men and women. Body composition, VO2max, and insulin sensitivity (M) by 3-hour hyperinsulinemic-euglycemic clamp with vastus lateralis muscle biopsies were measured. Women had lower body weight, VO2max, and fat-free mass than men. Men had lower M, lower change (insulin minus basal) in muscle glycogen synthase (GS) activity, and lower change in AKT protein expression than women. M was associated with the change (insulin-basal) in GS activity and the change in AKT protein expression. Sex differences (n = 60) were tested with 6-month weight loss or 3×/week aerobic exercise training. The postintervention minus preintervention change (insulin-basal) (∆∆) in GS activity (fractional, independent, total) was higher in men than women in the weight loss group and ∆∆ in GS fractional activity was higher in women than men in the aerobic exercise group. In all participants, ∆∆ in GS fractional and independent activities was related to ∆∆ in AKT expression and glycogen content. Sex differences in insulin sensitivity may be explained at the cellular muscle level, and to improve skeletal muscle insulin action in older adults, it may be necessary to recommend different behavioral strategies depending on the individual's sex.

HER2 phosphorylation induced by TGF-β promotes mammary morphogenesis and breast cancer progression.

Transforming growth factor β (TGF-β) and HER2 signaling collaborate to promote breast cancer progression. However, their molecular interplay is largely unclear. TGF-β can activate mitogen-activated protein kinase (MAPK) and AKT, but the underlying mechanism is not fully understood. In this study, we report that TGF-β enhances HER2 activation, leading to the activation of MAPK and AKT. This process depends on the TGF-β type I receptor TβRI kinase activity. TβRI phosphorylates HER2 at Ser779, promoting Y1248 phosphorylation and HER2 activation. Mice with HER2 S779A mutation display impaired mammary morphogenesis, reduced ductal elongation, and branching. Furthermore, wild-type HER2, but not S779A mutant, promotes TGF-β-induced epithelial-mesenchymal transition, cell migration, and lung metastasis of breast cells. Increased HER2 S779 phosphorylation is observed in human breast cancers and positively correlated with the activation of HER2, MAPK, and AKT. Our findings demonstrate the crucial role of TGF-β-induced S779 phosphorylation in HER2 activation, mammary gland development, and the pro-oncogenic function of TGF-β in breast cancer progression.

Downregulating DNA methyltransferase 3B by suppressing the PI3K/Akt signaling pathway enhances the chemosensitivity of glioblastoma to temozolomide

Glioblastoma (GBM) is the most common malignant brain tumor and has the poorest prognosis attributed to its chemoresistance to temozolomide (TMZ), the first-line drug for treating GBM. TMZ resistance represents a significant obstacle to successful GBM treatment, necessitating the development of new strategies to overcome this resistance and augment the chemosensitivity of GBM cells to TMZ. This study established a TMZ-resistant U251 (U251-TMZ) cell line by exposing it to increasing doses of TMZ in vitro. We focused on the DNA methyltransferase 3B (DNMT3B) gene, phosphorylated Akt (p-Akt), total Akt (t-Akt), phosphorylated PI3K (p-PI3K), and total PI3K (t-PI3K) protein expression. Results showed that the DNMT3B gene was significantly upregulated in the U251-TMZ cell line. The p-Akt and p-PI3K protein expression in U251-TMZ cells was also significantly elevated. Moreover, we found that DNMT3B downregulation was correlated with the increased chemosensitivity of GBM cells to TMZ. LY294002 suppressed the PI3K/Akt signaling pathway, leading to a notable inhibition of PI3K phosphorylation and a significant decrease in DNMT3B expression in U251-TMZ cells. Given that DNMT3B expression is mediated by the PI3K/Akt signaling pathway, its downregulation further increased the chemosensitivity of GBM cells to TMZ and therefore is a promising therapeutic for GBM treatment. Our results suggested that DNMT3B downregulation can inhibit the proliferation of GBM cells and induce GBM cell apoptosis in vitro. In addition, the PI3K/Akt signaling pathway plays an important role in the chemosensitivity of GBM cells to TMZ by regulating DNMT3B expression.

The novel miR-873-5p-YWHAE-PI3K/AKT axis is involved in non-small cell lung cancer progression and chemoresistance by mediating autophagy.

Non-small cell lung cancer (NSCLC) encompasses approximately 85% of all lung cancer cases and is the foremost cancer type worldwide; it is prevalent in both sexes and known for its high fatality rate. Expanding scientific inquiry underscores the indispensability of microRNAs in NSCLC. Here, we probed the impact of miR-873-5p on NSCLC development and chemoresistance. qRT‒PCR was used to measure the miR-873-5p level in NSCLC cells with or without chemoresistance. A model of miR-873-5p overexpression was constructed. The proliferation and viability of NSCLC cells were evaluated through CCK8 and colony formation experiments. Cell migration and invasion were monitored via Transwell assays. Western blotting was used to determine the levels of YWHAE, PI3K, AKT, EMT, apoptosis, and autophagy-related proteins. The sensitivity of NSCLC cells to the chemotherapeutic agent gefitinib was assessed. Additionally, the correlation of YWHAE with miR-873-5p was validated via a dual-luciferase reporter assay and RNA immunoprecipitation (RIP). Overexpressed miR-873-5p suppressed migration, proliferation, invasion, and EMT while concurrently stimulating apoptotic processes. miR-873-5p was downregulated in NSCLC cells resistant to gefitinib. Upregulating miR-873-5p reversed gefitinib resistance by inducing autophagy. YWHAE was confirmed to be a downstream target of miR-873-5p. YWHAE overexpression promoted the malignant behaviors of NSCLC cells and boosted tumor growth, while these effects were reversed following miR-873-5p overexpression. Subsequent investigations revealed that overexpressing YWHAE promoted PI3K/AKT pathway activation, with miR-873-5p displaying inhibitory effects on the YWHAE-mediated PI3K/AKT signaling cascade. miR-873-5p affects proliferation, invasion, migration, EMT, autophagy, and chemoresistance in NSCLC by controlling the YWHAE/PI3K/AKT axis.