Jiedu Xiaozheng Yin Inhibits the Progression of Colitis Associated Colorectal Cancer by Stimulating Macrophage Polarization Towards an M1 Phenotype via the TLR4 Pathway.

To investigate the effect of Jiedu Xiaozheng Yin (JXY) on the polarization of macrophages in colitis-associated colon cancer (CAC). An orthotopic model of CAC was established to monitor changes in the pathological state of mice. Colon length, number of colon tumors were recorded, and indices for liver, spleen, and thymus were calculated. Hematoxylin and eosin (H&E) staining was employed to observe intestinal mucosal injury and tumor formation. Immunohistochemistry (IHC) staining was utilized to investigate the effect of JXY on M1 and M2 polarization of macrophages in the colonic mucosa of CAC mice. For in vitro experiments, RT-qPCR (Reverse Transcription-quantitative PCR) and flow cytometry were used to observe the effect of JXY on various M1-related molecules such as IL-1ß, TNF-α, iNOS, CD80, CD86, and its phagocytic function as well as M2-related molecules including Arg-1, CD206, and IL-10. Subsequently, after antagonizing the TLR4 pathway with antagonists (TAK242, PDTC, KG501, SR11302, LY294002), the expression of IL-6, TNF-α, iNOS, and IL-1ß mRNA were detected by RT-qPCR. In vivo experiments, the results showed that JXY improved the pathological condition of mice in general. And JXY treatment decreased the shortening of colon length and number of tumors as compared to non-treated CAC mice. Additionally, JXY treatment improved the lesions in the colonic tissue and induced a polarization of intestinal mucosal macrophages towards the M1 phenotype, while inhibiting polarization towards the M2 phenotype. In vitro experiments further confirmed that JXY treatment promoted the activation of macrophages towards the M1 phenotype, leading to increased expression of IL-1ß, TNF-α, iNOS, CD80, CD86, as well as enhanced phagocytic function. JXY treatment concomitantly inhibited the expression of M2-phenotype related molecules Arginase-1 (Arg-1), CD206, and IL-10. Furthermore, JXY inhibited M1-related molecules such as IL-6, TNF-α, iNOS, and IL-1ß after antagonizing the TLR4 pathway. Obviously, JXY could exhibit inhibitory effects on the development of colon tumors in mice with CAC by promoting M1 polarization through TLR4-mediated signaling and impeding M2 polarization of macrophages.

Babao Dan inhibits the migration and invasion of gastric cancer cells by suppressing epithelial-mesenchymal transition through the TGF-ß/Smad pathway.

OBJECTIVE: To investigate the anti-metastatic effects of Babao Dan (BBD) on gastric cancer (GC) cells (AGS and MGC80-3) and explore the underlying molecular mechanisms by which it inhibits epithelial-mesenchymal transition (EMT). METHODS: AGS and MGC80-3 cells were treated with BBD. In addition, cells were treated with the EMT inducer transforming growth factor-ß1 (TGF-ß1). Cell viability was determined using the MTT assay, and the live cell ratio was calculated via cell counting. Cell invasion and migration were evaluated using the Transwell assay. Western blotting was performed to measure the protein expression of EMT biomarkers and related genes. RESULTS: BBD inhibited the viability, migration, and invasion of AGS and MGC80-3 cells, but it did not reduce the live cell ratio. Furthermore, BBD inhibited the expression of N-cadherin, vimentin, zinc finger E-box binding homeobox (ZEB)1, ZEB2, Twist1, matrix metalloproteinase (MMP)2, MMP9, TGF-ß1, and p-Smad2/3, whereas E-cadherin expression was increased in AGS and MGC80-3 cells to different degrees. Using a GC cell model of EMT induced by TGF-ß1, we proved that BBD inhibited p-Smad2/3 and N-cadherin expression, cell migration, and cell invasion. CONCLUSION: BBD suppressed cell migration and invasion by inhibiting TGF-ß-induced EMT and inactivating TGF-ß/Smad signaling in GC cells.

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.

Synergisic effect of APRIL knockdown and Jiedu Xiaozheng Yin, a Chinese medicinal recipe, on the inhibition of hepatocellular carcinoma cell proliferation.

It is well documented that A proliferation-inducing ligand (APRIL), a member of the tumor necrosis factor superfamily, plays a crucial role in the occurrence and development of tumors. In the present study, we evaluated the synergistic effect of APRIL knockdown and Jiedu Xiaozheng Yin (JXY), a Traditional Chinese Medicinal recipe, on the inhibition of hepatocellular carcinoma (HCC) cell proliferation and elucidated the underlying mechanism. The results demonstrated that both APRIL knockdown using small interfering RNA (siRNA) and JXY treatment could trigger cell cycle arrest and cell apoptosis, and suppress HCC cell proliferation through an NF-κB-related pathway. Synergism was further demonstrated between APRIL knockdown and JXY treatment. In conclusion, these results indicate that APRIL is a target gene for HCC and combination of siRNA-APRIL and JXY application holds great promise as a novel approach for the treatment of APRIL-positive HCC.

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.

Pien Tze Huang inhibits liver metastasis by targeting TGF-ß signaling in an orthotopic model of colorectal cancer.

Metastasis is the leading cause of cancer-related mortality in almost all types of cancers, including colorectal cancer (CRC). Epithelial-mesenchymal transition (EMT) is a critical process during the metastatic cascade. This process may be a potential target for the diagnosis and treatment of CRC. Pien Tze Huang (PZH), a well-known traditional Chinese formula, has been demonstrated to be clinically effective in treating various types of human malignancies, including CRC. Our published data suggest that PZH can induce apoptosis, as well as inhibit cell proliferation and tumor angiogenesis, thus suppressing CRC growth in vitro and in vivo. We evaluated the therapeutic efficacy of PZH against CRC metastasis using a CRC liver metastasis mouse model to further explore the mechanisms underlying the antitumor action of PZH. MTT, migration, and Matrigel invasion assays were used to assess the effect of PZH on cell viability, migration and invasion. We then established an orthotopic liver metastasis model of colon cancer using microsurgical techniques. Mice were intragastrically administered 234 mg/kg/day dose of either PZH or saline for 14 days. The body and tumor weights of the mice were measured after they were sacrificed. Moreover, we examined the effect of PZH inhibition on liver metastasis. Finally, EMT-related proteins and the TGF-ß signaling pathway were assessed using immunohistochemical staining (IHS). The present data revealed that PZH significantly inhibited the migration and invasion of CT-26 cells in a dose-dependent manner, which affirmed the inhibitory effect of PZH on CRC cell metastasis. No significant change was observed between the in vivo primary tumor growth and body weight. However, the control group had five cases of liver metastasis (5/6), whereas one case was found in the PZH group (1/6). Thus, PZH exhibited therapeutic efficacy against CRC metastasis without apparent toxicity. The inhibitory effect of PZH on EMT resulted in an increase in E-cadherin expression, as well as a decrease in N-cadherin expression. In addition, PZH significantly inhibited TGF-ß, as well as the phosphorylation of Smad2/3 and Smad4 in the tumor tissues, indicating its suppressive action on TGF-ß signaling. These molecular effects ultimately resulted in the inhibition of cancer cell EMT and tumor metastasis.

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.

Total alkaloids of Rubus alceifolius Poir shows anti-angiogenic activity in vivo and in vitro.

Total alkaloids is an active ingredient of the natural plant Rubus alceifolius Poir, commonly used for the treatment of various cancers. Antitumor effects may be mediated through anti-angiogenic mechanisms. As such, the goal of the present study was to investigate and evaluate the effect of total alkaloids in Rubus alceifolius Poir (TARAP) on tumor angiogenesis and investigate the underlying molecular mechanisms of TARAP action in vivo and in vitro. A chick embryo chorioallantoic membrane (CAM) assay was used to assess angiogenesis in vivo. An MTT assay was performed to determine the viability of human umbilical vein endothelial cells (HUVECs) with and without treatment. Cell cycle progression of HUVECs was examined by FACS analysis with propidium iodide staining. HUVEC migration was determined using a scratch wound method. Tube formation of HUVECs was assessed with an ECMatrix gel system, and mRNA and protein expression of VEGF-A in both HUVECs and HepG2 human hepatocellular carcinoma cells were examined by RT-PCR and ELISA, respectively. Our results showed that TARAP inhibited angiogenesis in the CAM model in vivo and inhibited HUVEC proliferation via blocking cell cycle G1 to S progression in a dose- and time-dependent manners in vitro. Moreover, TARAP inhibited HUVEC migration and tube formation and downregulated mRNA and protein expression of VEGF-A in both HepG2 cells and HUVECs. Our findings suggest that the anti-angiogenic activity of TARAP may partly contribute to its antitumor properties and may be valuable for the treatment of diseases involving pathologic angiogenesis such as cancer.

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.

Livistona chinensis seeds inhibit hepatocellular carcinoma angiogenesis in vivo via suppression of the Notch pathway.

Livistona chinensis seeds have been used for centuries to clinically treat various types of cancer. Our published data suggest that Livistona chinensis seeds are able to inhibit hepatocellular carcinoma (HCC) growth in vitro and in vivo via promotion of mitochondrial-dependent apoptosis. To further elucidate the molecular mechanisms of its antitumor activity, in the present study, we used an HCC xenograft mouse model to evaluate the effect of an ethanol extract of Livistona chinensis seeds (EELC) on tumor angiogenesis and on the activation of the Notch pathway. Intratumoral microvessel density (MVD) in HCC xenograft mouse tumors was evaluated via immunohistochemical (IHC) staining for CD31. The mRNA and protein expression of vascular endothelial growth factor A (VEGF-A), VEGFR-2, Notch, Dll4 and Jagged1 was evaluated using RT-PCR and IHC, respectively. We found that EELC profoundly reduced MVD in the HCC mouse tumors, demonstrating the in vivo inhibitory effect of EELC on tumor angiogenesis. In addition, EELC treatment reduced the expression of VEGF-A and VEGFR-2 in tumor tissues. Furthermore, EELC treatment inhibited the expression of Notch, Dll4 and Jagged1. Our findings suggest that Livistona chinensis seeds inhibit tumor angiogenesis through suppression of the Notch 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.

Spica prunellae promotes cancer cell apoptosis, inhibits cell proliferation and tumor angiogenesis in a mouse model of colorectal cancer via suppression of stat3 pathway.

BACKGROUND: Constitutive activation of STAT3 is one of the major oncogenic pathways involved in the development of various types of malignancies including colorectal cancer (CRC); and thus becomes a promising therapeutic target. Spica Prunellae has long been used as an important component in many traditional Chinese medicine formulas to clinically treat CRC. Previously, we found that Spica Prunellae inhibits CRC cell growth through mitochondrion-mediated apoptosis. Furthermore, we demonstrated its anti-angiogenic activities in vivo and in vitro. To further elucidate the precise mechanism of the potential tumoricidal activity of Spica Prunellae, using a CRC mouse xenograft model, in this study we evaluated its therapeutic efficacy against CRC and investigated the underlying molecular mechanisms. METHODS: CRC mouse xenograft model was generated by subcutaneous injection of human colon carcinoma HT-29 cells into nude mice. Animals were given intra-gastric administration with 6 g/kg of the ethanol extract of Spica Prunellae (EESP) daily, 5 days a week for 16 days. Body weight and tumor growth were measured every two days. Tumor growth in vivo was determined by measuring the tumor volume and weight. HT-29 cell viability was examined by MTT assay. Cell apoptosis and proliferation in tumors from CRC xenograft mice was evaluated via immunohistochemical staining (IHS) for TUNEL and PCNA, and the intratumoral microvessel density (MVD) was examined by using IHS for the endothelial cell-specific marker CD31. The activation of STAT3 was evaluated by determining its phosphorylation level using IHS. The mRNA and protein expression of Bcl-2, Bax, Cyclin D1, VEGF-A and VEGFR2 was measured by RT-PCR and IHS, respectively. RESULTS: EESP treatment reduced tumor volume and tumor weight but had no effect on body weight change in CRC mice; decreased HT-29 cell viability in a dose-dependent manner, suggesting that EESP displays therapeutic efficacy against colon cancer growth in vivo and in vitro, without apparent toxicity. In addition, EESP significantly inhibited the phosphorylation of STAT3 in tumor tissues, indicating its suppressive action on the activation of STAT3 signaling. Consequently, the inhibitory effect of EESP on STAT3 activation resulted in an increase in the pro-apoptotic Bax/Bcl-2 ratio, decrease in the expression of the pro-proliferative Cyclin D1 and CDK4, as well as down-regulation of pro-angiogenic VEGF-A and VEGFR-2 expression. Finally, these molecular effects led to the induction of apoptosis, the inhibition of cell proliferation and tumor angiogenesis. CONCLUSIONS: Spica Prunellae possesses a broad range of anti-cancer activities due to its ability to affect STAT3 pathway, suggesting that Spica Prunellae could be a novel potent therapeutic agent for the treatment of CRC.

Livistona chinensis seed suppresses hepatocellular carcinoma growth through promotion of mitochondrial-dependent apoptosis.

The Livistona chinensis seed has been used for centuries to clinically treat various types of cancer. However, the precise mechanism of its anticancer activity remains to be elucidated. In the present study, we evaluated the efficacy of the ethanol extract of Livistona chinensis seed (EELC) against tumor growth using a hepatocellular carcinoma (HCC) mouse xenograft model and an HCC cell line, HepG2, and investigated the molecular mechanisms mediating its biological activities. We found that EELC inhibited HCC growth both in vivo and in vitro, without apparent signs of toxicity. In addition, EELC treatment resulted in the induction of HCC cell apoptosis. Moreover, EELC-induced apoptosis was accompanied by the loss of mitochondrial membrane potential, activation of caspase-9 and caspase-3, and increase in the pro-apoptotic Bax/Bcl-2 ratio. Our findings suggest that promotion of mitochondrial-dependent apoptosis in cancer cells may be one of the mechanisms by which the Livistona chinensis seed is effective in cancer treatment.

Studies on the anti-angiogenic effect of Marsdenia tenacissima extract in vitro and in vivo.

Marsdenia tenacissima, which is widely used as an anticancer herb in traditional Chinese medicine, has been shown to possess anticancer activities. However, the underlying molecular mechanism(s) involved in the anticancer effect of this herb are poorly understood. Angiogenesis is important in the development of cancer. The main features of angiogenesis are increased vasculature and overexpression of vascular endothelial growth factor (VEGF). In the present study, the effects of M. tenacissima extract (MTE) on human umbilical vein endothelial cell (HUVEC) proliferation, migration and capillary-like tube formation were investigated in vitro and using the chick embryo chorioallantoic membrane (CAM) assay in vivo. It was observed that MTE inhibited the proliferation of HUVECs by blocking the cell cycle progression from G1 to S. In addition, MTE inhibited the migration and tube formation of HUVECs. MTE treatment decreased the VEGF-A expression in human hepatoma cells (HepG2), as well as the expression of VEGF-A and VEGF receptor (VEGFR)-2 in HUVECs. MTE exposure in the CAM was able to reduce the formation of blood vessels in chick embryos. Overall, the present data suggest that extracts of M. tenacissima may serve as potential anti-angiogenesis agents.

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.

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), cyclin­dependent 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.

Jiedu Xiaozheng Yin, a Chinese herbal formula, inhibits tumor angiogenesis via downregulation of VEGF-A and VEGFR-2 expression in vivo and in vitro.

Angiogenesis is crucial for cancer growth and metastasis and inhibition of angiogenesis has been recognized to be a promising strategy for the treatment of cancer. Traditional Chinese medicine (TCM) has been used for thousands of years to treat cancer. Jiedu Xiaozheng Yin (JXY), a polyherbal formula of TCM, has been used to treat various tumors in China. However, the mechanism of its anticancer activity has yet to be fully elucidated. Using human umbilical vein endothelial cells (HUVECs), chick chorioallantoic membrane (CAM) and a hepatoma mouse xenograft model, we investigated the underlying molecular mechanisms of ethanol extract of Jiedu Xiaozheng Yin (EE-JXY). EE-JXY treatment significantly inhibited tumor cell growth both in vitro and in the mouse xenograft model (P<0.05). Moreover, EE-JXY reduced tube formation of HUVECs and angiogenesis in the CAM (P<0.01) and microvessel density (MVD) of tumor in vivo (P<0.05). Further studies showed that EE-JXY was able to suppress the expression of vascular endothelial growth factor A (VEGF-A) and vascular endothelial growth factor receptor 2 (VEGFR-2) in both HepG2 cells and HUVECs (P<0.01) and in tumor (P<0.01). Thus, JXY suppressed tumor growth at least by inhibiting angiogenesis.

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.