Long-term di-(2-ethylhexyl) phthalate exposure reduces sorafenib treatment efficacy by enhancing mesenchymal transition in hepatocellular carcinoma.

Di(2-ethylhexyl) phthalate (DEHP) is a worldwide common plasticizer. Nevertheless, DEHP is easily leached out to the environment due to the lack of covalent bonds with plastic. High dose of DEHP exposure is often observed in hemodialysis patients because of the continual usage of plastic medical devices. Although the liver is the major organ that catabolizes DEHP, the impact of long-term DEHP exposure on the sensitivity of liver cancer to chemotherapy remains unclear. In this study, we established long-term DEHP-exposed hepatocellular carcinoma (HCC) cells and two NOD/SCID mice models to investigate the effects and the underlying mechanisms of long-term DEHP exposure on chemosensitivity of HCC. The results showed long-term DEHP exposure potentially increased epithelial-mesenchymal transition (EMT) in HCC cells. Next generation sequencing showed that long-term DEHP exposure increased cell adhesion/migratory related genes expression and blunted sorafenib treatment induced genes alterations. Long-term exposure to DEHP reduced the sensitivity of HCC cells to sorafenib-induced anti-migratory effect by enhancing the EMT transcription factors (slug, twist, and ZEB1) and mesenchymal protein (vimentin) expression. In NOD/SCID mice model, we showed that long-term DEHP-exposed HCC cells exhibited higher growth rate. Regarding the anti-HCC effects of sorafenib, subcutaneous HuH7 tumor grew slowly in sorafenib-treated mice. Nonetheless, the anti-tumor growth effect of sorafenib was not observed in long-term DEHP-exposed mice. Higher mesenchymal markers and proliferating cell nuclear antigen (PCNA) expression were found in sorafenib-treated long-term DEHP-exposed mice. In conclusion, long-term DEHP exposure promoted migratory activity in HCC cells and decreased sorafenib sensitivity in tumor-bearing mice.

Dietary rice bran attenuates hepatic stellate cell activation and liver fibrosis in mice through enhancing antioxidant ability.

Various endogenous and exogenous stimuli can result in an inflammatory response and collagen deposition in the liver, which affect liver function and increase the risk of developing liver cirrhosis and cancer. Rice bran, the main by-product of rice milling, contains various nutrients which possess hepatoprotective activities. In this study, we investigated the effects of rice bran on carbon tetrachloride (CCl4)-induced liver fibrosis in mice. Mice were fed a rice-bran-containing diet (10% rice bran w/w) or a standard diet with or without an injection of 20% CCl4 to induce liver fibrosis. Our results showed that feeding a rice-bran-containing diet could alleviate CCl4-induced liver damage, collagen deposition, and expressions of fibrosis-related genes, including α-smooth muscle actin (α-SMA), collagen 1a2 (COL1A2), and transforming growth factor-ß (TGF-ß) in liver tissues. Moreover, consumption of rice bran enhanced phase II detoxification and antioxidant gene expressions, including Gsta3, Gstp1, Catalase, SOD1, SOD2, and SOD3. Treatment with γ-oryzanol, the major bioactive compound in rice bran, decreased the sensitivity of hepatic stellate cells (HSCs) to TGF-ß1-induced α-SMA, COL1A2, and phosphorylated smad2 expressions. In conclusion, a rice-bran-containing diet may have beneficial effects on liver fibrogenesis through increased antioxidant and detoxification activities. γ-Oryzanol, the major bioactive compound of rice bran, can inhibit activation of HSCs.

Treatment with a new barbituric acid derivative suppresses diet-induced metabolic dysfunction and non-alcoholic fatty liver disease in mice.

INTRODUCTION: Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, often accompanied by obesity, diabetes, and increased risks of depression and anxiety. Currently, there are no FDA-approved drugs to treat NAFLD and its related systemic symptoms. Previously, we identified a new barbituric acid derivative (BA-5) that expressed effectiveness against fibrosis and drug-resistant hepatocellular carcinoma. AIMS: This study investigated the potential of BA-5 against high-fat diet (HFD)-induced NAFLD and mood disorders in mice. MAIN METHODS: Six-weeks-old male C57BL/6 mice were fed with a 45 % HFD for 8 weeks to induce NAFLD and associated metabolic disorders. Mice were treated with a BA-5 and the therapeutic effects and the underlying molecular mechanisms were investigated. KEY FINDINGS: Administration of BA-5 significantly reduced serum levels of alanine aminotransferase (ALT), low-density lipoprotein (LDL), fatty acids (FA), and triglycerides (TG) in HFD-fed mice. BA-5 treatment decreased expressions of hepatic lipogenesis-related markers (acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), and ATP-citrate lyase (ACLY)), increased fatty acid oxidation markers (carnitine palmitoyltransferase 1A (CPT1A) and acyl-CoA oxidase 1 (ACOX1)), and attenuated hepatic fat accumulation in HFD-fed mice. Moreover, HFD-induced adipocyte size enlargement and activation of lipolysis markers such as phosphorylated (p)-hormone-sensitive lipase (HSL) 565, p-HSL 660, and perilipin were inhibited in BA-5-treated mice. Notably, HFD-induced anxiety- and depression-like behaviors significantly improved in the BA-5 treated group through enhanced anti-inflammatory responses in the hippocampus. SIGNIFICANCE: This study provides new insights into clinical therapeutic strategies of barbituric acid derivatives for HFD-induced NAFLD and associated mood disturbances.

Isolation and Biological Evaluation of Alfa-Mangostin as Potential Therapeutic Agents against Liver Fibrosis.

The increased proliferation and activation of hepatic stellate cells (HSCs) are associated with liver fibrosis development. To date, there are no FDA-approved drugs for the treatment of liver cirrhosis. Augmentation of HSCs apoptosis is one of the resolutions for liver fibrosis. In this study, we extracted α-mangostin (1,3,6-trihydroxy-7-methoxy-2,8-bis(3-methyl-2-butenyl)-9H-xanthen-9-one) from the fruit waste components of mangosteen pericarp. The isolated α-mangostin structure was determined and characterized with nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS) and compared with those known compounds. The intracellular signaling pathway activities of α-mangostin on Transforming growth factors-beta 1 (TGF-ß1) or Platelet-derived growth factor subunit B (PDGF-BB) induced HSCs activation and were analyzed via Western blot and Real-time Quantitative Polymerase Chain Reaction (Q-PCR). α-Mangostin-induced mitochondrial dysfunction and apoptosis in HSCs were measured by seahorse assay and caspase-dependent cleavage. The in vivo anti-fibrotic effect of α-mangostin was assessed by carbon tetrachloride (CCl4) treatment mouse model. The data showed that α-mangostin treatment inhibited TGF-ß1-induced Smad2/3 phosphorylation and alpha-smooth muscle actin (α-SMA) expression in HSCs in a dose-dependent manner. Regarding the PDGF-BB-induced HSCs proliferation signaling pathways, α-mangostin pretreatment suppressed the phosphorylation of extracellular-signal-regulated kinase (ERK) and p38. The activation of caspase-dependent apoptosis and dysfunction of mitochondrial respiration (such as oxygen consumption rate, ATP production, and maximal respiratory capacity) were observed in α-mangostin-treated HSCs. The CCl4-induced liver fibrosis mouse model showed that the administration of α-mangostin significantly decreased the expression of the fibrosis markers (α-SMA, collagen-a2 (col1a2), desmin and matrix metalloproteinase-2 (MMP-2)) as well as attenuated hepatic collagen deposition and liver damage. In conclusion, this study demonstrates that α-mangostin attenuates the progression of liver fibrosis through inhibiting the proliferation of HSCs and triggering apoptosis signals. Thus, α-mangostin may be used as a potential novel therapeutic agent against liver fibrosis.

ß-HB treatment reverses sorafenib resistance by shifting glycolysis-lactate metabolism in HCC.

Hepatocellular carcinoma (HCC) is the most common primary malignant tumor. Although sorafenib and regorafenib have been approved for first-line and second-line treatment, respectively, of patients with advanced HCC, long-term treatment often results in acquired resistance. Given that glycolysis-mediated lactate production can contribute to drug resistance and impair HCC treatment efficacy, we investigated the effects of ketone body treatment on the metabolic shift in sorafenib-resistant HCC cells. We discovered differential expression of 3-hydroxymethyl glutaryl-CoA synthase 2 (HMGCS2) and the ketone body D-ß-hydroxybutyrate (ß-HB) in four sorafenib-resistant HCC cell lines. In sorafenib-resistant HCC cells, lower HMGCS2 and ß-HB levels were correlated with more glycolytic alterations and higher lactate production. ß-HB treatment enhanced pyruvate dehydrogenase (PDH) expression and decreased lactate dehydrogenase (LDHA) expression and lactate production in sorafenib-resistant HCC cells. Additionally, ß-HB combined with sorafenib or regorafenib promoted the antiproliferative and antimigratory abilities of sorafenib-resistant HCC cells by inhibiting the B-raf/mitogen-activated protein kinase pathway and mesenchymal N-cadherin-vimentin axis. Although the in vivo ß-HB administration did not affect tumor growth, the expression of proliferative and glycolytic proteins was inhibited in subcutaneous sorafenib-resistant tumors. In conclusion, exogenous ß-HB treatment can reduce lactate production and reverse sorafenib resistance by inducing a glycolytic shift; it can also synergize with regorafenib for treating sorafenib-resistant HCC.

Dietary oxidized frying oil activates hepatic stellate cells and accelerates the severity of carbon tetrachloride- and thioacetamide-induced liver fibrosis in mice.

Deep-frying is a common cooking practice worldwide, and after repeated heating's, the oil undergoes various chemical reactions, including hydrolysis, polymerization, lipid oxidation, and the Maillard reaction. Studies have pointed out that oxidized dietary frying oil may cause teratogenesis in mice and increase cancer and cardiovascular risks. The liver is the main organ involved in dietary nutrient catabolism, detoxification, bile production, and lipid metabolism. Nevertheless, the effects of oxidized frying oil exposure on the activation of hepatic stellate cells (HSCs) and liver fibrosis are still unclear. In this study, we showed that exposure to oxidized frying oil enhanced the sensitivity of HSCs to transforming growth factor (TGF)-ß1-induced α-smooth muscle actin (α-SMA), collagen 1a2, collagen 1a1, metalloproteinase-2, and phosphorylated smad2/3 activation. In both carbon tetrachloride (CCl4)- and thioacetamide (TAA)-induced liver fibrosis mouse models, we showed that long-term administration of a 10% fried oil-containing diet significantly upregulated fibrogenesis genes expression and deposition of hepatic collagen. Furthermore, long-term fried oil exposure not only promoted macrophage infiltration and increased inflammatory-related gene expression, but also accumulated excess cholesterol and lipid peroxidation in the liver tissues. In conclusion, our study demonstrated that feeding a fried oil-containing diet may trigger TGF-ß1-induced HSCs activation and thereby promote liver damage and fibrosis progression through enhancing the inflammatory response and lipid peroxidation.

HMGCS2 Mediation of Ketone Levels Affects Sorafenib Treatment Efficacy in Liver Cancer Cells.

Primary liver cancer is the fifth leading death of cancers in men, and hepatocellular carcinoma (HCC) accounts for approximately 90% of all primary liver cancer cases. Sorafenib is a first-line drug for advanced-stage HCC patients. Sorafenib is a multi-target kinase inhibitor that blocks tumor cell proliferation and angiogenesis. Despite sorafenib treatment extending survival, some patients experience side effects, and sorafenib resistance does occur. 3-Hydroxymethyl glutaryl-CoA synthase 2 (HMGCS2) is the rate-limiting enzyme for ketogenesis, which synthesizes the ketone bodies, ß-hydroxybutyrate (ß-HB) and acetoacetate (AcAc). ß-HB is the most abundant ketone body which is present in a 4:1 ratio compared to AcAc. Recently, ketone body treatment was found to have therapeutic effects against many cancers by causing metabolic alternations and cancer cell apoptosis. Our previous publication showed that HMGCS2 downregulation-mediated ketone body reduction promoted HCC clinicopathological progression through regulating c-Myc/cyclin D1 and caspase-dependent signaling. However, whether HMGCS2-regulated ketone body production alters the sensitivity of human HCC to sorafenib treatment remains unclear. In this study, we showed that HMGCS2 downregulation enhanced the proliferative ability and attenuated the cytotoxic effects of sorafenib by activating expressions of phosphorylated (p)-extracellular signal-regulated kinase (ERK), p-P38, and p-AKT. In contrast, HMGCS2 overexpression decreased cell proliferation and enhanced the cytotoxic effects of sorafenib in HCC cells by inhibiting ERK activation. Furthermore, we showed that knockdown HMGCS2 exhibited the potential migratory ability, as well as decreasing zonula occludens protein (ZO)-1 and increasing c-Myc expression in both sorafenib-treated Huh7 and HepG2 cells. Although HMGCS2 overexpression did not alter the migratory effect, expressions of ZO-1, c-Myc, and N-cadherin decreased in sorafenib-treated HMGCS2-overexpressing HCC cells. Finally, we investigated whether ketone treatment influences sorafenib sensitivity. We showed that ß-HB pretreatment decreased cell proliferation and enhanced antiproliferative effect of sorafenib in both Huh7 and HepG2 cells. In conclusion, this study defined the impacts of HMGCS2 expression and ketone body treatment on influencing the sorafenib sensitivity of liver cancer cells.

Secretory NPC2 Protein-Mediated Free Cholesterol Levels Were Correlated with the Sorafenib Response in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the most common primary malignant tumor in the world. Sorafenib is the first-line drug for patients with advanced HCC. However, long-term treatment with sorafenib often results in reduced sensitivity of tumor cells to the drug, leading to acquired resistance. Identifying biomarkers which can predict the response to sorafenib treatment may represent a clinical challenge in the personalized treatment era. Niemann-Pick type C2 (NPC2), a secretory glycoprotein, plays an important role in regulating intracellular free cholesterol homeostasis. In HCC patients, downregulation of hepatic NPC2 is correlated with poor clinical pathological features through regulating mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) activation. This study aimed to investigate the roles of secretory NPC2-mediated free cholesterol levels as biomarkers when undergoing sorafenib treatment and evaluate its impact on acquired sorafenib resistance in HCC cells. Herein, we showed that NPC2 downregulation and free cholesterol accumulation weakened sorafenib's efficacy through enhancing MAPK/AKT signaling in HCC cells. Meanwhile, NPC2 overexpression slightly enhanced the sorafenib-induced cytotoxic effect. Compared to normal diet feeding, mice fed a high-cholesterol diet had much higher tumor growth rates, whereas treatment with the free cholesterol-lowering agent, hydroxypropyl-ß-cyclodextrin, enhanced sorafenib's tumor-inhibiting ability. In addition, sorafenib treatment induced higher NPC2 secretion, which was mediated by inhibition of the Ras/Raf/MAPK kinase (MEK)/ERK signaling pathway in HCC cells. In both acquired sorafenib-resistant cell and xenograft models, NPC2 and free cholesterol secretion were increased in culture supernatant and serum samples. In conclusion, NPC2-mediated free cholesterol secretion may represent a candidate biomarker for the likelihood of HCC cells developing resistance to sorafenib.

Ketogenic Diet Enhances the Cholesterol Accumulation in Liver and Augments the Severity of CCl4 and TAA-Induced Liver Fibrosis in Mice.

Persistent chronic liver diseases increase the scar formation and extracellular matrix accumulation that further progress to liver fibrosis and cirrhosis. Nevertheless, there is no antifibrotic therapy to date. The ketogenic diet is composed of high fat, moderate to low-protein, and very low carbohydrate content. It is mainly used in epilepsy and Alzheimer's disease. However, the effects of the ketogenic diet on liver fibrosis remains unknown. Through ketogenic diet consumption, ß-hydroxybutyrate (bHB) and acetoacetate (AcAc) are two ketone bodies that are mainly produced in the liver. It is reported that bHB and AcAc treatment decreases cancer cell proliferation and promotes apoptosis. However, the influence of bHB and AcAc in hepatic stellate cell (HSC) activation and liver fibrosis are still unclear. Therefore, this study aimed to investigate the effect of the ketogenic diet and ketone bodies in affecting liver fibrosis progression. Our study revealed that feeding a high-fat ketogenic diet increased cholesterol accumulation in the liver, which further enhanced the carbon tetrachloride (CCl4)- and thioacetamide (TAA)-induced liver fibrosis. In addition, more severe liver inflammation and the loss of hepatic antioxidant and detoxification ability were also found in ketogenic diet-fed fibrotic mouse groups. However, the treatment with ketone bodies (bHB and AcAc) did not suppress transforming growth factor-ß (TGF-ß)-induced HSC activation, platelet-derived growth factor (PDGF)-BB-triggered proliferation, and the severity of CCl4-induced liver fibrosis in mice. In conclusion, our study demonstrated that feeding a high-fat ketogenic diet may trigger severe steatohepatitis and thereby promote liver fibrosis progression. Since a different ketogenic diet composition may exert different metabolic effects, more evidence is necessary to clarify the effects of a ketogenic diet on disease treatment.

Branched I antigen regulated cell susceptibility against natural killer cytotoxicity through its N-linked glycosylation and overall expression.

Cell surface glycosylation has been known as an important modification process that can be targeted and manipulated by malignant cells to escape from host immunosurveillance. We previously showed that the blood group branched I antigen on the leukemia cell surface can regulate the cell susceptibility against natural killer (NK) cell-mediated cytotoxicity through interfering target-NK interaction. In this work, we first identified N-linkage as the major glycosylation linkage type for branched I glycan formation on leukemia cells, and this linkage was responsible for cell sensitivity against therapeutic NK-92MI targeting. Secondly, by examining different leukemia cell surface death receptors, we showed death receptor Fas had highest expressions in both Raji and TF-1a cells. Mutations on two Fas extracellular N-linkage sites (118 and 136) for glycosylation impaired activation of Fas-mediated apoptosis during NK-92MI cytotoxicity. Last, we found that the surface I antigen expression levels enable leukemia cells to respond differently against NK-92MI targeting. In low I antigen expressing K-562 cell, reduction of I antigen presence greatly reduced leukemia cell susceptibility against NK-92MI targeting. But in other high I antigen expressing leukemia cells, similar reduction in I antigen expression did not affect cell susceptibility.

Alterations of Specific Lymphocytic Subsets with Aging and Age-Related Metabolic and Cardiovascular Diseases.

To investigate the association of immunosenescence with aged-related morbidity in the elderly, a clinical study was conducted to analyze and compare the alterations in peripheral blood (PB) T-cell subsets among young healthy (YH) controls, elderly healthy (EH) controls, and age-matched elderly patients with metabolic diseases (E-MDs), with cardiovascular diseases (E-CVDs) or with both (E-MDs/E-CVDs). The frequencies of CD3T, CD8T and invariant natural killer T (iNKT) cells were decreased in the EH, E-MD and E-CVD cohorts, indicating a decline in defense function. Although CD4T and regulatory T (Treg) cell frequencies tended to increase with aging, they were lower in patients with E-MDs and E-CVDs. Subset analyses of T-cells consistently showed the accumulation of senescent T-cell in aging and in patients with E-MDs and E-CVDs, compared with YH volunteers. These accumulated senescent T-cells were undergoing apoptosis upon stimulation due to the replicative senescence stage of T-cells. In addition, serum levels of cytokines, including interferon (IF)-γ, transforming growth factor (TGF)-ß and growth differentiation factor (GDF)-15, consistently reflected alterations in T-cell subsets. This study demonstrated that T-cell subset changes with paralleled alterations in cytokines were associated with aging and age-related pathogenesis. These altered T-cell subsets and/or cytokines can potentially serve as biomarkers for the prevention, diagnosis and treatment of age-related morbidities.

Characteristics of CD133-Sustained Chemoresistant Cancer Stem-Like Cells in Human Ovarian Carcinoma.

Cancer stem cells (CSCs) are considered to be the origin of ovarian cancer (OC) development, recurrence, and chemoresistance. We investigated changes in expression levels of the CSC biomarker, cluster of differentiation 133 (CD133), from primary OC cell lines to induction of CSC-spheres in an attempt to explore the mechanisms related to modulation of stemness, drug resistance, and tumorigenesis in CSCs, thus facilitating the search for new therapeutics for OC. The effect of CD133 overexpression on the induction of CSC properties was evaluated by sphere-forming assays, RT-qPCR, flow cytometry, cell viability assays, and in vivo xenograft experiments. Moreover, the potential signaling molecules that participate in CD133 maintenance of stemness were screened by RNA-sequencing. CD133 expression was upregulated during OCSC induction and chemotherapeutic drug treatment over time, which increased the expressions of stemness-related markers (SOX2, OCT4, and Nanog). CD133 overexpression also promoted tumorigenesis in NOD/SCID mice. Several signalings were controlled by CD133 spheres, including extracellular matrix receptor interactions, chemokine signaling, and Wnt signaling, all of which promote cell survival and cell cycle progression. Our findings suggest that CD133 possesses the ability to maintain functional stemness and tumorigenesis of OCSCs by promoting cell survival signaling and may serve as a potential target for stem cell-targeted therapy of OC.

Loss of HMGCS2 Enhances Lipogenesis and Attenuates the Protective Effect of the Ketogenic Diet in Liver Cancer.

Hepatocellular carcinoma (HCC) is the most common primary malignant liver tumor with limited treatment. The ketogenic diet (KD) emerged as a metabolic therapy for cancer; however, the antitumor effect on HCC remains controversial. We previously reported that the ketogenesis rate-limiting enzyme, 3-hydroxymethylglutaryl-CoA synthase 2 (HMGCS2), was downregulated in most patients with HCC. The knockdown of HMGCS2 enhanced the proliferation and metastasis ability of HCC cells. However, the role of HMGCS2 in affecting KD-mediated metabolic effects remains unclear. Here, we report that KD feeding upregulates HMGCS2 expression and inhibits HCC tumor growth, while a reverse correlation between tumor size and HMGCS2 expression was observed. We found that HCC cells with HMGCS2 downregulation possess altered lipid metabolism that increases fatty acid, triglyceride, and cholesterol synthesis. Under KD feeding, a higher tumor growth rate was observed in HMGCS2 knockdown tumors, which had increased lipid synthesis-related marker expression and a positive correlation between lipid quantity and tumor weight. In conclusion, these results demonstrate that the downregulation of HMGCS2 attenuates the protective effect of the KD by shifting ketone production to enhance de novo lipogenesis in HCC. Our study elucidates a new molecular mechanism underlying the crosstalk between HMGCS2 expression and the KD in cancer treatment, which provides more information for precision medicine in developing personalized treatment strategies.

Engineering Chimeric Antigen Receptor T Cells against Immune Checkpoint Inhibitors PD-1/PD-L1 for Treating Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a 5-year survival rate of 9%. Major obstacles to successful treatment of pancreatic cancer are the immunosuppressive tumor microenvironment (TME) and antigenic complexity or heterogeneity. Programmed death-ligand 1 (PD-L1) is expressed on PDAC and immunosuppressed cells within the TME, providing suitable immunotherapy targets. We applied a chimeric antigen receptor (CAR) strategy to target immune checkpoint programmed death-1 (PD-1)/PD-L1 interactions. Lentiviral vectors were used to express the extracellular domain of human PD-1 (PD-1-CD28-4-1BB activating chimeric receptor [PD1ACR]) or the single-chain variable fragment (scFv) region of anti-PD-L1 (PDL1CAR) that binds to PD-L1, and each was fused to intracellular signaling domains containing CD3 zeta, CD28, and 4-1BB (CD137). Both engineered CAR T cells recognized and eliminated PD-L1-overexpressing CFPAC1 cells efficiently at approximately 80% in vitro. Adoptive transfer of both CAR T cells enhanced T cell persistence and induced specific regression of established CFPAC1 cancer by >80% in both xenograft and orthotopic models. Ki67 expression in tumors decreased, whereas proinflammatory cytokines/chemokines increased in CAR T cell-treated mouse sera. PD1ACR and PDL1CAR obtained a similar therapeutic efficacy. Thus, these armed third-generation PD-L1-targeted CAR T cells confer antitumor activity and the ability to combat T cell exhaustion, providing a potentially new and innovative CAR T cell immunotherapy against pancreatic cancers.

Treatment with a New Barbituric Acid Derivative Exerts Antiproliferative and Antimigratory Effects against Sorafenib Resistance in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a common cause of cancer death worldwide. Sorafenib, a multikinase inhibitor, is the first-line drug approved by the Food and Drug Administration (FDA) for the treatment of patients with advanced HCC. However, most patients who continuously receive sorafenib may acquire resistance to this drug. Therefore, it is important to develop a new compound to treat liver cancer and sorafenib-resistant liver cancer. Barbituric acid derivatives have been used as antiasthmatic drugs in the clinic. We previously reported that a novel barbituric acid derivative inhibited carbon tetrachloride-induced liver fibrosis in mice, but its effects on liver cancer remain unknown. Thus, the purpose of this study was to investigate the antitumor effect of barbituric acid derivatives on HCC cells and sorafenib-resistant HCC cells (HCC-SRs). Our findings reveal that one of the barbituric acid derivatives, BA-5, significantly inhibited HCC and HCC-SR cell viability in a dose- and time-dependent manner. Therefore, compound BA-5 was selected for further experiments. Western blot data revealed that BA-5 treatment decreased the phosphorylation of AKT/p70s6k without affecting the MAPK pathway and increased cleaved PARP and cleaved caspase-7 in both HCC and HCC-SR cells. Since epithelial-mesenchymal transition plays a significant role in regulating cancer invasion and migration, we used the wound healing assay to evaluate the antimigratory effect of compound BA-5. The results showed that BA-5 treatment inhibited HCC and HCC-SR cell migration and reduced Vimentin protein expression. These results were confirmed by microarray analysis showing that BA-5 treatment influenced cancer cell motility and growth-related pathways. In the xenograft mouse model experiment, BA-5 administration significantly inhibited HCC cancer cell growth in mice. Furthermore, the combination of BA-5 with a low dose of regorafenib synergistically inhibited HCC-SR cell proliferation. In conclusion, our study showed that the barbituric acid derivative BA-5 is a new candidate for HCC and sorafenib-resistant HCC therapy.

Long-Term Exposure to Low-Dose Di-(2-ethylhexyl) Phthalate Impairs Cholesterol Metabolism in Hepatic Stellate Cells and Exacerbates Liver Librosis.

Phthalates are often added to plastic products to increase their flexibility. Di-(2-ethylhexyl) phthalate (DEHP) is one of the most common plasticizers. Previously, a major incident involving phthalate-contaminated foodstuffs occurred, where phthalates were deliberately added to foodstuffs as a substitute for emulsifiers, resulting in a threat to public health. DEHP exposure can cause liver damage and further lead to cancer; however, the effects of long-term exposure to low-dose DEHP on hepatic stellate cells (HSCs) and on liver fibrosis are still unclear. In this study, we showed that chronic exposure to low-dose DEHP results in an accumulation of cholesterol in HSCs by disturbing the cholesterol metabolism and enhancing endogenous cholesterol synthesis. In addition, long-term exposure to low-dose DEHP reduces the sensitivity of HSCs to platelet-derived growth factor BB (PDGF-BB)-induced proliferation by blocking the MAPK pathway. Dysfunction of mitochondrial respiration and induction of caspase 3/PARP-dependent apoptosis were observed in HSCs following chronic, low-dose exposure. The carbon tetrachloride (CCl4)-induced liver fibrosis mouse model showed that long-term administration of DEHP significantly promoted liver damage, inflammatory infiltration, cholesterol accumulation, and deposition of hepatic collagen. In conclusion, long-term exposure to low-dose DEHP may perturb the cholesterol metabolism in HSCs and accelerate liver damage and fibrosis.

Antifibrotic Effects of a Barbituric Acid Derivative on Liver Fibrosis by Blocking the NF-κB Signaling Pathway in Hepatic Stellate Cells.

Hepatic stellate cells (HSCs) are the major profibrogenic cells that promote the pathogenesis of liver fibrosis. The crosstalk between transforming growth factor-ß1 (TGF-ß1) signaling and lipopolysaccharide (LPS)-induced NF-κB signaling plays a critical role in accelerating liver fibrogenesis. Until now, there have been no FDA-approved drug treatments for liver fibrosis. Barbituric acid derivatives have been used as antiasthmatic drugs in the clinic; however, the effect of barbituric acid derivatives in treating liver fibrosis remains unknown. In this study, we synthesized a series of six barbituric acid (BA) derivatives, and one of the compounds, BA-5, exhibited the best ability to ameliorate TGF-ß1-induced HSC activation without overt cytotoxic effects. Then, we treated HSCs and RAW264.7 macrophages with BA-5 to analyze the cross-talk of anti-fibrotic and anti-inflammatory effects. Carbon tetrachloride (CCl4)-induced liver fibrosis mouse model was used to evaluate the therapeutic effects of BA-5. Treatment with BA-5 inhibited TGF-ß1-induced α-SMA, collagen1a2, and phosphorylated smad2/3 expression in HSCs. Furthermore, BA-5 treatment reversed the LPS-induced reduction in BAMBI protein and decreased IκBα and NF-κB phosphorylation in HSCs. NF-κB nuclear translocation, MCP-1 secretion, and ICAM-1 expression were also inhibited in BA-5-treated HSCs. Conditioned medium collected from BA-5-treated HSCs showed a reduced ability to activate RAW264.7 macrophages by inhibiting the MAPK pathway. In the mouse model, BA-5 administration reduced CCl4-induced liver damage, liver fibrosis, and F4/80 expression without any adverse effects. In conclusion, our study showed that the barbituric acid derivative BA-5 inhibits HSCs activation and liver fibrosis by blocking both the TGF-ß1 and LPS-induced NF-κB signaling pathways and further inhibits macrophages recruitment and activation.

Treatment with a new benzimidazole derivative bearing a pyrrolidine side chain overcomes sorafenib resistance in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a major cause of cancer-related death worldwide. Currently, sorafenib is the standard first-line drug for patients with advanced HCC. However, long-term exposure to sorafenib often results in reduced sensitivity of tumour cells to the drug, leading to acquired resistance. Therefore, developing new compounds to treat sorafenib resistance is urgently needed. Although benzimidazole and its derivatives have been reported to exert antimicrobial and antitumour effects, the anti-drug resistance potential of these molecules is still unknown. In this study, we established sorafenib-resistant (SR) cell lines and an acquired sorafenib resistance xenograft model. We showed that treatment with a benzimidazole derivative bearing a pyrrolidine side chain (compound 9a) inhibited the proliferation of SR cells by blocking the phosphorylation of AKT, p70S6 and the downstream molecule RPS6. In addition, caspase 3/PARP-dependent apoptotic signals were induced in 9a-treated cells. Regarding epithelial-mesenchymal transition (EMT) activities, 9a treatment significantly suppressed the migration of SR cells. In particular, the levels of EMT-related transcription factors (snail, slug and twist) and mesenchymal markers (vimentin and N-cadherin) were downregulated. In the acquired sorafenib resistance xenograft model, compound 9a administration decreased the growth of tumours with acquired sorafenib resistance and the expression of the HCC markers α-fetoprotein, glypican 3 and survivin. In conclusion, treatment with this compound may be a novel therapeutic strategy for patients with sorafenib resistance.

HMGCS2 Mediates Ketone Production and Regulates the Proliferation and Metastasis of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the most common primary malignant tumor worldwide; however, the traditional therapeutic approaches and survival rates are still limited. To improve current therapies, it is necessary to investigate the molecular mechanisms underlying liver cancer and to identify potential therapeutic targets. The aims of this study were to verify the mechanisms and therapeutic potential of the ketogenesis rate-limiting enzyme 3-Hydroxymethylglutaryl-CoA synthase 2 (HMGCS2) in HCC. Immunohistochemical staining of human liver disease tissue arrays showed that HMGCS2 is abundantly expressed in normal liver tissues but is downregulated in cirrhosis and HCC tissues. In HCC patients, lower HMGCS2 expression was correlated with higher pathological grades and clinical stages. In our investigation of the molecular mechanisms of HMGCS2 in HCC, we showed that knockdown of HMGCS2 decreased ketone production, which promoted cell proliferation, cell migration, and xenograft tumorigenesis by enhancing c-Myc/cyclinD1 and EMT signaling and by suppressing the caspase-dependent apoptosis pathway. Ketone body treatment reduced the proliferation- and migration-promoting effects of HMGCS2 knockdown in cells. In contrast, HMGCS2 overexpression increased the intracellular ketone level and inhibited cell proliferation, cell migration, and xenograft tumorigenesis. Finally, ketogenic diet administration significantly inhibited liver cancer cell growth in mice. Our studies highlight the potential therapeutic strategy of targeting HMGCS2-mediated ketogenesis in liver cancer.

TGF-ß regulated leukemia cell susceptibility against NK targeting through the down-regulation of the CD48 expression.

Transforming growth factor-ß (TGF-ß) is known to function as a dual role regulatory cytokine for being either a suppresser or promoter during tumor initiation and progression. In solid tumors, TGF-ß secreted from tumor microenvironment acts as a suppresser against host immunity, like natural killer (NK) cells, to favor tumor evasion. However, besides solid tumors, the underlying mechanism of how TGF-ß regulates leukemogenesis, tumor progression, immunoediting, and NK function is still not clear in detail. In this study, we found that TGF-ß induced leukemia MEG-01 and U937 cells to become less sensitive to NK-92MI targeting by down-regulating CD48, a ligand for NK activating receptor 2B4, but not down-regulating other tumor-associated carbohydrate antigens (TACAs). In CD48-knockdown cells, cells responding to NK-92MI targeting displayed a phenotype of less NK susceptibility and cell conjugation. On the other hand, when NK cells were treated with TGF-ß, TGF-ß suppressed NK recognition, degranulation, and killing activity in time-dependent manner by regulating ICAM-1 binding capacity instead of affecting expressions of activating and inhibitory receptors. Taken together, both leukemia cells and immune NK cells could be regulated by TGF-ß through suppressing leukemia cell surface CD48 to escape from host surveillance and down-regulating NK cell surface ICAM-1 binding activity to impair NK functions, respectively. Our results suggested that TGF-ß had effect in leukemia similar to that observed in solid tumors but through different regulatory mechanism.