Hu-Qi-Zheng-Xiao Decoction Inhibits the Metastasis of Hepatocellular Carcinoma Cells by Suppressing the HIF-1α Signaling Pathway to Inhibit EMT, LCSC, and Angiogenic Process.

BACKGROUND: Hepatocellular carcinoma (HCC) is a common clinical malignant tumor of the digestive system. Hu-Qi-Zheng-Xiao (HQZX) decoction has been clinically found to prolong the survival of patients with hepatocellular carcinoma and improve the quality of patients' survival, but its antitumor biological mechanism is still unclear. METHODS: A nude mouse hollow fiber hepatocellular carcinoma model was constructed to analyze the in vivo efficacy of HQZX decoction against 7 different hepatocellular carcinoma cells. The subcutaneous graft tumor model was again validated. In vitro, the effect of HQZX decoction on the growth and metastasis of the cell line with the highest growth inhibition was evaluated. The cell line with the best efficacy response screened was again used to construct a hollow fiber hepatocellular carcinoma model and hollow fiber conduit cells were extracted to detect the expression of HIF-1α, VEGF, EMT-related molecules, LCSCs-related molecules, and to observe the density of the subcutaneous vascular network of hollow fiber conduits. The liver metastasis model of splenic injection was constructed to observe the effect of HQZX decoction on tumor metastasis. RESULTS: The hollow fiber hepatocellular carcinoma model was evaluated for the efficacy of HQZX decoction, and it was found to have the highest growth inhibition of LM3-luc cells. In vitro, the CCK8 assay revealed that HQZX decoction could inhibit tumor migration and invasion and promote apoptosis. In addition, the mechanism study of extracting cells from hollow fiber tubes found that HQZX decoction could inhibit metastasis-associated HIF-1α, VEGF, EMT-related molecules, and LCSCs-related molecules expression. capillary network around subcutaneous fiber tubes was reduced in the HQZX decoction gavage group of mice. It inhibited tumor metastasis in nude mice. CONCLUSIONS: HQZX decoction inhibited the growth of a variety of hepatocellular carcinoma cells. HQZX decoction suppressed the expression of metastasis-associated VEGF, EMT-related molecules, and LCSCs-related molecules and inhibited tumor angiogenesis and growth and metastasis, which may be related to the inhibition of the HIF-1α signaling pathway. It reveals that HQZX decoction may be a promising herbal compound for anti-HCC therapy, and also reveals the accurate feasibility of the hollow fiber hepatocellular carcinoma model for in vivo pharmacodynamic evaluation and mechanism study.

TP53BP2: Roles in suppressing tumorigenesis and therapeutic opportunities.

Malignant tumor is still a major problem worldwide. During tumorigenesis or tumor development, tumor suppressor p53-binding protein 2 (TP53BP2), also known as apoptosis stimulating protein 2 of p53 (ASPP2), plays a critical role in p53 dependent and independent manner. Expression of TP53BP2 is highly correlated with the prognosis and survival rate of malignant tumor patients. TP53BP2 can interact with p53, NF-κB p65, Bcl-2, HCV core protein, PP1, YAP, CagA, RAS, PAR3, and other proteins to regulate cell function. Moreover, TP53BP2 can also regulate the proliferation, apoptosis, autophagy, migration, EMT and drug resistance of tumor cells through downstream signaling pathways, such as NF-κB, RAS/MAPK, mevalonate, TGF-ß1, PI3K/AKT, aPKC-ι/GLI1 and autophagy pathways. As a potential therapeutic target, TP53BP2 has been attracted more attention. We review the role of TP53BP2 in tumorigenesis or tumor development and the signal pathway involved in TP53BP2, which may provide more deep insight and strategies for tumor treatment.

Dysregulated hepatic lipid metabolism and gut microbiota associated with early-stage NAFLD in ASPP2-deficiency mice.

Background: Growing evidence indicates that lipid metabolism disorders and gut microbiota dysbiosis were related to the progression of non-alcoholic fatty liver disease (NAFLD). Apoptosis-stimulating p53 protein 2 (ASPP2) has been reported to protect against hepatocyte injury by regulating the lipid metabolism, but the mechanisms remain largely unknown. In this study, we investigate the effect of ASPP2 deficiency on NAFLD, lipid metabolism and gut microbiota using ASPP2 globally heterozygous knockout (ASPP2+/-) mice. Methods: ASPP2+/- Balb/c mice were fed with methionine and choline deficient diet for 3, 10 and 40 day to induce an early and later-stage of NAFLD, respectively. Fresh fecal samples were collected and followed by 16S rRNA sequencing. HPLC-MRM relative quantification analysis was used to identify changes in hepatic lipid profiles. The expression level of innate immunity-, lipid metabolism- and intestinal permeability-related genes were determined. A spearman's rank correlation analysis was performed to identify possible correlation between hepatic medium and long-chain fatty acid and gut microbiota in ASPP2-deficiency mice. Results: Compared with the WT control, ASPP2-deficiency mice developed moderate steatosis at day 10 and severe steatosis at day 40. The levels of hepatic long chain omega-3 fatty acid, eicosapentaenoic (EPA, 20:5 n-3) and docosahexaenoic (DHA, 22:6 n-3), were decreased at day 10 and increased at day 40 in ASPP+/- mice. Fecal microbiota analysis showed significantly increased alpha and beta diversity, as well as the composition of gut microbiota at the phylum, class, order, family, genus, species levels in ASPP2+/- mice. Moreover, ASPP-deficiency mice exhibited impaired intestinal barrier function, reduced expression of genes associated with chemical barrier (REG3B, REG3G, Lysozyme and IAP), and increased expression of innate immune components (TLR4 and TLR2). Furthermore, correlation analysis between gut microbiota and fatty acids revealed that EPA was significantly negatively correlated with Bifidobacterium family. Conclusion: Our findings suggested that ASPP2-deficiency promotes the progression of NAFLD, alterations in fatty acid metabolism and gut microbiota dysbiosis. The long chain fatty acid EPA was significantly negatively correlated with Bifidobacterial abundance, which is a specific feature of NAFLD in ASPP2-deficiency mice. Totally, the results provide evidence for a mechanism of ASPP2 on dysregulation of fatty acid metabolism and gut microbiota dysbiosis.

ASPP2 reduction attenuates HBV induced chronic liver damage: A hybrid mouse model study.

BACKGROUND & AIM: P53 Apoptosis Stimulating Protein 2 (ASPP2) is confirmed to participate in cellular activities including apoptosis, proliferation, autophagy, injury and so on. However, the role of ASPP2 in Hepatitis B virus (HBV) infection has not been reported in detail. The study explored the role of ASPP2 in HBV induced chronic liver damage. METHODS: Transcriptome profiling of ASPP2-konckdown mouse liver were analyzed by RNA-sequencing. HBV-ASPP2-knockdown mice was the hybrid offspring of HBV transgenic mice and ASPP2 knockdown mice. Liver tissues were taken for the experiments such as western Blot (WB), PCR, Hematoxylin and Eosin (HE), Immunohistochemistry and high throughput sequencing of transcriptome. RESULTS: Pathological and transcriptomic analysis of liver tissue from ASPP2 knockdown vs con mice showed that after ASPP2 knockdown, the pathological changes in the liver tissue of mice were not significant, but transcriptomics showed obvious changes in immune system process, and response to stimulus, metabolism, Human Diseases and other directions etc. In the HBV-ASPP2-knockdown mice, liver tissue HE staining found less cell swelling and necrosis foci; F4/80 and MPO staining showed less inflammatory cell infiltration; serum ALT and AST decreased than the HBV-ASPP2-con mice. Transcriptome results showed significantly changed in HBV-ASPP2-knockdown mice including immune system process, inflammatory response, and innate immune response etc. Further comparison of the two transcriptomes yielded 9 identical pathways related to inflammatory and cell injury. The PPAR pathway was verified, and found that the increase of PPARγ caused by the reduction of ASPP2 is likely to be the reason for the reduction of HBV-related liver injury. The expression of PPARγ was then analyzed by transcriptome and PCR, it was found that in the absence of HBV, ASPP2 knockdown resulted in a mild decrease in PPARγ, and in the presence of HBV infection, ASPP2 knockdown resulted in a marked increase in PPARγ.In addition, this study found that high expression of ASPP2 had opposite effects on HCC (HBV-none) and HCC (HBV-yes). CONCLUSION: This study demonstrated that reduction of ASPP2 reduces HBV-induced hepatocyte damage during chronic HBV infection. This phenomenon is related to the different regulation of PPARγ by ASPP2 in the presence or absence of HBV stimulation.

Knockout of ASPP2 promotes DEN-induced hepatocarcinogenesis via the NF-κB pathway in mice.

Apoptosis-stimulating protein p53 2 (ASPP2) is a member of the p53-binding protein family, which is closely related to tumor development. However, the precise mechanism of ASPP2 in liver inflammation and tumorigenesis remains largely unclear. We aimed to characterize the mechanistic significance and clinical implication of ASPP2 in hepatitis and hepatocellular carcinoma (HCC). In this study, ASPP2 knockout (APKO) mice were generated to confirm the role of ASPP2 in the development of hepatitis and HCC. Liver tissues from mice were analyzed by immunohistochemistry, Western blotting, proteomic analysis, ChIP-Seq, and qRT-PCR to evaluate the role of ASPP2 in DEN-induced hepatitis and HCC. We found that APKO promoted the formation of hepatitis/hepatocarcinoma and the increased expression of proinflammatory factors. The proteomics and Western blotting results showed that APKO activated the NF-κB signaling pathway. Further, ChIP-Seq results revealed that NF-κB target genes were dramatically increased in APKO mice. In contrast, blockade of the NF-κB pathway by QNZ reduced the expression of proinflammatory factors and the susceptibility of APKO mice to DEN-induced hepatocarcinogenesis. These results suggested that the absence of ASPP2 activates the NF-κB pathway to promote the occurrence of DEN-induced hepatocarcinogenesis, indicating that ASPP2 may be a potential target for the treatment of hepatocarcinoma.

ASPP2 inhibits hepatitis B virus replication by preventing nucleus translocation of HSF1 and attenuating the transactivation of ATG7.

Hepatitis B virus (HBV) is a kind of virus with the capability to induce autophagy, thereby facilitating its replication. Reducing hepatocyte autophagy is proved to be a useful way to inhibit HBV replication. Herein, we reported that p53-binding protein 2 (apoptosis-stimulating protein of p53-2, ASPP2) could attenuate HBV-induced hepatocyte autophagy in a p53-independent manner. Mechanistically, overexpressed ASPP2 binds to HSF1 in cytoplasm of HBV-infected cells, which prevents the translocation of HSF1 to nuclei, thereby inhibiting the transactivation of Atg7. By regulating the transcription of Atg7, ASPP2 reduces hepatocyte autophagy, thereby inhibiting HBV replication. Therefore, ASPP2 is a key regulator of cell autophagy, and overexpression of ASPP2 could be a novel method to inhibit HBV replication in hepatocytes.

Progress and prospects of biomarkers in primary liver cancer (Review).

Tumor biomarkers are important in the early screening, diagnosis, therapeutic evaluation, recurrence and prognosis prediction of tumors. Primary liver cancer is one of the most common malignant tumors; it has high incidence and mortality rates and seriously endangers human health. The main pathological types of primary liver cancer include hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC) and combined HCC­cholangiocarcinoma (cHCC­CC). In the present review, a systematic outline of the current biomarkers of primary liver cancer is presented, from conventional blood biomarkers, histochemical biomarkers and potential biomarkers to resistance­associated biomarkers. The important relationships are deeply elucidated between biomarkers and diagnosis, prognosis, clinicopathological features and resistance, as well as their clinical significance, in patients with the three main types of primary liver cancer. Moreover, a summary of several important biomarker signaling pathways is provided, which is helpful for studying the biological mechanism of liver cancer. The purpose of this review is to provide help for clinical or medical researchers in the early diagnosis, differential diagnosis, prognosis and treatment of HCC.

Antitumor Efficacy of Huqizhengxiao (HQZX) Decoction Based on Inhibition of Telomerase Activity in Nude Mice of Hepatocarcinoma Xenograft.

OBJECTIVE: Huqizhengxiao (HQZX) decoction is a mixture of traditional Chinese medicines comprising 10 herbs, with inhibitory effects on hepatocarcinoma. The aim of the study is to observe the antitumor efficacy and mechanism of HQZX decoction in nude mice with hepatocellular carcinoma xenografts. METHODS: HepG2-luc subcutaneous hepatocarcinoma was established in nude mice. The mice were divided into 5 groups: control, cinobufagin, HQZXS, HQZXM, and HQZXH with doses 13.52, 27.03, and 54.06 g/kg, respectively. HQZX decoction was prepared for intraperitoneal intragastric administration for 3 weeks. Tumor growth was measured with Vernier calipers and in vivo imaging system. α-Fetoprotein (AFP) was determined by radioimmunoassay. Tumor necrosis factor-α (TNF-α) was measured with enzyme-linked immunosorbent assay (ELISA) assay. Telomerase activity was measured with polymerase chain reaction-ELISA. Nuclear mitosis and necrosis were observed with hematoxylin-eosin stain. Apoptotic proteins of caspase-3, Bcl-2, and Bax were examined by Western blot. Signaling molecules of ERK, mTOR, and STAT3 were measured with Luminex assay. RESULTS: HQZX decoction showed good inhibition of HepG2-luc xenografts. Compared with control group, the relative tumor proliferation rate was less than 60% in the HQZXH and HQZXS. The tumor inhibition rate of HQZXH group reached 52% ± 15%. Relative average optical density values of the HQZXS and HQZXH groups decreased significantly. The mitotic index in HQZXS, HQZXM, and HQZXH groups decreased greatly. Telomerase activity of HQZXS was clearly reduced, and, the caspase-3 expression upregulated in HQZXH group. Bcl-2 expression was downregulated in HQZXS and HQZXH. The ratios of p-ERK/ERK and p-STAT3/STAT3 in HQZXS group were significantly downregulated. CONCLUSION: HQZX decoction can clearly inhibit the growth of hepatocellular carcinoma and induce tumor apoptosis. Its antitumor mechanism may be related to reducing telomerase activity and regulating the STAT3 and ERK signal pathway.

Extract of Stellerachamaejasme L(ESC) inhibits growth and metastasis of human hepatocellular carcinoma via regulating microRNA expression.

BACKGROUND: MicroRNAs(miRNAs)are involved in the initiation and progression of hepatocellular carcinoma. ESC, an extract of Stellerachamaejasme L, had been confirmed as a potential anti-tumor extract of Traditional Chinese Medicine. In light of the important role of miRNAs in hepatocellular carcinoma, we questioned whether the inhibitory effects of ESC on hepatocellular carcinoma (HCC) were associated with miRNAs. METHODS: The proliferation inhibition of ESC on HCC cells was measured with MTT assay. The migration inhibition of ESC on HCC cells was measured with transwell assay. The influences of ESC on growth and metastasis inhibition were evaluated with xenograft tumor model of HCC. Protein expressions were measured with western blot and immunofluorescence methods and miRNA profiles were detected with miRNA array. Differential miRNA and target mRNAs were verified with real-time PCR. RESULTS: The results showed that ESC could inhibit proliferation and epithelial mesenchymal transition (EMT) in HCC cells in vitro and tumor growth and metastasis in xenograft models in vivo. miRNA array results showed that 69 differential miRNAs in total of 429 ones were obtained in MHCC97H cells treated by ESC. hsa-miR-107, hsa-miR-638, hsa-miR-106b-5p were selected to be validated with real-time PCR method in HepG2 and MHCC97H cells. Expressions of hsa-miR-107 and hsa-miR-638 increased obviously in HCC cells treated by ESC. Target genes of three miRNAs were also validated with real-time PCR. Interestingly, only target genes of hsa-miR-107 changed greatly. ESC downregulated the MCL1, SALL4 and BCL2 gene expressions significantly but did not influence the expression of CACNA2D1. CONCLUSION: The findings suggested ESC regressed growth and metastasis of human hepatocellular carcinoma via regulating microRNAs expression and their corresponding target genes.

[Keratin 18 phosphorylation increases autophagy of colorectal cancer HCT116 cells and enhanced its sensitivity to oxaliplatin].

OBJECTIVE: To study the correlation between the phosphorylation of keratin 18 (K18) and the autophagy and apoptosis of HCT116 cells under the effect of oxaliplatin (OXA) and investigate its possible mechanism. METHODS: HCT116 cells were transfected with empty plasmid, wild-type K18 expression plasmid and 33, 52 phosphorylation site mutated K18 (Ser33/52A) expression plasmid separately, and all cells were then treated with 60 µmol/L OXA, followed by supplementation of autophagy inhibitor 3-methyladenine (3-MA) or autophagy inducer rapamycin. FITC-conjugated annexin V and propidium iodide (PI) double staining combined with flow cytometry, calcein-AM/PI staining were used to analyze the effects of K18 and its mutants on cell apoptosis; Western blotting was performed to detect the expressions of K18 phosphorylation, autophagy related proteins microtubule associated protein 1 light chain 3 (LC3) and beclin-1. RESULTS: Transfection of Ser33/52A plasmid significantly reduced the level of K18 phosphorylation. After treated with OXA, the apoptosis rate of K18 plasmid transfected group was significantly higher than that of empty plasmid transfected group, while the apoptosis rate of Ser33/52A plasmid transfected HCT116 cells was significantly lower than that of empty plasmid or K18 plasmid transfected group. Compared with empty plasmid group, the autophagy of K18 plasmid transfected group was significantly promoted, while the autophagy in Ser33/52A plasmid transfected group was significantly inhibited. CONCLUSION: K18 overexpression enhanced the autophagy in HCT116 cells and increased its sensitivity to OXA. The decrease of K18 ser33 and ser52 phosphorylation inhibited autophagy and decreased apoptosis of HCT116 cells.