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.

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.