The Promotion of Erythropoiesis via the Regulation of Reactive Oxygen Species by Lactic Acid.

The simultaneous increases in blood lactic acid and erythrocytes after intense exercise could suggest a link between lactate and the erythropoiesis. However, the effects of lactic acid on erythropoiesis remain to be elucidated. Here, we utilized a mouse model to determine the role of lactic acid in this process in parallel with studies using leukaemic K562 cells. Treatment of K562 cells in vitro with lactic acid increased the mRNA and protein expression of haemoglobin genes and the frequency of GPA+ cells. Also, increases in haematocrit and CD71-/Ter119+ erythroid cells were observed in lactic acid-treated mice, which showed a physiological increase in blood lactate. Mouse bone marrow CD34+/CD117- cells showed an increase in erythroid burst-forming units after stimulation with lactic acid in vitro. Furthermore, lactic acid increased the intracellular reactive oxygen species (ROS) content in bone marrow and in K562 cells. Erythroid differentiation induced in Haematopoietic Stem Cells (HSCs) and K562 cells by lactic acid was abolished by reducing ROS levels with SOD or 2-mercaptoethanol, which suggests that ROS is a critical regulator of this process. These findings provide a better understanding of the role of lactic acid in cellular metabolism and physiological functions.

Folate-Modified Lipoplexes Delivering the Interleukin-12 Gene for Targeting Colon Cancer Immunogene Therapy.

The incidence and mortality rate of colorectal cancer increase every year, making it a serious threat to human health. Targeted immunogene therapy is a novel method of treating this type of cancer. Colon cancer overexpresses folate receptor α (FRα) and folate-modified liposomes for colon cancer immunogene therapy may suppress tumor growth effectively. In this study, F-PLP/pIL12, an FRα-targeted lipoplex loading plasmid interleukin-12 (pIL12) was prepared and its physicochemical properties were characterized. Then the antitumor effect of F-PLP/pIL12 was studied in an in vivo model of CT-26 colon cancer. F-PLP/pIL12 was associated with about 56.6% tumor growth inhibition compared with the saline control. The production of malignant ascites was significantly less pronounced than in controls, and there were fewer tumor nodules and less overall tumor mass (P < 0.01). There was more IL12 expression and IFN-γ secretion in F-PLP/pIL12-treated tumor tissues, but there was less FRα expression. The antitumor mechanisms involved inducing tumor cell apoptosis, reducing microvessel density, and stimulating TNF-α secretion. In addition, there were fewer M2 macrophages in the tumor microenvironment of tissues stimulated with F-PLP/pIL12, which also activated the natural killer cells. H&E staining of vital organs suggested that F-PLP/pIL12 is safe for use in intraperitoneally administered cancer therapy. It was here concluded that F-PLP/plL12 may be a suitable targeting formulation for colon cancer immunogene therapy.

[REGgamma promotes malignant behaviors of lung cancer cells].

OBJECTIVE: To determine the expression of proteasome aotivator gamma (REGgamma) in human lung cancer tissues and cell lines and its association with malignant biological behaviors. METHODS: Immunohistochemistry (IHC) was used to detect the expression of REGgamma in lung cancer and normal lung tissues. The expressions of REGgamma in lung cancer cells and normal epithelial cells were determined by Western blot. The H1975 lung cancer stable cell lines with different levels of REGgamma expression were constructed and their proliferations were evaluated by MTT assay. PI staining was used to assess the influence of REGgamma on cell growth cycle. The effect of REGgamma on the migration of lung cancer cells were observed with the cell scratch experiment. RESULTS: Lung cancer tissues had significantly higher levels of REGgamma expression than normal tissues. Similarly, lung cancer cell lines showed higher levels of REGgamma expression than the normal epithelial cell line. The overexpression of REGgamma enhanced cancer cell proliferations (P < 0.05), promoted more cells into the S+G2/M phase (P < 0.05) and promoted the migration of cancer cells (P < 0.05). All of these effects were reversed after suppression of REGgamma. CONCLUSION: REGgamma facilitates malignant biological behaviors of lung cancers.

Enhanced efficacy of combination therapy with adeno­associated virus-delivered pigment epithelium-derived factor and cisplatin in a mouse model of Lewis lung carcinoma.

Pigment epithelium-derived factor (PEDF) is a potent inhibitor of angiogenesis, and the antitumor effect of adeno-associated virus (AAV)-mediated PEDF expression has been demonstrated in a range of animal models. The combined treatment of low-dose chemotherapy and gene therapy inhibits the growth of solid tumors more effectively than current traditional therapies or gene therapy alone. In the present study, the effect of treatment with an AAV2 vector harboring the human PEDF (hPEDF) gene in combination with low-dose cisplatin on the growth of Lewis lung carcinoma (LLC) in mice was assessed. LLC cells were infected with AAV-enhanced green fluorescent protein (EGFP) in the presence or absence of cisplatin, and then the effect of cisplatin on AAV-mediated gene expression was evaluated by image and flow cytometric analysis. Tumor growth, survival time, vascular endothelial growth factor (VEGF) expression, microvessel density (MVD) and apoptotic index were analyzed in C57BL/6 mice treated with AAV-hPEDF, cisplatin or cisplatin plus AAV-hPEDF. The results of the present study provide evidence that cisplatin treatment is able to enhance AAV-mediated gene expression in LLC cells. In addition, the combined treatment of cisplatin plus AAV­hPEDF markedly prolonged the survival time of the mice and inhibited tumor growth, resulting in significant suppression of tumor angiogenesis and induction of tumor apoptosis in vivo, and also protected against cisplatin-related toxicity. These findings suggest that combination of AAV-hPEDF and cisplatin has potential as a novel therapeutic strategy for lung cancer.

Folate-linked lipoplexes for short hairpin RNA targeting claudin-3 delivery in ovarian cancer xenografts.

Ovarian cancers highly overexpress folate receptor α (FRα) and claudin3 (CLDN3), both of which are associated with tumor progression and poor prognosis of patients. Downregulation of FRα and CLDN3 in ovarian cancer may suppress tumor growth and promote benign differentiation of tumor. In this study, F-P-LP/CLDN3, a FRα targeted liposome loading with short hairpin RNA (shRNA) targeting CLDN3 was prepared and the pharmaceutical properties were characterized. Then, the antitumor effect of F-P-LP/CLDN3 was studied in an in vivo model of advanced ovarian cancer. Compared with Control, F-P-LP/CLDN3 promoted benign differentiation of tumor and achieved about 90% tumor growth inhibition. In the meantime, malignant ascites production was completely inhibited, and tumor nodule number and tumor weight were significantly reduced (p<0.001). FRα and CLDN3 were downregulated together in tumor tissues treated by F-P-LP/CLDN3. The antitumor mechanisms were achieved by promoting tumor cell apoptosis, inhibiting tumor cell proliferation and reducing microvessel density. Finally, safety evaluation indicated that F-P-LP/CLDN3 was a safe formulation in intraperitoneally administered cancer therapy. We come to a conclusion that F-P-LP/CLDN3 is a potential targeting formulation for ovarian cancer gene therapy.

Systemically administered liposome-encapsulated Ad-PEDF potentiates the anti-cancer effects in mouse lung metastasis melanoma.

BACKGROUND: The use of adenoviral vector for gene therapy is still an important strategy for advanced cancers, however, the lack of the requisite coxsackie-adenovirus receptor in cancer cells and host immune response to adenovirus limit the application of adenoviral vector in vivo. METHOD: We designed the antiangiogenic gene therapy with recombinant PEDF adenovirus (Ad-PEDF) encapsulated in cationic liposome (Ad-PEDF/Liposome), and investigated the anti-tumor efficacy of Ad-PEDF/Liposome complex on inhibition of tumor metastasis. RESULTS: We found that systemic administration of Ad-PEDF/liposome was well tolerated and resulted in marked suppression of tumor growth, and was more potent than uncoated Ad-PEDF to induce apoptosis in B16-F10 melanoma cells and inhibit murine pulmonary metastases in vivo. After Ad-luciferase was encapsulated with liposome, its distribution decreased in liver and increased in lung. The anti-Ad IgG level of Ad-PEDF/Liposome was significantly lower than Ad-PEDF used alone. CONCLUSION: The present findings provide evidences of systematic administration of cationic liposome-encapsulated Ad-PEDF in pulmonary metastatic melanoma mice model, and show an encouraging therapeutic effect for further exploration and application of more complexes based on liposome-encapsulated adenovirus for more cancers.

AAV2-mediated gene transfer of VEGF-Trap with potent suppression of primary breast tumor growth and spontaneous pulmonary metastases by long-term expression.

Vascular endothelial growth factor (VEGF) is an important signaling protein and a predominant mediator of angiogenesis in tumor growth and metastasis. Therefore, antagonism of the VEGF pathway results in inhibition of abnormal angiogenesis, then suppression of tumor growth and metastasis. VEGF-Trap, a high-affinity soluble decoy receptor, is currently in phase II clinical trails, and has demonstrated more efficacy in different types of solid tumors by intravenous injection every two weeks. In our study, we used recombinant AAV2 as a delivery vehicle to achieve long-lasting expression of VEGF Trap protein in a mouse model for the first time. We report that AAV2-VEGF-Trap can be safely administered and sustained expression in vivo via a single intravenously administration, simultaneously suppressing primary tumor growth and preventing the pulmonary metastases of 4T1 tumors. Decreased microvessel density and increased tumor cell apoptosis were observed in the treatment group. AAV2-VEGF-Trap can obviously decrease not only the concentration of VEGF in sera, but also the concentration of other angiogenic factors, such as aFGF, bFGF, angiopoietin-1 and others. These studies suggest that AAV-mediated long-term expression of VEGF-Trap is a useful and safe tool to block tumor progression and inhibit spontaneous pulmonary metastases.

AAV-mediated human PEDF inhibits tumor growth and metastasis in murine colorectal peritoneal carcinomatosis model.

BACKGROUND: Angiogenesis plays an important role in tumor growth and metastasis, therefore antiangiogenic therapy was widely investigated as a promising approach for cancer therapy. Recently, pigment epithelium-derived factor (PEDF) has been shown to be the most potent inhibitor of angiogenesis. Adeno-associated virus (AAV) vectors have been intensively studied due to their wide tropisms, nonpathogenicity, and long-term transgene expression in vivo. The objective of this work was to evaluate the ability of AAV-mediated human PEDF (hPEDF) as a potent tumor suppressor and a potential candidate for cancer gene therapy. METHODS: Recombinant AAV2 encoding hPEDF (rAAV2-hPEDF) was constructed and produced, and then was assigned for in vitro and in vivo experiments. Conditioned medium from cells infected with rAAV2-hPEDF was used for cell proliferation and tube formation tests of human umbilical vein endothelial cells (HUVECs). Subsequently, colorectal peritoneal carcinomatosis (CRPC) mouse model was established and treated with rAAV2-hPEDF. Therapeutic efficacy of rAAV2-hPEDF were investigated, including tumor growth and metastasis, survival time, microvessel density (MVD) and apoptosis index of tumor tissues, and hPEDF levels in serum and ascites. RESULTS: rAAV2-hPEDF was successfully constructed, and transmission electron microscope (TEM) showed that rAAV2-hPEDF particles were non-enveloped icosahedral shape with a diameter of approximately 20 nm. rAAV2-hPEDF-infected cells expressed hPEDF protein, and the conditioned medium from infected cells inhibited proliferation and tube-formation of HUVECs in vitro. Furthermore, in CRPC mouse model, rAAV2-hPEDF significantly suppressed tumor growth and metastasis, and prolonged survival time of treated mice. Immunofluorescence studies indicated that rAAV2-hPEDF could inhibit angiogenesis and induce apoptosis in tumor tissues. Besides, hPEDF levels in serum and ascites of rAAV2-hPEDF-treated mice were significant higher than those in rAAV2-null or normal saline (NS) groups. CONCLUSIONS: Thus, our results suggest that rAAV2-hPEDF may be a potential candidate as an antiangiogenic therapy agent.

AAV-mediated gene transfer of human pigment epithelium-derived factor inhibits Lewis lung carcinoma growth in mice.

Pigment epithelium-derived factor (PEDF) is the most potent inhibitor of angiogenesis in the mammalian eye, and mechanisms through which PEDF exerts its antitumour activity have recently been defined. The aim of our research was to evaluate the ability of adeno-associated virus (AAV) vector-mediated transfer of human PEDF to inhibit lewis lung carcinoma (LCC) cell growth. Intratumoural injection of AAV-PEDF caused significant reduction of the tumour volume and prolonged the survival time of mice bearing LLC cells, which were associated with decreased microvessel density and increased apoptosis in the tumours. AAV vectors represent a very promising tool for cancer gene therapy. No noticeable toxicity concerning AAV was detected as inferred from monitoring changes in animal body weight as well as basic organ structure and histological morphology, and by analyzing mouse liver and kidney function. Our findings indicate that AAV-mediated PEDF gene expression may offer an active approach to inhibit LLC growth and that treatment with AAV-PEDF may provide a promising therapeutic strategy in lung cancer treatment.

bFGF peptide combined with the pVAX-8CpG plasmid as adjuvant is a novel anticancer vaccine inducing effective immune responses against Lewis lung carcinoma.

Due to the poor immunogenicity of subunit protein antigens, there is a need to use adjuvants in order to generate effective immune responses. Basic fibroblast growth factor (bFGF) is one of the best characterized pro-angiogenic cytokine and is a candidate target for anticancer therapy. We used truncated bFGF (tbFGF) combined with engineered pVAX-nCpG as novel adjuvant to immunize mice in order to inhibit tumor angiogenesis and suppress tumor growth. In our study, the results demonstrated that the mice immunized with tbFGF-alum-pVAX-8CpG produced a better tumor-suppression effect compared with the other groups, apart from the group treated with tbFGF-alum-CpG. In addition, the function of immune modulation of pVAX-8CpG was similar to CpG ODNs. The vaccine composed of tbFGF, alum and pVAX-8CpG effectively inhibited tumor angiogenesis and induced strong antitumor immune responses. The antitumor activity induced by the vaccine tbFGF-alum-pVAX-8CpG was not only associated with the antigen-specific antibody, but also with the killing activity of cytotoxic cells. This indicates that alum-pVAX-8CpG may be an innovative adjuvant for cancer vaccines.

A novel combined conjugate vaccine: enhanced immunogenicity of bFGF with CRM197 as a carrier protein.

Tumor growth is partly dependent on tumor-associated angiogenesis, which is regulated by angiogenic growth factors. As the first angiogenic growth factor to be identified, basic fibroblast growth factor (bFGF) plays a major role in angiogensis and tumor growth and has been an effective target for anti-tumor therapy. However, due to its low immunogenicity, injection with bFGF alone cannot stimulate the body to produce a strong immune response. In this study, we investigated the role of CF (containing bFGF and CRM197) assisted by CpG and alum in enhancing antigen-specific immune response and suppressing the growth of murine colon carcinoma. The results revealed that compared to bFGF, CF could not stimulate NIH-3T3 fibroblast proliferation even at a concentration of 10 µg/ml in vitro. In vivo, the CF-CpG-alum produced a stronger antigen-specific immune response and inhibited tumor growth. The anti-tumor activity was associated with generating antigen-specific antibody, suppressing angiogenesis, promoting the apoptosis of tumor cells and inducing the mixed Th1 and Th2 responses. This indicates that CRM197 may be an innovative intramolecular adjuvant and provides a rational preservation for mouse CT26 colon carcinoma.

Anti-tumor effect of adenovirus-mediated gene transfer of pigment epithelium-derived factor on mouse B16-F10 melanoma.

BACKGROUND: Angiogenesis plays an important role in tumor growth, invasion, and eventually metastasis. Antiangiogenic strategies have been proven to be a promising approach for clinical therapy for a variety of tumors. As a potent inhibitor of tumor angiogenesis, pigment epithelium-derived factor (PEDF) has recently been studied and used as an anticancer agent in several tumor models. METHODS: A recombined adenovirus carrying PEDF gene (Ad-PEDF) was prepared, and its expression by infected cells and in treated animals was confirmed with Western blotting and ELISA, respectively. Its activity for inhibiting human umbilical vein endothelial cell (HUVEC) proliferation was tested using the MTT assay. C57BL/6 mice bearing B16-F10 melanoma were treated with i.v. administration of 5 x 108 IU/mouse Ad-PEDF, or 5 x 108 IU/mouse Ad-Null, or normal saline (NS), every 3 days for a total of 4 times. Tumor volume and survival time were recorded. TUNEL, CD31 and H&E stainings of tumor tissue were conducted to examine apoptosis, microvessel density and histological morphology changes. Antiangiogenesis was determined by the alginate-encapsulated tumor cell assay. RESULTS: The recombinant PEDF adenovirus is able to transfer the PEDF gene to infected cells and successfully produce secretory PEDF protein, which exhibits potent inhibitory effects on HUVEC proliferation. Through inhibiting angiogenesis, reducing MVD and increasing apoptosis, Ad-PEDF treatment reduced tumor volume and prolonged survival times of mouse bearing B16-F10 melanoma. CONCLUSION: Our data indicate that Ad-PEDF may provide an effective approach to inhibit mouse B16-F10 melanoma growth.

[Comparison of TTR and CMV promoters in vivo and in vitro via a secreted luciferase reporter system].

GLuc (Gaussia luciferase) is a secreted luciferase with high sensitivity. In this study, we primarily compared expression character of PTTR with that of PCMV, relied on easy secretion, high sensitivity and simple and fast detection of GLuc. We firstly constructed two plasmids pAAV2-neo-TTR-GLuc and pAAV2-neo-CMV-GLuc. Then, 4 cell lines were transfected with the two plasmids in aid of Lipofectamine 2000, including Huh7 and HepG2, which are derived from liver cells, as well as HEK293 and HeLaS3 cells, which are non-liver cell lines. We monitored the expression of GLuc in the supernatant of these cell cultures at different time points post-transfection. Furthermore, we injected the two plasmids with different doses into BALB/c mice by the means of hydrodynamic delivery and monitored the GLuc expression in vivo with 2.5 microl tail tip blood since 2 h post-injection. The cell assay results suggested that the expression of GLuc driven by CMV promoter was significantly higher than that of GLuc driven by TTR promoter. And, the luciferase activity of GLuc driven by CMV promoter was 50-300 times higher than that of GLuc driven by TTR promoter in HEK293 and HeLaS3 cell lines, but less than 10 times higher than that of GLuc driven by TTR promoter in the HepG2 and Huh7 cell lines, indicating the relative liver-specificity of TTR promoter. In the animal assay, the higher luciferase activity was determined in CMV promoter group than in TTR promoter group at different doses of the two plasmids. But the expression patterns for the two promoters differed obviously. The expression of GLuc driven by CMV promoter reached the maximum 10 hours post-injection and declined rapidly; while the expression of GLuc driven by TTR promoter reached the maximum 48 hours after delivery, and declined very slowly. These results implied that PTTR could keep expression of driven gene in a long time although its expression intensity is lower than PCMV's. Thus, it is more suitable for maintaining longer expression of target genes in liver.