Fuzheng Qingjie Granules Inhibit Growth of Hepatoma Cells via Inducing Mitochondria-Mediated Apoptosis and Enhancing Immune Function.

Fuzheng Qingjie (FZQJ) granules, a compound Chinese medicine, have been used as an adjuvant therapy for alimentary tract cancers. However, the underlying anticancer mechanisms are still not well understood. In the present study, HepG2 cells were treated with FZQJ-containing serum. Cell proliferation was evaluated using MTT assay. Apoptosis was analyzed using a flow cytometer. Cell ultrastructure was observed under a transmission electron microscope. The mitochondrial membrane potential (Δψ) was examined with JC-1 dye. In H22 tumor-bearing mice, CD4+ T cells, CD8+ T cells, CD3+ T cells, and natural killer (NK) cells in peripheral blood were evaluated cytometrically. Interleukin (IL)-2 and tumor necrosis factor (TNF)-α levels were measured using radioimmunoassay.The mRNA levels of Bax and Bcl-2 were examined by reverse transcription-polymerase chain reaction. The protein levels of Bax, Bcl-2, cytochrome C, caspase 3 and 9, PARP, and CD69 were examined by Western blotting. The apoptotic cells in tissues were observed using TUNEL method. Alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN), and creatinine (CRE) were detected by an automatic biochemical analyzer. The results showed that FZQJ-containing serum remarkably inhibited proliferation of HepG2 cells in dose- and time-dependent manners, induced HepG2 cell apoptosis and caused a decrease of Δψ. Analysis of tumor tissue showed that FZQJ-induced apoptosis was accompanied by downregulation of Bcl-2 and upregulation of Bax, release of cytochrome c, activation of caspase 3 and 9, and cleavage of PARP. In addition, FZQJ increased the percentages of CD4+ T and NK cells, the ratio of CD4+/CD8+ T cells as well as the levels of serum TNF-α. FZQJ also increased CD69 expression in tumor tissue. No hepatorenal toxicity was observed in H22 tumor-bearing mice. These results indicated that FZQJ could inhibit the growth of hepatoma cells via regulating immune function and inducing mitochondria mediated apoptosis.

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.

The quality of reports of randomized clinical trials on traditional Chinese medicine treatments: a systematic review of articles indexed in the China National Knowledge Infrastructure database from 2005 to 2012.

BACKGROUND: The Consolidated Standards for Reporting Trials (CONSORT) are aimed to standardize clinical trial reporting. Our objective is to compare the quality of randomized clinical trials (RCTs) of traditional Chinese medicine (TCM) published in 2005-2009 and 2011-2012 according to the current CONSORT statements and Jadad scale. DATA SOURCES: Reports on RCTs of TCM in the China National Knowledge Infrastructure database (CNKI database) for manuscripts published from 2005 to 2009 and 2011-2012. Search terms included TCM and clinical trial. STUDY SELECTION: Manuscripts that reported RCTs of TCM were included. DATA EXTRACTION: Independent extraction of articles was done by 3 authors. Disagreement was discussed until agreement was reached. According to the CONSORT checklist, an item was scored as 1 when the item was described in the paper. Otherwise the item was scored as 0. RESULTS: A total of 4133 trials in 2005-2009 and 2861 trials in 2011-2012 were identified respectively. There was a significant increase in proportion of reports that included details of background (24.71% vs 35.20%, P < 0.001), participants (49.79% vs 65.26%, P < 0.001), the methods of random sequence generation (13.77% vs 19.85%, P < 0.001), statistical methods (63.00% vs 72.77%, P < 0.001) and recruitment date (70.14% vs 80.36%, P < 0.001) in 2011-2012 compared to 2005-2009. However, the percentage of reports with trial design decreased from 4.45% to 3.25% (P = 0.011). Few reports described the blinding methods, and there was a decreasing tendency (4.77% vs 2.48%, P < 0.001). There was a similar decreasing tendency on the reporting of funding (6.53% vs 5.00%, P = 0.007). There were no significant differences in the other CONSORT items. In terms of Jadad Score, the proportion of reports with a score of 2 was markedly increased (15.15% vs 19.71%, P < 0.001). CONCLUSIONS: Although the quality of reporting RCTs of TCM was improved in 2011-2012 compared to 2005-2009, the percentages of high-quality reports are both very low in terms of Jadad score. There is a need for improving standards for reporting RCTs in China.

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.

Ethanol extract of Chaenomeles speciosa Nakai induces apoptosis in cancer cells and suppresses tumor growth in mice.

Chaenomeles speciosa Nakai is commonly used in traditional Chinese medicine for a variety of health-promoting effects. The present study aimed to investigate the antitumor effects of Chaenomeles speciosa Nakai. The tumor-inhibitory activity of the ethanol extract of Chaenomeles speciosa Nakai (EEC) was evaluated by in vitro growth assays of tumor cells and in vivo H22 tumor formation assays in mice. Mitochondrial membrane potential and DNA ladder assays were used to detect tumor cell apoptosis in the presence of EEC. To investigate the cellular targets of EEC, the immunomodulatory genes PD-L1, Foxp3 and TGF-ß were detected in the tumor tissue using reverse transcription polymerase chain reaction (RT-PCR). Immune responses were determined by hemolysis and lymphocyte proliferation assays. EEC markedly inhibited the proliferation of the H22 cells in a dose-dependent manner. Moreover, it induced DNA fragmentation and decreased the mitochondrial membrane potential. In vivo, EEC inhibited tumor growth and enhanced the immune responses in mice, while the expression of PD-L1, Foxp3 and TGF-ß was inhibited in the tumor tissue. These results provide the first evidence that EEC may inhibit tumor growth by directly killing tumor cells and enhancing immune function. Thus, it is a natural source for safe anticancer medicine.