Anti-tumor Effect of Ginkgo biloba Exocarp Extracts on B16 Melanoma Bearing Mice Involving P I3K/Akt/HIF-1α/VEGF Signaling Pathways.

The objective of this study is to investigate the anti-tumor effect of Ginkgo biloba exocarp extracts (GBEE) on B16 melanoma bearing mice and its related molecular mechanisms. The B16-F10 melanoma solid tumor model was established in C57BL/6J mice. The tumor-bearing mice were treated with GBEE (50, 100, 200 mg/kg), taking cis-Dichlorodiamineplatinum (Ⅱ) (DDP, 3 mg/kg) as positive control and normal saline (NS) as model control. After 17 days of administration, the transplanted tumors was stripped and weighed, and the inhibition rate was calculated. Quantitative Reverse Transcription Polymerase chain reaction (qRT-PCR), Western Blot and immunohistochemistry were applied to detect mRNA and protein levels of related factors in B16 transplanted tumor tissues. The results indicated that GBEE (50, 100, 200 mg/kg) inhibited the growth of B16 transplanted solid tumor in C57BL/6J mice. Meanwhile, it inhibited the expression of CD34 and reduced microvessel density (MVD) in a dose-dependent manner. Moreover, GBEE dose-dependently down-regulated the mRNA and protein levels of hypoxia inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), and vascular endothelial growth factor receptor 2 (VEGFR2). The phosphoinositide 3-kinase (PI3K) and protein kinase B (Akt) proteins were not changed obviously, but the protein levels of p-PI3K and p-Akt were down-regulated. Overall, the inhibitory effect of GBEE on the growth of B16 melanoma transplant tumor in mice is related to inhibiting angiogenesis, and the mechanism involves the regulation of PI3K/Akt/ HIF-lα/VEGF signaling pathway.

Activation of cannabinoid receptor 2 enhances osteogenic differentiation of bone marrow derived mesenchymal stem cells.

Bone marrow derived mesenchymal stem cells (BM-MSCs) are considered as the most promising cells source for bone engineering. Cannabinoid (CB) receptors play important roles in bone mass turnover. The aim of this study is to test if activation of CB2 receptor by chemical agonist could enhance the osteogenic differentiation and mineralization in bone BM-MSCs. Alkaline phosphatase (ALP) activity staining and real time PCR were performed to test the osteogenic differentiation. Alizarin red staining was carried out to examine the mineralization. Small interference RNA (siRNA) was used to study the role of CB2 receptor in osteogenic differentiation. Results showed activation of CB2 receptor increased ALP activity, promoted expression of osteogenic genes, and enhanced deposition of calcium in extracellular matrix. Knockdown of CB2 receptor by siRNA inhibited ALP activity and mineralization. Results of immunofluorescent staining showed that phosphorylation of p38 MAP kinase is reduced by knocking down of CB2 receptor. Finally, bone marrow samples demonstrated that expression of CB2 receptor is much lower in osteoporotic patients than in healthy donors. Taken together, data from this study suggested that activation of CB2 receptor plays important role in osteogenic differentiation of BM-MSCs. Lack of CB2 receptor may be related to osteoporosis.

ISG15 inhibits IFN-α-resistant liver cancer cell growth.

Hepatocellular carcinoma (HCC) is one of the most prevalent tumors worldwide. Interferon-α (IFN-α) has been widely used in the treatment of HCC, but patients eventually develop resistance. ISG15 ubiquitin-like modifier (ISG15) is a ubiquitin-like protein transcriptionally regulated by IFN-α which shows antivirus and antitumor activities. However, the exact role of ISG15 is unknown. In the present study, we showed that IFN-α significantly induced ISG15 expression but failed to induce HepG2 cell apoptosis, whereas transient overexpression of ISG15 dramatically increased HepG2 cell apoptosis. ISG15 overexpression increased overall protein ubiquitination, which was not observed in cells with IFN-α-induced ISG15 expression, suggesting that IFN-α treatment not only induced the expression of ISG15 but also inhibited ISG15-mediated ubiquitination. The tumor suppressor p53 and p21 proteins are the key regulators of cell survival and death in response to stress signals such as DNA damage. We showed that p53 or p21 is only up regulated in HepG2 cells ectopically expressing ISG15, but not in the presence of IFN-α-induced ISG15. Our results suggest that ISG15 overexpression could be developed into a powerful gene-therapeutic tool for treating IFN-α-resistant HCC.

Polymorphisms of DNA repair genes XPD, XRCC1, and OGG1, and lung adenocarcinoma susceptibility in Chinese population.

Lung adenocarcinoma (ADC) is one of the major histological types of lung cancer. Genetic polymorphism in DNA repair genes and lung ADC susceptibility is well documented. In this case-control study, the association between the polymorphic sites of DNA repair genes XPD-751, XRCC1-399, and OGG1-326, and lung ADC susceptibility in ethnic Han Chinese population has been investigated. Genomic DNA was isolated from the peripheral blood of 201 healthy controls and 82 lung ADC patients from the people of Hunan Province, China. Polymorphisms of the investigated genes were analyzed by using polymerase chain reaction-restriction fragment length polymorphism. There was no significant difference between the samples from lung ADC patients and healthy controls about the genotype frequencies of XPD-751, XRCC1-399, and OGG1-326 sites. However, multifactor dimensionality reduction analysis showed that the genetic polymorphisms of the three-loci models of DNA repair genes (XPD-751/XRCC1-399/OGG1-326) are associated with lung ADC. Thus, this study reveals that a three-order interaction among the polymorphic sites of XPD-751, XRCC1-399, and OGG1-326 is associated with lung ADC risk in the studied population, although polymorphism in individual gene was not associated.

CYP2A6, CYP1A1, and CYP2D6 polymorphisms in lung cancer patients from central south China.

Lung cancer is a common cause of cancer-related death. The link between risk of lung cancer susceptibility and genetic polymorphisms in metabolic enzymes is well documented. In this study, the relationships between lung cancer susceptibility and polymorphisms in the phase I metabolic enzyme genes CYP1A1, CYP2D6, and CYP2A6 were investigated. Genomic DNA was isolated from the peripheral blood of 201 healthy controls and 168 lung carcinoma patients from the Han ethnic group of Hunan Province in Central South China. Polymorphisms of the investigated genes were analyzed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and two-step allelic-specific PCR assays. No significant differences were found between the frequencies in cases and controls for the genotypes wild-type (WW), heterozygous mutant, or homozygous mutant; for CYP1A1 or CYP2D6; or for the genotypes WW, heterozygous deletion, or null genotype for CYP2A6. The three-locus model (CYP2A6/CYP1A1/CYP2D6) had a maximum test sample accuracy that was significant (P < 0.001) with a cross-validation consistency of 10. These results indicated that the three-order interaction of CYP2A6, CYP1A1, and CYP2D6 polymorphisms might increase genetic susceptibility to lung cancer. We report the involvement of a three-order interaction between CYP1A1, CYP2A6, and CYP2D6 polymorphisms in lung cancer risk in people in Central South China, although no relationship between lung cancer risk and individual gene polymorphisms was found.

Regulatory effects of resveratrol on antioxidant enzymes: a mechanism of growth inhibition and apoptosis induction in cancer cells.

Resveratrol (RSV) is a natural polyphenol that is known as a powerful chemopreventive and chemotherapeutic anticancer molecule. This study focused on the effects of RSV on the activities and expression levels of antioxidant enzymes in the cancer cells. Prostate cancer PC-3 cells, hepatic cancer HepG2 cells, breast cancer MCF-7 cells and the non-cancerous HEK293T kidney epithelial cells were treated with a wide range of RSV concentrations (10-100 µM) for 24-72 h. Cell growth was estimated by trypan blue staining, activities of the antioxidant enzymes were measured spectrophotometrically, expression levels of the antioxidant enzymes were quantified by digitalizing the protein band intensities on Western blots, and the percentage of apoptotic cells was determined by flow cytometry. Treatment with a low concentration of RSV (25 µM) significantly increased superoxide dismutase (SOD) activity in PC-3, HepG2 and MCF-7 cells, but not in HEK293T cells. Catalase (CAT) activity was increased in HepG2 cells, but no effect was found on glutathione peroxidase (GPX) upon RSV treatment. RSV-induced SOD2 expression was observed in cancer cells, although the expression of SOD1, CAT and GPX1 was unaffected. Apoptosis increased upon RSV treatment of cancer cells, especially in PC-3 and HepG2 cells. Together, our data demonstrated that RSV inhibits cancer cell growth with minimal effects on non-cancerous cells. We postulate that the disproportional up-regulation of SOD, CAT and GPX expression and enzymatic activity in cancer cells results in the mitochondrial accumulation of H2O2, which in turn induces cancer cell apoptosis.

Identification of heptapeptides interacting with IFN-α-sensitive CML cells.

BACKGROUND: Interferon-alpha (IFN-α) is the traditional therapeutic agent for chronic myeloid leukemia (CML). The molecular mechanism of IFN-α efficacy in the treatment of CML is not fully clear. OBJECTIVES: To identify the peptides and/or proteins that bind to the proteins specifically expressed on the surface of IFN-α-sensitive CML cells by using a phage display library. DESIGN/METHODS: IFN-α-sensitive KT-1/A3 cells were used as the target, and IFN-α-resistant subline KT-1/A3R was used as absorber for phage display biopanning. The positive phage clones were identified by enzyme-linked immunosorbent assay and flow cytometry. The peptides were deduced from their DNA sequences. RESULTS: Multiple clones showed high binding efficiency to KT-1/A3 cells compared with that of the other leukemia cells. One of the peptides, KLWVIPQ, has a partial amino acid sequence homology with the C-terminal domain of E3 ubiquitin-protein ligase. CONCLUSIONS: This study presents the identification of specific heptapeptides that bind to IFN-α-sensitive KT-1/A3 cells. The cancer-selective ligands provide novel strategies for early and differential diagnoses, as well as potential targeted drug delivery.

Effects of a Shuangling Fuzheng anticancer preparation on the proliferation of SGC-7901 cells and immune function in a cyclophosphamide-treated murine model.

AIM: To study the inhibitory effects of a Shuangling Fuzheng anticancer preparation (SFAP) on the human gastric cancer cell line SGC-7901 in vitro as well as its immune-modulated effects in a cyclophosphamide-treated murine model. METHODS: MTT experiments and immunocytochemistry ABC experiments were performed for detecting the proliferation of SGC-7901 cells in vitro and protein expression of c-myc. The staphylococcal protein A (SPA) rosette test was utilized for measuring the ratio of T-lymphocyte subsets from peripheral blood in a cyclophosphamide-treated murine model. Enzyme-linked immunosorbant assay (ELISA) was performed for measuring the levels of serum sIL-2R in treated mice, while immunoturbidimetry was used for measuring the levels of immunoglobulins (Ig). RESULTS: SFAP (40-640 mg/L, 48 h) inhibited the proliferation of SGC-7901 cells, and a positive correlation was noted between inhibitory effects and dosage. At a dosage of 160-320 mg/L in cultured cells, the expression of c-myc was decreased. SFAP (50-200 mg/kg) increased the percentage of CD3+ and CD4+ T-lymphocytes, the ratio of CD4/CD8, and the contents of Ig such as IgM, IgG or IgA, but decreased the levels of serum sIL-2R in peripheral blood from cyclophosphamide-treated mice. CONCLUSION: SFAP can inhibit the proliferation of SGC-7901 cells via the c-myc gene. In addition, SFAP can modulat the cellular and humoral immunity in cyclophosphamide-induced immunosuppressed mice.

[Study of Shuangling Fuzheng anti-tumor preparation on proliferation and c-myc gene expression of SGC-7901 cells].

OBJECTIVE: To study the effect of Shuangling Fuzheng anti-tumor preparation (SLAP) five groups on proliferation and c-myc gene expression of SGC-7901 cells in vitro. METHOD: The inhibitory effect of single SLAP (40 -640 microg x mL(-1)) and combined therapy with adriamycin (0.4, 4.0 microg x mL(-1) or cisplatin (0.1,1.0 microg x mL(-1) on human gastric carcinoma SGC-7901 cells proliferation were observed by MTT colorimetric analysis method. Technique of flow cytometry in vitro was used to measure the rate of positive sign of SLAP (80 - 320 microg x mL(-1)) on c-myc gene protein of SGC-7901 cells. RESULT: SGC-7901 cells proliferation were inhibited by single SLAP in dose of 40 - 640 microg x mL(-1) 24 h. Its inhibitory rate was increased with increase of dose. The inhibitory rate on SGC-7901 cells could be increased by SLAP in dose of 40 - 640 microg x mL(-1) plus adriamycin in dose of 0.4 and 4.0 microg x mL(-1) or plus cisplatin in dose of 0.1 and 1.0 microg x mL(-1). At the same time, SLAP (80 - 320 microg x mL(-1)) also could inhibite the expression of c-myc gene of SGC -7901 cells. CONCLUSION: Single SLAP had inhibiting effect on human gastric carcinoma cells proliferation with a dose-effect relationship and synergic effect while combined with adriamycin or cisplatin. To inhibit the expression of c-myc gene of human gastric carcinoma cells might be one of action mechanisms of SLAP, which inhibited tumor cells proliferation.

Therapeutic mechanism of ginkgo biloba exocarp polysaccharides on gastric cancer.

AIM: To study the therapeutic mechanism of Ginkgo biloba exocarp polysaccharides (GBEP) on gastric cancer. METHODS: Thirty patients with gastric cancer were treated with oral GBEP capsules. The area of tumors was measured by electron gastroscope before and after treatment, then the inhibitory and effective rates were calculated. The ultrastructures of tumor cells were examined by transmissional electron microscope. Cell culture, MTT, flow cytometry were performed to observe proliferation, apoptosis and changes of relevant gene expression of human gastric cancer SGC-7901 cells. RESULTS: Compared with the statement before treatment, GBEP capsules could reduce the area of tumors, and the effective rate was 73.4%. Ultrastructural changes of the cells indicated that GBEP could induce apoptosis and differentiation in tumor cells of patients with gastric cancer. GBEP could inhibit the growth of human gastric cancer SGC-7901 cells following 24-72 h treatment in vitro at 10-320 mg/L, which was dose- and time-dependent. GBEP was able to elevate the apoptosis rate and expression of c-fos gene, but reduce the expression of c-myc and bcl-2 genes also in a dose-dependent manner. CONCLUSION: The therapeutic mechanism of GBEP on human gastric cancer may relate to its effects on the expression of c-myc, bcl-2 and c-fos genes, which can inhibit proliferation and induce apoptosis and differentiation of tumor cells.

[Clinical study on treatment of patients with upper digestive tract malignant tumors of middle and late stage with Ginkgo biloba exocarp polysaccharides capsule preparation].

OBJECTIVE: To observe the clinical efficacy of Ginkgo biloba exocarp polysaccharides (GBEP) capsule preparation in treating upper digestive tract malignant tumors of middle and late stage. METHODS: Eighty-six patients of the upper digestive tract malignant tumors were treated with GBEP capsule preparation taken orally. The clinical symptoms and the qualities of life of the patients with single GBEP and combined with operation, radiotherapy or intervention chemotherapy were observed. The tumor size was measured by electronic gastroscope before and after treatment with single GBEP. Objective response rate (RR) of the tumor was calculated. The survival period of patient was observed. The changes of blood routine examination in the patients treated with radiotherapy were observed. RESULTS: GBEP preparation could markedly improve the patients'clinical symptoms. Karnofsky scoring of the patients markedly increased after treatment. There were 2 CR (complete response, 6.3%), 22 PR (partial response, 68.8%)and 5 SD (stable disease, 15.6%) of 32 cases with single GBEP preparation. The survival periods of the 32 cases were markedly prolonged. The preparation could relieve the inhibited hematopietic function and the weight loss due to radiotherapy. CONCLUSION: GBEP capsule preparation has some definite therapeutic effects on upper digestive tract malignant tumors of middle and late stage.