Combined effect of sCD40L and PI3K siRNA on transplanted tumours growth and microenvironment in nude mice with gastric cancer

The objective of the paper is to investigate the combined effect of sCD40L and phosphatidylinositol 3-kinase (PI3K) siRNA on transplanted tumours growth and microenvironment in nude mice with gastric cancer. 48 h after modeling, the animals were randomly divided into saline + AGS group (A), sCD40L + AGS group (B), saline + PI3K siRNA group (C) and sCD40L + PI3K siRNA group (D), six mice in each group. The mouse in the groups was inoculated with exponential phase AGS cell or PI3K gene silencing cells (100 μl, 5 × 10(6)). After tumour size reaches 0.2-0.3 cm, Tumours in animals of groups were injected with sCD40L (100 μl, 10 mg/kg) or equal volume of saline, thrice each day, respectively. Microvessel density (MVD), apoptosis index, and expression levels of PI3K, Survivin and vascular endothelial growth factor (VEGF) proteins in transplanted tumor cells in gastric cancer nude mice were analyzed by utilizing Immunohistochemistry, western blot, terminal deoxynucleotidyl transferase dUTP nick end labeling assays. Results showed that combination of sCD40L with PI3K siRNA could significantly decrease tumour size, MVD, expression levels of PI3K, Survivin and VEGF proteins, and increase apoptosis index. It can be concluded that combination of sCD40L with PI3K siRNA provides a promising future for gastric cancer therapy.     

Screening and test of CD40 related signal transduction pathway in AGS cells and construction of gene silencing vector

This study is designed to screen the CD40 related signal transduction pathway in AGS cells and construction of gene silencing vector. Analysis results showed 414 differential genes expression, including upregulation of 209 genes and downregulation of 205 genes. Basing on the ratio of signal in experimental group to signal in control group, 45 genes (38 genes upregulation and seven genes downregulation) with significant (P < 0.01) change in expression levels were screened according to the screening standard (signal log ratio ≥1 or ≤?1). These genes involved into metabolism, cell cycle and apoptosis, signal transduction and stress response. Furthermore, PI3K mRNA expression level in PI3K siRNA transfected AGS cells was 0.2335 ± 0.0116 72 h after transfection. This value was significantly (P < 0.05) lower than that in blank and negative control groups. PI3K protein expression in PI3K siRNA transfected AGS cells was significantly (P < 0.05) lower than that in blank and PI3K siRNA/N transfected groups. Therefore, PI3K siRNA gene silencing vector can significantly inhibit PI3K mRNA and protein expression in AGS cells.     

Sodium butyrate induces differentiation of gastric cancer cells to intestinal cells via the PTEN/phosphoinositide 3‐kinase pathway

NaB (sodium butyrate) inhibits cell proliferation and induces differentiation in a variety of tumour cells. In this study, we aimed to determine whether NaB induced differentiation and regulated the expression of the mucosal factor MUC2 through the PTEN/PI3K (phosphoinositide 3‐kinase) pathway. BGC823 cells treated with NaB for 24–72 h showed marked inhibition of cell proliferation and alteration in cellular morphology. NaB treatment markedly increased the expression of PTEN and MUC2, but it decreased the expression of PI3K. These effects were enhanced by intervention with PI3K inhibitors and were reduced by intervention with PTEN siRNA. Hence, we conclude that NaB increased PTEN expression, promoted the expression of MUC2 and induced the differentiation of gastric cancer cells through the PTEN/PI3K signalling pathway.     

Ligation or cross-linking of CD40 has different effects on AGS gastric cancer cells

A gastric cancer (GC) cell line, AGS, has high-level expression of CD40, a tumor necrosis factor receptor (TNFR) family member. CD40 is present on the surfaces of a large variety of cells, including B cells, endothelial cells, dendritic cells and some carcinoma cells, and delivers signals regulating diverse cellular responses, such as proliferation, differentiation, growth suppression, and cell death. In this research, we studied the effects of different forms of CD40 stimulation on AGS cells by flow cytometry, Western blotting and siRNA transfection. We found that different forms of CD40 stimulation, either recombinant soluble CD40L (sCD40L, ligation) or agonist anti-CD40 antibody (cross-linking), induced different effects in AGS gastric cancer cells, proliferation or apoptosis. We also showed that VEGF provided a significant contribution to sCD40L-induced proliferation, while agonist anti-CD40 antibody induced GADD45 upregulation and promoted apoptosis.     

Inhibition of miR-21-5p suppresses high glucose-induced proliferation and angiogenesis of human retinal microvascular endothelial cells by the regulation of AKT and ERK pathways via maspin

The aim of the present study is to investigate the role of miR-21-5p in angiogenesis of human retinal microvascular endothelial cells (HRMECs). HRMECs were incubated with 5 mM glucose, 30 mM glucose or 30 mM mannitol for 24 h, 48 h or 72 h. Then, HRMECs exposed to 30 mM glucose were transfected with miR-21-5p inhibitor. We found that high glucose increased the expression of miR-21-5p, VEGF, VEGFR2 and cell proliferation activity. Inhibition of miR-21-5p reduced high glucose-induced proliferation, migration, tube formation of HRMECs, and reversed the decreased expression of maspin as well as the abnormal activation of PI3K/AKT and ERK pathways. Down-regulation of maspin by siRNA significantly increased the activities of PI3K/AKT and ERK pathways. In conclusion, inhibition of miR-21-5p could suppress high glucose-induced proliferation and angiogenesis of HRMECs, and these effects may partly dependent on the regulation of PI3K/AKT and ERK pathways via its target protein maspin.     

Influence of sCD40L on gastric cancer cell lines

The CD40 signaling pathway plays a key role in tumor cell proliferation, differentiation, and apoptosis. Gastric cancer usually possesses a higher level of CD40 expression than normal tissue. We evaluated inhibition of soluble CD40 ligand (sCD40L) in apoptosis induction of gastric cancer cells. Gastric cancer cells (AGS and BGC-823) were incubated with sCD40L. Cell viability and cell cycle were determined by methyl-tetrazolium (MTT) assay and flow cytometry, respectively. The results showed that sCD40L hindered cell growth, arrested cells at G0/G1 phase and induced apoptosis. In conclusion, sCD40L suppress growth of gastric cancer cells through apoptosis induction and cell cycle quiescence. This work will provided a new approach to gene therapy of gastric cancer.     

Targeted inhibition of phosphatidyl inositol-3-kinase p110β, but not p110α, enhances apoptosis and sensitivity to paclitaxel in chemoresistant ovarian cancers

The phosphatidylinositol 3-kinase (PI3K) pathway is one of the critical signaling cascades playing important roles in the chemoresistance of human cancer cells, including ovarian cancer. In this study, we investigated the potential of targeting the PI3K p110β-isoform as a novel approach to overcome the chemoresistance in ovarian cancer. The effects on apoptosis, cell viability, proliferation and migration in chemoresistant ovarian cancer cell were determined following targeted p110β inhibition by small interfering RNA (siRNA). Seven paclitaxel (PTX)-resistant sublines (SKpacs and A2780pac) were produced from SKOV3 and A2780 ovarian cancer cell lines. We, first, evaluated the expression of PI3K p110 isoforms in chemosensitive and chemoresistant ovarian cancer cell lines and patient specimens, and found that p110β-isoform was significantly overexpressed both in a panel of ovarian cancer samples, and in PTX-resistant sublines compared with their parent cell lines. RNA interference-mediated p110β silencing augmented PTX-mediated apoptosis (31.15 ± 13.88 %) and reduced cell viability (67 %) in PTX-resistant cells, whereas targeting p110α did not show a significant change in cell viability and apoptosis. In addition, p110β silencing impaired cell proliferation (60 %) in PTX-resistant SKpac cells. We also found the combined treatment group with p110β siRNA and PTX showed a significant inhibition of tumor growth of SKpac cells compared to the PTX-only treated group in a xenograft nude mouse model. Thus, the siRNA-mediated silencing of PI3K p110β resensitizes PTX-resistant ovarian cancer cells, and may be a useful therapeutic strategy for PTX-resistant ovarian cancers.     

CD40-dependent activation of phosphatidylinositol 3-kinase/Akt pathway mediates endothelial cell survival and in vitro angiogenesis

CD40 has been involved in tumor and inflammatory neoangiogenesis. In this study we determined that stimulation of endothelial CD40 with sCD154 induced resistance to apoptosis and in vitro vessel-like formation by human microvascular endothelial cells (HMEC). These effects were determined to be mediated by CD40-dependent signaling because they were inhibited by a soluble CD40-muIg fusion protein. Moreover, apoptosis of HMEC was associated with an impairment of Akt phosphorylation, which was restored by stimulation with sCD154. The anti-apoptotic effect as well as in vitro vessel-like formation and Akt phosphorylation were inhibited by treatment of HMEC with two unrelated pharmacological inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin and LY294002. CD40 stimulation induced a rapid increase in Akt enzymatic activity that was not prevented by cycloheximide, an inhibitor of protein synthesis. The enhanced Akt activity induced by stimulation of endothelial CD40 was temporarily correlated with the association of CD40 with TRAF6, c-Cbl, and the p85 subunit of PI3K. Expression of negative-dominant Akt inhibited the activation of endogenous Akt through CD40 stimulation, despite the observation that association of CD40 with TRAF6, c-Cbl, and PI3K was intact. The defective activation of Akt abrogated not only the anti-apoptotic effect of CD40 stimulation but also the proliferative response, the enhanced motility, and the in vitro formation of vessel-like tubular structures by CD40-stimulated HMEC. In conclusion, these results suggest that endothelial CD40, through activation of the PI3K/Akt signaling pathway, regulates cell survival, proliferation, migration, and vessel-like structure formation, all steps considered critical for angiogenesis.     

Roxarsone induces angiogenesis via PI3K/Akt signaling

Background

3-Nitro-4-hydroxy phenyl arsenic acid, roxarsone, is widely used as an organic arsenic feed additive for livestock and poultry, which may increase the level of arsenic in the environment and the risk of exposure to arsenic in human. Little information is focused on the angiogenesis roxarsone-induced and its mechanism at present. This paper aims to study the role of PI3K/Akt signaling in roxarsone-induced angiogenesis in rat vascular endothelial cells and a mouse B16–F10 melanoma xenograft model.

Results

The results showed that treatment with 0.1–10.0 µmol/L roxarsone resulted in an increase in the OD rate in the MTT assay, the number of BrdU-positive cells in the proliferation assay, the migration distance in the scratch test and the number of meshes in tube formation assay. Further, treatment with 1.0 µmol/L roxarsone was associated with significantly higher phosphorylation of PI3K/Akt and expression of VEGF than the control treatment. The PI3K inhibitor was found to significantly combat the effects of 1.0 µmol/L roxarsone. Furthermore, roxarsone treatment was observed to increase the weight and volume of B16–F10 xenografts and VEGF expression and PI3K/Akt phosphorylation in a dose-dependent manner, with the 25 mg/kg dose having significant effects.

Conclusions

These results demonstrate that roxarsone has the ability to promote growth and tube formation in vascular endothelial cells and the growth of mouse B16–F10 xenografts. Further, the findings also indicate that PI3K/Akt signaling plays a regulatory role in roxarsone-induced angiogenesis in vivo and in vitro.

     

Soluble CD40 Ligand Stimulates CD40-Dependent Activation of the β2 Integrin Mac-1 and Protein Kinase C Zeda (PKCζ) in Neutrophils: Implications for Neutrophil-Platelet Interactions and Neutrophil Oxidative Burst

Recent work has revealed an essential involvement of soluble CD40L (sCD40L) in inflammation and vascular disease. Activated platelets are the major source of sCD40L, which has been implicated in platelet and leukocyte activation, although its exact functional impact on leukocyte-platelet interactions and the underlying mechanisms remain undefined. We aimed to determine the impact and the mechanisms of sCD40L on neutrophils. We studied neutrophil interactions with activated, surface-adherent platelets as a model for leukocyte recruitment to the sites of injury. Our data show that CD40L contributes to neutrophil firm adhesion to and transmigration across activated surface-adherent platelets, possibly through two potential mechanisms. One involves the direct interaction of ligand-receptor (CD40L-CD40), i.e., platelet surface CD40L interaction with neutrophil CD40; another involves an indirect mechanism, i.e. soluble CD40L stimulates activation of the leukocyte-specific β2 integrin Mac-1 in neutrophils and thereby further promotes neutrophil adhesion and migration. Activation of the integrin Mac-1 is known to be critical for mediating neutrophil adhesion and migration. sCD40L activated Mac-1 in neutrophils and enhanced neutrophil-platelet interactions in wild-type neutrophils, but failed to elicit such responses in CD40-deficient neutrophils. Furthermore, our data show that the protein kinase C zeta (PKCζ) is critically required for sCD40L-induced Mac-1 activation and neutrophil adhesive function. sCD40L strongly stimulated the focal clustering of Mac-1 (CD11b) and the colocalization of Mac-1 with PKCζ in wild-type neutrophils, but had minimal effect in CD40-deficient neutrophils. Blocking PKCζ completely inhibited sCD40L-induced neutrophil firm adhesion. Moreover, sCD40L strongly stimulates neutrophil oxidative burst via CD40-dependent activation of PI3K/NF-KB, but independent of Mac-1 and PKCζ. These findings may contribute to a better understanding of the underlying mechanisms by which sCD40L/CD40 pathway contributes to inflammation and vascular diseases.     

Soluble CD40 Ligand in Sera of Subjects Exposed to Leishmania infantum Infection Reduces the Parasite Load in Macrophages

Background

While CD40L is typically a membrane glycoprotein expressed on activated T cells and platelets that binds and activates CD40 on the surface on antigen presenting cells, a soluble derivative (sCD40L) that appears to retain its biological activity after cleavage from cell membrane also exists. We recently reported that sCD40L is associated with clinical resolution of visceral leishmaniasis and protection against the disease. In the present study we investigated if this sCD40L is functional and exerts anti-parasitic effect in L. infantum-infected macrophages.

Methodology/Principal Findings

Macrophages from normal human donors were infected with L. infantum promastigotes and incubated with either sera from subjects exposed to L. infantum infection, monoclonal antibodies against human CD40L, or an isotype control antibody. We then evaluated infection by counting the number of infected cells and the number of parasites in each cell. We also measured a variety of immune modulatory cytokines in these macrophage culture supernatants by Luminex assay. The addition of sCD40L, either recombinant or from infected individuals’ serum, decreased both the number of infected macrophages and number of intracellular parasites. Moreover, this treatment increased the production of IL-12, IL-23, IL-27, IL-15, and IL1β such that negative correlations between the levels of these cytokines with both the infection ratio and number of intracellular parasites were observed.

Conclusions/Significance

sCD40L from sera of subjects exposed to L. infantum is functional and improves both the control of parasite and production of inflamatory cytokines of infected macrophages. Although the mechanisms involved in parasite killing are still unclear and require further exploration, these findings indicate a protective role of sCD40L in visceral leishmaniasis.     

Soluble CD40 Ligand Stimulates the Pro-Angiogenic Function of Peripheral Blood Angiogenic Outgrowth Cells via Increased Release of Matrix Metalloproteinase-9

The role of endothelial progenitor cells in vascular repair is related to their incorporation at sites of vascular lesions, differentiation into endothelial cells, and release of various angiogenic factors specifically by a subset of early outgrowth endothelial progenitor cells (EOCs). It has been shown that patients suffering from cardiovascular disease exhibit increased levels of circulating and soluble CD40 ligand (sCD40L), which may influence the function of EOCs. We have previously shown that the inflammatory receptor CD40 is expressed on EOCs and its ligation with sCD40L impairs the anti-platelet function of EOCs. In the present study, we aimed at investigating the effect of sCD40L on the function of EOCs in endothelial repair. Human peripheral blood mononuclear cell-derived EOCs express CD40 and its adaptor proteins, the tumor necrosis factor receptor-associated factors; TRAF1, TRAF2 and TRAF3. Stimulation of EOCs with sCD40L increased the expression of TRAF1, binding of TRAF2 to CD40 and phosphorylation of p38 mitogen activated protein kinase (MAPK). In an in vitro wound healing assay, stimulation of EOCs with sCD40L increased the release of matrix metalloproteinase 9 (MMP-9) in a concentration-dependent manner and significantly enhanced the angiogenic potential of cultured human umbilical vein endothelial cells (HUVECs). Inhibition of p38 MAPK reversed sCD40L-induced MMP-9 release by EOCs, whereas inhibition of MMP-9 reversed their pro-angiogenic effect on HUVECs. This study reveals the existence of a CD40L/CD40/TRAF axis in EOCs and shows that sCD40L increases the pro-angiogenic function of EOCs on cultured HUVECs by inducing a significant increase in MMP-9 release via, at least, the p38 MAPK signaling pathway.     

Hydroxysafflor Yellow A Protects Against Cerebral Ischemia–Reperfusion Injury by Anti-apoptotic Effect Through PI3K/Akt/GSK3β Pathway in Rat

Hydroxysafflor yellow A (HSYA) is the major active chemical component of the flower of the safflower plant, Carthamus tinctorius L. Previously, its neuroprotection against cerebral ischemia–reperfusion (I/R) injury was reported by anti-oxidant action and suppression of thrombin generation. Here, we investigate the role of HSYA in cerebral I/R-mediated apoptosis and possible signaling pathways. Male Wistar rats were subjected to transient middle cerebral artery occlusion for 2 h, followed by 24 h reperfusion. HSYA was administered via tail-vein injection just 15 min after occlusion. The number of apoptotic cells was measured by TUNEL assay, apoptosis-related proteins Bcl-2, Bax and the phosphorylation levels of Akt and GSK3β in ischemic penumbra were assayed by western blot. The results showed that administration of HSYA at the doses of 4 and 8 mg/kg significantly inhibited the apoptosis by decreasing the number of apoptotic cells and increasing the Bcl-2/Bax ratio in rats subjected to I/R injury. Simultaneously, HSYA treatment markedly increased the phosphorylations of Akt and GSK3β. Blockade of PI3K activity by wortmannin dramatically abolished its anti-apoptotic effect and lowered both Akt and GSK3β phosphorylation levels. Taken together, these results suggest that HSYA protects against cerebral I/R injury partly by reducing apoptosis via PI3K/Akt/GSK3β signaling pathway.     

The role of the CD95, CD38 and TGFbeta1 during active human cytomegalovirus infection in liver transplantation

AIM: Human cytomegalovirus (HCMV) has highly evolved mechanisms for avoiding detection by the host immune system. The aim of this study was to analyze the expression levels of TGFbeta1, soluble form of CD95, CD95 ligand (sCD95 and sCD95L, respectively) in plasma and CD95 expression on CD3(+) cells, CD38 expression on CD8(+) cells in liver transplanted recipients with active HCMV infection. METHODS: Blood samples were collected from 15 liver transplanted recipients with active HCMV infection and 15 recipients without HCMV infection. CD95 expression on CD3(+) cells and CD38 expression on CD8(+) cells were quantitatively detected with two-color fluorescence activated cell sorter (FACS) analysis. Lymphocyte surface phenotypes of CD4 and CD8 were detected with FACS analysis. Plasma sCD95, sCD95L and TGFbeta1 levels were determined with enzyme linked-immuno-sorbent assay (ELISA). The results were compared with that from 15 healthy individuals. RESULTS: CD95 expression on CD3(+) T-cells and CD38 expression on CD8(+) cells were significantly increased in active HCMV infection group compared with that in stable group or healthy group (P<0.01). No significant difference was seen between stable group and healthy group (P>0.05). The percentages of CD4(+) T-cell and CD4/CD8 ratio in active HCMV infection group were significantly lower than the values in stable group and healthy group (P<0.05). Plasma levels of TGFbeta1 and sCD95 were significantly increased in active HCMV infection group compared to stable group and healthy group (P<0.05). In contrast, plasma levels of sCD95L in healthy group were not significantly different from that expressed in active HCMV infection group and stable group (P>0.05). CONCLUSION: HCMV suppress proliferation of activated T cells by apoptosis and by releasing immunosuppressive cytokine TGFbeta1. This may provide an important clue to a better understanding of the pathogenesis in liver transplanted recipients with active HCMV infection.     

The skewed frequency of B-cell subpopulation CD19+CD24 hiCD38 hi cells in peripheral blood mononuclear cells is correlated with the elevated serum sCD40L in patients with active systemic lupus erythematosus

CD19⁺CD24^hiCD38^hi cells play an essential role in maintaining immune homeostasis. CD40 signaling is involved in regulating the induction and function of CD19⁺CD24^hiCD38^hi cells. Changes in B-cell subpopulations and CD19⁺CD24^hiCD38^hi cells have been observed in systemic lupus erythematosus (SLE) patients. Whether changes in the B-cell subpopulation are related to the aberrant CD40 signaling in SLE patients remains unclear. In this study, we examined changes in the levels of CD19⁺CD24^hiCD38^hi cells and CD19⁺CD24^hiCD38^low cells in peripheral blood mononuclear cells and the serum level of soluble CD40 ligand (sCD40L) in 30 patients with SLE. Through routine biochemical assays and flow cytometry assay, we found that (1) the CD19⁺CD24^hiCD38^hi cell subset was upregulated in SLE patients compared to that in healthy controls (HCs) (P < 0.05); (2) the CD19⁺CD24^hiCD38^low cell subset was downregulated in SLE patients compared with that in HCs; and (3) CD38 expression was positively correlated with SLE manifestations and the serum sCD40L level (P < 0.05). In conclusion, the relative level of Bregs is significantly higher in SLE patients than in HCs and is positively correlated with disease activity and sCD40L level.     

Differential induction of cytokine genes and activation of mitogen-activated protein kinase family by soluble CD40 ligand and TNF in a human follicular dendritic cell line.

Follicular dendritic cells (FDC)3 play crucial roles in germinal center (GC) formation and differentiation of GC B cells. Many aspects of FDC function are influenced by contact with B or T cells, and by cytokines produced in the GC, which involve stimulation of CD40 and TNF-alpha receptors on FDC. In this study, using an established FDC line, HK cells, we compared the effects of CD40 and TNF receptor triggering on cytokine induction and activation of mitogen-activated protein kinase family. We show that HK cells spontaneously produced IL-6, M-CSF, and G-CSF mRNA. Both the soluble form of CD40 ligand (sCD40L) and TNF increased the level of M-CSF and G-CSF mRNA. While TNF strongly induced IL-6 mRNA, its expression was not affected by sCD40L treatment, differing from the strong IL-6 induction in other cell types upon CD40 stimulation. In addition, sCD40L treatment resulted in activation of extracellular signal-related kinase 1 and 2 (ERK1/2) and p38 without significant increase in c-Jun N-terminal kinase (JNK) activity. Lack of JNK activation differs in that most B cells respond to CD40 stimulation by inducing JNK activity strongly, suggesting distinct characteristics of CD40 signaling in FDC. Compared with the effects of sCD40L, TNF was capable of inducing JNK activity in addition to the activation of ERK1/2 and p38. Furthermore, the proximal signaling elements activated by TNF differed from those activated by sCD40L, in that TNF did not require PMA-sensitive protein kinase C isoforms in the activation of ERK and p38, whereas sCD40L did. However, signals activated by these stimuli converged on cytokine gene expression in a synergistic manner, which may have implication in augmenting FDC function during GC reaction.     

CD40 ligand binds to alpha5beta1 integrin and triggers cell signaling

It was originally thought that the critical role of the CD40 ligand (CD40L) in normal and inflammatory immune responses was mainly mediated through its interaction with the classic receptor, CD40. However, data from CD40L(-/-) and CD40(-/-) mice suggest that the CD40L-induced inflammatory immune response involves at least one other receptor. This hypothesis is supported by the fact that CD40L stabilizes arterial thrombi through an alphaIIbbeta3-dependent mechanism. Here we provide evidence that soluble CD40L (sCD40L) binds to cells of the undifferentiated human monocytic U937 cell line in a CD40- and alphaIIbbeta3-independent manner. Binding of sCD40L to U937 cells was inhibited by anti-CD40L monoclonal antibody 5C8, anti-alpha5beta1 monoclonal antibody P1D6, and soluble alpha5beta1. The direct binding of sCD40L to purified alpha5beta1 was confirmed in a solid phase binding assay. Binding of sCD40L to alpha5beta1 was modulated by the form of alpha5beta1 expressed on the cell surface as the activation of alpha5beta1 by Mn(2+) or dithiothreitol resulted in the loss of sCD40L binding. Moreover, sCD40L induced the translocation of alpha5beta1 to the Triton X-100-insoluble fraction of U937 cells, the rapid activation of the MAPK pathways ERK1/2, and interleukin-8 gene expression. The binding of sCD40L to CD40 on BJAB cells, an alpha5beta1-negative B cell line, and the resulting activation of ERK1/2 was not inhibited by soluble alpha5beta1, suggesting that sCD40L can bind concomitantly to both receptors. These results document the existence of novel CD40L-dependent pathways of physiological relevance for cells expressing multiple receptors (CD40, alpha5beta1, and alphaIIbbeta3) for CD40L.     

The TLR3, PI3K,survin, FasL,and Fas genes as major risk factors of occurrence and development of cervical cancer disease

To investigate the role of TLR3/PI3K signals in the occurrence and development of cervical cancer disease, TLR3–siRNA was used to block key signaling pathways involved in cervical cancer metastasis that are pivotal to metastatic tumor cells but not to normal cells under ordinary physiologic conditions. Results show that tumor U14 cell growth, migration and invasion in TLR3–siRNA treatment group were significantly decreased. Through LY294002 suppressing targeted gene, the LY294002 treatment specifically and significantly knocked down the expressions of tumor TLR3 and PI3K proteins in cervical cancer mice. Furthermore, expressions of tumor Survivin and FasL proteins were markedly suppressed, whereas expressions of Fas protein were upregulated in LY294002 treatment group mice. LY294002 treatment suppressed tumor growth and increased the thymus and spleen indeces and survival days of cervical cancer mice. This study demonstrates that TLR3–siRNA and LY294002 treatments can markedly suppress cervical cancer cell invasion and tumor growth and increase survival life by silencing targeted genes.