Polyphyllin I ameliorates gefitinib resistance and inhibits the VEGF/VEGFR2/p38 pathway by targeting HIF-1a in lung adenocarcinoma.

BACKGROUND: Lung adenocarcinoma (LUAD) is the most common pathological type of lung cancer. Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have been administered as the first-line therapy for patients with EGFR mutations in LUAD, but it is almost inevitable that resistance to EGFR-TKIs therapy eventually arises. Polyphyllin I (PPI), derived from Paris polyphylla rhizomes, has been shown to have potent anti-cancer properties in a range of human cancer types including LUAD. However, the role of PPI in gefitinib resistance and the underlying mechanism remain elusive. PURPOSE: To evaluate the antitumor impacts of PPI on gefitinib resistance cells and investigate its molecular mechanism. METHODS: CCK-8, wound healing, transwell assay, and xenograft model were performed to determine the anti-cancer effects of PPI as well as its ability to overcome gefitinib resistance. Immunoblotting, co-immunoprecipitation, phospho-RTK antibody array, qRT-PCR, and immunofluorescence were utilized to explore the mechanism by which PPI overrides gefitinib resistance. RESULTS: PPI inhibited cell survival, growth, and migration/invasion in both gefitinib-sensitive (PC9) and -resistant (PC9/GR) LUAD cells (IC50 at 2.0 µM). Significantly, treatment with PPI at 1.0 µM resensitized the resistant cells to gefitinib. Moreover, cell-derived xenograft experiments revealed that the combination of PPI and gefitinib overcame gefitinib resistance. The phospho-RTK array and immunoblotting analyses showed PPI significant inhibition of the VEGFR2/p38 pathway. In addition, molecular docking suggested the interaction between PPI and HIF-1α. Mechanistically, PPI reduced the protein expression of HIF-1α in both normoxia and hypoxia conditions by triggering HIF-1α degradation. Moreover, HIF-1α protein but not mRNA level was elevated in gefitinib-resistant LUAD. We further demonstrated that PPI considerably facilitated the binding of HIF-1α to VHL. CONCLUSIONS: We present a novel discovery demonstrating that PPI effectively counteracts gefitinib resistance in LUAD by modulating the VEGF/VEGFR2/p38 pathway. Mechanistic investigations unveil that PPI facilitates the formation of the HIF-1α /VHL complex, leading to the degradation of HIF-1α and subsequent inhibition of angiogenesis. These findings uncover a previously unidentified mechanism governing HIF-1α expression in reaction to PPI, providing a promising method for therapeutic interventions targeting EGFR-TKI resistance in LUAD.

Modified Biejia Jianwan decoction restrains PD-L1-mediated immune evasion through the HIF-1α/STAT3/NF-κB signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Modified Biejia Jianwan (M-BJJW), a Traditional Chinese Medicine (TCM) decoction, has exhibited great potential in treating hepatocellular carcinoma (HCC). However, its underlying functional mechanism still remains unknown. AIM OF THE STUDY: The study aimed to explore the anti-hepatocarcinogenic effects of M-BJJW, specifically its influence on PD-L1-mediated immune evasion in hypoxic conditions, and elucidate the related molecular mechanisms in HCC. MATERIALS AND METHODS: To investigate the therapeutic efficacy and mechanisms underlying M-BJJW's effects on HCC, we employed a diethylnitrosamine (DEN)-induced rat model maintained for 120 days. Following model establishment, flow cytometry was utilized to assess the distribution of immune cell populations in peripheral blood, spleens, and tumor tissues after M-BJJW administration. Simultaneously, enzyme-linked immunosorbent assays (ELISA) were conducted to analyze cytokine profiles in serum samples. Immunohistochemistry was employed to determine the expression levels of crucial proteins within tumor tissues. Furthermore, HCC cells exposed to CoCl2 underwent Western blot analysis to validate the expression levels of HIF-1α, PD-L1, STAT3, and nuclear factor kappa B (NF-κB) p65. The modulatory effects of STAT3 and NF-κB p65 were investigated using specific inhibitors and activators in wild-type cell lines. High-performance liquid chromatography coupled with mass spectrometry (HPLC/MS) was utilized to identify the chemical constituents present in M-BJJW-medicated serum. The immunomodulatory properties and the anti-tumor activities of M-BJJW were evaluated by co-culturing with peripheral blood mononuclear cells (PBMC) and the CCK-8 assay. Additionally, we assessed M-BJJW's impact on hypoxia-induced alterations in HCC cell lines using immunofluorescence and Western blot assessments. RESULTS: M-BJJW exhibited substantial therapeutic advantages by effectively alleviating pathological deterioration within the HCC microenvironment. In the DEN-induced rat model, M-BJJW administration notably reduced tumor growth. Flow cytometry analyses revealed an increased proportion of Cytotoxic T lymphocytes (CTLs) accompanied by a simultaneous decrease in regulatory T cells (Tregs). ELISA data supported a marked decrease in pro-inflammatory cytokines, including interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor α (TNF-α). Immunohistochemistry confirmed the suppressive effect of M-BJJW on the expression of HIF-1α and PD-L1. Notably, western blotting unveiled the role of HIF-1α in regulating PD-L1 expression via the STAT3 and NF-κB signaling pathways in HCC cell lines, which was validated using activators and inhibitors of STAT3 and NF-κB. The CCK-8 assay and co-culture techniques demonstrated the anti-tumor activity of M-BJJW. Immunofluorescence and western blotting further confirmed that M-BJJW-containing serum dose-dependently inhibited HIF-1α, PD-L1, p-STAT3, and p-p65 in hypoxic HCC cell lines. CONCLUSIONS: M-BJJW demonstrates significant therapeutic potential against HCC by influencing the hypoxic microenvironment, thereby regulating the immunosuppressive milieu. Specifically, M-BJJW modulates the HIF-1α/STAT3/NF-κB signaling pathway, leading to reduced PD-L1 expression and an elevated ratio of cytotoxic T lymphocytes (CTLs), while concurrently decreasing T regulatory cells (Tregs) and immunosuppressive factors. These synergistic effects aid in countering PD-L1-mediated immune evasion, presenting compelling pharmacological evidence supporting the clinical application of M-BJJW as a therapeutic approach for HCC.

Insights into the role of nucleotide methylation in metabolic-associated fatty liver disease.

Metabolic-associated fatty liver disease (MAFLD) is a chronic liver disease characterized by fatty infiltration of the liver. In recent years, the MAFLD incidence rate has risen and emerged as a serious public health concern. MAFLD typically progresses from the initial hepatocyte steatosis to steatohepatitis and then gradually advances to liver fibrosis, which may ultimately lead to cirrhosis and carcinogenesis. However, the potential evolutionary mechanisms still need to be clarified. Recent studies have shown that nucleotide methylation, which was directly associated with MAFLD's inflammatory grading, lipid synthesis, and oxidative stress, plays a crucial role in the occurrence and progression of MAFLD. In this review, we highlight the regulatory function and associated mechanisms of nucleotide methylation modification in the progress of MAFLD, with a particular emphasis on its regulatory role in the inflammation of MAFLD, including the regulation of inflammation-related immune and metabolic microenvironment. Additionally, we summarize the potential value of nucleotide methylation in the diagnosis and treatment of MAFLD, intending to provide references for the future investigation of MAFLD.