Based on the epidermal growth factor receptor(EGFR)/mitogen-activated protein kinase(MAPK) signaling pathway-mediated cell proliferation, this study explores the effect of cisplatin combined with Guiqi Yiyuan Ointment on Lewis lung cancer-bearing mice. A total of 60 male C57BL/6 mice were randomly divided into a blank group with 10 mice and a modeling group with 50 mice. After modeling, they were randomly divided into the model group, cisplatin group, and low-, medium-, and high-dose groups of cisplatin combined with Guiqi Yiyuan Ointment, with 10 mice in each group. After 14 days of medication, the general condition of the mice was observed; body weight was measured, and organ index and tumor inhibition rate were calculated. Hematoxylin-eosin(HE) staining was used to observe the pathological morphology changes in tumor tissue. Immunohistochemistry was used to detect the positive rate of Ki-67 antigen(Ki-67) and proliferating cell nuclear antigen(PCNA) in tumor tissue. Western blot and real time-quantitative polymerase chain reaction(qPCR) were used to detect the expression of related proteins and mRNA in tumor tissue. Flow cytometry was used to detect the cell cycle of tumor cells in tumor tissue. The results showed that compared with that in the blank group, the general condition of mice in the model group deteriorated; the body weight, as well as thymus and spleen index of mice in the model group decreased after 14 days of medication. Compared with that in the model group, the general condition of mice in the cisplatin group deteriorated, while the condition of mice in the combined groups improved; the body weight, as well as thymus and spleen index of mice in the cisplatin group decreased, while the three indicators in the combined groups increased; the tumor weight of each medication group decreased, and the tumor inhibition rate increased; there were varying degrees of necrosis in tumor cells of each medication group, and the tightness of tumor cells, the increase in the number of cell nuclei and chromatin, and mitosis all decreased. The positive rate of Ki-67 and PCNA, as well as the protein expression and ratio of p-EGFR/EGFR, rat sarcoma viral oncogene homolog(Ras), phosphorylated Raf-1 protein kinase(p-Raf-1)/Raf-1, phosphorylated mitogen-activated protein kinase kinase(p-MEK)/MEK, phosphorylated extracellular signal-regulated kinase(p-ERK)/ERK and the mRNA expression of EGFR, Ras, Raf-1, MEK, and ERK all decreased. The proportion of tumor cells in the G_0/G_1 phase of each medication group increased, and that in the S phase decreased. In addition, there was no significant difference in the G_2/M phase. Compared with that of the cisplatin group, the tumor weight of the combined groups decreased, and the tumor inhibition rate increased. The necrosis and mitosis of tumor cells in the combined groups were more pronounced; the positive rate of Ki-67 and PCNA, the protein expression and ratio of p-EGFR/EGFR, Ras, p-Raf-1/Raf-1, p-MEK/MEK, and p-ERK/ERK, as well as the mRNA expression of EGFR, Ras, Raf-1, MEK, and ERK in the combined groups all decreased. The proportion of tumor cells in the G_0/G_1 phase of the combined medium-and high-dose groups increased, and that in the S phase decreased. There was no significant difference in the proportion of tumor cells of the combined groups in the G_2/M phase. This indicates that the combination of cisplatin and Guiqi Yiyuan Ointment can enhance the anti-tumor effect of cisplatin on tumor-bearing mice, and the mechanism may be associated with the inhibition of the EGFR/MAPK pathway, which accelerates the arrest of tumor cells in the G_0/G_1 phase, thereby inhibiting the proliferation of tumor cells. At the same time, the study also indicates that Guiqi Yiyuan Ointment may reduce the damage of tumors to mice and the toxic side effects brought by cisplatin chemotherapy.
Animals ; Male ; Carcinoma, Lewis Lung/metabolism* ; Drugs, Chinese Herbal/administration & dosage* ; ErbB Receptors/genetics* ; Mice ; Cisplatin/administration & dosage* ; Mice, Inbred C57BL ; Cell Proliferation/drug effects* ; Ointments/administration & dosage* ; MAP Kinase Signaling System/drug effects* ; Humans ; Antineoplastic Agents/administration & dosage* ; Lung Neoplasms/metabolism*

The aim of this paper was to observe the effect of Feiliuping Gao and its combination with different types of drugs intervention on the expression of PI3K/AKT/NF-κB in lung metastatic microenvironment, and to reveal the advantage of Chinese medicine intervention time on the key molecule in lung metastatic microenvironment. The mouse model of Lewis lung carcinoma was established, and lung tissues were collected at 14 days, 21 days and 28 days after the intervention of Feiliuping Gao, and the expressions of PI3K, AKT and NF-κB were detected by immunohistochemistry and Western blot. At 14 days, there was no significant difference in PI3K expression between each group and the control group. The expression of AKT protein was significantly inhibited in the celecoxib (CLB) group, the Feiliuping Gao (FLP) combination with cyclophosphamide (FLP+CTX) group, and the Feiliuping Gao combination with celecoxib (FLP+CLB) group (<0.05). The inhibition of AKT protein expression in FLP+CLB group was superior. The FLP+CLB group can inhibit the expression of NF-κB protein (<0.05). At 21 days, compared with the control group, the expression of PI3K was inhibited in FLP group and the FLP+CTX group (<0.05), while the expression of PI3K was best inhibited in the FLP+CLB group (<0.001). Only the FLP+CLB group could significantly inhibit the expression of AKT protein (<0.01). The FLP+CTX group had the best effect in inhibiting the expression of NF-κB protein (<0.001). At 28 days, compared with the control group, the expression of PI3K and AKT was inhibited in the FLP+CLB group (<0.001). Feiliuping ointment combination with celecoxib has an advantage in regulating the expression of PI3K/AKT/NF-κB molecules in lung metastatic microenvironment.
Animals ; Carcinoma, Lewis Lung ; pathology ; Drugs, Chinese Herbal ; pharmacology ; Lung ; Mice ; NF-kappa B ; metabolism ; Neoplasm Metastasis ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Signal Transduction

OBJECTIVETo observe the effect of alcohol extracts from Pharbitidis Semen on the proliferation and metastasis of Lewis lung cancer, and study its anti-tumor mechanism.

METHODIn vitro, MTT assay and scratch assay were adopted to detect the effect of alcohol extracts from Pharbitidis Semen on the proliferation and metastasis of Lewis lung cancer cells. The cell autophagy was detected by the acridine orange staining. The gap-junction intercellular communication (GJIC) was investigated by the fluorescent yellow transfer. The expression of aquaporin 1 (AQP1) was analyzed by the Western blotting. In vivo, the subcutaneous implant model and the experimental pulmonary metastasis model of Lewis lung cancer in mice were established to evaluate the anti-tumor and anti-metastasis effects of alcohol extract from Pharbitidis Semen. The serum carcinoembryonic antigen (CEA) and beta2 microglobulin (beta2-MG) of mice bearing Lewis lung cancer were detected by the electrochemiluminesence immunoassay. The expressions of lung AQP1 and Connexin 43 (Cx43) were examined by the immunohistochemical method.

RESULTIn vitro, alcohol extracts from Pharbitidis Semen inhibited the cell proliferation in a dose-dependent matter, significantly prevented the cell migration, down-regulated AQP1 proteins of cells, promoted GJIC, and decreased the serum-free autophagy of tumor cells. In vivo, compared with untreated model mice, alcohol extracts from Pharbitidis Semen inhibited the tumor growth in a dose-dependent matter, prevented the tumor metastasis and prolonged the life span of mice bearing Lewis lung cancer, while decreasing serum CEA and beta2-MG of mice bearing Lewis lung cancer, enhancing the immumohistochemical staining intensity of Cx43 and weakening aquaporins AQP1 positive intensity.

CONCLUSIONAlcohol extracts from Pharbitidis Semen could prevent the proliferation and metastasis in Lewis lung cancer cells. Its mechanism may be related to the promotion of GJIC and the down-regulation of AQP1.

Animals ; Antineoplastic Agents ; administration & dosage ; Aquaporin 1 ; genetics ; metabolism ; Carcinoma, Lewis Lung ; drug therapy ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Connexin 43 ; genetics ; metabolism ; Disease Models, Animal ; Drugs, Chinese Herbal ; administration & dosage ; Humans ; Ipomoea ; chemistry ; Lung Neoplasms ; drug therapy ; genetics ; metabolism ; pathology ; Male ; Mice ; Mice, Inbred C57BL ; Neoplasm Metastasis ; Seeds ; chemistry

OBJECTIVETo explore the radiosensitization effect of black garlic extract (BGE) on lung cancer cell line Lewis cells.

METHODSThe inhibition rate of lung cancer cells after BGE action was detected by MTT. Effect of BGE combined radiotherapy on the colony formation rate was observed by cloning formation assay. Changes of the cell morphology were observed by Hoechst staining. Changes of the cell cycle were detected by flow cytometry. Real time PCR was used to detect mRNA expressions of bcl-2 and bax.

RESULTSBGE could have significant inhibitory action on the growth of lung cancer Lewis cells. The combination of BGE and radiotherapy (by 60Co gamma) significantly induced Lewis cells' apoptosis in G2/M stage, obviously decreased the expression of bcl-2, and up-regulated the expression of bax.

CONCLUSIONSBGE could sensitize the lung cancer Lewis cells to ionizing irradiation. This effect might be probably caused by changing the cell cycles and affecting expressions of bax and bcl-2.

Animals ; Apoptosis ; Carcinoma, Lewis Lung ; metabolism ; pathology ; Cell Line, Tumor ; drug effects ; radiation effects ; Garlic ; Mice ; Plant Extracts ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Radiation-Sensitizing Agents ; pharmacology ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo observe the effect of Yifei Qinghua Granule (YQG) on vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), angiostatin, and endostatin in tumor tissue of Lewis Lung cancer mice, and to explore its anti-tumor mechanisms.

METHODSTotally 70 C57BL/6 mice were randomly divided into the model group, the low, medium, and high dose YQG groups, the gefitinib group, the gefitinib plus medium dose YQG group, and the cyclophosphamide (CTX) group, 10 in each group. The models were established by subcutaneously injecting Lewis lung cancer cells from the right axilla of C57BL/6 mice. Mice in the model group were given with 0.4 mL pure water by gastrogavage, once daily. Mice in the low and medium dose YHG groups were given with YHG at the daily dose of 5 and 10 g/kg by gastrogavage, once daily. Those in the high dose YHG group were given with YHG at 10 g/kg by gastrogavage, twice daily. Those in the gefitinib group were given with gefitinib 100 mg/ kg by gastrogavage, once daily. Those in the gefitinib plus medium dose YHG group were given with gefitinib at 100 mg/kg by gastrogavage in the morning and YHG at 10 g/kg by gastrogavage in the afternoon. All medication was started from the 2nd day of inoculation, lasting 14 successive days. Those in the CTX group were given CTX at 60 mg/kg by peritoneal injection on the 3rd and the 7th day of the experiment. Mice were sacrificed at the fifteenth day of the experiment. Tumors were taken out. Expressions of VEGF, bFGF, angiostatin, and endostatin in the tumor tissue were detected using immunohistochemical assay.

RESULTSCompared with the model group, the expression of VEGF significantly decreased, expressions of angiostatin and endostatin significantly increased in each group (P < 0.01). The expression of bFGF significantly decreased in the gefitinib group (P < 0.05). There was no statistical difference in VEGF among all groups (P > 0.05). The angiostatin expression was significantly higher in the CTX group than in the low dose YQG group (P < 0.01). The expression of endostatin was significantly higher in the high dose YQG group and the gefitinib plus medium dose YQG group than in the low and the medium dose YQG groups (P < 0.01). The expression of endostatin was significantly higher in the gefitinib plus medium dose YQG group than in the gefitinib group (P < 0.05).

CONCLUSIONThe action mechanism of YQG in treating lung cancer might be achieved through reducing the expression of angiogenesis promoting factor VEGF and increasing expressions of angiogenesis inhibitors angiostatin and endostatin.

Angiostatins ; metabolism ; Animals ; Carcinoma, Lewis Lung ; drug therapy ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Endostatins ; metabolism ; Fibroblast Growth Factor 2 ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Phytotherapy ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVETo explore the mechanism of polypeptide extract from scorpion venom (PESV) on promoting anti-tumor effects of cyclophosphamide (CTX).

METHODSThe Lewis lung tumor model was established by subcutaneously implanting Lewis lung cells into C57BL/6 mice. The tumor-bearing mice were randomly divided into 4 groups, i. e., the model group, the cyclophosphamide (CTX) group, the polypeptide extract from scorpion venom (PESV) group, and the combination group (CTX + PESV), 10 mice in each group. The tumor growth curve was recorded. Changes of vascular endothelial growth factor-A (VEGF-A) and transforming growth factor-beta1 (TGF-beta1) expressions in the tumor microenvironment were detected using reverse transcription PCR and immunohistochemical assay. Changes of dendritic cells (DCs) phenotype CD80 and CD86 expressions in the tumor tissue were detected using immunofluorescence chemical assay.

RESULTSAfter 21 successive days of treatment, the growth of Lewis lung cancer transplantation tumor in the combination group was obviously inhibited (P<0.05). Compared with the model group,the expressions of CD80 and CD86 in the PESV group was somewhat enhanced, while those in the CTX group was somewhat lowered. Compared with the CTX group, the fluorescent signal strength and expressions in the combination group somewhat increased. Compared with the model group, the expressions of TGF-beta1 and VEGF-A mRNA decreased in the PESV group and the CTX group (both P<0.05). Compared with the PESV group and the CTX group, the expressions of TGF-beta1 and VEGF-A in the combination group both decreased (both P<0.05).

CONCLUSIONPESV could inhibit the expressions of VEGF and TGF-beta1, promote the maturation of DCs, recover its antigen uptake presentation function, and reverse the immune injury to the body by CTX, thus playing a role in inducing the tumor cell apoptosis.

Animals ; B7-1 Antigen ; B7-2 Antigen ; Carcinoma, Lewis Lung ; immunology ; metabolism ; pathology ; Cyclophosphamide ; pharmacology ; Dendritic Cells ; immunology ; Lung Neoplasms ; metabolism ; pathology ; Male ; Mice ; Mice, Inbred C57BL ; Peptides ; pharmacology ; Scorpion Venoms ; pharmacology ; Transforming Growth Factor beta1 ; metabolism ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVETo reveal the different molecular mechanisms between Chinese drugs for activating blood (CDAB) and Chinese drugs for nourishing qi and activating blood (CDNQAB) in the metastasis process of Lewis lung carcinoma, thus providing experimental reliance for Chinese drugs to reverse immune escape.

METHODSThe inhibition rate of lung metastasis was observed in each group. The dynamic percentage and ratio changes of Th17 and Treg cells in spleen CD4+ T lymphocytes were detected using flow cytometry. The dynamic levels of IL-17, IL-23, and gamma interferon (IFN-gamma) in the culture supernatant of CD4+ T lymphocytes were detected by ELISA. The dynamic mRNA expressions of Foxp3 and RORgammat in CD4+ T lymphocytes were detected by RT-PCR.

RESULTSCDNQAB (sapanwood +astragalus) showed better lung metastasis inhibiting rate than CDAB (sapanwood alone) (P<0.05), similar to the effects of cyclophosphamide (P>0.05). Except the CDNQAB group, spleen Th17 and Treg cells showed a rising tendency in mice of each tumor-bearing group. The effectors of Th17 and Treg cells (IL-17, IL-23, and IFN-gamma) and key transcription molecules of Th17 and Treg cells (RORgammat and Foxp3) showed dynamic changes corresponding to Th17 and Treg cells.

CONCLUSIONSThe immune inflammatory reactions of CDNQAB (sapanwood +astragalus) were superior to those of CDAB (sapanwood alone) and of cyclophosphamide during the process of inhibiting tumor immunotolerance and of the formation of tumor. All drugs showed certain inhibition on the mechanisms for neoplasm metastasis. But CD-NQAB was superior to CDAB and chemotherapeutics.

Animals ; Carcinoma, Lewis Lung ; drug therapy ; immunology ; pathology ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Forkhead Transcription Factors ; metabolism ; Interferon-gamma ; immunology ; Interleukin-17 ; immunology ; Interleukin-23 ; immunology ; Male ; Mice ; Mice, Inbred C57BL ; Neoplasm Metastasis ; Nuclear Receptor Subfamily 1, Group F, Member 3 ; metabolism ; T-Lymphocytes, Helper-Inducer ; immunology ; T-Lymphocytes, Regulatory ; immunology

OBJECTIVETo observe the effects of Feiyanning Recipe's (FR) components on the microvessel density (MVD), the mRNA and protein expressions of matrix metalloproteinase-2 (MMP-2) and MMP-9 in Lewis tumors loaded in C57BL/6 mice.

METHODSC57BL/6 Lewis lung cancer mouse model was established. Mice were randomly divided into five groups, i.e., the model group, the FR group, the qi benefiting group, the Shen-tonifying group, and the anti-cancer group. The mice were killed on the 22nd day after 21-day gastrogavage. Tumors were extracted. The MVD of Lewis tumor was detected by immunohistochemical SP method. The mRNA and protein expressions of MMP-2 and MMP-9 were measured using reverse transcription-polymerase chain reaction (RT-PCR) and SP method.

RESULTSCompared with the model group and the qi benefiting group, the MVD was significantly reduced in the FR group, the Shen-tonifying group, and the anti-cancer group (P<0.01, P<0.05). But there was no significant difference between the qi benefiting group and the model group (P>0.05). The mRNA and protein expressions of MMP-2 in the FR group, the Shen-tonifying group, and the anti-cancer group were also significantly less than those in the model group (P<0.05, P<0.01). At the same time the expression of MMP-2 mRNA in the Shen-tonifying group and the anti-cancer group was also less than that in the qi benefiting group (P<0.05). The mRNA and protein expressions of MMP-9 in the FR group, the Shen-tonifying group, and the anti-cancer group were significantly lower than those in the model group and the qi benefiting group (P<0.05, P<0.01).

CONCLUSIONThe target for Shen-tonifying and anti-cancer Chinese herbs to inhibit tumor angiogenesis might be correlated with restraining expressions of MMP-2 and MMP-9, and protecting tumor microenvironment.

Animals ; Carcinoma, Lewis Lung ; blood supply ; drug therapy ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Male ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Mice ; Mice, Inbred C57BL ; Neovascularization, Pathologic ; pathology ; RNA, Messenger ; genetics

OBJECTIVETo investigate the effects of Yiqi Chutan Recipe (YCR, a Chinese herbal prescription for invigorating qi and removing phlegm) on the growth and metastasis of tumor, survival time, and the expressions of peroxiredoxin (PRDX-1 and PRDX-6) in tumor tissue of C57BL/6J mice bearing Lewis lung carcinoma.

METHODSLewis lung carcinoma cells were transplanted to 90 C57BL/6J mice receiving preconditioning for inducing Pi-deficiency syndrome and divided into three groups treated respectively with saline, high dose YCR (3.0 g/kg) and low dose YCR (1.0 g/kg) once a day via gastric infusion. Besides, a group of 30 healthy mice simply received tumor cell transplantation was set up for controls. Ten mice selected from each group were sacrificed 21 days later, the size, weight and lung metastasis foci of tumor in mice were measured, and expressions of PRDX-1 and PRDX-6 in tumor tissue were detected using immunohistochemical method. The survival time of the remained 20 mice in each groups was observed.

RESULTSTumor size, weight and the numbers of lung metastatic foci were (1.14 +/- 0.30) cm3, (0.83 +/- 0.26) g, (6.20 +/- 2.53) foci in the high dose YCR treated group, which were significantly lower than those in the control group [(2.83 +/- 0.35) cm3, (2.08 +/- 0.28) g, and (8.60 +/- 1.84) foci] respectively, also lower than those in the saline treated group [(2.29 +/- 0.49) cm3, (1.67 +/- 0.33) g and (8.70 +/- 2.00) foci]. The median survival time in the three groups, in above order, were 29.00 +/- 0.89 days, 22.00 +/- 0.75 days and 21.00 +/- 0.53 days; the average survival time in them 29.60 +/- 0.53 days, 22.90 +/- 0.50 days and 20.95 +/- 0.44 days; the PRDX-1 expression were 0.15 +/- 0.03, 0.52 +/- 0.07 and 0.61 +/- 0.09; and the PRDX-6 expression were 0.12 +/- 0.02, 0.43 +/- 0.06 and 0.56 +/- 0.07, all showed significant difference in comparing the indices in the high dose treated group with those in the control group and in the saline treated group (P < 0.05 or P < 0.01). The tumor growth inhibition rate was 50.30% in the high dose YCR group with life prolongation rate of 41.29%, all better than those in the low dose YCR treated group (P < 0.05).

CONCLUSIONSYCR can remarkably inhibit the growth and metastasis of Lewis lung carcinoma in mice with Pi-asthenia syndrome, prolong their survival period, and its mechanism is possibly related to the reduction of over expressed PRDX-1 and PRDX-6.

Animals ; Carcinoma, Lewis Lung ; drug therapy ; metabolism ; pathology ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Lung Neoplasms ; drug therapy ; metabolism ; pathology ; Male ; Mice ; Mice, Inbred C57BL ; Peroxiredoxin VI ; metabolism ; Peroxiredoxins ; metabolism

BACKGROUND AND OBJECTIVEStudies have shown that nitric oxide (NO) derived from endothelial nitric oxide synthase (eNOS) is expressed widely in tumor tissues and regulates tumor angiogenesis. However, the results are controversial. This study was to investigate the effect of NO on tumor angiogenesis and its mechanism.

METHODSC57BL/6 mice inoculated with Lewis lung cancer cells were randomly divided into three groups. Mice in the NO group were inoculated with lung cancer cells transfected with eNOS gene, mice in the L-NAME group with L-NAME, an eNOS antagonist, and mice in the control group with normal saline. Plasma concentration of NO and the number of endothelial progenitor cells (EPCs) in peripheral blood were detected . Tumor vessel density, CD133+ cells, and the expression of VEGF-VEGFR in tumor tissues were also measured.

RESULTSFour weeks after inoculation of Lewis cells, tumor volume was significantly larger in control group [ (3022 +/- 401) mm(3)] than in the L-NAME group [ (1204 +/-97) ) mm(3)] and in the eNOS group [(1824 +/- 239) mm(3)] (P<0.01). eNOS protein and NO production increased significantly in Lewis lung cancer cells transfected with eNOS gene. But the number of CD133-positive cells and vessel density in tumors were significantly lower in the eNOS group than in the control group [(48+/-19) / HPF vs. ( 103 +/- 27)/ HPF, (19+/- 7) HPF vs. (31 +/- 9) HPF, P<0.05]. The number of EPCs in peripheral blood was not statistically different between each group. The levels of NO in blood and tumor tissue significantly decreased after the treatment of L-NAME, while the tumor vessel density reduced to 12+/- 5/ HPF (P<0.01, vs. the control group; P<0.05, vs the eNOS transfected group). The number of EPCs in blood and that of CD133-positive cells in tumor tissue were significantly smaller in the L-NAME group than in the control group (P<0.05).

CONCLUSIONNo derived from eNOS inhibits angiogenesis and tumor growth, which may be due to its suppression on either the mobilization or homing of EPCs via VEGF binding to VEGFR.

AC133 Antigen ; Animals ; Antigens, CD ; metabolism ; Carcinoma, Lewis Lung ; blood supply ; metabolism ; pathology ; Cell Count ; Cells, Cultured ; Endothelium, Vascular ; pathology ; Enzyme Inhibitors ; pharmacology ; Female ; Glycoproteins ; metabolism ; Mice ; Mice, Inbred C57BL ; Microvessels ; pathology ; NG-Nitroarginine Methyl Ester ; pharmacology ; Neoplasm Transplantation ; Neovascularization, Pathologic ; Nitric Oxide ; blood ; metabolism ; Nitric Oxide Synthase Type III ; antagonists & inhibitors ; genetics ; metabolism ; Peptides ; metabolism ; Plasmids ; Random Allocation ; Stem Cells ; metabolism ; pathology ; Transfection ; Tumor Burden ; Vascular Endothelial Growth Factor A ; blood ; metabolism ; Vascular Endothelial Growth Factor Receptor-2 ; metabolism