S670, an amide derivative of 3-O-acetyl-11-keto-ß-boswellic acid, induces ferroptosis in human glioblastoma cells by generating ROS and inhibiting STX17-mediated fusion of autophagosome and lysosome.

Glioblastoma (GBM) is the most common malignant tumor in the brain with temozolomide (TMZ) as the only approved chemotherapy agent. GBM is characterized by susceptibility to radiation and chemotherapy resistance and recurrence as well as low immunological response. There is an urgent need for new therapy to improve the outcome of GBM patients. We previously reported that 3-O-acetyl-11-keto-ß-boswellic acid (AKBA) inhibited the growth of GBM. In this study we characterized the anti-GBM effect of S670, a synthesized amide derivative of AKBA, and investigated the underlying mechanisms. We showed that S670 dose-dependently inhibited the proliferation of human GBM cell lines U87 and U251 with IC50 values of around 6 µM. Furthermore, we found that S670 (6 µM) markedly stimulated mitochondrial ROS generation and induced ferroptosis in the GBM cells. Moreover, S670 treatment induced ROS-mediated Nrf2 activation and TFEB nuclear translocation, promoting protective autophagosome and lysosome biogenesis in the GBM cells. On the other hand, S670 treatment significantly inhibited the expression of SXT17, thus impairing autophagosome-lysosome fusion and blocking autophagy flux, which exacerbated ROS accumulation and enhanced ferroptosis in the GBM cells. Administration of S670 (50 mg·kg-1·d-1, i.g.) for 12 days in a U87 mouse xenograft model significantly inhibited tumor growth with reduced Ki67 expression and increased LC3 and LAMP2 expression in the tumor tissues. Taken together, S670 induces ferroptosis by generating ROS and inhibiting STX17-mediated fusion of autophagosome and lysosome in GBM cells. S670 could serve as a drug candidate for the treatment of GBM.

Dissecting the multifaced function of transcription factor EB (TFEB) in human diseases: From molecular mechanism to pharmacological modulation.

The transcription factor EB (TFEB) is a transcription factor of the MiT/TFE family that translocations from the cytoplasm to the nucleus in response to various stimuli, including lysosomal stress and nutrient starvation. By activating genes involved in lysosomal function, autophagy, and lipid metabolism, TFEB plays a crucial role in maintaining cellular homeostasis. Dysregulation of TFEB has been implicated in various diseases, including cancer, neurodegenerative diseases, metabolic diseases, cardiovascular diseases, infectious diseases, and inflammatory diseases. Therefore, modulating TFEB activity with agonists or inhibitors may have therapeutic potential. In this review, we reviewed the recently discovered regulatory mechanisms of TFEB and their impact on human diseases. Additionally, we also summarize the existing TFEB inhibitors and agonists (targeted and non-targeted) and discuss unresolved issues and future research directions in the field. In summary, this review sheds light on the crucial role of TFEB, which may pave the way for its translation from basic research to practical applications, bringing us closer to realizing the full potential of TFEB in various fields.

Recent research of BTK inhibitors: Methods of structural design, pharmacological activities, manmade derivatives and structure-activity relationship.

Protein kinases constitute the largest group within the kinase family, and mutations and translocations of protein kinases due to genetic alterations are intimately linked to the pathogenesis of numerous diseases. Bruton's tyrosine kinase (BTK) is a member of the protein kinases and plays a pivotal role in the development and function of B cells. BTK belongs to the tyrosine TEC family. The aberrant activation of BTK is closely associated with the pathogenesis of B-cell lymphoma. Consequently, BTK has always been a critical target for treating hematological malignancies. To date, two generations of small-molecule covalent irreversible BTK inhibitors have been employed to treat malignant B-cell tumors, and have exhibited clinical efficacy in hitherto refractory diseases. However, these drugs are covalent BTK inhibitors, which inevitably lead to drug resistance after prolonged use, resulting in poor tolerance in patients. The third-generation non-covalent BTK inhibitor Pirtobrutinib has obtained approval for marketing in the United States, thereby circumventing drug resistance caused by C481 mutation. Currently, enhancing safety and tolerance constitutes the primary issue in developing novel BTK inhibitors. This article systematically summarizes recently discovered covalent and non-covalent BTK inhibitors and classifies them according to their structures. This article also provides a detailed discussion of binding modes, structural features, pharmacological activities, advantages and limitations of typical compounds within each structure type, providing valuable references and insights for developing safer, more effective and more targeted BTK inhibitors in future studies.

A review of Brucea javanica: metabolites, pharmacology and clinical application.

This review examines advances in the metabolites, pharmacological research, and therapeutic applications of the medicinal fruit of Brucea javanica (L.) Merr. Brucea javanica (BJ) is derived from the fruit of the Brucea javanica (L.) Merr. There are nearly 200 metabolites present in BJ, and due to the diversity of its metabolites, BJ has a wide range of pharmacological effects. The traditional pharmacological effects of BJ include anti-dysentery, anti-malaria, etc. The research investigating the contemporary pharmacological impacts of BJ mainly focuses on its anti-tumor properties. In the article, the strong monomeric metabolites among these pharmacological effects were preliminarily screened. Regarding the pharmacological mechanism of action, current research has initially explored BJ's pharmacological agent and molecular signaling pathways. However, a comprehensive system has yet to be established. BJ preparations have been utilized in clinical settings and have demonstrated effectiveness. Nevertheless, clinical research is primarily limited to observational studies, and there is a need for higher-quality research evidence to support its clinical application. There are still many difficulties and obstacles in studying BJ. However, it is indisputable that BJ is a botanical drugs with significant potential for application, and it is expected to have broader global usage.

Advances in RIPK1 kinase inhibitors.

Programmed necrosis is a new modulated cell death mode with necrotizing morphological characteristics. Receptor interacting protein 1 (RIPK1) is a critical mediator of the programmed necrosis pathway that is involved in stroke, myocardial infarction, fatal systemic inflammatory response syndrome, Alzheimer's disease, and malignancy. At present, the reported inhibitors are divided into four categories. The first category is the type I ATP-competitive kinase inhibitors that targets the area occupied by the ATP adenylate ring; The second category is type Ⅱ ATP competitive kinase inhibitors targeting the DLG-out conformation of RIPK1; The third category is type Ⅲ kinase inhibitors that compete for binding to allosteric sites near ATP pockets; The last category is others. This paper reviews the structure, biological function, and recent research progress of receptor interaction protein-1 kinase inhibitors.

Self-Delivery Janus-Prodrug for Precise Immuno-Chemotherapy of Colitis-Associated Colorectal Cancer.

Aromatized thioketal (ATK) linked the immunoregulatory molecule (budesonide, Bud) and the cytotoxic molecule (gemcitabine, Gem) to construct a ROS-activated Janus-prodrug, termed as BAG. Benefiting from the hydrogen bonding, π-π stacking, and other intermolecular interactions, BAG could self-assemble into nanoaggregates (BAG NA) with a well-defined spherical shape and uniform size distribution. Compared to the carrier-based drug delivery system, BAG NA have ultrahigh drug loading content and ROS concentration-dependent drug release. Colitis-associated colorectal cancer (CAC) is a typical disease in which chronic inflammation transforms into tumors. BAG NA can be internalized by colon cancer C26 cells and then triggered by excessive intracellular ROS to release nearly 100% of the drugs. Based on this, BAG NA showed a stronger pro-apoptotic effect than free Bud combined with free Gem. What is gratifying is that orally administered BAG NA can precisely accumulate in the diseased colon tissues of CAC mice induced by AOM/DSS and simultaneously release Bud and Gem. Bud can regulate the tumor immune microenvironment to restore and enhance the cytotoxicity of Gem. Therefore, BAG NA maximizes the synergistic therapeutic effect through co-delivery of Bud and Gem. This work provided a cutting-edge method for constructing self-delivery Janus-prodrug based on ATK and confirmed its potential application in inflammation-related carcinogenesis.

Discovery of 1,8-naphthalidine derivatives as potent anti-hepatic fibrosis agents via repressing PI3K/AKT/Smad and JAK2/STAT3 pathways.

Liver fibrosis is one of the most common pathological consequences of chronic liver diseases (CLD). To develop effective antifibrotic strategies, a novel class of 1-(substituted phenyl)-1,8-naphthalidine-3-carboxamide derivatives were designed and synthesized. By means of the collagen type I α 1 (COL1A1)-based screening and cytotoxicity assay in human hepatic stellate cell (HSC) line LX-2, seven compounds were screened out from total 60 derivatives with high inhibitory effect and relatively low cytotoxicity for further COL1A1 mRNA expression analysis. It was found that compound 17f and 19g dose-dependently inhibited the expression of fibrogenic markers, including α-smooth muscle actin (α-SMA), matrix metalloprotein 2 (MMP-2), connective tissue growth factor (CTGF) and transforming growth factor ß1 (TGFß1) on both mRNA and protein levels. Further mechanism studies indicated that they might suppress the hepatic fibrogenesis via inhibiting both PI3K/AKT/Smad and non-Smad JAK2/STAT3 signaling pathways. Furthermore, 19g administration attenuated hepatic histopathological injury and collagen accumulation, and reduced fibrogenesis-associated protein expression in liver tissues of bile duct ligation (BDL) rats, showing significant antifibrotic effect in vivo. These findings identified 1,8-naphthalidine derivatives as potent anti-hepatic fibrosis agents, and provided valuable information for further structure optimization.

Association between CYP2B6 c.516G >T variant and acute leukaemia: A protocol for systematic review and meta-analysis.

BACKGROUND: Acute leukemia (AL) is a kind of malignant tumor of hematopoietic system. A number of studies have suggested that Single Nucleotide Polymorphisms are significantly associated with risk of AL. Present study performs meta-analysis to evaluate the association between CYP2B6 c.516G>T variant and AL risk. METHODS: Databases including PubMed, EMBASE, Chinese National Knowledge Infrastructure (CNKI), and Wanfang were searched for literatures to September 30, 2019, both in English and Chinese. Relative risk and its 95% confidence intervals were used to assess the associations. Statistical analyses of this meta-analysis were conducted by using STATA 13.0. software. RESULTS: A total of 7 studies, including 1038 cases and 1648 controls, were analyzed. Our results indicated that CYP2B6 c.516G>T variant was significantly related to an increased the risk of AL under dominant model, recessive model, homozygote model, and allelic model. In addition, subgroup analyses were also performed by disease classification, country, and study design. No significant associations were obtained between CYP2B6 c.516G>T variant and the risk of AL under the recessive model in the design of hospital-based (relative risk = 0.98; 95% confidence interval: 0.95-1.01; P  = 0.118). CONCLUSION: Our meta-analysis indicated that the CYP2B6 variant is significantly associated with AL risk, in which CYP2B6 c.516G>T is related to an increased risk of AL.

Physalin B attenuates liver fibrosis via suppressing LAP2α-HDAC1-mediated deacetylation of the transcription factor GLI1 and hepatic stellate cell activation.

BACKGROUND AND PURPOSE: Liver fibrosis is one of the leading causes of morbidity and mortality worldwide but lacks any acceptable therapy. The transcription factor glioma-associated oncogene homologue 1 (GLI1) is a potentially important therapeutic target in liver fibrosis. This study investigates the anti-fibrotic activities and potential mechanisms of the phytochemical, physalin B. EXPERIMENTAL APPROACH: Two mouse models (CCl4 challenge and bile duct ligation) were used to assess antifibrotic effects of physalin B in vivo. Mouse primary hepatic stellate cells (pHSCs) and human HSC line LX-2 also served as in vitro liver fibrosis models. Liver fibrogenic genes, GLI1 and GLI1 downstream genes were examined using Western blot and quantitative real-time PCR (qRT-PCR). GLI1 acetylation and LAP2α-HDAC1 interaction were analysed by co-immunoprecipitation. KEY RESULTS: In vivo, physalin B administration attenuated hepatic histopathological injury and collagen accumulation and decreased expression of fibrogenic genes. Physalin B dose-dependently suppressed fibrotic marker expression in LX-2 cells and mouse pHSCs. Mechanistic studies showed that physalin B inhibited GLI activity by non-canonical Hedgehog signalling. Physalin B blocked formation of lamina-associated polypeptide 2α (LAP2α)/histone deacetylase 1 (HDAC1) complexes, thus inhibiting HDAC1-mediated GLI1 deacetylation. Physalin B up-regulated acetylation of GLI1, down-regulated expression of GLI1 and subsequently inhibited HSC activation. CONCLUSION AND IMPLICATIONS: Physalin B exerted potent antifibrotic effects in vitro and in vivo by disrupting LAP2α/HDAC1 complexes, increasing GLI1 acetylation and inactivating GLI1. This indicates that the phytochemical physalin B may be a potential therapeutic candidate for the treatment of liver fibrosis.

Nrf2 signalling pathway and autophagy impact on the preventive effect of green tea extract against alcohol-induced liver injury.

OBJECTIVES: To explore the potential molecular mechanism underlying the effect of green tea extract (TE), rich in tea polyphenols (TPs), on improving alcohol-induced liver injury. METHODS: Mice were intragastrically treated with 50% (v/v) alcohol administration (15 ml/kg BW) with or without three doses of TE (50, 120 and 300 mg TPs/kg BW) daily for 4 weeks, and biological changes were tested. KEY FINDINGS: The TE improved the functional and histological situations in the liver of the mice accepted alcohol administration, including enzymes for alcohol metabolism, oxidative stress and lipid accumulation. Interestingly, the TE increased the nuclear translocation of nuclear factor erythroid-2-related factor 2 (Nrf2), with the decreasing expression of kelch-like ECH-associated protein 1 (Keap1), indicating the association between the effect of TE with Nrf2-mediated antioxidant signalling. Moreover, the TE restored the activity of autophagy, showing as lifted Beclin-1 expression, LC3B-II/LC3B-I ratio, and decreased p62 expression. Importantly, all these effects were dose-dependent. CONCLUSIONS: These findings provide a new notion for the first time that the TE preventing against alcohol-induced liver injury is closely related to accelerated metabolism of alcohol and relieved oxidative stress, which is associated with Nrf2 signalling activation and autophagy restoration, thus the reduction of lipid accumulation in liver.

Association analysis between chronic obstructive pulmonary disease and polymorphisms in circadian genes.

BACKGROUND: Circadian genes have been suggested to play an important role in lung pathology. However, it remains unknown whether polymorphisms of these genes are associated with chronic obstructive pulmonary disease (COPD). Here, we aimed to investigate the association of circadian genes polymorphisms with COPD in a case-control study of 477 COPD patient and 323 control Han Chinese persons. METHODS: Genotyping assays were carried out for nine single nucleotide polymorphisms (SNPs) from five circadian genes (PER3, CLOCK, RORB, BMAL1 and CRY2) that were previously identified in lung pathology. Age, sex, BMI and smoking status and comorbidities were recorded for all subjects. RESULTS: No significant association was found in all SNP sites in overall subjects and no significant difference was found in age, sex, smoking status stratification analysis. DISCUSSION: The findings of this investigation indicated the effect of circadian genes polymorphisms on COPD susceptibility may only be small and possibly dependent on the subject factors, such as age and sex.

Probiotics modulate the microbiota-gut-brain axis and improve memory deficits in aged SAMP8 mice.

ProBiotic-4 is a probiotic preparation composed of Bifidobacterium lactis, Lactobacillus casei, Bifidobacterium bifidum, and Lactobacillus acidophilus. This study aims to investigate the effects of ProBiotic-4 on the microbiota-gut-brain axis and cognitive deficits, and to explore the underlying molecular mechanism using senescence-accelerated mouse prone 8 (SAMP8) mice. ProBiotic-4 was orally administered to 9-month-old SAMP8 mice for 12 weeks. We observed that ProBiotic-4 significantly improved the memory deficits, cerebral neuronal and synaptic injuries, glial activation, and microbiota composition in the feces and brains of aged SAMP8 mice. ProBiotic-4 substantially attenuated aging-related disruption of the intestinal barrier and blood-brain barrier, decreased interleukin-6 and tumor necrosis factor-α at both mRNA and protein levels, reduced plasma and cerebral lipopolysaccharide (LPS) concentration, toll-like receptor 4 (TLR4) expression, and nuclear factor-κB (NF-κB) nuclear translocation in the brain. In addition, not only did ProBiotic-4 significantly decreased the levels of γ-H2AX, 8-hydroxydesoxyguanosine, and retinoic-acid-inducible gene-I (RIG-I), it also abrogated RIG-I multimerization in the brain. These findings suggest that targeting gut microbiota with probiotics may have a therapeutic potential for the deficits of the microbiota-gut-brain axis and cognitive function in aging, and that its mechanism is associated with inhibition of both TLR4-and RIG-I-mediated NF-κB signaling pathway and inflammatory responses.

Novel HER2-Targeting Antibody-Drug Conjugates of Trastuzumab Beyond T-DM1 in Breast Cancer: Trastuzumab Deruxtecan(DS-8201a) and (Vic-)Trastuzumab Duocarmazine (SYD985).

Targeted drug delivery has improved cancer treatment significantly in recent years, although it is difficult to achieve. Different approaches have been developed to apply targeted drug delivery. Among which, antibody-drug conjugate (ADC) provides a potentially ideal solution to such a challenge. ADC is an innovative drug treatment model with three key components: payload, monoclonal antibody, and linker. The monoclonal antibody targets the antigen-expressing tumor cells and internalizes the payload linked by the linker to the target cells to reduce the side effects of the traditional chemotherapy drugs. The off-target effect has an excellent therapeutic prospect. Among them, ado-trastuzumab emtansine (T-DM1) is a successful example of targeting human epidermal growth factor receptor-2 (HER2). Its antibody (trastuzumab) is derived from Herceptin with annual sales of more than $6 billion. It has excellent targeting and specific anti-tumor activity against HER2. Its linker is not cleavable and releases the Lys-linker-payload to kill the cells. The two ADCs described here use the same antibody as T-DM1, but the cleavable linker and the more toxic payload allow them to have the not only targeting of T-DM1, but also the reduce T-DM1 resistance and improve efficacy in heterogeneous tumors. This paper describes the mechanism of action and the biochemical characteristics of different parts and preclinical and clinical progress of trastuzumab deruxtecan(DS-8201a) and (vic-)trastuzumab duocarmazine (SYD985).

The association between maternal methylenetetrahydrofolate reductase C677T and A1298C polymorphism and birth defects and adverse pregnancy outcomes.

Published studies indicate the MTHFR C677T and A1298C polymorphisms are associated with abnormal homocysteine levels, which may cause various pregnancy complications and birth defects. However, the results obtained from different studies have been inconsistent. Therefore, this meta-analysis explores the association between MTHFR polymorphisms and birth defects and adverse pregnancy outcomes. The PubMed, ScienceDirect, Embase, and China Biology Medicine literature databases and ClinicalTrials were searched. Analyses of public bias, meta-regression, subgroups, and sensitivity were used to ensure the robustness of our results. MTHFR C677T was significantly associated with recurrent pregnancy loss in developing countries (odds ratio [OR], 1.34; 95% confidence interval [CI], 1.20-1.50) but not in developed countries (OR, 0.87; 95% CI, 0.68-1.11). No significant relationship was found between MTHFR A1298C and recurrent pregnancy loss (OR, 1.04; 95% CI, 0.93-1.18). MTHFR C677T and A1298C were not associated with preeclampsia (OR, 1.06; 95% CI, 0.97-1.16 and OR, 1.16; 95% CI, 0.97-1.39, respectively), and C677T was not associated with placental abruption (OR, 1.03; 95% CI, 0.87-1.21), intrauterine growth retardation (OR, 1.02; 95% CI, 0.90-1.15), or congenital heart disease (OR, 1.05; 95% CI, 0.89-1.25). MTHFR C677T, but not A1298C, was associated with neural tube defects (OR, 1.24; 95% CI, 1.08-1.42) and Down syndrome (OR, 1.65; 95% CI, 1.39-1.95). CONCLUSION: Although MTHFR C677T and A1298C are significantly associated with some types of congenital defects and adverse pregnancy outcomes, the impact of these polymorphisms is moderate.

A novel ASBT inhibitor, IMB17-15, repressed nonalcoholic fatty liver disease development in high-fat diet-fed Syrian golden hamsters.

The manipulation of bile acid (BA) homeostasis by blocking the ileal apical Na+-dependent bile salt transporter (ASBT/SLC10A2) may have therapeutic effects in nonalcoholic fatty liver disease. We developed a novel ASBT inhibitor, an N-(3,4-o-dichlorophenyl)-2-(3-trifluoromethoxy) benzamide derivative referred to as IMB17-15, and investigated its therapeutic effects and the molecular mechanisms underlying the effects. Syrian golden hamsters were challenged with high-fat diet (HFD) to induce NAFLD and were subsequently administered 400 mg/kg IMB17-15 by gavage daily for 21 days. Serum, liver, and fecal samples were collected for further analysis. Plasma concentration-time profiles of IMB17-15 were also constructed. The human hepatocyte cell line HL-7702 was treated with Oleic acid (OA) with or without IMB17-15. Western blotting and real-time PCR were used to study the molecular mechanisms of IMB17-15. We found that IMB17-15 inhibited ASBT and subsequently suppressed ileal farnesoid X receptor (FXR) and FXR-activated fibroblast growth factor15/19 (FGF15/19) expression, which reduced the hepatic phosphorylated extracellular regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) levels and upregulated the cholesterol 7α-hydroxylase (CYP7A1) activity. Additionally, IMB17-15 stimulated adenosine monophosphate (AMP)-activated protein kinase (AMPKα) phosphorylation and enhanced peroxisome proliferator activated receptor α (PPARα) expression and thus promoted triglyceride (TG) oxidation and high-density lipoprotein cholesterol (HDL-c) metabolism through an ASBT-independent mechanism. In conclusion, a novel ASBT inhibitor known as IMB17-15 protected hamsters against HFD-induced NFALD by manipulating BA and lipid homeostasis. IMB17-15 also reduced lipid deposition in human hepatic cell lines, indicating that it may be useful as a therapy for NAFLD patients.

Na+-taurocholate cotransporting polypeptide (NTCP/SLC10A1) ortholog in the marine skate Leucoraja erinacea is not a physiological bile salt transporter.

The Na+-dependent taurocholate cotransporting polypeptide (NTCP/SLC10A1) is a hepatocyte-specific solute carrier, which plays an important role in maintaining bile salt homeostasis in mammals. The absence of a hepatic Na+-dependent bile salt transport system in marine skate and rainbow trout raises a question regarding the function of the Slc10a1 gene in these species. Here, we have characterized the Slc10a1 gene in the marine skate, Leucoraja erinacea The transcript of skate Slc10a1 (skSlc10a1) encodes 319 amino acids and shares 46% identity to human NTCP (hNTCP) with similar topology to mammalian NTCP. SkSlc10a1 mRNA was mostly confined to the brain and testes with minimal expression in the liver. An FXR-bile salt reporter assay indicated that skSlc10a1 transported taurocholic acid (TCA) and scymnol sulfate, but not as effectively as hNTCP. An [3H]TCA uptake assay revealed that skSlc10a1 functioned as a Na+-dependent transporter, but with low affinity for TCA (Km = 92.4 µM) and scymnol sulfate (Ki = 31 µM), compared with hNTCP (TCA, Km = 5.4 µM; Scymnol sulfate, Ki = 3.5 µM). In contrast, the bile salt concentration in skate plasma was 2 µM, similar to levels seen in mammals. Interestingly, skSlc10a1 demonstrated transport activity for the neurosteroids dehydroepiandrosterone sulfate and estrone-3-sulfate at physiological concentration, similar to hNTCP. Together, our findings indicate that skSlc10a1 is not a physiological bile salt transporter, providing a molecular explanation for the absence of a hepatic Na+-dependent bile salt uptake system in skate. We speculate that Slc10a1 is a neurosteroid transporter in skate that gained its substrate specificity for bile salts later in vertebrate evolution.

The anti-fibrotic effects of epigallocatechin-3-gallate in bile duct-ligated cholestatic rats and human hepatic stellate LX-2 cells are mediated by the PI3K/Akt/Smad pathway.

AIM: (-)-Epigallocatechin-3-gallate (EGCG) is one of the most abundant polyphenols in green tea with strong antioxidant activity and various therapeutic effects. In this study, we investigated the anti-fibrotic effects of EGCG and underlying mechanisms in bile duct-ligated (BDL) rats and a liver fibrosis model in vitro. METHODS: BDL rats were treated with EGCG (25 mg·kg(-1)·d(-1), po) for 14 d, and then the serum, bile and liver samples were collected. Liver fibrosis was assessed by serum, urine and bile biochemistry analyses and morphological studies of liver tissues. TGF-ß1-stimulated human hepatic stellate LX-2 cells were used as a liver fibrosis model in vitro. The expression of liver fibrogenic genes and signaling proteins in the PI3K/Akt/Smad pathway was examined using Western blotting and/or real-time PCR. RESULTS: In BDL rats, EGCG treatment significantly ameliorates liver necrosis, inflammation and fibrosis, and suppressed expression of the genes associated with liver inflammation and fibrogenesis, including TNF-α, IL-1ß, TGF-ß1, MMP-9, α-SMA, and COL1A1. In LX-2 cells, application of EGCG (10, 25 µmol/L) dose-dependently suppressed TGF-ß1-stimulated expression of COL1A1, MMP-2, MMP-9, TGF-ß1, TIMP1, and α-SMA. Furthermore, EGCG significantly suppressed the phosphorylation of Smad2/3 and Akt in the livers of BDL rats and in TGF-ß1-stimulated LX-2 cells. Application of LY294002, a specific inhibitor of PI3K, produced similar effects as EGCG did in TGF-ß1-stimulated LX-2 cells, but co-application of EGCG and LY294002 did not produce additive effects. CONCLUSION: EGCG exerts anti-fibrotic effects in BDL rats and TGF-ß1-stimulated LX-2 cells in vitro via inhibiting the PI3K/Akt/Smad pathway.

Lidamycin inhibits tumor initiating cells of hepatocellular carcinoma Huh7 through GSK3ß/ß-catenin pathway.

Recently, tumor initiating cells are considered as the central role of tumorigenicity in hepatocellular carcinoma. Enediyne anticancer antibiotic lidamycin with great potential antitumor activity is currently evaluated in Phase II clinical trials. In this study, we evaluated the effect of lidamycin on tumor initiating cells of hepatocellular carcinoma Huh7 and identified the potential mechanism. Flow cytometry analysis and sorting assay, surface marker assay, sphere formation assay, and aldefluor assay were used to evaluate the effect of lidamycin on Huh7 tumor initiating cells in vitro. To investigate the potential mechanism, the activity of GSK3ß/ß-catenin pathway was detected by Western blot and T cell factors transcriptional activity assay. Subcutaneous tumor model in nude mice was used to observe in vivo effect of lidamycin on Huh7 cells. Lidamycin decreased the proportion of EpCAM+ cells and the expression of EpCAM protein. Lidamycin inhibited sphere formation of sorted EpCAM+ cells in 7 d, and of parental cells in three serial passages. The population of aldehyde dehydrogenase-positive cells was reduced by lidamycin. In addition, lidamycin restrained tumor volume and incidence in vivo. Lidamycin activated GSK3ß, and degraded the activity of ß-catenin. Consequently, transcriptional activity of ß-catenin/T cell factors was decreased. In brief, these results suggest that lidamycin suppressed Huh7 tumor initiating cells via GSK3ß/ß-catenin pathway. These findings reveal the potential mechanism of lidamycin on tumor initiating cells and the benefit for further clinical evaluation.

RasGAP-derived peptide GAP159 enhances cisplatin-induced cytotoxicity and apoptosis in HCT116 cells.

To increase the efficacy of currently used anti-cancer genotoxins, one of the current efforts is to find agents that can sensitize cancer cells to genotoxins so that the efficacious doses of genotoxins can be lowered to reduce deleterious side-effects. In this study, we reported that a synthetic RasGAP-derived peptide GAP159 could enhance the effect of chemotherapeutic agent cisplatin (CDDP) in human colon carcinoma HCT116 cells. Our results showed that GAP159 significantly increased the CDDP-induced cytotoxicity and apoptosis in HCT116 cells. This synergistic effect was associated with the inhibitions of phospho-AKT, phospho-ERK and NF-κB. In mouse colon tumor CT26 animal models, GAP159 combined with CDDP significantly suppressed CT26 tumor growth, and GAP159 alone showed slight inhibitory effect. Our data suggests that co-treatment of GAP159 and chemotherapeutics will become a potential therapeutic strategy for colon cancers.

The blockage of Ras/ERK pathway augments the sensitivity of SphK1 inhibitor SKI II in human hepatoma HepG2 cells.

The treatment of hepatocellular carcinoma (HCC) remains a challenge and the future of cancer therapy will incorporate rational combinations directed to molecular targets that cooperate to drive critical pro-survival signaling. Sphingosine kinase 1 (SphK1) has been shown to regulate various processes important for cancer progression. Given the up-regulated expression of SphK1 in response to the silence of N-ras and other interactions between Ras/ERK and SphK1, it was speculated that combined inhibition of Ras/ERK and SphK1 would create enhanced antitumor effects. Experimental results showed that dual blockage of N-ras/ERK and SphK1 resulted in enhanced growth inhibitions in human hepatoma cells. Similarly, MEK1/2 Inhibitor U0126 potentiated SKI II-induced apoptosis in hepatoma HepG2 cells, consistently with the further attenuation of Akt/ERK/NF-κB signaling pathway. It was also shown that the combination of SKI II and U0126 further attenuated the migration of hepatoma HepG2 cells via FAK/MLC-2 signaling pathway. Taken together, the dual inhibition of SphK1 and Ras/ERK pathway resulted in enhanced effects, which might be an effective therapeutic approach for the treatment of HCC.