PI3K As A Co-target On Angiogenesis And Vasculogenic Mimicry In Lung Cancer

Objective:The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway plays an important role in cancer cell proliferation, survival and angiogenesis and so on. While the relationship between PI3K/Akt pathway and formation of vasculogenic mimicry remains unclear. In this study, we will investgate the role of PI3K in the formation of VM in lung cancer.Methods:58 resected tissues were collected including 47 patients with lung cancer and 11 patients with lung benign disease during from Jan,2007 to Apr,2008 in the Union Hospital, tongji Medical College, Huazhong University of Science and Technology. Immunohistochemistry was used to detect the expression of Akt and p-Akt in pathological tissue sections. Immunohistochemistry and PAS histochemistry double staining was used to observe the occurrence of VM in lung cancer tissues. The correlation of p-Akt and VM in lung cancer tissues was analyzed statistically.Results:Akt was expressed in 38 cases (80.8%), p-Akt in 36 cases (76.5%) in lung cancer tissue sections. Akt was expressed in 3 cases (27.3%) and p-Akt in 2 cases (18.2%) in lung benign disease tissue sections. The expression of Akt and p-Akt is higher in lung cancer than that in lung benign disease. VM existed in 6 cases (12.7%) with lung cancer, while the expression of p-Akt was positive. The occurrence of VM was 0%in those cases in lung cancer which the expression of p-Akt was negative.Conclusion:PI3K/Akt signal pathway might play an important part in the formation of VM in lung cancer. The advanced research is needed to investigate the precise mechanism of the formation of VM in lung cancer.Objective:The phosphatidylinositol 3-kinase (PI3K) signaling axis plays an important role in angiogenesis in tumor. LY294002 is a kind of PI3K specific inhibitor. The effect and precise molecular mechanism of LY294002 on angiogenesis in lung cancer remains unclear.Methods:Fluorescence-immunocytochemistry was used to detect the expression of factorⅧantigen in EA.hy926 to identify whether the cell line is endothelial cell. The cytotoxic effect and the inhibition of adhesion of LY294002 on EA.hy926 cell were measured by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Western blot assay was used to investigate the expression of p-Akt in EA.hy926 cells after treated with LY294002, which is used to evaluate the activity of PI3K. Morphological change of EA.hy926 cells was detected by 2% Coomassie Brilliant Blue R250. The migration activity of EA.hy926 cells was detected by Transwell chamber assay. The effect of LY294002 on the tube formation of EA.hy926 was observed by the tube formation assay. Nude mice inoculated subcutaneously with A549 cells were randomLy divided into three groups:the blank control group, the negative control group and the treatment group. The volume of the tumors was measured every one day after inoculation until the mice were sacrificed. Then the expression of CD31, p-Akt, and VEGF in tumor tissues was assessed by Immunohistochemistry.Results:The expression of factorⅧantigen in EA.hy926 was positive. This cell line could be used to advanced experiment. PI3K specific inhibitor LY294002 inhibited proliferation and adhesion activity of EA.hy926 cells in a time and concentration-dependent manner. The IC50 of LY294002 to EA.hy926 cell was (38.4±4.9)μmol/L at 24h, (24.6±5.1)μmol/L at 48h and (16±3.7)μmol/L at 72h. LY294002 changed cell morphology, decreased the migration activity. After treated with LY294002 with the dose of IC50 at 24h, the expression of p-Akt was down-regulated by western blot assay. The tumor volume and micro vessel density in the treatment group were significantly decreased compared with the control group (p<0.05). The expression of p-Akt and VEGF in the treatment group was significantly lower than that in the bank control group and the negative control group.Conclusion:Our findings indicate that PI3K inhibitor LY294002 might inhibit angiogenesis of lung cancer though inhibiting cell proliferation, adhesion, migration, the tube formation of EA.hy926 cell and down-regulating the expression of VEGF in lung cancer cell A549.Objective:PI3K are being extensively studied as a potential target to tumor.In part I, our results showed that PI3K might play an important role in the formation of VM in lung cancer. In the present study, we studied the effect of PI3K inhibitor LY294002 on the formation of vasculogenic mimicry channels of lung cancer cell in vitro and in vivo.Methods:MTT assay was used to evaluate the effect of PI3K inhibitor LY294002 on the proliferation of A549 cell, and to calculate the IC50 of LY294002 to A549 cell at 72h. The effect of LY294002 on the adhesion activity of A549 cells was detected by MTT, too. Then we examined the expression of p-Akt in A549 cell after addition LY294002 to assess the PI3K activity by western blot. Cytoskeleton staining and tube formation assay were used to observe the effect of LY294002 on the actin cytoskeleton and the ability to form vascular networks in A549 cells. Wound healing assay was used to evaluate the effect of LY294002 on migration activity of A549 cells. The invasion activity of A549 cells was detected by Transwell chamber assay. The activity of MMPs of A549 cells treated with LY294002 at different doses was measured by Gelatin Zymography. RT-PCR and western blot were used to investigate the expression of VEGF of A549 cells after treated with LY294002. Nude mice inoculated subcutaneously with A549 cells were randomLy divided into three groups: the blank control group, the negative control group and the treatment group. The volume of the tumors was measured every one day after inoculation until the mice were sacrificed. Then the expression of p-Akt and VEGF in tumor was assessed by immunohistochemical. The density of VM tube was evaluated by immunohistochemical and PAS histochemical double-Staining.Results:MTT assay showed that LY294002 inhibits the proliferation of A549 cell in a dose-dependent and time-dependent manner. The IC50 of LY294002 to A549 cell was (62.5±6.9)μmol/L at 24h, (38±4.1)μmol/L at 48h and (23.3±4.7)μmol/L at 72h. The PI3K activity of A549 cells incubated with LY294002 for 24h decreased by western blot. The adhesion activity of A549 cells was inhibited by LY294002. Human lung carcinoma cell line A549 had the ability to form vascular network in vitro. After addition LY294002, the actin cytoskeleton of A549 cells were broken, the activity of migration and invasion were inhibited, the ability to form vascular network was inhibited significantly, the activity of MMP-2 and MMP-9 secreted by A549 cell were significantly inhibited compared with the control group. Furthermore, the expression of VEGF of A549 cells down-regulated after treated with LY294002. The tumor volume and the density of VM in the treatment group were significantly decreased compared with the control groups (p<0.05). The expression of p-Akt and VEGF in the treatment group was significantly.lower than that in the bank control group and the negative control group.Conclusion:A549 cells can form cords and vascular networks when cultured on 3-D collagen matrices. PI3K inhibitor LY294002 can inhibit the ability to form vascular networks of A549 cells though changing the action cytoskeleton, inhibiting cell proliferation, the activity of adhesion, migration and invasion and the ability of tube formation and down-regulating the expression of MMP-2/9 and VEGF in lung cancer cells A549. Objective:In partⅢ, Our results showed that PI3K specific inhibitor LY294002 inhibited the formation of VM though down-regulating the expressiong of VEGF in lung cancer cell line. VEGF can induce angiogenesis in solid tumor. Our present study will explore the relationship between VEGF expression and vasculogenic mimicry of tumor.Methods We observed the ability to form vasculogenic mimicry in several cell lines follow as LO2 cell,hepG2 cell,SMMC-7721 cell and A549 cell in three-dimensional cell cultures. Then, we detected the expression of VEGF in the level of mRNA and protein by RT-PCR and western blot in those cell lines. The cell lines including A549A transfected with empty plasmids and A549B transfected with Eukaryotic expression plasmids pcDNA3.0-VEGF165b were constructed. The ability of tube formation of two cell lines was observed in the 3-dimensional culture.Results HepG2 cell and A549 cell had the ability to form vasculogenic mimicry in three-dimensional cell cultures, while LO2 cell and SMMC-7721 cell hadn't the ability. The expression of VEGF gene in the level of mRNA and protein in HepG2 cell,LO2 cell and A549 cell were significantly higher than that in SMMC-7721 cell(P<0.01). VEGF protein expression in A549A group was significantly higher than that in A549B group, while the expression of VEGF165b in A549A was lower (p<0.05). Both A549A and A549B cells had the ability to form vascular networks in vitro.Conclusion The cell lines which the expression of VEGF gene is low don't have the ability to form vasculogenic mimicry and the high expression of VEGF can't form vasculogenic mimicry at all, while the expression of VEGF gene in the cell lines is high in the cell lines which can form vasculogenic mimicry. VEGF has impact on the formation of vasculgenic mimicry. Only the cells which the expression of VEGF is high have the potential to form vasculogenic mimicry. But, it doesn't play a unique key role in vasculogenic mimicry. VEGF165b doesn't inhibit the formation of vasculogenic mimicry of lung carcinoma in vitro.