12-Deoxyphorbol 13-palmitate inhibit VEGF-induced angiogenesis via suppression of VEGFR-2-signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: 12-Deoxyphorbol 13-palmitate (G) is one toxic compound isolated from Euphorbia fischeriana, an Asian spice used for cancer treatment as a folk remedy. However, whether 12-deoxyphorbol 13-palmitate affects angiogenesis remains unclear. AIM OF THE STUDY: To explore the in vitro and in vivo antiangiogenic effects of 12-deoxyphorbol 13-palmitate and its underlying mechanisms. MATERIALS AND METHODS: We explored antigenic functions in human umbilical vein endothelial cells (HUVEC) by 12-deoxyphorbol 13-palmitate, including proliferation, migration and metastasis through matrigel plug assay, chorioallantoic membrane assay, in vitro migration assay, tube formation assay, motility assay. Antibody chip was applied to screen differentially expressed proteins modulated by 12-deoxyphorbol 13-palmitate, and was further confirmed by RT-PCR and western blot analysis. Tumor xenograft mice were applied to investigate whether 12-deoxyphorbol 13-palmitate could inhibit microvessel density in vivo. RESULTS: 12-Deoxyphorbol 13-palmitate inhibited vascular endothelial growth factor (VEGF)-induced angiogenic processes in vitro, such as proliferation, in vitro migration, and tube formation of HUVEC. In chorioallantoic membrane assay, 12-deoxyphorbol 13-palmitate significantly inhibited neovessel formation. Antibody chip technology demonstrated decreased expression of TIMP-1, TIMP-2, VEGF, basic fibroblast growth factor (bFGF), matrix metalloproteinases (MMP)-2, VEGFR-2 and VEGFR-3 proteins in HUVEC after 24h. In addition, 12-deoyphorbol 13-palmitate inhibited the in vivo growth of MCF-7 cells in grafted mouse model. Immunohistochemistry staining showed decreased microvessel density (CD31) and attenuated VEGFR-2 signaling pathways by 12-deoxyphorbol 13-palmitate. CONCLUSION: 12-Deoxyphorbol 13-palmitate may be utilized to target active angiogenesis through VEGF/VEGFR2 signal pathway for cancer.

Isolation of diallyl trisulfide inducible differentially expressed genes in human gastric cancer cells by modified cDNA representational difference analysis.

Extensive epidemiologic studies indicated protective effects of consumption of garlic on reducing human gastric cancer (HGC) incidence. Diallyl trisulfide (DATS), a critical organic allyl sulfur component of garlic, was reported to have chemopreventive effects in inhibiting tumor process. We used DATS to treat HGC cell line BGC823 cells, and showed that DATS induces G1/S arrest and apoptosis in BGC823 cells demonstrated by a flow cytometric analysis. To further isolate DATS inducible differentially expressed genes in BGC823 cells, we combined a highly specific subtractive hybridization of cDNA representational difference analysis (cDNA RDA) with a sensitive bidirectional radioactive detection of mRNA differential display (mRNA DD) to develop a subtractive hybridization differential display (SHDD) method. This modified method adopted a first round of bidirectional subtractive hybridization between two sample cDNAs and a second round of bidirectional subtractive hybridization between the two resultant first-round difference products. Bidirectional subtractive hybridizations magnified the differences between the two sample cDNAs and favored isolating mRNA species with very small expression differences. We employed the SHDD method to detect DATS inducible differentially expressed genes in BGC823 cells. A total of 14 cDNA fragments (11 upregulated and 3 downregulated by DATS treatment) were isolated and confirmed by reverse Northern blot analysis. Our data show that SHDD is a powerful technique for identifying differentially expressed mRNA species between two sample cDNAs and provide useful cellular and molecular information for understanding the effects of garlic against human gastric cancer.

Construction of cDNA representational difference analysis based on two cDNA libraries and identification of garlic inducible expression genes in human gastric cancer cells.

AIM: To elucidate molecular mechanism of chemopreventive efficacies of garlic against human gastric cancer (HGC). METHODS: HGC cell line BGC823 was treated with Allitridi (a kind of garlic extract) and Allitridi-treated and parental BGC823 cDNA libraries were constructed respectively by using lambdaZAP II vector. cDNA Representational Difference Analysis (cDNA RDA) was performed using Bam H I cutting-site and abundant cDNA messages provided by the libraries. Northern blot analysis was applied to identify the obtained difference products. RESULTS: Two specific cDNA fragments were obtained and characterized to be derived from homo sapiens folate receptor alpha (FRalpha) gene and calcyclin gene respectively. Northern blot results showed a 4-fold increase in FRalpha gene expression level and 9-fold increase in calcyclin mRNA level in BGC823 cells after Allitridi treatment for 72h. CONCLUSION: The method of cDNA RDA based on cDNA libraries combines the high specificity of cDNA RDA with abundant cDNA messages in cDNA library; this expands the application of cDNA library and increases the specificity of cDNA RDA. Up-regulation of FRalpha gene and calcyclin gene expressions induced by Allitridi provide valuable molecular evidence for the efficacy garlic in treating HGC as well as other diseases.

Applying a highly specific and reproducible cDNA RDA method to clone garlic up-regulated genes in human gastric cancer cells.

AIM: To develop and optimize cDNA representational difference analysis (cDNA RDA) method and to identify and clone garlic up-regulated genes in human gastric cancer (HGC) cells. METHODS: We performed cDNA RDA method by using abundant double-stranded cDNA messages provided by two self-constructed cDNA libraries (Allitridi-treated and paternal HGC cell line BGC823 cells cDNA libraries respectively). Bam H I and Xho I restriction sites harbored in the library vector were used to select representations. Northern and Slot blots analyses were employed to identify the obtained difference products. RESULTS: Fragments released from the cDNA library vector after restriction endonuclease digestion acted as good marker indicating the appropriate digestion degree for library DNA. Two novel expressed sequence tags (ESTs) and a recombinant gene were obtained. Slot blots result showed a 8-fold increase of glia-derived nexin/protease nexin 1 (GDN/PN1) gene expression level and 4-fold increase of hepatitis B virus x-interacting protein (XIP) mRNA level in BGC823 cells after Allitridi treatment for 72h. CONCLUSION: Elevated levels of GDN/PN1 and XIP mRNAs induced by Allitridi provide valuable molecular evidence for elucidating the garlic's efficacies against neurodegenerative and inflammatory diseases. Isolation of a recombinant gene and two novel ESTs further show cDNA RDA based on cDNA libraries to be a powerful method with high specificity and reproducibility in cloning differentially expressed genes.