Xiancheng Chen

A Tumor-selective Biotherapy With Prolonged Impact on Established Metastases Based on Cytokine Gene-engineered MSCs

The poor prognosis for patients with advanced malignancy relates partly to the inability to reverse cancer metastasis. In this study we have investigated an integrated immunotherapy method against pre-established metastases in three kinds of advanced cancer models including B16 melanoma, 4T1 breast tumor, and Hca hepatoma. The progression of metastases into multistep lymph nodes (LN) and internal organs was, markedly impeded in the midway stage and reversed in the ultimate stage following a 20-day course of intravenous immunotherapy [with interleukin-12 (IL-12) gene-engineered mesenchymal stem cells (MSCs), administered once every 5 days P < 0.05)]; the therapy was without systemic toxic effects. As the control, obvious systemic toxicity was observed in the free AdIL-12 group, yet metastasis was partly delayed only in the midway stage but not in the ultimate stage. Enzyme-linked immunosorbent assay (ELISA) showed that the intratumoral expression levels of IL-12 were enhanced by cytokine-engineered MSCs to be tenfold greater than that of free AdIL-12 groups in the ultimate stage; conversely, free AdIL-12 groups showed elevated serum, but not intratumoral levels of IL-12, during the midway stage. Furthermore, histomorphometric analysis revealed a reductive tendency toward reversion of tumor-associated lymphatic sprouts and an increased tumor apoptosis index in engineered MSC groups (P < 0.05). These data indicate the potential of cytokine-engineered MSCs to be considered as an integrated therapeutic weapon for targeting advanced malignancies.

IL-37 Ameliorates the Inflammatory Process in Psoriasis by Suppressing Proinflammatory Cytokine Production

IL-37 is a potent inhibitor of innate immunity by shifting the cytokine equilibrium away from excessive inflammation. Psoriasis is thought to be initiated by abnormal interactions between the cutaneous keratinocytes and systemic immune cells, triggering keratinocyte hyperproliferation. In the current study, we assessed IL-37 in two well-known psoriasis models: a human keratinocyte cell line (HaCaT) and the keratin 14 VEGF-A–transgenic mouse model. First, we used the HaCaT cell line, which was transiently transfected with an overexpressing IL-37 vector, and tested the effect of IL-37 on these cells using a mixture of five proinflammatory cytokines. IL-37 was effective in suppressing the production of CXCL8, IL-6, and S100A7, which were highly upregulated by the mixture of five proinflammatory cytokines. Keratin 14 VEGF-A–transgenic mice were treated with plasmid coding human IL-37 sequence–formulated cationic liposomes, and we observed potent immunosuppressive effects over the 18-d period. In this model, we observed reduced systemic IL-10 levels, local IFN-γ gene transcripts, as well as mild mast cell infiltration into the psoriatic lesions of the mice. Immunohistochemical analysis indicated that IL-37 was expressed by effector memory T cells, as well as macrophages, in human psoriatic plaques. In conclusion, our studies strongly indicate that IL-37 plays a potent immunosuppressive role in the pathogenesis of both experimental psoriasis models in vitro and in vivo by downregulating proinflammatory cytokines. Importantly, our findings highlight new therapeutic strategies that can be designed to use this immunosuppressive anti-inflammatory cytokine in psoriasis and other inflammatory cutaneous diseases.

Quantitative proteomics identification of phosphoglycerate mutase 1 as a novel therapeutic target in hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with poor prognosis due to resistance to conventional chemotherapy and limited efficacy of radiotherapy. There is an urgent need to develop novel biomarkers for early diagnosis, as well as to identify new drug targets for therapeutic interventions.Patients and methods54 paired HCC samples and 21 normal liver tissues were obtained from West China Hospital of Sichuan University. Informed consent was obtained from all the patients or their relatives prior to analysis, and the project was approved by the Institutional Ethics Committee of Sichuan University. Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC)-based proteomics was employed to profile the differentially expressed proteins between a HepG2 human hepatoma cell line and an immortal hepatic cell line L02. Validation of PGAM1 expression was performed by semi-quantitative RT-PCR, immunoblot and immunohistochemistry using clinical samples. shRNA expressing plasmids specifically targeting PGAM1 were designed and constructed by GenePharma Corporation (Shanghai, China), and were utilized to silence expression of PGAM1 in vitro and in vivo. Cell proliferation was measured by a combination of colony formation assay and Ki67 staining. Apoptosis was examined by flow cytometry and TUNEL assay.ResultsA total of 63 dysregulated proteins were identified, including 51 up-regulated proteins, and 12 down-regulated proteins (over 2-fold, p < 0.01). Phosphoglycerate mutase 1 (PGAM1) was found markedly upregulated. Clinico-pathological analysis indicated that overexpression of PGAM1 was associated with 66.7% HCC, and strongly correlated with poor differentiation and decreased survival rates (p < 0.01). shRNAs-mediated repression of PGAM1 expression resulted in significant inhibition in liver cancer cell growth both in vitro and in vivo.ConclusionOur studies suggested that PGAM1 plays an important role in hepatocarcinogenesis, and should be a potential diagnostic biomarker, as well as an attractive therapeutic target for hepatocellular carcinoma.

Human α-defensin-1 inhibits growth of human lung adenocarcinoma xenograft in nude mice

Human α-defensin-1 (HNP1), a small antimicrobial peptide, shows cytotoxicity to tumor cells in vitro and inhibitory activity for pathologic neovascularization in vivo. Here, we did a gene therapy with a plasmid that expresses a secretable form of HNP1 for assaying its antitumor activity. The expression and secretion of HNP1 were determined by reverse transcription-PCR and ELISA in vitro. We found that expression of HNP1 in A549 tumor cells caused significant growth inhibition. This effect is most likely cell autonomous, as a significant amount of recombinant HNP1 protein was found to be accumulated in the cytoplasm by immunohistochemical staining using an anti-HNP1 antibody and the supernatant containing secreted HNP1 failed to produce any noticeable antitumor activity. Flow cytometry and Hoechst 33258 staining showed that the number of apoptotic cells among the A549 cells expressing recombinant HNP1 proteins was significantly greater than that of the nontransfected control cultures, suggesting that this growth-inhibitory activity was due to an apoptotic mechanism triggered by the intracellular HNP1. The antitumor activity of intracellularly expressed HNP1 was also shown in vivo. Decreased microvessel density and increased lymphocyte infiltration were observed in tumor tissue from HNP1-treated mice through histologic analysis. These results indicate that intracellularly expressed HNP1 induces tumor cell apoptosis, which inhibits tumor growth. The antiangiogenesis effect of HNP1 may contribute to its inhibitory activity in vivo, and HNP1 might involve the host immune response to tumor. These findings provide a rationale for developing HNP1-based gene therapy for cancer. [Mol Cancer Ther 2008;7(6):1588–97]

A systemic administration of liposomal curcumin inhibits radiation pneumonitis and sensitizes lung carcinoma to radiation

Radiation pneumonitis (RP) is an important dose-limiting toxicity during thoracic radiotherapy. Previous investigations have shown that curcumin is used for the treatment of inflammatory conditions and cancer, suggesting that curcumin may prevent RP and sensitize cancer cells to irradiation. However, the clinical advancement of curcumin is limited by its poor water solubility and low bioavailability after oral administration. Here, a water-soluble liposomal curcumin system was developed to investigate its prevention and sensitizing effects by an intravenous administration manner in mice models. The results showed that liposomal curcumin inhibited nuclear factor-κB pathway and downregulated inflammatory factors including tumor necrosis factor-α, interleukin (IL)-6, IL-8, and transforming growth factor-β induced by thoracic irradiation. Furthermore, the combined treatment with liposomal curcumin and radiotherapy increased intratumoral apoptosis and microvessel responses to irradiation in vivo. The significantly enhanced inhibition of tumor growth also was observed in a murine lung carcinoma (LL/2) model. There were no obvious toxicities observed in mice. The current results indicate that liposomal curcumin can effectively mitigate RP, reduce the fibrosis of lung, and sensitize LL/2 cells to irradiation. This study also suggests that the systemic administration of liposomal curcumin is safe and deserves to be investigated for further clinical application.

Distinct contributions of angiogenesis and vascular co-option during the initiation of primary microtumors and micrometastases

Primary tumors and metastases have been thought to initiate avascularly as multicellular aggregates and later induce angiogenesis or initiate vascularly by co-opting pre-existing host blood vessels without inducing angiogenesis. These two distinct concepts of microtumor vascularization have raised significant controversies. To clarify intratumoral vascularization and tumor cell behaviors at single-cell level during the earliest stage of microtumor initiation, we established primary and metastatic microtumor models in Tg(flk1:EGFP) transgenic zebrafish. We found that tumor cells preferred to initiate avascularly as multicellular aggregates and only later (50-100 cells in size) induced angiogenesis in blood-supply-sufficient microenvironments. In blood-supply-deficient microenvironments, less tumor cells (20-30 cells per fish) managed to co-opt and migrate along host vessels, whereas more tumor cells (100-300 cells per fish) could immediately induce angiogenesis without obvious cell migration. In a metastatic model, we clearly observed that tumor cells co-opted, migrated along and proliferated on the surface of host vessels at an early stage after they extravasated from host vessels and induced angiogenesis later when micromatastases comprised only 15-30 tumor cells. Moreover, the inducement of neovessels accelerated the growth of micromatastases in size, meanwhile, decreased the migration of tumor cells on the surface of host vessels. These results suggest that vessel co-option and angiogenesis have distinct contributions during the initiation of microtumors. Microtumors initiated reasonably through co-opting host vessels or inducing angiogenesis, depending on the differences of local microenvironments and cell numbers in microtumors. The results in this study may have important implications for the therapeutic application of antiangiogenic strategies.

Liposomal honokiol inhibits VEGF-D-induced lymphangiogenesis and metastasis in xenograft tumor model

Lymph nodes metastasis of tumor could be a crucial early step in the metastatic process. Induction of tumor lymphangiogenesis by vascular endothelial growth factor‐D may play an important role in promoting tumor metastasis to regional lymph nodes and these processes can be inhibited by inactivation of the VEGFR‐3 signaling pathway. Honokiol has been reported to possess potent antiangiogenesis and antitumor properties in several cell lines and xenograft tumor models. However, its role in tumor‐associated lymphangiogenesis and lymphatic metastasis remains unclear. Here, we established lymph node metastasis models by injecting overexpressing VEGF‐D Lewis lung carcinoma cells into C57BL/6 mice to explore the effect of honokiol on tumor‐associated lymphangiogenesis and related lymph node metastasis. The underlying mechanisms were systematically investigated in vitro and in vivo. In in vivo study, liposomal honokiol significantly inhibited the tumor‐associated lymphangiogenesis and metastasis in Lewis lung carcinoma model. A remarkable delay of tumor growth and prolonged life span were also observed. In in vitro study, honokiol inhibited VEGF‐D‐induced survival, proliferation and tube‐formation of both human umbilical vein endothelial cells (HUVECs) and lymphatic vascular endothelial cells (HLECs). Western blotting analysis showed that liposomal honokiol‐inhibited Akt and MAPK phosphorylation in 2 endothelial cells, and downregulated expressions of VEGFR‐2 of human vascular endothelial cells and VEGFR‐3 of lymphatic endothelial cells. Thus, we identified for the first time that honokiol provided therapeutic benefit not only by direct effects on tumor cells and antiangiogenesis but also by inhibiting lymphangiogenesis and metastasis via the VEGFR‐3 pathway. The present findings may be of importance to investigate the molecular mechanisms underlying the spread of cancer via the lymphatics and explore the therapeutical strategy of honokiol on antilymphangiogenesis and antimetastasis.

Immunity against Tumor Angiogenesis Induced by a Fusion Vaccine with Murine β-Defensin 2 and mFlk-1

Purpose: Previous studies indicated that humoral or cellular immunity against murine vascular endothelial growth factor 2 (mFlk-1) was elicited to inhibit tumor growth. Here we describe a genetic fusion vaccine, pMBD2-mFlk-1, based on the targeting of a modified mFlk-1 to antigen-presenting cells by a murine β-defensin 2 (MBD2) protein to induce both humoral and cellular immunity against mFlk-1, with the targeting especially focused on immature dendritic cells. Experimental Design: The protective and therapeutic antitumor immunity of the fusion vaccine was investigated in mouse models. Antiangiogenesis effect was detected by immunohistochemical staining and alginate-encapsulate tumor cell assay. The mechanisms of the fusion vaccine were primarily explored by detection of autoantibodies and CTL activity and confirmed by the deletion of immune cell subsets. Results: The fusion vaccine elicited a strong protective and therapeutic antitumor immunity through antiangiogenesis in mouse models, and this worked through stimulation of an antigen-specific CD8+ T-cell response as well as a specific B-cell response against mFlk-1. The findings were confirmed by depletion of immune cell subsets and in knockout mice. Conclusion: Our study showed that a fusion vaccine based on self immune peptide (MBD2) and self antigen (mFlk-1) induced autoimmunity against endothelial cells, resulting in inhibition of tumor growth, and could be further exploited in clinical applications of cancer immunotherapy.

Deguelin—An inhibitor to tumor lymphangiogenesis and lymphatic metastasis by downregulation of vascular endothelial cell growth factor-D in lung tumor model

Deguelin, a rotenoid of the flavonoid family, has been reported to possess antiproliferative and anticarcinogenic activities in several cell lines and tumor models. However, it is still unclear whether deguelin effectively inhibits tumor‐associated lymphangiogenesis and lymphatic metastasis. Since tumor production of vascular endothelial cell growth factor (VEGF)‐D was associated with tumor lymphangiogenesis and lymphatic metastasis, we established the mouse lymphatic metastasis model by transfecting high expression VEGF‐D into LL/2 Lewis lung cells (VEGF‐D‐LL/2) and explored the effects of deguelin on lymphatic metastasis in the immunocompetent C57BL/6 mice. Our results indicated that deguelin inhibited proliferation, migration of VEGF‐D‐LL/2 cells via downregulating AKT and mitogen‐activated protein kinase pathway and interfered tube formation of lymphatic vascular endothelial cells on matrigel at nanomolar concentrations. Deguelin significantly downregulated the expression of VEGF‐D both at mRNA and protein levels in VEGF‐D‐LL/2 cells in a dose‐dependent manner. In the in vivo study, intraperitoneal administration of deguelin (4 mg/kg) remarkably inhibited the tumor‐associated lymphangiogenesis and lymphatic metastasis. The rates of lymph node and lung metastasis in deguelin‐treated mice were 0 and 16.7% compared with 58.3 and 83.3% in control group mice, respectively. Deguelin also resulted in a remarkable delay of tumor growth and prolongation of life span. Immunohistochemical staining with antibodies against VEGF‐D, LYVE‐1 and VEGFR‐3 revealed fewer positive vessel‐like structures in deguelin‐treated mice compared with control group mice. Taken together, we demonstrate for the first time that deguelin suppresses tumor‐associated lymphangiogenesis and lymphatic metastasis by downregulation of VEGF‐D both in vitro and in vivo.

Efficient inhibition of ovarian cancer by short hairpin RNA targeting claudin-3.

Ovarian cancer is one of the most lethal gynecologic neoplasms. Even though various new chemotherapeutics have been developed for the treatment of ovarian cancer, drug resistance and undesired serious side effects remain unavoidable obstacles for chemotherapeutic approaches. New strategies to overcome the therapeutic dilemma are needed. Claudin-3 (CLDN3) is a recently discovered gene generally overexpressed in human ovarian cancers but not in normal ovarian tissue. Its high expression has been identified to associate with the invasion, proliferation and survival of cancer cells, making it a promising target for gene therapy of ovarian cancer. However, in gene therapy, traditional gene carriers such as virus or cationic liposomes suffer from distressing shortcomings of potential carcinogenicity, obvious cytotoxicity and immunogenicity. Nanoparticles (NPs) based on PLGA are a novel gene delivery system with good biodegradability, excellent biocompatibility and low toxcity for in vivo gene delivery compared with traditional gene carriers. We constructed a plasmid expressing shRNA targeted CLDN3 (pshCLDN3) encapsulated with PLGA-NPs, and administered it by i.p. injection to nude mice bearing intraperitoneal SKOV3 ovarian cancer, to investigate the antitumor potential of knocking down CLDN3. After 12 times of administration, the tumors of each group were compared. The underlying antitumor mechanisms were revealed by immunostaining of CD31, Ki-67 and TUNEL assay, to exhibit possible alterations in microvessel density, cell proliferation and cell apoptosis. Our study demonstrated that i.p. administration of pshCLDN3 effectively suppressed the expression of CLDN3 and, thus, inhibited the growth of ovarian tumors, significantly reducing tumor weight by 67.4% compared with blank controls (p<0.05). Immunostaining of CD31, Ki-67 and TUNEL assay demonstrated decreased angiogenesis (p<0.05), reduced proliferation (p<0.05) and increased apoptosis (p<0.05) in the pshCLDN3 treated group compared with controls. No obvious toxicity of PLGA-NPs was observed either in vitro or in vivo. Our results indicated that knockdown of CLDN3 by pshCLDN3 encapsulated in PLGA NPs may provide a promising approach for the treatment of ovarian cancer.