Effect of Babao Dan on angiogenesis of gastric cancer in vitro by regulating VEGFA/VEGFR2 signaling pathway.

BACKGROUND: To further elucidate the anti-angiogenesis effect of Babao Dan (BBD) in vitro, gastric cancer (GC) cells and human umbilical vein endothelial cells (HUVECs) were used to evaluate the regulation role of BBD by vascular endothelial growth factor A (VEGFA)/vascular endothelial growth factor receptor 2 (VEGFR2) signaling pathway. METHODS: After induced by VEGFA, GC cells (AGS, MGC80-3 and BGC823) were treated by different concentrations of BBD and then were detected cell viability, migration and VEGFA level. And the anti-angiogenesis effect of BBD was evaluated with HUVECs. To furtherly mimic the tumor microenvironment of angiogenesis, VEGFA as an inducer (10 ng/mL) was used to trigger a cascade of angiogenesis of HUVECs in vitro. RESULTS: The viability and migration of GC cells with VEGFA-induced or non-induced and VEGFA levels in GC cells were significantly inhibited by BBD with concentration-dependent manner (P<0.01). BBD significantly inhibited the HUVECs viability with concentration-dependent manner (P<0.01), which was consistent with the inhibitory action on augmentation of cell viability induced by VEGFA (P<0.01). BBD exhibited the similar inhibitory trend on cyto behavioral variability such as wound repairing (P<0.05), migration (P<0.01) and tube formation (P<0.01) and activation effect on cell apoptosis rate (P<0.01) with VEGFA-induced or non-induced. Moreover, BBD notably regulated the levels of VEGFA, VEGFR2, matrix metalloprotein 2 (MMP2) and matrix metalloprotein 9 (MMP9) of HUVECs on present or absent of VEGFA with dose-dependent manner. CONCLUSIONS: BBD inhibited GC growth against VEGFA-induced angiogenesis of HUVECs by VEGFA/VEGFR2 signaling pathway in vitro.

[Cone-beam computed tomography-synthesized cephalograms for evaluating the vertical dimension of occlusions].

OBJECTIVE: The accuracy of the occlusion vertical dimensions of edentulous Han patients from Yunnan province was compared and analyzed on the basis of cone-beam computed tomography (CBCT)-synthesized cephalograms, closest speaking space method, and interocclusal distance. METHODS: A database correlating the CBCT head lateral images of Han patients from Yunnan province with normal occlusal conditions was first constructed. Then, five edentulous Han patients aged 63-78 years old from Yunnan Province were selected. NNT.View software was used to measure and analyze hard tissue cephalometric radiographs that had been transformed by the CBCT marker. The radiographs were then combined with the normal population database for the assessment of occlusion vertical dimensions. The occlusion vertical dimensions determined on the basis of CBCT-synthesized cephalograms, the closest speaking space method, and the free-way space were analyzed. RESULTS: The closest speaking space method was used as the standard control group, the differences between seven methods and the closest speaking space method were analyzed. The seven methods include free-way space method and six CBCT-synthesized cephalograms methods (N-ANS/ANS-Me, S-Go/N-Me, ANS-Gn/N-ANS, ANS-FH/Me-FH, ANS-Xi-Pm, and CA/LA). The seven methods were highly consistent with the closest speaking space method (intraclass correlation coefficient>0.986). The absolute values of the differences between the methods of free-way space, N-ANS/ANS-Me, S-Go/N-Me and the closest speaking space method were lower than those of the other four groups (P<0.05), while the differences between ANS-FH/Me-FH and the closest speaking space method was higher than those other groups (P<0.05). CONCLUSIONS: CBCT-synthesized cephalograms, with the exception of ANS-FH/Me-FH, can provide references for the clinical evaluation of the occlusion vertical dimensions of patients with edentulous jaws.

Fuzheng Qingjie granules potentiate the anticancer effect of cyclophosphamide by regulating cellular immune function and inducing apoptosis in Hepatoma 22 tumor-bearing mice.

Fuzheng Qingjie (FZQJ) is a polyherbal Chinese medicine that has previously been implemented as an adjuvant therapy for gastrointestinal cancer. The present study investigated whether FZQJ is able to potentiate the anticancer effect of cyclophosphamide (CTX). Hepatoma 22 tumor-bearing mice were randomly divided into a vehicle group, CTX group, FZQJ group and combination (CTX+FZQJ) group. In addition, untreated mice without H22 cells served as blank controls. Seven days post-treatment, the mice were sacrificed and the tumors were weighed. Blood cells were evaluated using an automatic hemocytometer analyzer and flow cytometer. The expression levels of interleukin (IL)-2 and tumor necrosis factor (TNF)-α were evaluated using a radioimmunoassay. Apoptotic cells were observed using a terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. Alanine transaminase, aspartate aminotransferase, blood urea nitrogen and creatinine were examined using an automatic biochemical analyzer. The results demonstrated that the tumor inhibitory rate and apoptosis index were higher in the combination group, compared with those in the CTX group. Notably, FZQJ was able to alleviate CTX-induced decreases in the numbers of white blood cells and platelets, CD3+ and CD4+ T lymphocyte subsets, and the concentration of hemoglobin, body weight and thymus index, and increase serum TNF-α and IL-2 levels without overt hepatorenal toxicity. These results suggest that FZQJ granules may enhance the anticancer effect of CTX, in addition to alleviating the side effects.

Ethyl acetate extract from Jiedu Xiaozheng Yin inhibits the proliferation of human hepatocellular carcinoma cells by suppressing polycomb gene product Bmi1 and Wnt/ß-catenin signaling.

Jiedu Xiaozheng Yin (JXY) is a Chinese herbal decoction used to treat hepatocellular carcinoma (HCC). Previous studies have demonstrated that JXY can inhibit HCC cell proliferation via induction of G0/G1 phase arrest. In this study, we investigated whether the inhibitory effect of JXY on HCC cells is associated with the inhibition of the Wnt/ß­catenin pathway and the polycomb gene product Bmi1. Ethyl acetate extract from JXY (EE-JXY) was prepared. Methyl thiazolyl tetrazolium (MTT) and colony formation assays were used to measure cell proliferation. Immunofluorescence was used to analyze the expression and location of ß-catenin and Bmi1. Immunohistochemistry was used to examine the expression of proliferating cell nuclear antigen (PCNA), c-myc and cyclin D1. ß-catenin, Bmi1, c-myc, cyclin D1 and p16INK4A mRNA levels were detected by RT-PCR. The results demonstrated that EE-JXY inhibited the expression of PCNA, c-myc, cyclin D1 and Bmi1, and upregulated the expression of p16INK4A. We also found that EE-JXY could facilitate ß-catenin translocation from the cytoplasm and nuclei to the cytomembrane. Finally, suppression of cell proliferation and expression of Bmi1 and Wnt/ß-catenin by EE-JXY was confirmed in a mouse xenograft model of HCC. Thus, EE-JXY can inhibit the proliferation of HCC partially via suppression of the Bmi1 and Wnt/ß-catenin signaling pathways.

[Effect of bear bile powder on STAT3 pathway in hepatocellular carcinoma xenograft].

OBJECTIVE: To observe the effect of bear bile powder (BBP) on the STAT3 pathway and its downstream target genes of nude mice hepatocellular carcinoma (HCC) xenograft, and to explore its mechanism for treating HCC. METHODS: The subcutaneous xenograft model was established using HepG2 cells. When the subcutaneous transplanted tumor was formed, naked mice were randomly divided into two groups, the BBP group and the control group. Mice in the BBP group were administered with BBP by gastrogavage, once daily for 3 consecutive weeks, while mice in the control group were administered with normal saline by gastrogavage, once daily for 3 consecutive weeks. The body weight and the tumor volume were measured once per week. By the end of medication, the tumor weight was weighed and the tumor inhibition ratio calculated. The apoptosis of the tumor tissue was detected by TdT-mediated dUTP nick end labeling (TUNEL). The expression of Bcl2-associated X protein (Bax), B cell lymphoma/eukemina-2 (Bcl-2), cyclin-dependent protein kinase (CDK4), cyclinD1 were detected by reverse transcription-polymerase chain reaction (RT-PCR). The protein expression levels of signal transducers and transcription activators 3 (p-STAT3), proliferating cell nuclear antigen (PCNA), Bax, Bcl-2, CDK4, and cyclinD1 were determined by immunohistochemistry. RESULTS: BBP could inhibit the tumor volume and tumor weight, showing statistical difference when compared with the control group (P < 0.01). Results of TUNEL showed that BBP could significantly induce the apoptosis of hepatoma carcinoma cells. Results of RT-PCR showed that BBP could up-regulate the expression of Bax and down-regulate mRNA expression of Bcl-2, CDK4, and cyclinD1. Immunohistochemical results showed that BBP could up-regulate the expression of Bax and inhibit the protein expression of p-STAT3, PCNA, Bcl-2, CDK4, and cyclinD1. CONCLUSION: BBP could induce the apoptosis of hepatoma carcinoma cells and inhibit their proliferation by regulating STAT3 pathway.

Fuzheng Qingjie recipe induces apoptosis in HepG2 cells via P38 MAPK activation and the mitochondria-dependent apoptotic pathway.

Fuzheng Qingjie (FZQJ) recipe is a polyherbal Chinese medicine capable of suppressing tumor growth and is used as an adjuvant therapy for various types of cancer. However, its anticancer mechanisms are yet to be fully elucidated. In the present study, we explored whether p38 mitogen-activated protein kinase (MAPK) was involved in FZQJ-mediated mitochondria-dependent apoptosis in human hepatocellular carcinoma cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were used to measure the viability of HepG2 cells. 4,6-Diamidino-2-phenylindole (DAPI) and Annexin-V fluorescein isothiocyanate (FITC) were used to analyze the apoptosis of HepG2 cells. The mitochondrial membrane potential (∆ψ) and phosphorylated P38 MAPK protein were examined by a flow cytometer following 5,5',6,6'-tetrachloro­1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1) and Alexa Fluor® 647 mouse anti-phosphorylated P38 MAPK antibody staining, respectively. The activation of caspase-9 and caspase-3 were measured using colorimetric assays. Additionally, Bcl-2 and Bax expression were examined using reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis. The results demonstrated that water extract of FZQJ was able to induce apoptosis of HepG2 cells in vitro. FZQJ-induced apoptosis was accompanied by the loss of ∆ψ, downregulation of Bcl-2 and upregulation of Bax expression, and the activation of caspase-3, -9 and P38 MAPK. These results indicated that FZQJ induced apoptosis in HepG2 cells at least via P38 MAPK activation and the mitochondria-dependent apoptotic pathway.

Application of serum pharmacology in evaluating the antitumor effect of Fuzheng Yiliu Decoction from Chinese medicine.

OBJECTIVE: To investigate the feasibility of serum pharmacology in evaluating the antitumor effect of Chinese medicine (CM) of Fuzheng Guben (supporting the healthy energy and strengthening the body's resistance to pathogens), the effects of Fuzheng Yiliu Decoction (FYD), a typical prescription of Fuzheng Guben, on proliferation and apoptosis of hepatoma cells in vitro were observed by two methods with serum pharmacology and traditional pharmacology, respectively. METHODS: HepG2 cells were treated with FYD-containing serum or crude FYD extract in vitro. The proliferation rate was determined by methyl thiazolyl tetrazolium (MTT) assay. Cell cycle and apoptosis rate was performed by flow cytometry. And the levels of interleukin-2 (IL-2) and tumor necrosis factor α (TNF-α) in FYD-containing serum were detected by radioimmunoassay. RESULTS: FYD-containing serum remarkably inhibited proliferation and induced apoptosis of hepatoma cells at least by promoting the production of IL-2 and TNF-α in vivo. On the contrary, crude FYD extract promoted the proliferation and did not induce cell apoptosis. CONCLUSION: The results by serum pharmacology were accordant with those of our previous animal and clinical trials which indicates that serum pharmacology is a reasonable and feasible method for the evaluation of the antitumor effect of herbs of Fuzheng Guben.

Water extract of Hedyotis Diffusa Willd suppresses proliferation of human HepG2 cells and potentiates the anticancer efficacy of low-dose 5-fluorouracil by inhibiting the CDK2-E2F1 pathway.

Hedyotis Diffusa Willd (HDW), a Chinese herbal medicine, has been widely used as an adjuvant therapy against various cancers, including hepatocellular carcinoma (HCC). However, the underlying anticancer mechanisms are yet to be elucidated. In the present study, the anticancer effects of HDW were evaluated and the efficacy and safety of HDW combined with low-dose 5-fluorouracil (5-FU) were investigated. HepG2 cells were cultured in vitro and nude mouse xenografts were established in vivo. The proliferation of HepG2 cells was measured using the MTT method and flow cytometry. The mRNA and protein expression levels of cyclin-dependent kinase 2 (CDK2), cyclin E and E2F1 were examined using relative quantitative real-time PCR and western blot analysis, respectively. The results showed that water extract of HDW remarkably inhibited HepG2 cell proliferation in a dose-dependent manner via arrest of HepG2 cells at the G0/G1 phase and induction of S phase delay. This suppression was accompanied by a great decrease of E2F1 and CDK2 mRNA expression. In addition, HDW remarkably potentiated the anticancer effect of low-dose 5-FU in the absence of overt toxicity by downregulating the mRNA and protein levels of CDK2, cyclin E and E2F1. Our findings support the use of HDW as adjuvant therapy of chemotherapy and suggest that HDW may potentiate the efficiency of low-dose 5-FU in treating HCC.

Fuzheng Yiliu Granule inhibits the growth of hepatocellular cancer by regulating immune function and inducing apoptosis in vivo and in vitro.

OBJECTIVE: To study the inhibitory effect of Fuzheng Yiliu Granule (FYG) on hepatocellular cancer (HCC) and investigate the mechanism mediating its bioactivity. METHODS: H22 tumor-bearing ICR mice were treated with FYG [3.6 g/(kg·d)] for 5 days. Tumor volume and tumor weight, percentages of CD3(+), CD4(+), CD8(+), and natural killer (NK) cells in peripheral blood, tumor apoptosis and serum levels of interleukin-2 (IL-2), and tumor necrosis factor-α (TNF-α) were evaluated. FYG-containing serum was prepared from SD rats treated for 7 days [high dose 3.6 g/(kg·d); middle dose 1.8 g/(kg·d); low dose 0.9 g/(kg·d)]. Cell cycle, cell viability, and apoptosis were evaluated after HepG2 cell line was cultured in FYG-containing serum for 48 h. The levels of IL-2 and TNF-α in FYG-containing serum were also determined. RESULTS: FYG produced a potent antitumor effect (P<0.01) and induced marked apoptosis of the tumor tissue (P<0.05). Mice treated with FYG had higher percentages of CD3(+) and CD4(+) (P<0.05), and more NK cells (P<0.01) in the peripheral blood than those in the animals treated with normal saline. Mice receiving FYG had the highest serum levels of IL-2 and TNF-α (P<0.01). High-dose FYG-containing serum significantly decreased HepG2 cell viability, inhibited cell proliferation (P<0.05), and induced apoptosis (P<0.01). In addition, the levels of IL-2 and TNF-α of high-dose-containing serum were higher than the blank serum (P<0.01). CONCLUSION: FYG could inhibit HCC growth by regulating immune function and inducing apoptosis of tumor cells in vivo and in vitro.

Effective components of Chinese herbal compound decoction and Maillard reaction.

This paper intends to explore the color changes considered to be Maillard reaction during the process of Chinese herbal medicine. The Maillard reaction products (MRPs) are often in substantial proportions of Chinese herbal compound decoctions but their effects are often neglected. By considering the effects of MRPs in studies of effective components on Chinese herbal compounds, a new perspective is established in future researches of Chinese herbal compound decoctions.