Pioglitazone, a peroxisome proliferator­activated receptor γ agonist, induces cell death and inhibits the proliferation of hypoxic HepG2 cells by promoting excessive production of reactive oxygen species.

Hypoxia is a hallmark of solid tumors. Hypoxic cancer cells adjust their metabolic characteristics to regulate the production of cellular reactive oxygen species (ROS) and facilitate ROS-mediated metastasis. Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor that regulates the transcription of fatty acid metabolism-related genes that have a key role in the survival and proliferation function of hypoxic cancer cells. In the present study, mRNA expression in HepG2 cells under chemically induced hypoxia was assessed. The protein expression levels of hypoxia-inducible factor 1α (HIF-1α) were measured using western blotting. Following treatment with the PPARγ agonist pioglitazone, cell viability was assessed using a Cell Counting Kit-8 assay, whilst cell proliferation and death were determined using 5-ethynyl-2'-deoxyuridine incorporation staining, and calcein-acetoxymethyl ester and propidium iodide staining, respectively. Cellular ROS production was assessed using dihydroethidium staining. Cobalt chloride was used to induce hypoxia in HepG2 cells, which was evaluated using HIF-1α expression. The results revealed that the mRNA expression of PPARγ, CD36, acetyl-co-enzyme A dehydrogenase (ACAD) medium chain (ACADM) and ACAD short-chain (ACADS) was downregulated in hypoxic HepG2 cells. The PPARγ agonist pioglitazone decreased the cell viability of hypoxic HepG2 cells by inhibiting cell proliferation and inducing cell death. Following treatment with the PPARγ agonist pioglitazone, hypoxic HepG2 cells produced excessive ROS. ROS-mediated cell death induced by the PPARγ agonist pioglitazone was rescued with the antioxidant N-acetyl-L-cysteine. The downregulated mRNA expression of PPARγ, CD36, ACADM and ACADS was not reverted by a PPARγ agonist in hypoxic HepG2 cells. By contrast, the PPARγ agonist suppressed the mRNA expression of BCL2, which was upregulated in hypoxic HepG2 cells. In summary, the PPARγ agonist stimulated excessive ROS production to inhibit cell proliferation and increase the death of hypoxic HepG2 cells by decreasing BCL2 mRNA expression, suggesting a negative association between PPARγ and BCL2 in the regulation of ROS production in hypoxic HepG2 cells.

The C-type lectin COLEC10 is predominantly produced by hepatic stellate cells and involved in the pathogenesis of liver fibrosis.

Hepatic stellate cell is one of the major nonparenchymal cell types in liver. It has been proved the hepatic stellate cells are activated upon liver injury and produce excessive extracellular matrix to induce liver fibrosis. Single-cell RNA sequencing has been introduced to identify the subpopulations and function of hepatic stellate cells for its remarkable resolution of representation of single-cell transcriptome. According to the re-analysis of single-cell RNA sequencing data and pseudotime trajectory inference, we have found the C-type lectins including Colec10 and Colec11 are not produced by hepatocytes but predominantly produced by hepatic stellate cells, especially quiescent ones in the mice livers. In addition, the expression of Colec10 is decreased in the fibrotic livers of CCl4-challenged mice. COLEC10 is also mainly expressed in the hepatic stellate cells of human livers and the expression of COLEC10 is decreased with the progression of liver fibrosis. The bulk RNA sequencing data of the lentivirus transfected LX-2 cells indicates the function of COLEC10 is associated with inflammation, angiogenesis and extracellular matrix alteration. Surprisingly, the in vitro overexpression of COLEC10 in LX-2 cells promotes the mRNA expression of extracellular matrix components including COL1A1, COL1A2 and COL3A1 and the extracellular matrix degradation enzyme MMP2. To further investigate the role of COLEC10 in the pathogenesis of liver fibrosis, the serum concentration of COLEC10 in patients with chronic liver disease and healthy donors is measured. The serum concentration of COLEC10 is elevated in the patients with chronic liver disease compared to the healthy donors and positively correlated with serum concentration of the D-dimer but not the most of liver function markers. Altogether, we conclude that the C-type lectin COLEC10 is predominantly produced by the hepatic stellate cells and involved in the pathogenesis of liver fibrosis.

The impact of drug-eluting bead (vs. conventional) transarterial chemoembolization on hepatic fibrosis in treating intermediate or advanced hepatocellular carcinoma.

OBJECTIVE: Limited studies have reported the impact of drug-eluting bead transarterial chemoembolization (DEB-TACE) on hepatic fibrosis in hepatocellular carcinoma (HCC). This study evaluated multiple hepatic fibrosis indicators, aiming to comprehensively compare the influence of DEB-TACE and conventional transarterial chemoembolization (cTACE) on hepatic fibrosis in treating HCC patients. METHODS: Intermediate/advanced HCC patients (N = 121) were divided into the DEB-TACE group (n = 62) and the cTACE group (n = 59) based on their chosen treatment. Serum hyaluronic acid (HA), pro-collagen type-III (PC-III), collagen type-IV (IV-C), and laminin (LN) were detected; aminotransferase to platelet ratio index (APRI) and fibrosis index based on the four factors (FIB-4) were calculated; liver stiffness measurement (LSM) was assessed by real-time shear wave elastography. RESULTS: HA, PC-III, IV-C, and LN at 1 month after the second TACE and at 12 months after the first TACE were all decreased in DEB-TACE group compared with cTACE group (all P < .050). Then, APRI, FIB-4, and LSM were further assessed, which also showed a decreasing trend at aforementioned timepoints in DEB-TACE group compared with cTACE group (all P < .050). Additionally, the multivariate logistic regression analysis revealed that DEB-TACE (vs. cTACE) was independently associated with reduced occurrence of severe hepatic fibrosis at 12 months (OR = 0.215, 95%CI: 0.058-0.802, P = .022). Concerning the liver function indexes, alanine aminotransferase, aspartate aminotransferase, and total bilirubin after treatment were not different between the two groups (all P > .050). CONCLUSION: DEB-TACE displays attenuated hepatic fibrosis progression and noninferior tolerance compared to cTACE in treating intermediate- or advanced-stage HCC patients.

The addition of camrelizumab is effective and safe among unresectable hepatocellular carcinoma patients who progress after drug-eluting bead transarterial chemoembolization plus apatinib therapy.

OBJECTIVE: Camrelizumab synergizes with apatinib or transarterial chemoembolization via tumor immunity and chemosensitivity. This study aimed to investigate the efficacy and safety of camrelizumab plus apatinib with or without drug-eluting bead transarterial chemoembolization (DEB-TACE) in unresectable hepatocellular carcinoma (HCC) patients after progression to DEB-TACE plus apatinib. METHODS: Eighty-nine unresectable HCC patients accepted previous DEB-TACE plus apatinib therapy, then further received second-line camrelizumab plus apatinib with or without DEB-TACE treatment. Treatment responses were calculated at 3 months (M3) and 6 months (M6). Survival and treatment-related adverse events were documented. RESULTS: Objective response rate and disease control rate were 39.3% and 80.9% at M3; meanwhile, they were 22.4% and 54.1% at M6. Furthermore, the median progression-free survival (PFS) (95% confidence interval (CI)) was 7.0 (6.2-7.8) months with a 1-year PFS rate of 18.4%; the median overall survival (OS) (95% CI) was 17.0 (15.3-18.7) months with a 1-year OS rate of 73.9%. Multivariable Cox's proportional hazards regression analysis presented that 3-4 times (vs. 0 time) of DEB-TACE, apatinib dose duration> 4 months, and camrelizumab dose duration> 5 months independently predicted longer PFS (all P<0.05); meanwhile, declined ECOG PS score, new lesions as progression pattern, 1-2 and 3-4 times (vs. 0 time) of DEB-TACE, apatinib dose duration> 4 months independently predicted prolonged OS (all P<0.05). Moreover, treatment-related adverse events mainly included grade 1-2 fever, gastrointestinal reaction, fatigue, hand-foot skin reaction, and hypertension. CONCLUSION: After progression to DEB-TACE plus apatinib treatment, the addition of camrelizumab is effective and safe among unresectable HCC patients.

Nrf2-siRNA Enhanced the Anti-Tumor Effects of As2O3 in 5-Fluorouracil-Resistant Hepatocellular Carcinoma by Inhibiting HIF-1α/HSP70 Signaling.

Purpose: Chemoresistance is a major factor contributing to the failure of cancer treatment. The conventional chemotherapy agent 5-fluorouracil (5-FU) has been used for cancer treatment for decades. However, its use is limited in the treatment of hepatocellular carcinoma (HCC) due to acquired resistance. Nrf2 (NF-E2-related factor 2) is known to be associated with drug resistance across a wide range of cancer types. Also, since arsenic trioxide (As2O3) showed antitumor effects on HCC, the purpose of this study was to determine whether As2O3 and Nrf2-siRNA could inhibit HCC synergistically. Methods: We generated two separate 5-FU-resistant HCC cell lines (SNU-387/5-FU and Hep3B/5-FU). Western blotting was used to determine protein levels. An efficient lentiviral delivery system was used to establish stable knockdown or overexpression of Nrf2 and HIF-1α. In vitro and in vivo analyses of the effects of Nrf2 gene knockdown and As2O3 on 5-FU-resistant HCC cells were conducted. Results: The expression of Nrf2 was higher in the 5-FU-resistant HCC cell lines than in the parental cell lines. When coupled with Nrf2 knockdown, As2O3 treatment significantly decreased 5-FU-resistant SNU-387 and Hep3B cell viability, migration, and invasion, inactivated HIF-1α/HSP70 signaling, inhibited anti-apoptotic B-cell lymphoma (Bcl-2) activity, and increased the expression of pro-apoptotic Bcl-2-associated X protein (BAX) along with caspase-3. The synergistic effect was also confirmed using a 5-FU-resistant Hep3B mouse xenograft model in vivo. Conclusion: Nrf2 knockdown could improve the effect of As2O3 on reversing drug resistance in 5-FU-resistant HCC cells.

Covalent organic polymer induces apoptosis of liver cancer cells via photodynamic and photothermal effects.

The purpose of this study was to explore the photodynamic and photothermal effects of the supramolecular material Purp@COP and to test the anti-cancer effect on HepG2 cells in vitro. Materials and methods: Purp@COP is a covalent organic polymer (COP) with robust tailoring heteroatom incorporation, plentiful pore structure, and multiple functions similar to the metal-organic framework (MOF). Hepatocellular carcinoma cell line HepG2 was cultured with Purp@COP for 24 h and treated with near-infrared 808-nm laser 1 W/cm2 for 10 min. Cell Counting Kit-8 (CCK-8) assay, colony formation assay, live-dead cell fluorescence staining, and Annexin V/propidium iodide (PI) staining flow cytometry were performed to detect the viability, proliferation, and apoptosis of the HepG2 cells. Results: The supramolecular material Purp@COP exhibited significant photothermal performance under near-infrared 808-nm laser irradiation in vitro. With the treatment of Purp@COP and near-infrared 808-nm laser irradiation on HepG2 cells, cell viability and colony formation capacity were decreased, and the number and proportion of apoptotic cells were increased. Conclusions: The supramolecular material Purp@COP has both photothermal and photodynamic effects and can significantly induce cancer cell death and inhibit the proliferation of cancer cells in vitro.

Removal of p-arsanilic acid and phenylarsonic acid from water by Fenton coagulation process: influence of substituted amino group.

Phenylarsonic acid compounds, which were widely used in poultry and swine production, are often introduced to agricultural soils with animal wastes. Fenton coagulation process is thought as an efficient method to remove them. However, the substituted amino group could apparently influence the removal efficiency in Fenton coagulation process. Herein, we investigated the optimal conditions to treat typical organoarsenic contaminants (p-arsanilic acid (p-ASA) and phenylarsonic acid (PAA)) in aqueous solution based on Fenton coagulation process for oxidizing them and capturing the released inorganic arsenic, and elucidated the influence mechanism of substituted amino group on removal. Results showed that the pH value and the dosage of H2O2 and Fe2+ significantly influenced the performance of the oxidation and coagulation processes. The optimal conditions for removing 20 mg L-1-As in this research were 40mg L-1 Fe2+ and 60mg L-1 H2O2 (the mass ratio of Fe2+/H2O2 = 1.5), initial solution pH of 3.0, and final solution pH of 5.0 adjusting after 30-min Fenton oxidation reaction. Meanwhile, the substituted amino group made p-ASA much more easily be attacked by ·OH than PAA and supply one more binding sites for forming complexes with Fe3+ hydrolysates, resulting in 36% higher oxidation rate and 7% better coagulation performance at the optimal conditions.

[Photocatalytic Oxidation of p-arsanilic Acid by TiO2].

The p-arsanilic acid (ASA) is an important organoarsenical compound and its removal is more difficult compared to inorganic arsenic, however, little attention has been paid to the removal of ASA in aqueous environment. The influence of P25 on the adsorption of ASA, effect of P25 dosage, pH and illumination intensity on the photo-catalysis, the production analysis and main mechanism of photo-degradation were investigated in this study. The results showed that in the P25 catalysis process, simulated natural light could degrade ASA into As (V) by oxidation. The total As was reduced to about 0.34 mg x L(-1) within 0.5 h under the following condition: the initial concentration of ASA was 2 mg x L(-1) and the dosage of TiO2 was 1 g x L(-1). The result showed that the removal rate of ASA in acidic conditions was much higher than that in alkaline conditions. The optimal strength of light was 68.5 mW x Cm(-2). Hydroxide radical played a major role in photocatalytic oxidation of ASA by P25.