Inhibitory Effect Of Lidamycin On Tumor Angiogenesis And Its Mechanism

Tumor growth and metastasis are angiogenesis-dependent. Blocking angiogenesis that could arrest tumor growth has been proved an effective approach to tumor therapy. Currently, the study of anti-angiogenesis has become one of the focal points in cancer research.Tumor vessels grow by three possible mechanisms: (1) the host vascular network expands by budding of endothelial sprouts or formation of bridges (angiogenesis); (2) endothelial cell precursors (angioblasts) home from the bone marrow or peripheral blood in tumor site contribute to the endothelial lining of tumor vessels (vasculogenesis); and (3) the marrow stem cells (MSC) differentiate to endothelial cells and participate in tumor vessels. As known, a panel of activators and inhibitors regulates tumor angiogenesis. One of the most important angiogenic activators is vascular endothelial growth factor (VEGF).Lidamycin (LDM, C-1027), a macromolecular peptide anticancer antibiotic produced by Streptomyces globisporus nov. sp. C-1027, displayed extremely potent cytotoxicity to cancer cells. This study investigated the anti-angiogenesis effect of LDM and its mechanisms both in vitro and in vivo.1. Establishing various methods for culture of primary cells and tumor cell lines which can express GFP stablyIn order to investigate the anti-angiogenesis effect and mechanism of LDM, a series of methods and techniques were established for separation and culture of various cells, including human umbilical vein endothelial cells (HUVEC), rat cerebral endothelial cells (rCMEC), endothelial progenitor cells (EPC), human marrow stem cells (hMSC) and rat marrow stem cells (rMSC). By using transfection and cloning technique, four stably GFP-expressing tumor cell lines, which included SSMC-7721-GFP, HCT116-GFP, A549-GFP, and C6-GFP, were established, laying the foundation for subsequent experiments and studies on the co-culture of tumor cells with endothelial cells, mesenchymal stem cells, and fibroblasts.2. The anti-angiogenesis effects of LDM in vitro and its mechanism2.1. The effect of LDM on endothelial cellsMTT assay showed that LDM suppressed the proliferation of HUVEC, rCMEC, and EPC with IC₅₀ values of 3.88×10^-12 M, 4.33×10^-11 M, and 7.63×10^-12 M, respectively. The IC₅₀ values of LDM are 10³-10⁶ fold lower than that of adriamycin, mitomycin, pingyangmycin, and taxol.Three methods were employed to examine the apoptosis of endothelial cells: Annexin V-FITC/PI dyeing analysis, PI dyeing analysis, and Hoechst 33342 dyeing analysis. LDM concentrations between 0.1 nM and 2 nM significantly induced apoptosis in endothelial cells. Especially, LDM of 2 nM led to early apoptosis of HUVEC at the rate of 77.68%. Comparative results indicated that endothelial cells were more sensitive to LDM than tumor cells and other normal cells.The effects of LDM on the proliferation and apoptosis of HUVEC were observed by cultivating HUVEC under different conditions. Experiment results revealed that VEGF had a stronger effect than bFGF did on promoting HUVEC proliferation. The suppression of HUVEC proliferation and induction of apoptosis by LDM were partly reversed by VEGF.In tube formation assay, LDM ranging from 0.1 nM to 2 nM blocked endothelial tube formation of HUVEC and EPC. LDM significantly inhibited the migration of endothelial cells, as examined by Wound Healing Assay, Transwell assay, and the secretion of MMP-2 and MMP-9.LDM ranging from 0.1 nM to 2 nM decreased the mitochondrial membrane potential (△ψm) of HUVEC, but showed no effect on the level of calcium ion in HUVEC cells.Gene chip technique was employed to analyze the effect of LDM on HUVEC mRNA. LDM caused changes in a number of genes that are relevant to apoptosis, cell cycle progression, angiogenesis, cell adhesion molecules, DNA polymerase, and RNA polymerase; in addition, LDM also had certain impact on some genes of the signaling pathway of MAPK, TGF-β, calcium, Jak-STAT, insulin, and Wnt.LDM activated the signaling pathway of apoptosis in endothelial cells. LDM increased the level of p53, suppressed the expression of Bcl-2 and RB, activated caspase-9 and caspase-3. LDM also reduced Cyclin B1 and CDC2 of endothelial cells, which were associated with LDM-induced S and G2/M arrest in the cell cycle. Furthermore, LDM exerted down-regulation of the signaling pathway of ERK/VEGF in endothelial cells.The above-mentioned results show that LDM can directly affect endothelial cells, including DNA damage, induction of apoptosis, down-regnlation of VEGF signaling pathway, and reduction of gelatinase secretion. In a word, LDM inhibits the proliferation and migration of endothelial cells and plays an effective role in anti-angiogenesis.2.2. The effect of LDM on tumor cellsBy MTT assay, LDM suppressed the proliferation of tumor cell lines including human hepatoma HepG2 cells, human prostatic carcinoma DU 145 cells, human colon carcinoma HCT116 cells with IC₅₀ values of 7.74×10^-11 M, 2.06×10¹¹ M, 7.66×10^-11 M, respectively. In terms of IC₅₀ values, the cytotoxicity of LDM was 10³-fold to 10⁵-fold more potent than that of adriamycin, mitomycin, pingyangmycin, and taxol.Three methods were employed to examine apoptosis: Annexin V-FITC/PI dyeing analysis, PI dyeing analysis, and Hoechst 33342 dyeing analysis. LDM ranging from 0.1 nM to 2 nM had substantial effect on inducing apoptosis of HepG2 cells and DU145 cells; in addition, LDM caused S arrest and G2/M arrest dose-dependently in these two tumor cell lines.LDM ranging from 0.5 nM to 2 nM markedly suppressed the migration and invasion of HepG2 cells and DU145 cells in Transwell assay and Matrigel assay and significantly inhibited the secretion of MMP-2 and MMP-9 in DU145 cells. Furthermore, LDM at concentrations between 0.1 nM and 2 nM decreased the mitochondrial membrane potential (△ψm) in DU145 cell.Gene chip examination in tumor cells showed that LDM caused some changes in a number of genes that are relevant to apoptosis, cell cycle progression, angiogenesis, cell adhesion molecules, DNA polymerase and RNA polymerase; moreover, LDM also had certain impact on some genes of the signaling pathway of MAPK, TGF-β, calcium, Jak-STAT, insulin, and Wnt.In HepG2 cell with wild p53, LDM activated the apoptosis related signaling pathway by increasing p53, reducing RB, and suppressed the ERK signaling pathway. In DU145 cells with mutant p53 and mutant RB, LDM reduced the expression of p53, but showed no effect on the ERK signaling pathway. LDM caused DNA damage in different tumor cell lines that have different genetic mutation; as well as, induced apoptosis in different ways. As found, LDM reduced the expression of Cyclin B1 and CDC2, which were associated with the S arrest and G2/M arrest in HepG2 cells and DU145 cells. Evidently, LDM reduced the expression of VEGF in several tumor cell lines, such as HepG2, DU145, SSMC-7721, MCF-7, and C6 cells. Furthermore, LDM caused down-regulation of the signaling pathway of VEGF/VEGFR2 in HepG2 cells and DU145 cells.The above-mentioned results show that LDM can inhibit the proliferation of tumor cells and induce apoptosis. There are different paths and patterns when LDM takes effect on different tumor cells. In particular, LDM can inhibit the expression of VEGF in various tumor cells. Therefore, besides acting on endothelial cells, LDM may play an important role in blocking the paracrine network of tumor angiogenesis.2.3. The effect of LDM on marrow stem cells and fibroblastsIn co-culture condition, hMSC and 2BS cells were less sensitive to LDM than endothelial cells and tumor cells as determined by MTT assay. This finding is in accordance with the results from in vivo experiment that LDM showed less myelotoxicity. LDM can suppress the migration of hMSC and inhibit its differentiation into endothelial cells.3. The inhibitory effect of LDM on tumor angiogenesis in vivo and its mechanism. LDM at the dosage of 0.02 mg/kg, 0.04 mg/kg, and 0.08 mg/kg suppressed the growth of human hepatoma HepG2 xenografts in nude mice by 37.1%, 65.0%, and 72.8%, respectively. As to the human prostatic carcinoma DU145 xenografts, LDM at 0.02 mg/kg and 0.04 mg/kg inhibited tumor growth by 53.3% and 76.5%, respectively. Immunohistochemical (IHC) examination showed that LDM significantly reduced the microvessel density (MVD) and VEGF level in the tumors.PKH2-labeled EPC and hMSC were injected into the tail vein of nude mice bearing with HepG2 xenografts and the effects of LDM on the tumor and vessel were investigated. No evidence was found that EPC and MSC involved in angiogenesis of the tumor; however, it was found that MVD increased after injection of EPC and it was suppressed by LDM,.The above-mentioned results show that LDM is highly active in suppressing tumor growth in vivo and inhibiting tumor angiogenesis.4. Intraceilular localization of LDMBy hybridoma technology, the monoclonal antibody directed against LDM was prepared. Through fluorescence microscope and confocal laser scanning microscope, the study found that LDM occurred in the cytoplasm of tumor cells and endothelial cells. However, no LDM was found in the nucleus. The putative explanation is that LDM can enter into the cell; then, its apoprotein stays in the cytoplasm and its enediyne chromophore may further penetrates into the nucleus to execute its biological effects.To sum up, LDM inhibits tumor angiogenesis by acting on endothelial cells to exert direct anti-angiogenesis effect. At the same time, LDM may exert an indirect anti-angiogenesis effect by reducing the expression of VEGF in cancer cells and blocking the paracrine network related to tumor angiogenesis.