Guan-Xin Shen

miR‐142‐3p restricts cAMP production in CD4+CD25− T cells and CD4+CD25+ TREG cells by targeting AC9 mRNA

Cyclic AMP (cAMP) is a ubiquitous second messenger that regulates diverse cellular functions. It has been found that CD4+CD25+ regulatory T (TREG) cells exert their suppressor function by transferring cAMP to responder T cells. Here, we show that miR‐142‐3p regulates the production of cAMP by targeting adenylyl cyclase (AC) 9 messenger RNA in CD4+CD25− T cells and CD4+CD25+ TREG cells. miR‐142‐3p limits the level of cAMP in CD4+CD25− T cells by inhibiting AC9 production, whereas forkhead box P3 (FOXP3) downregulates miR‐142‐3p to keep the AC9/cAMP pathway active in CD4+CD25+ TREG cells. These findings reveal a new molecular mechanism through which CD4+CD25+ TREG cells contain a high level of cAMP for their suppressor function, and also suggest that the microRNA controlling AC expression might restrict the final level of cAMP in various types of cells.

Listeria monocytogenes Promotes Tumor Growth via Tumor Cell Toll-Like Receptor 2 Signaling

The contribution of bacterial infection to tumorigenesis is usually ascribed to infection-associated inflammation. An alternate view is that direct interaction of bacteria with tumor cells promotes tumor progression. Here, we show that the microenvironment of large tumors favors bacterial survival, which in turn directly accelerates tumor growth by activating tumor cell Toll-like receptors (TLR). Listeria monocytogenes (Lm) survives in the microenvironment of large but not small tumors, resulting in the promotion of tumor growth. Lm did not affect the percentage of regulatory T cells or myeloid suppressor cells in the tumor. Through TLR2 signaling, Lm activated mitogen-activated protein kinases and nuclear factor-kappaB in tumor cells, resulting in the increased production of nitric oxide and interleukin-6 and increased proliferation of tumor cells. All of these effects were abrogated by silencing expression of TLR2, but not TLR4. The interaction of Helicobacter pylori with tumor cells from gastric carcinoma patients resulted in similar effects. These findings provide a new insight into infection-associated tumorigenesis and illustrate the importance of antibiotic therapy to treat tumors with bacterial infiltration.

Delivery of chemotherapeutic drugs in tumour cell-derived microparticles

Cellular microparticles are vesicular plasma membrane fragments with a diameter of 100-1,000 nanometres that are shed by cells in response to various physiological and artificial stimuli. Here we demonstrate that tumour cell-derived microparticles can be used as vectors to deliver chemotherapeutic drugs. We show that tumour cells incubated with chemotherapeutic drugs package these drugs into microparticles, which can be collected and used to effectively kill tumour cells in murine tumour models without typical side effects. We describe several mechanisms involved in this process, including uptake of drug-containing microparticles by tumour cells, synthesis of additional drug-packaging microparticles by these cells that contribute to the cytotoxic effect and the inhibition of drug efflux from tumour cells. This study highlights a novel drug delivery strategy with potential clinical application.

Mast cells mobilize myeloid-derived suppressor cells and Treg cells in tumor microenvironment via IL-17 pathway in murine hepatocarcinoma model.

Tumor immunosuppression is commonly braided with chronic inflammation during tumor development. However, the relationship between immunosuppression and inflammation in tumor microenvironment is still unclear. We have demonstrated that mast cells are accumulated and exacerbate the inflammation and immunosuppression in tumor microenvironment via SCF/c-kit signaling pathway. Here, we further elucidate the underlying mechanism, which involves both myeloid-derived suppressor cells (MDSCs) and regulatory T (Treg) cells. Our data showed that mast cells mobilized the infiltration of MDSCs to tumor and induced the production of IL-17 by MDSCs; MDSCs-derived IL-17 indirectly attracted Treg cells, enhanced their suppressor function, and induced the IL-9 production by Treg cells; in turn, IL-9 strengthened the survival and protumor effect of mast cells in tumor microenvironment. Our findings disclose a closed loop among mast cells, MDSCs and Treg cells in tumor microenvironment, which provides a new insight into the paralleled developments of inflammation and immunosuppression in tumor microenvironment. Based on these findings, we propose that targeting tumor inflammation might be a potential strategy to reverse the immunosuppression of tumor microenvironment, thus facilitating cancer immunotherapy.

Microparticles mediate enzyme transfer from platelets to mast cells: A new pathway for lipoxin A4 biosynthesis

The inflammation-resolving lipid mediator lipoxin A4 (LXA4), which is derived from arachidonic acid in the context of inflammation, can be generated physiologically in vivo. However, the mechanism of physiologic formation of LXA4 remains elusive. In this report, we provide evidence that platelet-derived microparticles contain lipoxygenase 12 (12-LO) protein and act as a mediator in transferring 12-LO to mast cells, leading to the production of LXA4 by mast cells. Absence of either leukotriene, the precursor for LXA4, in mast cells or 12-LO in microparticles abolished LXA4 production. Using a mouse model, we demonstrated that platelet-derived microparticles were taken up by peritoneal mast cells in vivo and triggered LXA4 production. We also found that similar to LXA4, platelet-derived microparticles attenuated LPS- or dextran sulfate sodium-induced inflammation by regulating inflammatory cytokines. Together, these data suggest a critical role of platetlet-derived microparticles as a signal mediator, at least in LXA4 production, resulting in significant immunoregulatory consequences.

Local Accumulation of FOXP3+ Regulatory T Cells: Evidence for an Immune Evasion Mechanism in Patients with Large Condylomata Acuminata

Condylomata acuminata derived from the infection of human papillomavirus is a common sexually transmitted disease. Although T cell-mediated cellular immunity is considered as the main arm against such infection, the regulation of T cell immune responses in genital condylomata is unclear to date. In this study, we analyzed FOXP3+ regulatory T cells in genital condylomata of patients. The results show that FOXP3+ regulatory T cells with suppressive function accumulated in large warts. Consistently, the immunosuppressive milieu in large warts was characterized by high expression of IL-10 and TGF-β1 and low expression of IL-2 and IFN-γ. The responsiveness of wart-infiltrating T cells both in vitro and in vivo can be increased by depleting FOXP3+ T cells. The accumulation of FOXP3+ regulatory T cells in large warts can be partly ascribed to the chemotaxis of CCL17 and CCL22, derived from Langerhans cells and macrophages in wart. Although such accumulation favors the local immunosuppression, it seems not to influence the systemic immunity. In conclusion, these findings demonstrate that FOXP3+ regulatory T cells play an important role in genital condylomata, which has multiple implications in the comprehensive treatment of condylomata acuminata.

Transforming growth factor-β1 upregulates the expression of CXC chemokine receptor 4 (CXCR4) in human breast cancer MCF-7 cells

AbstractAim:To investigate whether rhTGF-β1 or a recombinant vector encoding a fusion protein comprising an extracellular domain of TGF-β receptor II and an IgG Fc fragment) affects the regulation of CXC chemokine receptor 4 (CXCR4) expression in MCF-7 human breast cancer cells.Methods:MCF-7 breast cancer cells were treated with rhTGF-β1 or transfected with a recombinant vector, pIRES2-EGFP-TβRII-Fc. Expression of CXCR4 in these cells was then analyzed at the mRNA and protein levels by quantitative RT-PCR and flow cytometry assay, respectively. A transwell assay was used to measure the chemotactic response of these cells to SDF-1α.Results:CXCR4 mRNA and protein expression were upregulated in TGF-β1-treated MCF-7 cells. These cells also demonstrated an enhanced chemotactic response to SDF-1α. In MCF-7 cells transiently transfected with pIRES2-EGFP-TβRII-Fc, a fusion protein named TβRII-Fc (approximately 41 kDa) was produced and secreted. In these transfected cells, there was a reduction in CXCR4 expression and in the SDF-1α-mediated chemotactic response.Conclusion:TGF-β1 upregulated CXCR4 expression in MCF-7 cells, which subsequently enhanced the SDF-1α-induced chemotactic response. The results suggest a link between TGF-β1 and CXCR4 expression in MCF-7 human breast cancer cells, which may be one of the mechanisms of TGF-β1-mediated enhancement of metastatic potential in breast cancer cells.

Rapamycin inhibits lung metastasis of B16 melanoma cells through down-regulating alphav integrin expression and up-regulating apoptosis signaling

Currently available data indicate the potential application of rapamycin and its analogues in the clinic as anticancer therapeutic agents through inhibiting tumor cell growth and tumor angiogenesis. However, whether rapamycin can directly suppress tumor metastasis remains unclear. In the present study, we demonstrated that rapamycin treatment results in reduced formation of metastatic nodules in the lung by B16 cells. This is due to two mechanisms. First, the expression of αv integrin is down‐regulated by rapamycin treatment, and subsequently, the phosphorylation of focal adhesion kinase (FAK) is reduced. Second, rapamycin promotes apoptosis by up‐regulating the proapoptotic molecules Bid and Bax and down‐regulating Bcl‐xL. Blocking the apoptosis pathway by pan‐caspase inhibitor zVAD partially reversed the suppression of rapamycin in B16 metastasis. Interestingly, rapamycin up‐regulates Bax and Bid in B16 cells via the S6K1 pathway and down‐regulates the expression of αv integrin via other pathway(s). In addition, our data showed that autophagy was not involved in the mechanisms of rapamycin‐mediated metastasis suppression. Our findings demonstrate a potential anti‐metastatic effect of rapamycin via down‐regulating αv integrin expression and up‐regulating apoptosis signaling, suggesting that rapamycin might be worthy of clinical evaluation as an antimetastatic agent. (Cancer Sci 2009)

The antiproliferative effects of somatostatin receptor subtype 2 in breast cancer cells.

AbstractAim:Somatostatin receptor subtype 2 (SSTR2) is the principal mediator of somatostatins (SST) antiproliferative effects on normal and cancer cells. Therefore, we investigated whether the enhanced expression of SSTR2 could inhibit the proliferation of tumor cells, and, if so, the mechanisms that might be involved.Methods:SSTR2 expression levels were determined by qRT-PCR in several tumor cell lines. Then, a plasmid pIRES2-EGFP-SSTR2 (pSIG) was constructed and stably transfected into MCF-7 cells (MCF-7/pSIG). After SSTR2 overexpression was identified by qRT-PCR, immunofluorescence staining and a receptor binding assay, the MCF-7/pSIG cells were analyzed by PI staining for apoptosis and cell cycle arrest was tested by flow cytometry for epidermal growth factor receptor (EGFR) expression. The EGF-stimulated proliferation of MCF-7 cells was assayed by MTT.Results:The human breast cancer cell line MCF-7 expresses a lower level of SSTR2, thereby partly accounting for the decreased response to SST. The overexpression of SSTR2 in MCF-7 cells resulted in apoptosis, cytostasis and G1/S cell cycle arrest. Furthermore, the expression of EGFR, together with EGF-stimulated proliferation, was markedly decreased in the MCF-7/pSIG cells.Conclusion:Enhanced SSTR2 expression played an antiproliferative role in MCF-7 cells through inducing apoptosis and G1/S cell cycle arrest, and also by decreasing EGFR expression, thereby counteracting the growth-stimulating effect of EGF. Our data seem to indicate that developing a new therapeutic agent capable of upregulating SSTR expression could potentially be a way to block tumor progression.

The immunosuppressive and protective ability of glucose-regulated protein 78 for improvement of alloimmunity in β cell transplantation

An insulinoma cell line, NIT‐1, transfected with glucose‐regulated protein 78 (GRP78) was established, namely NIT‐GRP78, and used to study the immunosuppressive and protective ability of GRP78. In extended cytotoxic T lymphocyte (CTL) killing assay, NIT‐1‐primed lymphocytes were more cytotoxic in killing β cells than NIT‐GRP78‐primed lymphocytes. Severe necrosis was observed only when the NIT‐1‐primed lymphocytes were cultured with NIT‐1 β cells, but not with NIT‐GRP78 cells. In addition, an increase of interleukin (IL)‐4 secretion from β cell‐primed splenocytes when GRP78 presence was observed in cytokine enzyme‐linked immunosorbent assay (ELISA). Diabetic mice reached normoglycaemia promptly and gained weight after transplantation of either NIT‐1 or NIT‐GRP78 cells. However, the recipient mice transplanted with NIT‐GRP78 cells lived much longer than those recipients transplanted with NIT‐1 cells, which was due apparently to prolonged insulin production by the transplanted NIT‐GRP78 cells. In fact, we observed a significant increase of insulin concentration after glucose stimulation of diabetic mice received NIT‐GRP78 cells at day 7 post‐transplantation. From the results we propose that GRP78 could have a dual function in both protecting NIT‐1 cells from CTL‐mediated lysis and stimulating a population of T helper 2 cells to down‐regulate the immune response to the transplanted β cells.