| Background Our previous studies have shown that a synthetic peptide encompassing both residues sequence 74-98 and residues sequence 185-203 of tumstatin, named recombinant vascular basement membrane derived multifunctional peptide (rVBMDMP), significantly suppresses the growth of Lewis lung carcinoma xenografts and decreases the tumor metastasis number of Lewis lung carcinoma. In the present study, we furtherly demonstrate the antitumor activities of rVBMDMP using assays in vitro and in vivo. In addition, phosphorylation changes of signaling molecules in human lung carcinoma cell line (A549) were detected in order to initially elucidate the molecular mechanisms for therapeutic human lung carcinoma by rVBMDMP.Methods MTT assay and cell counting in Trypan Blue staining were used to determined the cytotoxic effect of rVBMDMP on lung cancer cell line (A549) and normal cell line (KMB-17). Human lung cancer xenografts in nude mouse model were employed to evaluate the therapeutic effect of human lung cancer by rVBMDMP in vivo. To assess the effect of rVBMDMP on tumor angiogenesis, immunohistochemistry for CD34 was used and the microvessel density (MVD) of CD34 immunostained sections was counted. A549 cells cultured in vitro were tread with rVBMDMP(10.0 μ M) or vehicle (sterile PBS) as control, followed by preparation of whole cell extracts. Signal transduction antibodyarrayTM(Hypromatrix) was employed for screening protein tyrosine (serine and threonine) phosphorylation. Results Assays in vitro showed that rVBMDMP selectively inhibited proliferation of A549 in a dose dependent manner, with no effect on KMB-17.In addition, rVBMDMP also inhibited in vivo tumor growth in xenograft mouse model. Tertian intraperitoneal injection of rVBMDMP suppressed the growth of A549 xenograft in a dose dependent manner, rVBMDMP (5 mg/kg) decreased the tumor volume and tumor weight by 74％and 77％respectively in A549 xenograft, and this inhibitory effect on tumor growth is more potent than Cyclophosphamide (100mg/kg) and angiogenesis inhibitor TNP-470 (20mg/kg) as well.The microvessel density in rVBMDMP -treatment (5mg/kg) and TNP-470 (20mg/kg) positive control group was (38.56±4.64) strips per 200 magnification and (55.73±5.28) /strips per 200 magnification, respectively, which was significantly lower than that in NS(0.1ml/10g) control group (94.82±6.48) /strips per 200 magnification (P＜0.05).In addition,the anti-angiogenic effect of rVBMDMP (5mg/kg) is more potent than TNP-470 (20mg/kg).Proteins phosphorylation. of which were upregulated in rVBMDMP-treaed as compared to control included those in cell cycle regulation such as cyclin B, cyclin E, CDK2; proteins involved in apoptosis such as Fas, FasL, caspase 6; kinases such as JNK1, 14—3—3; cytokines such as IL—1R, IL—2Rβ, IL—4Rα, IFN—2Rα, IFN—γRαand G—CSFR. In contrast, proteins phosphorylation of which were downregulated included those associated with apoptosis and proliferation such as Bcl-2, NF-kB, and nuclear factor of activated T cells (NFAT).Conclusion1. rVBMDMP signalificantly inhibited proliferation of lung carcinoma cell line(A549) in a selective manner2. rVBMDMP signalificantly suppressed tumor growth of A549 cells xenografts, and tumor angiogenesis as well.3. The possible molecular mechanisms of rVBMDMP's anti-tumor activity included following(1) rVBMDMP inhibited proliferation of hunman lung carcinoma cell line A549 via intricate signaling transduction pathways triggered by the binding of rVBMDMP to intergrin alap(V)beta(3).The interaction between rVBMDMP and alap(V)beta(3) possibly decreased the level of phosphorylation of the latter, resulting in changes in confirmation and function of alap(V)beta(3).(2) rVBMDMP dephosphorylated Bcl-2 through the interaction between rVBMDMP and intergrins, leading to inactivation of Bcl-2 and cell apoptosis.(3) rVBMDMP dephosphorylated NF-kB and led to inactivation of NF-kB, and as a result, caused cell death. |
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