Xuzheng Chen

Ethyl acetate extraction from a Chinese herbal formula, Jiedu Xiaozheng Yin, inhibits the proliferation of hepatocellular carcinoma cells via induction of G0/G1 phase arrest in vivo and in vitro

Jiedu Xiaozheng Yin (JXY), a polyherbal formula of traditional Chinese medicine (TCM), has been used to treat various kinds of cancer in China. However, the mechanism of its anticancer activity has yet to be elucidated. Air-dried herbs were extracted with reagents of different polarity. HepG2 cells were treated with different doses of ethyl acetate extract (EE-JXY) and chloroform extract (CE-JXY) for 24 h. Cell viability was detected by MTT assay. Colony formation ability was also evaluated. Cell cycle was evaluated by FACS. Tumor bearing BALB/c nude mice was treated with EE-JXY (0.06 g/kg) for 20 days. Tumor volume and weight were monitored. The percentage of PCNA-positive cells and the level of G1 phase proteins [cyclin-dependent kinase2 (CDK2), cyclin‑dependent kinase4 (CDK4), cyclin D and cyclin E and G2 phase proteins [cyclin-dependent kinase1 (CDK1), cyclin A and cyclin B] were detected by immunohistochemistry and western blotting. EE-JXY and CE-JXY dose-dependently inhibited the growth of HepG2 cells (P<0.01 for both). Furthermore, EE-JXY inhibited the formation of cell colonies and blocked the cell cycle to G1 phase in a dose-dependent manner (P<0.01 for all). EE-JXY showed an obviously antitumor effect in vivo (P<0.05). Further investigation showed that EE-JXY decreased the proliferation index of tumors (P<0.01) through increasing the expression of G1-related proteins (cyclin D and cyclin E, P<0.05 and P<0.01). These results suggested that JXY inhibits the growth of HepG2 cells at least via arresting the cell cycle at the G0/G1 phase.

Total alkaloids of Rubus aleaefolius Poir inhibit hepatocellular carcinoma growth in vivo and in vitro via activation of mitochondrial-dependent apoptosis

The aim of this study was to evaluate the therapeutic efficacy of Rubus aleaefolius Poir total alkaloids (TARAP) against hepatocellular carcinoma growth in vivo and in vitro, and to investigate the possible molecular mechanisms mediating its biological activity. Nude mice were implanted with HepG2 human hepatocellular carcinoma cells and fed with vehicle (physiological saline) or 3 g/kg/d dose of TARAP, 5 days per week, for 21 days. The in vivo efficacy of TARAP against tumor growth was investigated by evaluating its effect on tumor volume and tumor weight in mice with HCC xenografts and its adverse effect was determined by measuring the body weight gain. The in vitro effect of TARAP on the viability of HepG2 cells was determined by MTT assay. HepG2 cell morphology was observed via phase-contrast microscopy. Apoptosis in tumor tissues or in HepG2 cells was analyzed by TUNEL assay or FACS analysis with Annexin V/PI, respectively. The loss of mitochondrial membrane potential in HepG2 cells was determined via JC-1 staining followed by FACS analysis. Activation of caspase-9 and -3 in HepG2 cells was examined by a colorimetric assay. The mRNA and protein expression of Bcl-2 and Bax in tumor tissues were measured by RT-PCR and immunohistochemistry. TARAP reduced tumor volume and tumor weight, but had no effect on the body weight gain in HCC mice. TARAP decreased the viability of HepG2 cells and induced cell morphological changes in vitro in a dose- and time-dependent manner. In addition, TARAP induced apoptosis both in tumor tissues and in HepG2 cells. Moreover, TARAP treatment resulted in the collapse of mitochondrial membrane potential in HepG2 cells, as well as the activation of caspase-9 and -3. Furthermore, administration of TARAP increased the pro-apoptotic Bax/Bcl-2 ratio in HCC mouse tumors, at both transcriptional and translational levels. TARAP inhibits hepatocellular carcinoma growth both in vivo and in vitro probably through the activation of mitochondrial-dependent apoptosis, which may, in part, explain its anticancer activity. These results suggest that total alkaloids in Rubus aleaefolius Poir may be a potential novel therapeutic agent for the treatment of hepatocellular carcinoma and other cancers.

Fuzheng Yiliu Granule (扶正抑瘤颗粒) inhibits the growth of hepatocellular cancer by regulating immune function and inducing apoptosis in vivo and in vitro

ObjectiveTo study the inhibitory effect of Fuzheng Yiliu Granule (扶正抑瘤颗粒, FYG) on hepatocellular cancer (HCC) and investigate the mechanism mediating its bioactivity.MethodsH22 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.ResultsFYG 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).ConclusionFYG could inhibit HCC growth by regulating immune function and inducing apoptosis of tumor cells in vivo and in vitro.

Enhancement of Antitumor Activity of Low-Dose 5-Fluorouracil by Combination With Fuzheng-Yiliu Granules in Hepatoma 22 Tumor-Bearing Mice

Objective. The adverse effects of 5-fluorouracil (5-FU) are well recognized. Fuzheng-Yiliu granule (FYG) is capable of enhancing the immune function and suppressing tumor growth. In the present study, the authors evaluated if FYG could synergize with low-dose 5-FU in inhibiting tumor growth. Methods. Hepatoma 22 (H22) tumor-bearing mice were treated with FYG (18 g/kg, ig), 5-FU (10 mg/kg, ip), or 5-FU plus FYG for 5 days. The relative tumor proliferation rates, tumor weight and apoptosis of tumor tissue were measured. White blood cell (WBC) and lymphocyte (LY) were counted. Interleukin-2 (IL-2) and tumor necrosis factor (TNF-a) in the serum were measured. Results. FYG alone had antitumor effect. Combination of 5-FU and FYG produced a more potent antitumor effect and caused more marked apoptosis in tumor tissue (compared with vehicle, P < 0.01; compared with 5-FU or FYG, P < 0.05). Mice treated with 5-FU plus FYG had higher thymus index (P < 0.05) compared with the vehicle group. The numbers of both WBC and LY were decreased by 5-FU (compared with vehicle, P < 0.01), which was significantly reversed after FYG was administered (5-FU + FYG vs 5-FU, P < 0.01 and P < 0.05). Mice receiving FYG alone or FYG plus 5-FU had higher serum levels of TNF-a (P <0.01) compared with the vehicle. Conclusions. Traditional Chinese medical herbs capable of strengthening the body’s vital energy have great potential to be used as an adjuvant therapy for cancer patients who cannot tolerate the adverse effects of chemotherapy.

Application of serum pharmacology in evaluating the antitumor effect of Fuzheng Yiliu Decoction (扶正抑瘤方) from Chinese medicine

ObjectiveTo investigate the feasibility of serum pharmacology in evaluating the antitumor effect of Chinese medicine (CM) of Fuzheng Guben (扶正固本, supporting the healthy energy and strengthening the body’s resistance to pathogens), the effects of Fuzheng Yiliu Decoction (扶正抑瘤方, 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.MethodsHepG2 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 apoptosisConclusionThe 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.

Ethyl acetate extract from Jiedu Xiaozheng Yin inhibits the proliferation of human hepatocellular carcinoma cells by suppressing polycomb gene product Bmi1 and Wnt/β-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/β‑catenin 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 β-catenin and Bmi1. Immunohistochemistry was used to examine the expression of proliferating cell nuclear antigen (PCNA), c-myc and cyclin D1. β-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 β-catenin translocation from the cytoplasm and nuclei to the cytomembrane. Finally, suppression of cell proliferation and expression of Bmi1 and Wnt/β-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/β-catenin signaling pathways.

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.

Active hexose correlated compound potentiates the antitumor effects of low-dose 5-fluorouracil through modulation of immune function in hepatoma 22 tumor-bearing mice

BACKGROUND/OBJECTIVES A variety of immunomodulators can improve the efficacy of low-dose chemotherapeutics. Active hexose correlated compound (AHCC), a mushroom mycelia extract, has been shown to be a strong immunomodulator. Whether AHCC could enhance the antitumor effect of low-dose 5-fluorouracil (5-FU) via regulation of host immunity is unknown. MATERIALS/METHODS In the current study Hepatoma 22 (H22) tumor-bearing mice were treated with PBS, 5-FU (10 mg·kg-1·d-1, i.p), or AHCC (360 mg·kg-1·d-1, i.g) plus 5-FU, respectively, for 5 d. CD3+, CD4+, CD8+, and NK in peripheral blood were detected by flow cytometry. ALT, AST, BUN, and Cr levels were measured by biochemical assay. IL-2 and TNFα in serum were measured using the RIA kit and apoptosis of tumor was detected by TUNEL staining. Bax, Bcl-2, and TS protein levels were measured by immunohistochemical staining and mRNA level was evaluated by RT-PCR. RESULTS Diet consumption and body weight showed that AHCC had no apparent toxicity. AHCC could reverse liver injury and myelosuppression induced by 5-FU (P < 0.05). Compared to mice treated with 5-FU, mice treated with AHCC plus 5-FU had higher thymus index, percentages of CD3+, CD4+, and NK cells (P < 0.01), and ratio of CD4+/CD8+ (P < 0.01) in peripheral blood. Radioimmunoassay showed that mice treated with AHCC plus 5-FU had the highest serum levels of IL-2 and TNFα compared with the vehicle group and 5-FU group. More importantly, the combination of AHCC and 5-FU produced a more potent antitumor effect (P < 0.05) and caused more severe apoptosis in tumor tissue (P < 0.05) compared with the 5-FU group. In addition, the combination of AHCC and 5-FU further up-regulated the expression of Bcl-2 associated X protein (Bax) (P < 0.01), while it down-regulated the expression of B cell lymphoma 2 (Bcl-2) (P < 0.01). CONCLUSIONS These results support the claim that AHCC might be beneficial for cancer patients receiving chemotherapy.

Jiedu Xiaozheng Yin decoction inhibits hepatoma cell proliferation by inducing apoptosis via the mitochondrial-mediated pathway.

Jiedu Xiaozheng Yin decoction (JXY) is a type of Chinese traditional medicine, which has been used to treat various types of cancer. The present study explored the mechanisms underlying the anticancer activity of JXY. The effects of ethyl acetate extraction of JXY (EE-JXY) were evaluated on the HepG2 human hepatoma cell line in vitro and in vivo. Following treatment of the HepG2 cells with EE-JXY for 24 h, cell viability, apoptosis, mitochondrial membrane potential, caspase enzyme activity and the expression levels of apoptotic-associated proteins (Bcl-2 and Bax) were detected by MTT, flow cytometry, ELISA and western blotting respectively. In addition, HepG2 cells were subcutaneously transplanted into BALB/c nude mice, and the tumor bearing mice were treated with either EE-JXY (0.06 g/kg) or normal saline for 21 days. Tumor volume and weight were measured and recorded. The apoptotic index, and the expression levels of Bax and cytochrome c were determined with immunohistochemical staining. Treatment with EE-JXY inhibited the proliferation of HepG2 cells, and reduced cell viability in a dose-and time-dependent manner. Furthermore, EE-JXY induced HepG2 cell apoptosis, as demonstrated by a loss of plasma membrane asymmetry and externalization of phosphatidylserine, collapse of mitochondrial membrane potential, activation of caspase-9 and caspase-3, and an increased ratio of pro-apoptotic Bax to anti-apoptotic Bcl-2. Furthermore, EE-JXY inhibited tumor growth and increased the apoptotic index of tumors in tumor-bearing mice. In conclusion, the results of the present study suggest that JXY inhibits HepG2 cell proliferation through mitochondrion-mediated apoptosis, which may partially explain its anticancer activity.

Bear bile powder (熊胆粉) induces apoptosis of human hepatocellular carcinoma cells via mitochondrion-dependent pathway

ObjectiveTo evaluate the effect of Bear Bile Powder(熊胆粉, BBP) on the growth and apoptosis of HepG2 human hepatocellular carcinoma cells, and investigate the possible molecular mechanisms mediating its anti-cancer activity.MethodsHepG2 cells were treated with 0.4–1.0 mg/mL of BBP for 24, 48 and 72 h. The viability of HePG2 cells was determined by MTT assay. Cellular morphology was observed via phase-contrast microscopy. Fluorescence-activated cell sorting analysis with Annexin-V/propidium idodide and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazol-carbocyanine iodide (JC-1) staining was performed to determine cell apoptosis and the loss of mitochondrial membrane potential, respectively. Activation of caspase-9 and -3 was evaluated by a colorimetric assay.ResultsThe treatment with 0.4–1 mg/mL of BBP for 24, 48, or 72 h respectively reduced cell viability significantly by 7%–60%, 20%–90% or 25%–98%, compared with the untreated control cells (P<0.01). In addition, BBP treatment induced morphological changes in HepG2 cells. Furthermore, after treated with 0, 0.4, 0.6, 0.8 and 1.0 mg/mL of BBP, apoptosis cells (including early and late apoptotic cells) were 18.0%±1.3%, 34.9%±2.2%, 33.9%±2.8%, 37.4%±2.8% and 46.0%±2.5%, respectively (P<0.05); and the percentage of cells with reduced JC-1 red fluorescence were 6.6%±0.8%, 8.5%±0.8%, 13.5%±1.6%, 17.6%±2.3% and 46.7%±3.6%, respectively (P<0.01). Finally, BBP treatment significantly and dose-dependently induced activation of both caspase-9 and caspase-3 in HepG2 cells (P<0.05).ConclusionsBBP could inhibit the growth of HepG2 hepatocellular cancer cells through mitochondrion-mediated apoptosis, which may, in part, explain its anti-cancer activity. BBP may be a potential novel therapeutic agent for the treatment of hepatocellular carcinoma.