Abstract. Aim: The aim of the present study was to investigate the efficacy of the traditional Chinese medicine (TCM), astragaloside IV (AS-IV) and curcumin on tumor growth and angiogenesis in an orthotopic nude-mouse model of human hepatocellular carcinoma (HCC (FGF2), matrix metalloproteinase 2 (MMP2), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), thrombosis-related factor tissue factor (TF) and coagulation factor VII (FVII), as well as microRNAs miR-122 and miR-221. Results: AS-IV and curcumin alone and in combination significantly reduced mean tumor weight compared to vehicle control (p<0.05). Tumor microvessel count was reduced by AS-IV and curcumin alone. Expression of FGF2, MMP2, VEGF, HGF, TF and FVII was reduced by AS-IV and curcumin alone. AS-IV and curcumin alone up-regulated expression of miR-122 and down-regulated that of miR-221. The combination of AS-IV and curcumin demonstrated significant synergistic effects on microvessel count as well as on expression of angiogenic and thrombosis-related factors and microRNAs. Conclusion: The present study indicates future clinical potential of combination therapy with AS-IV and curcumin for HCC.Hepatocellular carcinoma (HCC) is a highly aggressive and hypervascular tumor, associated with high morbidity (1), and is recalcitrant to therapy (2). Targeting angiogenesis could be an effective therapeutic strategy for controlling the progression of HCC.Traditional Chinese Medicine (TCM) has been widely used for anticancer treatment in China with a long history. Astragaloside IV (AS-IV) is a representative constituent of Astragalus membranaceus (Fisch.) Bge. Var. mongholicus (Bge.) Hsiao and has various pharmacological activities, including anti-tumor, anti-inflammatory and anti-diabetic (3, 4). Curcumin (diferuloylmethane) is the chief component of the spice turmeric and is derived from the rhizome of the East Indian plant Curcuma longa. Curcumin exhibits anti inflammatory, antioxidant and anticancer efficacy (5). AS-IV was reported to reduce invasiveness and angiogenesis of gastric cancer cells and down-regulate the expression of fibroblast growth factor-2 (FGF-2) and suppress angiogenesis of HCT-116 colon cancer cells (6, 7). Curcumin showed anti-metastatic and anti-angiogenesis efficacy against small-cell lung cancer 465

It has been reported that miR-615-5p was upregulated in hepatocellular carcinoma (HCC) preventing both growth and migration. However, the underlying mechanism by which miR-615-5p played a role in HCC remains unknown. Here, in our present study, to investigate the mechanism of miR-615-5p, bioinformatic prediction and luciferase reporter assay were employed to ascertain the downstream target of miR-615-5p finding that the serine hydromethyltransferase 2 (SHMT2) was the direct downstream target. Knockdown or overexpression of miR-615-5p can lead to increasing or decreasing expression of SHMT2 in HCC cells. Besides, knockdown or overexpression of SHMT2 can suppress or promote both proliferation and migration of HCC cells, indicating that miR-615-5p can directly and negatively regulate the SHMT2 in HCC cells. In addition, to understand the clinicopathological significance of SHMT2 expression in HCC, immunohistochemistry was performed. It was found that SHMT2 expression was significantly associated with poor prognosis and TNM stage. Together, our results for the first time showed that miR-615-5p prevents proliferation and migration through negatively regulating SHMT2 in HCC.

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Mutations were evaluated in workers in an iron foundry with exposure to polycyclic aromatic hydrocarbons (PAHs), measured by personal and area monitoring, ranging from < 5 to 60 ng/m3 of benzo[a]pyrene (B[a]P). Mutation at the hypoxanthine guanine phosphoribosyl transferase (HPRT) and glycophorin A (GPA) loci (measures of molecular effect in lymphocytes and erythrocytes respectively) were assessed to demonstrate their relationship to external exposure at lower levels than previously analyzed in foundry workers at this plant (< 50-200 ng/m3). The relationship between mutations and PAH-DNA adducts measured by immunoassay (as a measure of the biologically effective dose) was also investigated. The markers were analyzed for dose-response and interindividual variability. Workers were classified into three exposure categories (low, medium and high). PAH-DNA adduct values for the low, medium and high exposure groups were 5.19, 6.10 and 9.57 x 10(-8) nucleotides respectively (r = 0.28; P = 0.08). HPRT mutant frequencies (adjusted for age and cloning efficiency) for the low, medium and high exposure groups were 1.04, 1.13 and 1.82 x 10(-6) cells respectively and demonstrated an upward trend with increasing exposure that was of borderline significance (r = 0.46, P = 0.06). In contrast, HPRT mutations were highly correlated with PAH-DNA adducts (r = 0.67; P = 0.004). Interindividual variability in mutant frequencies ranged from 1.5- to 4.5-fold within the three exposure categories. With respect to GPA variants, NN frequency (Vf) in erythrocytes (which reflects chromosomal loss and duplication, recombination or gene conversion) was not positively correlated with PAH exposure. The level of N0 Vf (arising from small-scale structural mutations in the GPA gene or from larger-scale chromosomal rearrangements or deletions) increased slightly, but not significantly, over the three exposure groups from 8.2 to 10.7 to 11.8/10(6) cells (P = 0.32). Interindividual variation in GPA NN Vf ranged from 2- to 18-fold and in GPA N0 from 4- to 5-fold. NN and N0 Vf were highly correlated (P = 0.001) but no correlation was seen between GPA and HPRT or between GPA and PAH-DNA adducts. Thus, the most interesting and novel finding is that, even at relatively low exposures to PAH, HPRT mutations were increased in parallel with PAH-DNA adducts. The observed association between PAH-DNA adducts and HPRT gene mutation in humans is consistent with experimental data for PAHs. These results support the use of both biomonitoring and personal ambient monitoring in further molecular epidemiology studies.

Altered gut microbiota and a damaged colon mucosal barrier have been implicated in the development of colon cancer. Astragalus mongholicus Bunge-Curcuma aromatica Salisb. (ACE) is a common herbal drug pair that widely used clinically to treat cancer. However, whether the anti-cancer effect of ACE is related to gut microbiota remains unclear yet. We standardized ACE and investigated the effects of ACE on tumour suppression and analyze the related mechanisms on gut microbiota in CT26 colon cancer-bearing mice in the present study. Firstly, four flavonoids (calycosin-7-glucoside, ononin, calycosin, formononetin) and three astragalosides (astragaloside A, astragaloside II, astragaloside I) riched in Astragalus mongholicus Bunge, three curcumins (bisdemethoxycurcumin, demethoxycurcumin, curcumin) and four essential oils (curdione, curzerene, germacrone and β-elemene) from Curcuma aromatica Salisb., in concentrations from 0.08 to 2.07 mg/g, were examined in ACE. Then the results in vivo studies indicated that ACE inhibited solid tumours, liver and spleen metastases of colon cancer while simultaneously reducing pathological tissue damage. Additionally, ACE regulated gut microbiota dysbiosis and the short chain fatty acid content in the gut, repaired intestinal barrier damage. ACE treatment suppressed the overgrowth of conditional pathogenic gut bacteria, including Escherichia-Shigella, Streptococcus and Enterococcus, while the probiotic gut microbiota like Lactobacillus, Roseburia, Prevotellaceae_UCG-001 and Mucispirillum were increased. More interestingly, the content level of SCFAs such as propionic acid and butyric acid was increased after ACE administration, which further mediates intestinal SDF-1/CXCR4 signalling pathway to repair the integrity of the intestinal barrier, decrease Cyclin D1 and C-myc expressions, eventually suppress the tumor the growth and metastasis of colon cancer. To sum up, the present study demonstrated that ACE could efficiently suppress colon cancer progression through gut microbiota modification, which may provide a new explanation of the mechanism of ACE against colon cancer.

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Normalizing the disordered tumor vasculature, rather than blocking it, is a novel method for anticancer therapy. Astragali polysaccharide (APS) and curcumin were reported to be active against carcinomas. However, the effect and mechanism of the combination of APS and curcumin on vascular normalization in hepatocellular carcinoma (HCC) was not clear. In the present study, effects of combined APS and curcumin on tumor vascular normalization were evaluated in HepG2 tumor-bearing mice. Photoacoustic tomography (PAT) was performed to observe the morphological structure of tumor vessels in vivo. The microstructure of the tumor vessels was also analyzed through scanning electron microscopy. Additionally, the expression of CD31 and NG2 was analyzed by immunohistochemical staining. Tumor vessels of HepG2 tumor-bearing mice treated with the combination were sparse with uniform growth, morphology rules, and complete vascular walls, which had fewer branches and sprouts. ECs of tumor vessels were arranged regularly and were tightly connected, tending toward normalization. The expression of CD31 was reduced while NG2 was increased significantly by the combination of APS and curcumin. The results indicated that APS and curcumin in combination showed a better effect on inhibiting tumor growth in an orthotopic nude-mouse model of HCC. More important, the combination induced normalization of tumor vascular better than APS or curcumin administration alone, improving the morphological structure of tumor vessels and promoting maturation of tumor vessels. The results of the present study provided a reasonable possibility for combination therapy of APS and curcumin in the treatment of HCC via tumor vascular normalization.

Background/Aim: The aim of the present study was to investigate the vascular normalization effect of traditional Chinese medicine Astragalus membranaceus (AM) and Curcuma wenyujin (CW) on tumor-derived endothelial cells (TECs). Materials and Methods: TECs were isolated from the xenografted HCC cell line HepG2 expressing red fluorescent protein (RFP). The effect of AM and CW on TECs proliferation was measured using the CCK8 assay. The vascular normalization potential of AM and CW was assessed using a tube formation assay. Immunocytochemistry was performed to assess the effect of AM and CW on the expression of angiogenic maker CD34 and hypoxia-inducible factor HIF1a. Results: The isolated TECs and endothelioma (EOMA) cells did not differ with regard to the expression levels of endothelial markers CD34, CW, AM+CW and Nintedanib (Nin) showed a dose-dependent increasing inhibition effect on either TECs or EOMA cells. AM, CW and AM+CW significantly reduced HIF1a expression, increased CD34 expression and enhanced endothelial network formation in TECs or EOMA cells compared to the control. Conclusion: AM and CW promoted vascular normalization in tumor-derived endothelial cells of HCC, through increased expression of CD34 and reduced expression of HIF1a. Zang et al: Effect of Astragalus membranaceus and Curcuma wenyujin on Tumor Vascular Normalization