Andrographolide causes p53-independent HCC cell death through p62 accumulation and impaired DNA damage repair.

BACKGROUND: Hepatocellular carcinoma (HCC) is a highly lethal cancer characterized by dominant driver mutations, including p53. Consequently, there is an urgent need to search for novel therapeutic agents to treat HCC. Andrographolide (Andro), a clinically available anti-inflammatory phytochemical agent, has shown inhibitory effects against various types of cancer, including HCC. However, the underlying molecular mechanisms of its action remain poorly understood. PURPOSE: This study aims to investigate the molecular mechanisms by which p53 and p62 collectively affect Andro-induced HCC cell death, using both in vitro and in vivo models. METHODS: In vitro cellular experiments were conducted to examine the effects of Andro on cell viability and elucidate its mechanisms of action. In vivo xenograft experiments further validated the anti-cancer effects of Andro. RESULTS: Andro induced dose- and time-dependent HCC cell death while sparing normal HL-7702 hepatocytes. Furthermore, Andro caused DNA damage through the generation of reactive oxygen species (ROS), a critical event leading to cell death. Notably, HCC cells expressing p53 exhibited greater resistance to Andro-induced cell death compared to p53-deficient cells, likely due to the ability of p53 to induce G2/M cell cycle arrest. Additionally, Andro-induced p62 aggregation led to the proteasomal degradation of RAD51 and 53BP1, two key proteins involved in DNA damage repair. Consequently, silencing or knocking out p62 facilitated DNA damage repair and protected HCC cells. Importantly, disruption of either p53 or p62 did not affect the expression of the other protein. These findings were further supported by the observation that xenograft tumors formed by p62-knockout HCC cells displayed increased resistance to Andro treatment. CONCLUSION: This study elucidates the mechanistic basis of Andro-induced HCC cell death. It provides valuable insights for repurposing Andro for the treatment of HCC, regardless of the presence of functional p53.

Sodium Butyrate Pre-Treatment Enhance Differentiation of Bone Marrow Mesenchymal Stem Cells (BM-MSCs) into Hepatocytes and Improve Liver Injury.

OBJECTIVE: The treatment of liver failure by stem cell transplantation has attracted growing interest. Herein, we aim to explore the role of sodium butyrate (NaB) in the hepatic differentiation of bone marrow mesenchymal stem cells (BM-MSCs) under liver-specific factors induction in vitro and vivo. MATERIALS & METHODS: We isolated BM-MSCs from the mononuclear cell fraction of rabbit bone marrow samples and identified the cells by Immunophenotypic analysis. We investigated the effects of different concentrations and induction conditions. The histone deacetylase inhibitor NaB induced hepatic differentiation of BM-MSCs under liverspecific factors induction in vitro. Morphological features, liver-specific gene and protein expression, and functional analyses in vitro and vivo were performed to evaluate the hepatic differentiation of BM-MSCs. RESULTS: Our results showed that pre-treated NaB inhibited the expression of the liverspecific protein in a dose-dependent manner. The induction efficiency of NaB with 24h pre-treatment was higher than that of NaB continuous intervention. 0.5 mM 24h NaB pre-treated cells can improve liver tissue damage in vivo. The liver ALB, AAT, and the serum TP were significantly increased, while the serum ALT was significantly reduced. CONCLUSION: Continuous NaB treatment can inhibit BM-MSCs proliferation in a dosedependent manner at a certain concentration range. 0.5 mM 24h pre-treatment of NaB enhanced differentiation of BM-MSCs into hepatocytes and improved liver injury in vitro and vivo.

Zebularine Promotes Hepatic Differentiation of Rabbit Bone Marrow Mesenchymal Stem Cells by Interfering with p38 MAPK Signaling.

Demethylating agent zebularine is reported to be capable of inducing differentiation of stem cells by activation of methylated genes, though its function in hepatocyte differentiation is unclear. p38 signal pathway is involved in differentiation of hepatocytes and regulating of DNA methyltransferases 1 (DNMT1) expression. However, little is known about the impact of zebularine on bone marrow mesenchymal stem cells (BMMSCs) and p38 signaling during hepatic differentiation. The present study investigated the effects of zebularine on hepatic differentiation of rabbit BMMSCs, as well as the role of p38 on DNMT1 and hepatic differentiation, with the aim of developing a novel strategy for improving derivation of hepatocytes. BMMSCs were treated with zebularine at concentrations of 10, 20, 50, and 100 µM in the presence of hepatocyte growth factor; changes in the levels of hepatic-specific alpha-fetoprotein and albumin were detected and determined by RT-PCR, WB, and immunofluorescence staining. Expression of DNMT1 and phosphorylated p38 as well as urea production and ICG metabolism was also analyzed. Zebularine at concentrations of 10, 20, and 50 µM could not affect cell viability after 48 h. Zebularine treatment leads to an inhibition of DNMT activity and increase of hepatic-specific proteins alpha-fetoprotein and albumin in BMMSCs in vitro; zebularine addition also induced expression of urea production of and ICG metabolism. p38 signal was activated in BMMSCs simulated with HGF; inhibition of p38 facilitated the synthesis of DNMT1 and albumin in cells. Zebularine restrained DNMT1 and phosphorylated p38 which were induced by HGF. Therefore, this study demonstrated that treatment with zebularine exhibited terminal hepatic differentiation of BMMSCs in vitro in association with hepatocyte growth factor; p38 pathway at least partially participates in zebularine-induced hepatic differentiation of rabbit BMMSCs.

Continuous zebularine treatment enhances hepatic differentiation of mesenchymal stem cells under liver-specific factors induction in vitro.

AIMS: To investigate the effect of zebularine, a stable inhibitor of DNA methylation, on hepatic differentiation of bone marrow-derived mesenchymal stem cells (BM-MSCs) under liver-specific factors induction in vitro. MAIN METHODS: BM-MSCs were isolated from the mononuclear cell fraction of rabbit bone marrow samples. The identification of these cells was carried out by immunophenotype analysis. The three hepatic differentiation protocols of BM-MSCs were as follows: liver-specific factors (hepatocyte growth factor and epidermal growth factor) without zebularine, liver-specific factors combined with a 24 h zebularine pre-treatment, and liver-specific factors combined with continuous zebularine treatment. BM-MSCs cultured in basic medium without the differentiation stimuli were set as the control. Morphological features, liver-specific gene and protein expression, and functional analyses were assessed to evaluate hepatic differentiation of BM-MSCs. Global DNA methylation status was tested for investigating the underlying mechanism. KEY FINDINGS: Flow cytometry immunophenotyping proved the isolated cells with plastic adherence and a spindle shape were CD29, CD90 positive and CD34, CD45 negative. Albumin (ALB) and alpha-fetoprotein (AFP) messenger RNA and protein expression, glycogen storage and urea production were significantly higher in the continuous zebularine-treated group than the other groups while the differences between the zebularine-untreated group and 24 h zebularine pre-treated group were not significant. Meanwhile, significant decrease of global DNA methylation was observed in the continuous zebularine-treated group. SIGNIFICANCE: We conclude that continuous zebularine treatment can improve hepatic differentiation of BM-MSCs under liver-specific factors induction in vitro, and the decrease of global DNA methylation maybe involved in this process.

RSK4 knockdown promotes proliferation, migration and metastasis of human breast adenocarcinoma cells.

RSK4 has been shown to inhibit the growth of certain cancer cells. The aim of this study was to construct a lentiviral vector of RSK4-shRNA (Lenti-RSK4-shRNA) to specifically block the expression of RSK4 in the human breast adenocarcinoma cell line MCF-7, and investigate the effect of the RSK4 gene on cell proliferation and invasion in vitro and in vivo. Lenti-RSK4-shRNA was stably transfected into MCF-7 cells. RSK4 mRNA and protein expression were measured by fluorescence quantitative RT-PCR and western blot analysis. Cell proliferation was evaluated by MTT assays and flow cytometric analysis. Invasion was evaluated by Transwell assays and xenograft nude mouse models. The expression of RSK4 mRNA, Ki-67 mRNA, cyclin D1 mRNA, CXCR4 mRNA and E-cadherin mRNA of tumor xenografts were detected by fluorescence quantitative RT-PCR. Significant decreases in RSK4 mRNA and protein expression was detected in MCF-7 cells carrying lentiviral RSK4-shRNA vector. The cell proliferation was significantly promoted in the RSK4-shRNA group as compared to that in the negative and blank control group. In addition, the number of cells in the S phase in the RSK4­shRNA group was significantly greater than the blank and negative control groups (P<0.05). Furthermore, the number of invading cells was increased in the RSK4-shRNA (P<0.05). In vivo, we also found that the knockdown of RSK4 promoted tumorigenicity and migration in the xenograft nude mouse model. In addition, we showed that the RSK4 mRNA and E-cadherin mRNA expression were significantly lower in the RSK4-shRNA group compared to that in negative and blank control group (both P<0.05), while the Ki-67 mRNA, cyclin D1 mRNA and CXCR4 mRNA were higher in the shRNA group compared to that in negative and blank control group (both P<0.05). In conclusion, downregulation of RSK4 expression is indicated to be associated with tumor cell proliferation and invasion, and silencing of the RSK4 may be involved in the development and progression of breast cancer through the changes of Ki-67, cyclin D1, CXCR4 and E-cadherin, and suggesting that RSK4 may act as a potential cancer suppressor gene and therapeutic target for the treatment of breast cancer.

Three-component domino reactions for selective formation of indeno[1,2-b]indole derivatives.

Efficient three-component domino strategies for the synthesis of multifunctionalized tetracyclic indeno[1,2-b]indole derivatives with different substituted patterns have been established successfully. The first pathway involves a novel sequential methyl migration, aromatization, and esterification, while a second reaction in HOAc leads to compounds 6 with high syn diastereoselectivity. Both reactions showed attractive features including mild conditions, convenient one-pot operation, short reaction times of 15-32 min, and excellent regio- and/or stereoselectivity.

Efficient domino approaches to multifunctionalized fused pyrroles and dibenzo[b,e][1,4]diazepin-1-ones.

Efficient domino approaches for the synthesis of multifunctionalized tricyclic fused pyrroles and dibenzo[b,e][1,4]diazepin-1-ones have been established. The reaction pathways were controlled by varying enaminones with different substituted patterns to give a series of new fused pyrroles and dibenzo[b,e][1,4]diazepin-1-ones selectively. The complete anti diastereoselectivity was achieved for the first reaction.

[Phase I study of docetaxel combined with carboplatin in patients with non-small cell lung cancer].

OBJECTIVE: To determine the maximum tolerated dose (MTU) and dose-limiting toxicity (DLT) of Docetaxel and Carboplatin in patients with non-small cell lung cancer. METHODS: Docetaxel was given at escalating doses until MTD was determined from the initial dose of 65 mg/m2 to 75 mg/m2, 85 mg/ m2 on dl. Carboplatin was targeted to an area under the plasma concentration curve of 5 using Calver's equation on dl. The treatment cycle was repeated every 3 weeks. RESULTS: 16 patients received TXT and CBP for total of 54 courses (median four courses). Neutropenia was the dose-limiting toxicity. The MTD of TXT is 85 mg/m2. CONCLUSION: We recommend TXT 75 mg/m2 on d1 and CBP with a target AUC of 5 on d1, 3weeks repeated for chemotherapy in naïve patients with NSCLC.