Piperine induces autophagy of colon cancer cells: Dual modulation of AKT/mTOR signaling pathway and ROS production.

BACKGROUND: Colorectal cancer (CRC) is a prevalent malignancy and poses a significant clinical challenge. Piperine, an alkaloid molecule extracted from Piper nigrum and Piper longum, has emerged as a promising anticancer agent. However, the molecular mechanisms of piperine' antitumor effects in CRC need to be further elucidated. METHODS: Human colorectal cancer cells were treated with piperine in vitro. CCK-8 and clone formation assays were adopted to detect cell viability. The accumulation of autophagosomes was assessed by Western blotting and immunofluorescence. Apoptosis and reactive oxygen species (ROS) levels were analyzed by flow. In vivo, a xenograft tumor mouse model was constructed using CT26 cells. RESULTS: Piperine inhibited CRC cell viability and suppressed tumor weight and volume in a mouse model. Additionally, piperine treatment induced the accumulation of autophagosomes in CRC cells. This effect was attributed to the inhibition of the AKT/mTOR pathway and the accumulation of ROS. activation of AKT or clearance of ROS attenuated piperine-mediated tumor suppression. CONCLUSION: This study shows that piperine induces autophagy-dependent cell death in CRC cells by increasing ROS production and inhibiting Akt/mTOR signaling.

Knockdown of NUF2-derived exosomes can inhibit the migration and autophagy of BC cells and improve resistance to doxorubicin.

Breast cancer (BC) is the most common type of fatal cancer in women. New therapeutic strategies need to be explored to enhance the efficacy of doxorubicin by overcoming the resistance of BC cells. NUF2 is a component of the Ndc80 centromere complex and is a key substance in mediating mitosis and affects the progression of multiple tumors. However, the role as well as mechanisms of NUF2 resistance in BC remain unclear. This study aims to reveal the role of NUF2 in drug resistance in BC. We here revealed that NUF2 was highly expressed in human BC. NUF2 depletion-derived exosomes blocked the growth of BC cells. Further, NUF2 ablation-derived exosomes inhibited autophagy in BC cells. Also, NUF2 ablation-derived exosomes improved doxorubicin resistance in BC cells. Mechanically, NUF2 ablation-derived exosomes blocked PI3K/AKT/mTOR axis in BC cells. In summary, NUF2 ablation-derived exosomes blocked the autophagy of BC cells and improved doxorubicin resistance via mediating PI3K/AKT/mTOR axis.

A Lipid-Sensitive Spider Peptide Toxin Exhibits Selective Anti-Leukemia Efficacy through Multimodal Mechanisms.

Anti-cancer peptides (ACPs) represent a promising potential for cancer treatment, although their mechanisms need to be further elucidated to improve their application in cancer therapy. Lycosin-I, a linear amphipathic peptide isolated from the venom of Lycosa singorensis, shows significant anticancer potential. Herein, it is found that Lycosin-I, which can self-assemble into a nanosphere structure, has a multimodal mechanism of action involving lipid binding for the selective and effective treatment of leukemia. Mechanistically, Lycosin-I selectively binds to the K562 cell membrane, likely due to its preferential interaction with negatively charged phosphatidylserine, and rapidly triggers membrane lysis, particularly at high concentrations. In addition, Lycosin-I induces apoptosis, cell cycle arrest in the G1 phase and ferroptosis in K562 cells by suppressing the PI3K-AKT-mTOR signaling pathway and activating cell autophagy at low concentrations. Furthermore, intraperitoneal injection of Lycosin-I inhibits tumor growth of K562 cells in a nude mouse xenograft model without causing side effects. Collectively, the multimodal effect of Lycosin-I can provide new insights into the mechanism of ACPs, and Lycosin-I, which is characterized by high potency and specificity, can be a promising lead for the development of anti-leukemia drugs.

Modulation of PI3K/AKT/mTOR signaling pathway in the ovine liver and duodenum during early pregnancy.

The liver and intestine play a critical role in nutrient absorption, storage, and metabolism. The aim of this study was to evaluate expression pattern of phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of the rapamycin (mTOR) signaling pathway that included PI3K, AKT1, mTOR, FoxO1, SREBP-1, PPARα, PTEN and FXR in the maternal liver and duodenum. Ovine livers and duodenums were sampled at day 16 of the estrous cycle, and at days 13, 16 and 25 of gestation, and RT-qPCR, western blot and immunohistochemistry analysis were used to detect mRNA and protein expression. The results showed that expression of PI3K, AKT1, p-mTOR, FoxO1, SREBP-1 and PTEN upregulated in the maternal liver, and PPARα upregulated in the duodenum. However, expression of FoxO1, SREBP-1 and PTEN in the duodenum downregulated during early pregnancy. In addition, expression levels of SREBP-1, PTEN and PPARα in the maternal liver, and PI3K in the duodenum peaked at day 13 of pregnancy. In addition, expression levels of PI3K, p-mTOR and FoxO1 in the liver, and AKT1 and p-mTOR in the duodenum peaked at day 16 of pregnancy. Nevertheless, expression levels of FXR both in the maternal liver duodenum downregulated at days 13 and 16 of pregnancy. In conclusion, early pregnancy regulated expression pattern of PI3K/AKT/mTOR signaling pathway in the ovine liver and duodenum in a pregnancy stage-specific and tissue-specific manner, which may be necessary for the adaptations in maternal hepatic nutrient metabolism and intestinal nutrient absorption early pregnancy.

Bta-miR-330 promotes bovine intramuscular pre-adipocytes adipogenesis via targeting SESN3 to activate the Akt-mTOR signaling pathway.

Consumers are more inclined to choose beef with a high intramuscular fat content (IMF), which regulated by lots of factors. It is very significant to find a miRNA that plays a key role in the accumulation of IMF. In our study, we found that bta-miR-330 was highly expressed in Japanese black cattle and differentially expressed at intramuscular pre-adipocytes differentiation processes. Furthermore, we transfected the bta-miR-330 mimic & inhibitor in intramuscular pre-adipocytes. The results showed that bta-miR-330 inhibits the proliferation but promotes the adipogenesis of intramuscular pre-adipocytes. Subsequently, our study showed that bta-miR-330 binds to SESN3, which inhibits the adipogenesis of intramuscular pre-adipocytes. Moreover, we established the mechanism that bta-miR-330 promotes the adipogenesis of intramuscular pre-adipocytes by targeting SESN3 to activate the Akt-mTOR signaling pathway. Overall, our results revealed that bta-miR-330-SESN3-Akt-mTOR axis plays an important role in adipogenesis of intramuscular pre-adipocytes, which provides a molecular basis for increasing IMF content in beef cattle.

Methylprednisolone Modulates the Tfr/Tfh ratio in EAE-Induced Neuroinflammation through the PI3K/AKT/FoxO1 and PI3K/AKT/mTOR Signalling Pathways.

Methylprednisolone (MP) is a potent glucocorticoid that can effectively inhibit immune system inflammation and brain tissue damage in Multiple sclerosis (MS) patients. T follicular helper (Tfh) cells are a subpopulation of activated CD4 + T cells, while T follicular regulatory (Tfr) cells, a novel subset of Treg cells, possess specialized abilities to suppress the Tfh-GC response and inhibit antibody production. Dysregulation of either Tfh or Tfr cells has been implicated in the pathogenesis of MS. However, the molecular mechanism underlying the anti-inflammatory effects of MP therapy on experimental autoimmune encephalomyelitis (EAE), a representative model for MS, remains unclear. This study aimed to investigate the effects of MP treatment on EAE and elucidate the possible underlying molecular mechanisms involed. We evaluated the effects of MP on disease progression, CNS inflammatory cell infiltration and myelination, microglia and astrocyte activation, as well as Tfr/Tfh ratio and related molecules/inflammatory factors in EAE mice. Additionally, Western blotting was used to assess the expression of proteins associated with the PI3K/AKT pathway. Our findings demonstrated that MP treatment ameliorated clinical symptoms, inflammatory cell infiltration, and myelination. Furthermore, it reduced microglial and astrocytic activation. MP may increase the number of Tfr cells and the levels of cytokine TGF-ß1, while reducing the number of Tfh cells and the levels of cytokine IL-21, as well as regulate the imbalanced Tfr/Tfh ratio in EAE mice. The PI3K/AKT/FoxO1 and PI3K/AKT/mTOR pathways were found to be involved in EAE development. However, MP treatment inhibited their activation. MP reduced neuroinflammation in EAE by regulating the balance between Tfr/Tfh cells via inhibition of the PI3K/AKT/FoxO1 and PI3K/AKT/mTOR signalling pathways.

Plumbagin ameliorates LPS-induced acute lung injury by regulating PI3K/AKT/mTOR and Keap1-Nrf2/HO-1 signalling pathways.

Acute lung injury (ALI) is a major pathophysiological problem characterized by severe inflammation, resulting in high morbidity and mortality. Plumbagin (PL), a major bioactive constituent extracted from the traditional Chinese herb Plumbago zeylanica, has been shown to possess anti-inflammatory and antioxidant pharmacological activities. However, its protective effect on ALI has not been extensively studied. The objective of this study was to investigate the protective effect of PL against ALI induced by LPS and to elucidate its possible mechanisms both in vivo and in vitro. PL treatment significantly inhibited pathological injury, MPO activity, and the wet/dry ratio in lung tissues, and decreased the levels of inflammatory cells and inflammatory cytokines TNF-α, IL-1ß, IL-6 in BALF induced by LPS. In addition, PL inhibited the activation of the PI3K/AKT/mTOR signalling pathway, increased the activity of antioxidant enzymes CAT, SOD, GSH and activated the Keap1/Nrf2/HO-1 signalling pathway during ALI induced by LPS. To further assess the association between the inhibitory effects of PL on ALI and the PI3K/AKT/mTOR and Keap1/Nrf2/HO-1 signalling, we pretreated RAW264.7 cells with 740Y-P and ML385. The results showed that the activation of PI3K/AKT/mTOR signalling reversed the protective effect of PL on inflammatory response induced by LPS. Moreover, the inhibitory effects of PL on the production of inflammatory cytokines induced by LPS also inhibited by downregulating Keap1/Nrf2/HO-1 signalling. In conclusion, the results indicate that the PL ameliorate LPS-induced ALI by regulating the PI3K/AKT/mTOR and Keap1-Nrf2/HO-1 signalling, which may provide a novel therapeutic perspective for PL in inhibiting ALI.

SZC010 suppresses breast cancer development by regulating the PI3K/Akt/NF-κB signaling pathway.

BACKGROUND: Breast cancer has become one of the leading causes of cancer deaths and is the most frequently diagnosed cancer among females worldwide. Despite advances in breast cancer therapy, metastatic disease in most patients will eventually progress due to the development of de novo or secondary resistance. Thus, it is extremely important to seek novel drugs with high effectiveness and low toxicity for systematic therapy. METHODS: We applied a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in this study to analyze and evaluate the cytotoxic activity of oleanolic acid (OA) and its derivatives in three types of breast cancer cell lines (MDA-MB-231, MCF-7, and MDA-MB-453). A flow cytometry assay was performed to access the mechanisms of apoptosis and cell cycle analysis in SZC010 in MDA-MB-453 cells. Apoptosis- and cyclin-related proteins were evaluated by western blot. The key proteins of the NF-κB and PI3K-Akt-mTOR signaling pathway were also evaluated by western blot. RESULTS: Our results revealed that all OA derivatives were more effective than OA in three types of breast cancer cell lines (MCF-7, MDA-MB-231, and MDA-MB-453). Among these seven OA derivatives, SZC010 exhibited the most potent cytotoxicity in MDA-MB-453 cells. Additionally, we observed that SZC010 treatment induced dose-and time-dependent growth inhibition in MDA-MB-453 cells. Furthermore, we demonstrated that SZC010 induced growth arrest in the G2/M phase and apoptosis by inhibition of NF-κB activation via the PI3K/Akt/mTOR signaling pathway. CONCLUSIONS: Our data indicate that the novel OA derivative, SZC010, has great potential in breast cancer therapy.

PLAUR facilitates the progression of clear cell renal cell carcinoma by activating the PI3K/AKT/mTOR signaling pathway.

Background: PLAUR has been found upregulated in various tumors and closely correlated with the malignant phenotype of tumor cells. The aim of this study was to investigate the relationship between PLAUR and clear cell renal cell carcinoma (ccRCC) and its potential mechanism of promoting tumor progression. Methods: The expression levels and clinical significance of PLAUR, along with the associated signaling pathways, were extensively investigated in ccRCC samples obtained from The Cancer Genome Atlas (TCGA). PLAUR expression in 20 pairs of ccRCC tumor tissues and the adjacent tissues was assessed using qRT-PCR and IHC staining. Additionally, a series of in vitro experiments were conducted to investigate the impact of PLAUR suppression on cellular proliferation, migration, invasion, cell cycle progression, and apoptosis in ccRCC. The Western blot analysis was employed to investigate the expression levels of pivotal genes associated with the PI3K/AKT/mTOR signaling pathway. Results: The expression of PLAUR was significantly upregulated in ccRCC compared to normal renal tissues, and higher PLAUR expression in ccRCC was associated with a poorer prognosis than low expression. The in-vitro functional investigations demonstrated that knockdown of PLAUR significantly attenuated the proliferation, migration, and invasion capabilities of ccRCC cells. Concurrently, PLAUR knockdown effectively induced cellular apoptosis, modulated the cell cycle, inhibited the EMT process, and attenuated the activation of the PI3K/AKT/mTOR signaling pathway. PLAUR may represent a key mechanism underlying ccRCC progression. Conclusions: The involvement of PLAUR in ccRCC progression may be achieved through the activation of the PI3K/AKT/mTOR signaling pathway, making it a reliable biomarker for the identification and prediction of ccRCC.

Integrative analyses identified gap junction beta-2 as a prognostic biomarker and therapeutic target for breast cancer.

Background: Increasing evidence has shown that connexins are involved in the regulation of tumor development, immune escape, and drug resistance. This study investigated the gene expression patterns, prognostic values, and potential mechanisms of connexins in breast cancer. Methods: We conducted a comprehensive analysis of connexins using public gene and protein expression databases and clinical samples from our institution. Connexin mRNA expressions in breast cancer and matched normal tissues were compared, and multiomics studies were performed. Results: Gap junction beta-2 mRNA was overexpressed in breast cancers of different pathological types and molecular subtypes, and its high expression was associated with poor prognosis. The tumor membrane of the gap junction beta-2 mutated group was positive, and the corresponding protein was expressed. Somatic mutation and copy number variation of gap junction beta-2 are rare in breast cancer. The gap junction beta-2 transcription level in the p110α subunit of the phosphoinositide 3-kinase mutant subgroup was higher than that in the wild-type subgroup. Gap junction beta-2 was associated with the phosphoinositide 3-kinase-Akt signaling pathway, extracellular matrix-receptor interaction, focal adhesion, and proteoglycans in cancer. Furthermore, gap junction beta-2 overexpression may be associated with phosphoinositide 3-kinase and histone deacetylase inhibitor resistance, and its expression level correlated with infiltrating CD8+ T cells, macrophages, neutrophils, and dendritic cells. Conclusions: Gap junction beta-2 may be a promising therapeutic target for targeted therapy and immunotherapy and may be used to predict breast cancer prognosis.

ROR2 promotes invasion and chemoresistance of triple-negative breast cancer cells by activating PI3K/AKT/mTOR signaling.

Objective: This study aimed to investigate the role of receptor tyrosine kinase-like orphan receptor 2 (ROR2) in triple-negative breast cancer (TNBC). Methods: ROR2 expression in primary TNBC and metastatic TNBC tissues was analyzed by immunohistochemical staining and PCR. ROR2 expression in TNBC cell lines was detected by PCR and Western blot analysis. The migration, invasion and chemosensitivity of TNBC cells with overexpression or knockdown of ROR2 were examined. Results: ROR2 expression was high in metastatic TNBC tissues. ROR2 knockdown suppressed the migration, invasion and chemoresistance of TNBC cells. ROR2 overexpression in MDA-MB-435 cells promoted the migration, invasion, and chemoresistance. Moreover, ROR2 knockdown in HC1599 and MDA-MB-435 adriamycin-resistant cells enhanced chemosensitivity to adriamycin. ROR2 could activate PI3K/AKT/mTOR signaling in TNBC cells. Conclusion: ROR2 is upregulated and promotes metastatic phenotypes of TNBC by activating PI3K/AKT/mTOR signaling.

The effect and mechanism of palmar ginseng in type 2 diabetic cognitive impairment.

Objective: To investigate the therapeutic effect of palmar ginseng on cognitive impairment in rats with type 2 diabetes, evaluate its neuroprotective effects, and explore its underlying mechanism. Methods: A rat model of diabetic cognitive impairment (DCI) was established by feeding with homemade high-fat, high-sugar chow combined with intraperitoneal injection of streptozotocin (STZ). Rats were continually fed high-fat, high-sugar chow for 60 days after successful induction of the model. Palmar ginseng was administered via gavage. The Morris test was performed after 30 days of treatment. At the end of the test, blood samples were collected, and the activities of IL-6, IL-10, TNF-α, and IL-1ß in rat serum. Pathological changes in hippocampal tissues were observed by Haematoxylin-eosin (HE) staining of the brain, activation of microglia in hippocampal tissues was detected by immunofluorescence, and the expression of PI3K/Akt/mTOR and JAK2/STAT3 proteins in the hippocampal tissues by Western blot. Results: During the administration of palmar Ginseng, the body weight and blood glucose levels of DCI rats were measured weekly, with results showing that Palmar Ginseng effectively reduced blood glucose levels and body weight of DCI rats. Behavioural tests in the water maze indicated that palmar ginseng effectively improved the learning and memory ability of DCI rats. HE and immunofluorescence staining showed that palmar ginseng improved DCI in rats, ameliorated hippocampal neuronal damage, and improved microglial activation. ELISA showed that palmar ginseng significantly reduced the expression of pro-inflammatory factors in the serum of DCI rats. Increased expression of anti-inflammatory factors was observed, and Western blot analysis showed that Palmar Ginseng regulated PI3K/Akt/mTOR and JAK2/STAT3 protein expression, promoted the phosphorylation of PI3K/Akt/mTOR, and inhibited JAK2/STAT3 protein phosphorylation in rat hippocampal tissues as well as in BV2 cells. Conclusions: Palmar ginseng may improve the onset and development of DCI by upregulating the phosphorylation of proteins in the PI3K/Akt/mTOR pathway.

Targeting the PI3K/AKT/mTOR pathway offer a promising therapeutic strategy for cholangiocarcinoma patients with high doublecortin-like kinase 1 expression.

BACKGROUND: Cholangiocarcinoma (CCA), characterized by high heterogeneity and extreme malignancy, has a poor prognosis. Doublecortin-like kinase 1 (DCLK1) promotes a variety of malignant cancers in their progression. Targeting DCLK1 or its associated regulatory pathways can prevent the generation and deterioration of several malignancies. However, the role of DCLK1 in CCA progression and its molecular mechanisms remain unknown. Therefore, we aimed to investigate whether and how DCLK1 contributes to CCA progression. METHODS: The expression of DCLK1 in CCA patients was detected using Immunohistochemistry (IHC). We established DCLK1 knockout and DCLK1 overexpression cell lines for Colony Formation Assay and Transwell experiments to explore the tumor-promoting role of DCLK1. RT-PCR, Western blot and multiple fluorescent staining were used to assess the association between DCLK1 and epithelial-mesenchymal transition (EMT) markers. RNA sequencing and bioinformatics analysis were performed to identify the underlying mechanisms by which DCLK1 regulates CCA progression and the EMT program. RESULTS: DCLK1 was overexpressed in CCA tissues and was associated with poor prognosis. DCLK1 overexpression facilitated CCA cell invasion, migration, and proliferation, whereas DCLK1 knockdown reversed the malignant tendencies of CCA cells, which had been confirmed both in vivo and in vitro. Furthermore, we demonstrated that DCLK1 was substantially linked to the advancement of the EMT program, which included the overexpression of mesenchymal markers and the downregulation of epithelial markers. For the underlying mechanism, we proposed that the PI3K/AKT/mTOR pathway is the key process for the role of DCLK1 in tumor progression and the occurrence of the EMT program. When administered with LY294002, an inhibitor of the PI3K/AKT/mTOR pathway, the tumor's ability to proliferate, migrate, and invade was greatly suppressed, and the EMT process was generally reversed. CONCLUSIONS: DCLK1 facilitates the malignant biological behavior of CCA cells through the PI3K/AKT/mTOR pathway. In individuals with cholangiocarcinoma who express DCLK1 at high levels, inhibitors of the PI3K/AKT/mTOR signaling pathway may be an effective therapeutic approach.

BI-7273, a BRD9 inhibitor, reduces lipid accumulation by downregulating the AKT/mTOR/SREBP1 signaling pathway.

Increases in de novo lipogenesis that disturbed lipid homeostasis and caused lipid accumulation are a major cause of NAFLD and obesity. SREBP1 is a crucial regulatory factor controlling the expression of rate-limiting enzymes of lipid synthesis. A reduction in SREBP1expression can reduce lipid accumulation. Thus, we utilized an SREBP1-luciferase-KI HEK293 cell line constructed by our lab to screen 200 kinds of epigenetic drugs for their ability to downregulate SREBP1expression. BI-7273, an inhibitor of bromodomain-containing protein 9 (BRD9), was screened and found to decrease SREBP1 expression. What is more, BI-7273 has been confirmed that it could reduce lipid accumulation in HepG2 cells by BODIPY staining, and significantly decrease the protein expression of SREBP1 and FASN. To explore the potential mechanism BI-7273 reducing lipid accumulation, RNA sequencing (RNA-seq) was performed and demonstrated that BI-7273 reduced lipid accumulation by downregulating the AKT/mTOR/SREBP1 pathway in vitro. Finally, these results were verified in NAFLD and obesity mouse model induced by high fat diet (HFD). The results indicated that BI-7273 could decrease mouse body weight and improve insulin sensitivity, but also exhibited a strong negative correlation with serum lipid levels, and also demonstrated that BI-7273 reduced lipid accumulation via AKT/mTOR/SREBP1 pathway in vivo. In conclusion, our results revealed that BI-7273 decreases lipid accumulation by downregulating the AKT/mTOR/SREBP1 pathway in vivo and in vitro. This is the first report demonstrating the protective effect of this BRD9 inhibitor against NAFLD and obesity. BRD9 may be a novel target for the discovery of effective drugs to treat lipid metabolism disorders.

Arnicolide D Inhibits Proliferation and Induces Apoptosis of Osteosarcoma Cells through PI3K/Akt/mTOR Pathway.

BACKGROUND: Osteosarcoma is considered as the most prevalent form of primary malignant bone cancer, prompting a pressing need for novel therapeutic options. Arnicolide D, a sesquiterpene lactone derived from the traditional Chinese herbal medicine Centipeda minima (known as E Bu Shi Cao in Chinese), showed anticancer efficacy against several kinds of cancers. However, its effect on osteosarcoma remains unclear. OBJECTIVE: This study aimed to investigate the anticancer activity of arnicolide D and the underlying molecular mechanism of its action in osteosarcoma cells, MG63 and U2OS. METHODS: Cell viability and proliferation were evaluated through MTT assay and colony formation assay following 24 h and 48 h treatment with different concentrations of arnicolide D. Flow cytometry was employed to examine cell cycle progression and apoptosis after 24 h treatment of arnicolide D. Western blotting was performed to determine the expression of the PI3k, Akt and m-TOR and their phosphorylated forms. RESULTS: Our findings revealed that arnicolide D treatment resulted in a significant reduction in cell viability, the inhibition of proliferation, and the induction of apoptosis and cell cycle arrest in the G2/M phase. Furthermore, arnicolide D could inhibit the activation of PI3K/Akt/mTOR pathway in osteosarcoma cells. CONCLUSION: Based on our results, arnicolide D demonstrated significant anti-osteosarcoma activity and held the potential to be considered as a therapeutic candidate for osteosarcoma in the future.

G-CSF promotes the development of hepatocellular carcinoma by activating the PI3K/AKT/mTOR pathway in TAM.

OBJECTIVE: This investigation seeks to elucidate the role of the Granulocyte Colony-Stimulating Factor (G-CSF) in the progression of hepatocellular carcinoma (HCC), as well as the impact of the substance on related signaling pathways within the disease matrix. METHODS: Nude mouse tumor-bearing assay was used to detect tumor progression. Levels of Mannose/CD68 and CD34/Mannose within these samples and the concentrations of Mannose and inducible Nitric Oxide Synthase (iNOS) in macrophages were quantified using immunofluorescence techniques. The angiogenic capability was assessed via tube formation assays, and protein expressions of G-CSF, Vascular Endothelial Growth Factor (VEGF), Transforming Growth Factor-beta (TGF-ß), Matrix Metalloproteinases 2 and 9 (MMP2/9), SH2-containing protein tyrosine phosphatase-2 (SHP-2), phosphorylated PI3K/total PI3K (P-PI3K/t-PI3K), phosphorylated AKT/total AKT (P-AKT/t-AKT), and phosphorylated mTOR/total mTOR (P-mTOR/t-mTOR) were measured through Western Blot analysis in both tumor tissues and macrophages. RESULTS: Administration of G-CSF resulted in a marked augmentation of tumor volume. Macrophage Mannose expression was significantly elevated upon G-CSF treatment, while iNOS levels were conspicuously diminished. G-CSF substantially enhanced the secretion of VEGF, TGF-ß, and MMPs in tumor tissues. Macrophage parameters, following incubation in G-CSF pre-treated conditioned medium, indicated enhanced tube-forming capabilities relative to the control, an effect mitigated by the introduction of specific inhibitors. Furthermore, the G-CSF group exhibited a notable reduction in SHP-2 expression, alongside a substantial elevation in the phosphorylation levels of the PI3K/AKT/mTOR pathway proteins across all tumor-bearing paradigms. CONCLUSION: G-CSF ostensibly facilitates the advancement of hepatocellular carcinoma by activating the PI3K/AKT/mTOR signaling cascade within Tumor-Associated Macrophages (TAM).

Structural properties of glucan from Russula griseocarnosa and its immunomodulatory activities mediated via T cell differentiation.

The polysaccharide, RGP2, was isolated from Russula griseocarnosa and its immunostimulatory effects were confirmed in cyclophosphamide (CTX)-induced immunosuppressed mice. Following purification via chromatography, structural analysis revealed that RGP2 had a molecular weight of 11.82 kDa and consisted of glucose (Glc), galactose (Gal), mannose, glucuronic acid and glucosamine. Bond structure analysis and nuclear magnetic resonance characterization confirmed that the main chain of RGP2 was formed by →6)-ß-D-Glcp-(1→, →3)-ß-D-Glcp-(1→ and →6)-α-D-Galp-(1→, which was substituted at O-3 of →6)-ß-D-Glcp-(1→ by ß-D-Glcp-(1→. RGP2 was found to ameliorate pathological damage in the spleen and enhance immune cell activity in immunosuppressed mice. Based on combined multiomics analysis, RGP2 altered the abundance of immune-related microbiota (such as Lactobacillus, Faecalibacterium, and Bacteroides) in the gut and metabolites (uridine, leucine, and tryptophan) in the serum. Compared with immunosuppressed mice, RGP2 also restored the function of antigen-presenting cells, promoted the polarization of macrophages into the M1 phenotype, positively affected the differentiation of helper T cells, and inhibited regulatory T cell differentiation through the protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) pathway, ultimately exerting an immune boosting function. Overall, our findings highlight therapeutic strategies to alleviate CTX-induced immunosuppression in a clinical setting.

FAT10 induces immune suppression by upregulating PD-L1 expression in hepatocellular carcinoma.

The upregulation of programmed death ligand 1 (PD-L1) plays a crucial role in facilitating cancer cells to evade immune surveillance through immunosuppression. However, the precise regulatory mechanisms of PD-L1 in hepatocellular carcinoma (HCC) remain undefined. The correlation between PD-L1 and ubiquitin-like molecules (UBLs) was studied using sequencing data from 20 HCC patients in our center, combined with TCGA data. Specifically, the association between FAT10 and PD-L1 was further validated at both the protein and mRNA levels in HCC tissues from our center. Subsequently, the effect of FAT10 on tumor progression and immune suppression was examined through both in vivo and in vitro experiments. Utilizing sequencing data, qPCR, and Western blotting assays, we confirmed that FAT10 was highly expressed in HCC tissues and positively correlated with PD-L1 expression. Additionally, in vitro experiments demonstrated that the overexpression of FAT10 fostered the proliferation, migration, and invasion of HCC cells. Furthermore, the overexpression of FAT10 in HCC cells led to an increase in PD-L1 expression, resulting in the inhibition of T cell proliferation and the enhancement of HCC cell resistance to T cell-mediated cytotoxicity. Moreover, in vivo experiments utilizing the C57BL/6 mouse model revealed that overexpression of FAT10 effectively suppressed the infiltration of CD8 + GZMB + and CD8 + Ki67 + T cells, as well as reduced serum levels of TNF-α and IFN-γ. Mechanistically, we further identified that FAT10 upregulates PD-L1 expression via activating the PI3K/AKT/mTOR pathway, but not in a ubiquitin-like modification. In conclusion, our findings indicate that FAT10 promotes immune evasion of HCC via upregulating PD-L1 expression, suggesting its potential as a novel target to enhance the efficiency of immunotherapy in HCC.

Fibroblast growth factor 21 alleviates idiopathic pulmonary fibrosis by inhibiting PI3K-AKT-mTOR signaling and stimulating autophagy.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive pulmonary disease with an unclear pathogenesis and no available specific drug treatment. The principal etiological factors are lung inflammation caused by environmental factors, damage to alveolar epithelial cells, leading to epithelial-mesenchymal transition (EMT), and the abnormal proliferation of fibroblasts. Here, we have demonstrated that fibroblast growth factor 21 (FGF21) ameliorates IPF via the autophagy pathway. We administered FGF21 to bleomycin (BLM)-treated mice, which ameliorated their defects in lung function, reduced the accumulation of collagen, restored tissue structure, reduced the deposition of hydroxyproline, reduced the expression of collagen I and α-SMA and increased the expression of E-cadherin. The expression of LC3BII and the number of autophagosomes were significantly higher in the lungs. The expression of AKT and mTOR was significantly reduced by FGF21 treatment. We also determined the effects of FGF21 in A549 cells treated with TGF-ß, and found that FGF21 significantly inhibits activation of the AKT signaling pathway, thereby reducing TGF-ß-induced EMT and preventing the uncontrolled proliferation of fibroblasts. We conclude that FGF21 ameliorates IPF by inhibiting the PI3K-AKT-mTOR signaling pathway and activating autophagy, which provides a theoretical basis for FGF21 to be used for the treatment of IPF.

The combination of Curcumin and Doxorubicin on targeting PI3K/AKT/mTOR signaling pathway: an in vitro and molecular docking study for inhibiting the survival of MDA-MB-231.

The process of tumorigenesis is highly associated with the disruption of cell-cycle regulators and derangement of various signaling pathways, which end up with the inhibition of apoptosis and hyper-activation of survival pathways. The PI3K medicated AKT/mTOR pathway is the widely explained mechanism for cancer cell survival which causes the overexpression of MDM2 and downregulates the p53-BAX mediated apoptotic pathway. Curcumin (CUR), the phyto-compound, derived from Curcuma longa is currently being focused on for its anticancer activities against breast cancer cells, MDA-MB-231, not only because of its minimal cytotoxicity against healthy cells (HEK293) but also because it synergistically sensitizes the activity of Doxorubicin (DOXO) in lower doses, which can be a promising source for complementary drug development. This study aims to investigate the combinatorial effect of CUR and DOXO on PI3K/AKT/mTOR pathway proteins by sequential molecular docking analysis and MD simulation studies. The lower binding affinity of the sequentially docked protein-ligand complex proves the increasing binding affinity of CUR and DOXO in the combinatorial dose. The mRNA expressions of different genes of this pathway are observed and quantified using rt-qPCR, where the decreasing fold change (2-∆∆Ct) indicates the suppression of the AKT/mTOR pathway after co-treatment of CUR and DOXO against MDA-MB-231 cells. These in silico and in vitro findings can be a new horizon for further in vitro and clinical trials of breast cancer treatment. Supplementary Information: The online version contains supplementary material available at 10.1007/s40203-024-00231-2.