Rosiglitazone ameliorates skeletal muscle insulin resistance by decreasing free fatty acids release from adipocytes.

Skeletal muscle and white adipose tissue are important organs of glucose-lipid metabolism. However, excessive lipolysis and free fatty acids (FFA) release in adipocytes elevate plasma FFA, leading to insulin resistance in skeletal muscle. Here, we investigated effects of insulin-resistant adipocytes on skeletal muscle in vitro by simulating body environment using a transwell coculture method. Insulin-resistant 3T3-L1 adipocytes increased lipolysis and FFA release, which reduced insulin sensitivity in the cocultured C2C12 myotubes. Rosiglitazone (RSG) decreased excessive lipolysis by reducing expression of adipose triglyceride lipase (ATGL) and activity of hormone-sensitive lipase (HSL), which led to decrease of FFA release from insulin-resistant 3T3-L1 adipocytes. Meanwhile, insulin resistance in C2C12 myotubes cocultured with insulin-resistant 3T3-L1 adipocytes was ameliorated after RSG treatment. Taken together, our present study provided direct evidence to better understand insulin resistance between skeletal muscle and adipose tissue in type 2 diabetes.

miR-125b-5p inhibits cell proliferation, migration, and invasion in hepatocellular carcinoma via targeting TXNRD1.

BACKGROUND: Thioredoxin reductase 1 (TXNRD1) is an antioxidant enzyme reportedly overexpressed in hepatocellular carcinoma (HCC); however, the detailed function and mechanisms of TXNRD1 in HCC remain obscure. In this study, we investigated the miR-125b-5p-specific regulation of TXNRD1 levels and its effect on HCC cells. METHODS: We detected miR-125b-5p levels in human HCC tissue samples through quantitative reverse transcription polymerase chain reaction (qRT-PCR), and in vitro experiments were employed to investigate the effect of miR-125b-5p on HCC cell proliferation, migration, and invasion. Additionally, we examined miR-125b-5p-mediated changes in TXNRD1 levels by qRT-PCR and western blotting, and a dual luciferase-reporter assay was conducted to confirm direct targeting of the 3' untranslated region of TXNRD1 mRNA by miR-125b-5p. RESULTS: miR-125b-5p expression was reduced in HCC tissues relative to that in matched para-carcinoma tissues; this finding was verified in HCC cohorts from the Gene Expression Omnibus and The Cancer Genome Atlas. Additionally, low miR-125b-5p expression was associated with poor prognosis in HCC patients, and gene-set enrichment analysis indicated that miR-125b-5p levels were associated with HCC proliferation and metastasis. As predicted, overexpressing miR-125b-5p restrained the proliferation, migration, and invasion of Huh7 and SK-Hep-1 cells and forced expression of the miR-125b-5p-downregulated TXNRD1 mRNA and protein levels in HCC cells. Moreover, dual luciferase-reporter assays revealed that miR-125b-5p targets TXNRD1 to directly regulate its expression, whereas TXNRD1 overexpression abolishes the inhibitory effect of miR-125b-5p on HCC cell proliferation, migration, and invasion. CONCLUSIONS: These results demonstrated miR-125b-5p as a tumor suppressor in HCC through its inhibition of TXNRD1, thereby suggesting it as a potential target for the clinical treatment of HCC.

Dihydroartemisinin induces apoptosis preferentially via a Bim-mediated intrinsic pathway in hepatocarcinoma cells.

This report is designed to dissect the detail molecular mechanism by which dihydroartemisinin (DHA), a derivative of artemisinin, induces apoptosis in human hepatocellular carcinoma (HCC) cells. DHA induced a loss of the mitochondrial transmemberane potential (ΔΨm), release of cytochrome c, activation of caspases, and externalization of phosphatidylserine indicative of apoptosis induction. Compared with the modest inhibitory effects of silencing Bax, silencing Bak largely prevented DHA-induced ΔΨm collapse and apoptosis though DHA induced a commensurable activation of Bax and Bak, demonstrating a key role of the Bak-mediated intrinsic apoptosis pathway. DHA did not induce Bid cleavage and translocation from cytoplasm to mitochondria and had little effects on the expressions of Puma and Noxa, but did increase Bim and Bak expressions and decrease Mcl-1 expression. Furthermore, the cytotoxicity of DHA was remarkably reduced by silencing Bim, and modestly but significantly reduced by silencing Puma or Noxa. Silencing Bim or Noxa preferentially reduced DHA-induced Bak activation, while silencing Puma preferentially reduced DHA-induced Bax activation, demonstrating that Bim and to a lesser extent Noxa act as upstream mediators to trigger the Bak-mediated intrinsic apoptosis pathway. In addition, silencing Mcl-1 enhanced DHA-induced Bak activation and apoptosis. Taken together, our data demonstrate a crucial role of Bim in preferentially regulating the Bak/Mcl-1 rheostat to mediate DHA-induced apoptosis in HCC cells.

Tumor cuproptosis and immune infiltration improve survival of patients with hepatocellular carcinoma with a high expression of ferredoxin 1.

Background: Cuproptosis is a novel cell death pathway dependent on cellular copper ions and ferredoxin 1 (FDX1). Hepatocellular carcinoma (HCC) is derived from healthy liver as a central organ for copper metabolism. It remains no conclusive evidence whether cuproptosis is involved in survival improvement of patients with HCC. Method: A 365-liver hepatocellular carcinoma (LIHC) cohort with RNA sequencing data and paired clinical and survival information was obtained from the The Cancer Genome Atlas (TCGA) dataset. A retrospective cohort of 57 patients with HCC with stages I/II/III was collected by Zhuhai People's Hospital from August 2016 to January 2022. Low- or high-FDX1 groups were divided according to the median value of FDX1 expression. Cibersort, single-sample gene set enrichment analysis, and multiplex immunohistochemistry analyzed immune infiltration in LIHC and HCC cohorts. Cell proliferation and migration of HCC tissues and hepatic cancer cell lines were evaluated using the Cell Counting Kit-8. Quantitative real-time PCR and RNA interference measured and downregulated FDX1 expression. Statistical analysis was conducted by R and GraphPad Prism software. Results: High FDX1 expression significantly enhanced survival of patients with LIHC from the TCGA dataset, which was also demonstrated through a retrospective cohort with 57 HCC cases. Immune infiltration was different between the low- and high-FDX1 expression groups. Natural killer cells, macrophages, and B cells were significantly enhanced, and PD-1 expression was low in the high-FDX1 tumor tissues. Meanwhile, we found that a high expression of FDX1 decreased cell viability in HCC samples. HepG2 cells with FDX1 expression are sensitive to Cu2+, and interference of FDX1 promoted proliferation and migration of tumor cells. The consistent results were also demonstrated in Hep3B cells. Conclusion: This study reveals that cuproptosis and tumor immune microenvironment were together involved in improvement of survival in patients with HCC with a high expression of FDX1.

Cullin3 deficiency shapes tumor microenvironment and promotes cholangiocarcinoma in liver-specific Smad4/Pten mutant mice.

Cholangiocarcinoma (CC), the most lethal type of liver cancer, remains very difficult to treat due to an incomplete understanding of the cancer initiation and progression mechanisms and no effective therapeutic drugs. Thus, identification of genomic drivers and delineation of the underlying mechanisms are urgently needed. Here, we conducted a genome-wide CRISPR-Cas9 screening in liver-specific Smad4/Pten knockout mice (Smad4co/co;Ptenco/co;Alb-Cre, abbreviated as SPC), and identified 15 putative tumor suppressor genes, including Cullin3 (Cul3), whose deficiency increases protein levels of Nrf2 and Cyclin D1 that accelerate cholangiocytes expansion leading to the initiation of CC. Meanwhile, Cul3 deficiency also increases the secretion of Cxcl9 in stromal cells to attract T cells infiltration, and increases the production of Amphiregulin (Areg) mediated by Nrf2, which paracrinely induces inflammation in the liver, and promotes accumulation of exhausted PD1high CD8 T cells at the expenses of their cytotoxic activity, allowing CC progression. We demonstrate that the anti-PD1/PD-L1 blockade inhibits CC growth, and the effect is enhanced by combining with sorafenib selected from organoid mediated drug sensitive test. This model makes it possible to further identify more liver cancer suppressors, study molecular mechanisms, and develop effective therapeutic strategies.

Patient-Derived Organoids Can Guide Personalized-Therapies for Patients with Advanced Breast Cancer.

Most breast cancers at an advanced stage exhibit an aggressive nature, and there is a lack of effective anticancer options. Herein, the development of patient-derived organoids (PDOs) is described as a real-time platform to explore the feasibility of tailored treatment for refractory breast cancers. PDOs are successfully generated from breast cancer tissues, including heavily treated specimens. The microtubule-targeting drug-sensitive response signatures of PDOs predict improved distant relapse-free survival for invasive breast cancers treated with adjuvant chemotherapy. It is further demonstrated that PDO pharmaco-phenotyping reflects the previous treatment responses of the corresponding patients. Finally, as clinical case studies, all patients who receive at least one drug predicate to be sensitive by PDOs achieve good responses. Altogether, the PDO model is developed as an effective platform for evaluating patient-specific drug sensitivity in vitro, which can guide personal treatment decisions for breast cancer patients at terminal stage.

Anti-Cancer Nanomedicines: A Revolution of Tumor Immunotherapy.

Immunotherapies have been accelerating the development of anti-cancer clinical treatment, but its low objective responses and severe off-target immune-related adverse events (irAEs) limit the range of application. Strategies to remove these obstacles primarily focus on the combination of different therapies and the exploitation of new immunotherapeutic agents. Nanomedicine potentiates the effects of activating immune cells selectively and reversing tumor induced immune deficiency microenvironment through multiple mechanisms. In the last decade, a variety of nano-enabled tumor immunotherapies was under clinical investigation. As time goes by, the advantages of nanomedicine are increasingly prominent. With the continuous development of nanotechnology, nanomedicine will offer more distinctive perspectives in imaging diagnosis and treatment of tumors. In this Review, we wish to provide an overview of tumor immunotherapy and the mechanisms of nanomaterials that aim to enhance the efficacy of tumor immunotherapy under development or in clinic treatment.

Resveratrol bidirectionally regulates insulin effects in skeletal muscle through alternation of intracellular redox homeostasis.

AIMS: Reactive oxygen species (ROS) bidirectionally regulate insulin sensitivity in skeletal muscle. Insulin-induced ROS generation elevates insulin-regulated metabolic effects; however, chronic oxidative stress causes severe insulin resistance in skeletal muscle. Resveratrol (RV), as a natural antioxidant, eliminates intracellular ROS. It's unclear that whether it has different roles in insulin signaling pathway in skeletal muscle. MAIN METHODS: C57BL/6J mice and C2C12 myotubes were used to assess metabolic regulation effects of RV. Protein activation was detected using Immunofluorescence and Western Blot analysis. ROS were analyzed using confocal microscope and flow cytometry sorting (FACS). Intracellular reducing molecules were detected using an enzymatic method. Glucose uptake was measured using a fluorescent deoxyglucose analog (2-NBDG). KEY FINDINGS: We found that RV attenuated insulin-stimulated AKT phosphorylation via elimination of insulin-induced ROS generation in skeletal muscle, suggesting that RV decreased activation of the insulin-induced AKT signaling. In skeletal muscle of insulin resistance, RV reduced oxidative stress, restored intracellular glutathione (GSH) level, and enhanced insulin-induced AKT activation and glucose absorption. These results suggested that RV ameliorated insulin resistance by change of redox levels in skeletal muscle. SIGNIFICANCE: This study revealed bidirectional regulation effects of RV on insulin-stimulated metabolism in skeletal muscle through alternation of intracellular redox homeostasis, which might provide a guidance role for treatment of metabolic diseases.

Identification of hub genes in hepatocellular carcinoma using integrated bioinformatic analysis.

The molecular mechanisms underlying hepatocellular carcinoma (HCC) progression remain largely undefined. Here, we identified 176 commonly upregulated genes in HCC tissues based on three Gene Expression Omnibus datasets and The Cancer Genome Atlas (TCGA) cohort. We integrated survival and methylation analyses to further obtain 12 upregulated genes for validation. These genes were overexpressed in HCC tissues at the transcription and protein levels, and increased mRNA levels were related to higher tumor grades and cancer stages. The expression of all markers was negatively associated with overall and disease-free survival in HCC patients. Most of these hub genes can promote HCC proliferation and/or metastasis. These 12 hub genes were also overexpressed and had strong prognostic value in many other cancer types. Methylation and gene copy number analyses indicated that the upregulation of these hub genes was probably due to hypomethylation or increased gene copy numbers. Further, the methylation levels of three genes, KPNA2, MCM3, and LRRC1, were associated with HCC clinical features. Moreover, the levels of most hub genes were related to immune cell infiltration in HCC microenvironments. Finally, we identified three upregulated genes (KPNA2, TARBP1, and RNASEH2A) that could comprehensively and accurately provide diagnostic and prognostic value for HCC patients.

Modulating the tumor immune microenvironment with sunitinib malate supports the rationale for combined treatment with immunotherapy.

Small molecule inhibitors have proven useful in the treatment of a variety of tumors, but they are often limited by unsustainable benefits and confer resistance quickly. Immunotherapy can result in durable clinical responses, but activity only occurs in a minority of patients. The unfavorable tumor microenvironment (TME) is an important factor limiting immunotherapy. An appropriate understanding of how small molecule inhibitors modulate the TME may optimize the combination of targeted treatment and immunotherapy in managing tumors. In this study, we found that transient treatment with sunitinib malate inhibited the disorganized extension of tumor vessels, pericytes and collagen IV but increased the relative ratio of pericyte-wrapping blood vessels with alleviated hypoxia in tumors, which resulted from tumor vascular normalization. Sunitinib malate increased infiltration of CD8+ T cells and decreased regulatory T cells (Tregs), accompanied by inhibited expression of TGF-ß1 and IL-10 and increased CCL-28, IFN-γ and IL-12, but no significant inhibition of myeloid-derived suppressor cells (MDSCs) was observed. In addition, sunitinib malate increased the levels of PD-1 and PD-L1 in TME, combined with anti-PD-1 therapy showed a significant reduction in tumor burden compared with either monotherapy, suggesting that anti-PD-1 therapy is reasonable after sunitinib malate treatment.

Exosome-transmitted circular RNA hsa_circ_0051443 suppresses hepatocellular carcinoma progression.

Extracellular communication in the tumor microenvironment is critical. Results of qRT-PCR show that circ-0051443 is significantly lower in the plasma exosomes and tissues from patients with hepatocellular carcinoma (HCC) than healthy controls. Compared with the producer cells, circ-0051443 is mainly packaged into exosomes. A receiver operating characteristic curve (ROC) shows that the patients with HCC can be distinguished from the controls by exosomal circ-0051443. The role of exosomal circ-0051443 in HCC was determined by animal and cell analyses. Circ-0051443 is transmitted from normal cells to HCC cells via exosomes and suppresses the malignant biological behaviors by promoting cell apoptosis and arresting the cell cycle. Exosomal circ-0051443 decreases the weight and volume of the xenograft tumors in nude mice via BAK1 upregulation in these tumors. BAK1 expression is mediated by exosomal circ-0051443 through competitive bound to miR-331-3p. Therefore, exosomal circ-0051443 can serve as a predictor and potential therapeutic target for HCC.

Monitoring of tumor vascular normalization: the key points from basic research to clinical application.

Tumor vascular normalization alleviates hypoxia in the tumor microenvironment, reduces the degree of malignancy, and increases the efficacy of traditional therapy. However, the time window for vascular normalization is narrow; therefore, how to determine the initial and final points of the time window accurately is a key factor in combination therapy. At present, the gold standard for detecting the normalization of tumor blood vessels is histological staining, including tumor perfusion, microvessel density (MVD), vascular morphology, and permeability. However, this detection method is almost unrepeatable in the same individual and does not dynamically monitor the trend of the time window; therefore, finding a relatively simple and specific monitoring index has important clinical significance. Imaging has long been used to assess changes in tumor blood vessels and tumor changes caused by the oxygen environment in clinical practice; some preclinical and clinical research studies demonstrate the feasibility to assess vascular changes, and some new methods were in preclinical research. In this review, we update the most recent insights of evaluating tumor vascular normalization.

Bidirectional modulation of insulin action by reactive oxygen species in 3T3­L1 adipocytes.

Reactive oxygen species (ROS) serve an important role in glucose­lipid metabolic regulation. In the present study, the results demonstrated that there was bidirectional regulation of insulin action in 3T3­L1 adipocytes treated with ROS. Transient and acute ROS exposure improved insulin­induced metabolic effects in 3T3­L1 adipocytes. Hydrogen peroxide (H2O2), as a stable and diffusible ROS, diffused into adipocytes and altered intracellular redox homeostasis, resulting in oxidation and inactivation of phosphatase and tensin homologue deleted on chromosome 10 (PTEN). Inactivation of PTEN enhanced the activation of insulin­induced protein kinase B (AKT), leading to increased glucose transporter 4 (GLUT4) redistribution and glucose uptake in 3T3­L1 adipocytes. However, chronic ROS treatment induced insulin resistance in 3T3­L1 adipocytes. It was also revealed that insulin­induced AKT activation, GLUT4 translocation to cell membrane and glucose uptake were significantly inhibited in chronic ROS­treated 3T3­L1 adipocytes. Taken together, the present study provided further demonstration that transient ROS treatment improved insulin sensitivity; however, chronic ROS exposure induced insulin resistance in 3T3­L1 adipocytes.

Peroxynitrite dominates sodium nitroprusside-induced apoptosis in human hepatocellular carcinoma cells.

This study aims to explore which radicals dominate sodium nitroprusside (SNP)-induced cytotoxicity in human hepatocellular carcinoma (HCC) cells (HepG2 and Hep3B). Exposure of SNP to cell medium produced abundant nitric oxide (NO), superoxide anion (O2•-), hydrogen peroxide (H2O2) and iron ions. SNP potently induced caspases activation, mitochondrial membrane permeabilization and apoptosis in HCC cells. In Hep3B cells, pretreatment with NO scavenger (PTIO) did not prevent SNP-induced cytotoxicity. However, in HepG2 cells, SNP-induced cytotoxicity was prevented significantly by pretreatment with PTIO and O2•- scavenger, and especially was almost completely blocked by pretreatment with FeTPPS (peroxynitrite scavenger). In contrast, although H2O2 scavenger potently scavenged SNP-induced H2O2 production, it did not prevent SNP-induced cytotoxicity in HepG2 cells. In addition, pretreatment with DFO (iron ions chelator) and iron-saturated DFO respectively completely prevented SNP-induced cytotoxicity in HepG2 cells. Collectively, peroxynitrite from the reaction between NO and O2•- elicited from SNP dominates the SNP-induced apoptosis of HepG2 cells, in which both iron ions and H2O2 are not involved.

Gold Nanoparticles of Diameter 13 nm Induce Apoptosis in Rabbit Articular Chondrocytes.

Gold nanoparticles (AuNPs) have been widely used in biomedical science including antiarthritic agents, drug loading, and photothermal therapy. In this report, we studied the effects of AuNPs with diameters of 3, 13, and 45 nm, respectively, on rabbit articular chondrocytes. AuNPs were capped with citrate and their diameter and zeta potential were measured by dynamic light scattering (DLS). Cell viability was evaluated by Cell Counting Kit-8 (CCK-8) assay after the rabbit articular chondrocytes were pre-incubated with 3, 13, and 45 nm AuNPs, respectively, for 24 h. Flow cytometry (FCM) analysis with annexin V/propidium iodide (PI) double staining and fluorescence imaging with Hoechst 33258 staining were used to determine the fashion of AuNPs-induced chondrocyte death. Further, 13 nm AuNPs (2 nM) significantly induced chondrocyte death accompanying apoptotic characteristics including mitochondrial damage, externalization of phosphatidylserine and nuclear concentration. However, 3 nm AuNPs (2 nM) and 45 nm (0.02 nM) AuNPs did not induce cytotoxicity in chondrocytes. Although 13 nm AuNPs (2 nM) increased the intracellular reactive oxygen species (ROS) level, pretreatment with Nacetyl cysteine (NAC), a ROS scavenger, did not prevent the cytotoxicity induced by 13 nm AuNPs, indicating that 13 nm AuNPs (2 nM) induced ROS-independent apoptosis in chondrocytes. These results demonstrate the size-dependent cytotoxicity of AuNPs in chondrocytes, which must be seriously considered when using AuNPs for treatment of osteoarthritis (OA).

Dominant roles of Fenton reaction in sodium nitroprusside-induced chondrocyte apoptosis.

Sodium nitroprusside (SNP) has been widely used as an exogenous nitric oxide (NO) donor to explore the molecular mechanism of NO-mediated chondrocyte apoptosis during the latest two decades. We have recently found that NO-independent ROS play a key role in SNP-induced apoptosis in rabbit chondrocytes. This study aims to investigate what kind of ROS and how the reliable ROS mediators mediate the SNP-induced apoptosis. Data shows that SNP and NO-exhausted SNP (SNPex) induced ROS production or cytotoxicity to identically degree. SNP induced a marked increase in iron ions, superoxide anion (O2(•-)), hydrogen peroxide (H2O2) and hydroxyl radical ((•)OH) level. H2O2 scavenger (CAT) and (•)OH scavenger (DMSO) significantly inhibited SNP-induced chondrocyte apoptosis. Iron ions chelator (DFO) entirely prevented SNP-induced chondrocyte apoptosis. In contrast, O2(•-) scavenger (SOD) and glutathione depletion agent (BSO) promoted SNP-induced cytotoxicity. K3[Fe(CN)6] exhibited no cytotoxicity, and H2O2 alone up to 250µM or iron ions alone up to 90µM is non-cytotoxic to chondrocytes. Combination of 25µM FeSO4 and 100µM H2O2 in the presence of BSO induced chondrocyte death similar to SNP treatment. Fetal bovine serum (FBS) enhanced iron ions release from SNP and the cytotoxicity of SNP. Our data shows that the extracellular Fenton reaction between iron ions released from SNP and H2O2 induced by SNP plays a key role in SNP-induced chondrocyte apoptosis. Overall, our results indicate that the potential of SNP to increase iron ions and ROS should be especially considered for some biological functions and, possibly, also for clinical applications of this drug.