Expression of TGF-β1 and the mechanism of invasiveness and metastasis induced by TGF-β1 in breast cancer

Objective To investigate the expression of MMP-2,TIMP-2,TGF-β1 and TGF-βRI and the relationship among them in breast cancer.Methods The protein expression of MMP-2,TIMP-2,TGF-β1 and TGF-β1R1 was detected on tissue chips by S-P immunohistochemical staining in 160 cases of breast carcinoma.Results The positive rates of TGF-β1,TGF-β1 mRNA,MMP-2,MMP-9,TIMP-1 and TIMP-2 expression were 73.7%,56.2%,96.9%,95.0%,87.5% and 89.4%,respectively.Axillary lymph node metastasis and TNM staging(P ＜0.01 and P ＜0.01,respectively)were positively correlated to the expression of TGF-β1.Relase-free survival of TGF-β1 positive group was lower than that of TGF-β1 negative group(P = 0.023).The expression of M MP-2 or M M P-9 was positively correlated to that of TGF-β1 (r=0.170,P＜0.05;r =0.221,P＜0.01)and was negatively correlated to that of TGF-β1 mRNA(r =-0.126,P ＞0.05;r = 0.019,P ＞ 0.05).Conclusion The expression of TGF-β1 may be closely correlated with the invasion and metastasis of breast cancer.TGF-β1-induced invasiveness and metastasis of breast cancer cells are mediated by MMP-2 and MMP-9.     

Induction of apoptosis by quercetin is mediated through AMPKα1/ASK1/p38 pathway

Effective strategies for cancer prevention and treatment can be identified by understanding the mechanism of apoptotic pathways. In this study, we investigated the regulatory mechanism of quercetin-induced apoptosis through apoptosis signal-regulating kinase (ASK)-1 and mitogen-activated protein kinase pathways. Our results showed that quercetin increased apoptotic cell death through reactive oxygen species (ROS) generation and was responsible for ASK1 activation. Increasing ASK1 activity was accompanied by p38 activation. Interestingly, AMP-activated protein kinase (AMPK) seemed to be a critical controller of quercetin-regulated ASK1/p38 activation. Blocking AMPKα1 activity using Compound C, a synthetic inhibitor or siRNA showed that quercetin-activated ASK1 could not stimulate p38 activity. Thus, we suggested that quercetin-exerted apoptotic effects involve ROS/AMPKα1/ASK1/p38 signaling pathway, and AMPKα1 is a necessary element for apoptotic event induced by ASK1.     

Role of RNase L in apoptosis induced by 1-(3-C-ethynyl-β-d-ribo-pentofuranosyl)cytosine

Purpose  1-(3-C-Ethynyl-β-d-ribo-pentofuranosyl)cytosine (ECyd), a ribonucleoside analog, has a potent cytotoxic activity against cancer cells. The present studies have been performed to elucidate the overall mechanisms of ECyd-induced apoptotic cell death. Methods  Cultured cells of mouse mammary carcinoma FM3A and human fibrosarcoma HT 1080 lines were used. The efficacy of RNA synthesis inhibition by ECyd was assessed by kinetic analysis using nuclei isolated from FM3A cells. RNA status in ECyd-treated cells was investigated by Northern blots, and the cleavage sites of RNA were identified by rapid amplification of 5′ cDNA ends (5′-RACE). The effect of protein functions on the ECyd-induced apoptotic pathway was analyzed by siRNA and immunohistochemical techniques. Apoptotic cells were detected by TdT-mediated dUTP-biotin Nick End Labeling (TUNEL) assay. Results  ECyd induces inhibition of RNA synthesis in vitro and in vivo, which appears to be a major cause for the apoptosis. It is known that ECyd is converted inside the cell into its 5′-triphosphate (ECTP). We have now found in test-tube experiments that ECTP strongly inhibits the activity of RNA polymerase I by competing with CTP. In the absence of robust RNA synthesis, the cellular RNAs would be destined to break down. RNase L was found to be playing a role in the breakdown: thus, the 28S rRNA-fragmentation pattern observed for the ECyd-treated cells was very similar to that observable in an in vitro treatment of the 28S ribosomes with RNase L. Association of RNase L with the cytotoxic action of ECyd was confirmed by use of the siRNA-mediated suppression of the cellular RNase L. Thus, the cells in which the RNase L was knocked-down were highly resistant to the cytotoxic action of ECyd. Further events, downstream of the RNase L action that can lead to the eventual apoptosis, would conceivably involve the phosphorylation of c-jun N-terminal kinase and subsequent decrease in mitochondrial membrane-potential. Evidence to support this flow of events was obtained by siRNA-experiments. Conclusion  The results from this study demonstrated that RNase L is activated after the inhibition of RNA polymerase, and induces mitochondria-dependent apoptotic pathway. We propose this new role for RNase L in the apoptotic mechanism. These findings may open up the possibility of finding new targets for anticancer agents. Tomoharu Naito and Tatsushi Yokogawa have contributed equally to the main findings of the paper.     

miR-183 inhibits TGF-β1-induced apoptosis by downregulation of PDCD4 expression in human hepatocellular carcinoma cells

Background  

In recent years, some miRNAs have been reported to be connected closely with the development of human hepatocellular carcinoma. In our previous studies, a set of miRNAs were revealed to be dysregulated in HCC tissues. However, the functions of these miRNAs in HCC remain largely undefined.     

The cytotoxic effect of TGF-β1 on mesothelial cells via apoptosis in early peritoneal carcinomatosis

Peritoneal dissemination is one of the main causes of death in gastric cancer patients. We have previously reported that gastric cancer cells can induce peritoneal apoptosis, lead to damage of peritoneum integrity, and therefore promote peritoneal metastasis. However, the soluble factors secreted by cancer cells to trigger the damaging cascade remain unclear. TGF-β1, a cytokine known for its capacity to induce proliferative and transformative changes of cells is found in significantly higher quantities correlated with peritoneal metastasis and TNM stages of gastric cancer. High levels of TGF-β1 in the subperitoneal milieu may affect the morphology and function of mesothelial cells, so that the resulting environment becomes favorable for peritoneal metastases. We observed apoptosis induced by TGF-β1 in mesothelial cells in peritoneal carcinomatosis. Knockdown of the smad2 gene by siRNA silencing can partially inhibit these effects. TGF-β1 could upregulate the expressions of Bax and suppress Bcl-2 in mesothelial cells. We conclude that TGF-β1 could induce apoptosis of mesothelial cells, which involves the smad2 signaling pathway in peritoneal carcinomatosis. Bcl-2 and Bax may contribute to this phenomenon.     

The role of TGF-β1–miR-21–ROS pathway in bystander responses induced by irradiated non-small-cell lung cancer cells

Background:

Many studies have indicated an important implication of radiation-induced bystander effects (RIBEs) in cancer radiotherapy, but the detailed signalling remains unclear.

Methods:

The roles of tumour growth factor-beta1 (TGF-β1) and miR-21 in medium-mediated RIBEs in H1299 non-small-cell lung cancer cells were investigated using DNA damage, changes in proliferation and levels of reactive oxygen species (ROS) as end points. SB431542, a specific inhibitor of TGF-β type 1 receptor kinases, was used to inhibit TGF-β1 pathways in irradiated and bystander cells. Exogenous miR-21 regulation was achieved through inhibitor or mimic transfection.

Results:

Compared with relative sham-radiation-conditioned medium, radiation-conditioned medium (RCM) from irradiated cells 1 h post radiation (1-h RCM) caused an increase in ROS levels and DNA damage in bystander cells, while 18-h RCM induced cell cycle delay and proliferation inhibition. All these effects were eliminated by TGF-βR1 inhibition. One-hour RCM upregulated miR-21 expression in bystander cells, and miR-21 inhibitor abolished bystander oxidative stress and DNA damage. Eighteen-hour RCM downregulated miR-21 of bystander cells, and miR-21 mimic eliminated bystander proliferation inhibition. Furthermore, the dysregulation of miR-21 was attenuated by TGF-βR1 inhibition.

Conclusions:

The TGF-β1–miR-21–ROS pathway of bystander cells has an important mediating role in RIBEs in H1299 cells.     

T cell- but not tumor cell-produced TGF-β1 promotes the development of spontaneous mammary cancer

During their development, tumors acquire multiple capabilities that enable them to proliferate, disseminate and evade immunosurveillance. A putative mechanism is through the production of the cytokine TGF-β1. We showed in our recent studies that T cell-produced TGF-β1 inhibits antitumor T cell responses to foster tumor growth raising the question of the precise function of TGF-β1 produced by tumor cells in tumor development. Here, using a transgenic model of mammary cancer, we report that deletion of TGF-β1 from tumor cells did not protect mice from tumor development. However, ablation of TGF-β1 from T cells significantly inhibited mammary tumor growth. Additionally, absence of TGF-β1 in T cells prevented tumors from advancing to higher pathological grades and further suppressed secondary tumor development in the lungs. These findings reveal T cells but not tumor cells as a critical source of TGF-β1 that promotes tumor development.     

Glioma Cell Invasion: Regulation of Metalloproteinase Activity by TGF-β

Matrix metalloproteinases (MMPs) are a family of extracellular endopeptidases that selectively degrade components of the extracellular matrix. MMPs are implicated in tumor cell invasion because they mediate the breakdown of the basal membrane. In addition, they seem to be important for the creation and maintenance of a microenvironment that facilitates tumor cell survival. Among the essential characteristics of human malignant gliomas are infiltrative growth, angiogenesis and suppression of antitumor immune surveillance. Transforming growth factor-beta (TGF-) is intimately involved in the regulation of these processes. We have previously demonstrated that TGF- promotes the migration of LN-18 and LN-229 glioma cells via a process that may involve the upregulation of _V3 integrin expression. Furthermore, we have defined a novel pathway for hepatocyte growth factor (HGF)-induced glioma cell migration and invasion which requires the induction of TGF-₂ expression. Here, we demonstrate that TGF-₂ induces MMP-2 expression and suppresses tissue inhibitor of metalloproteinases (TIMP)-2 expression and that concentration-dependently promotes the invasion of U87MG and LN-229 glioma cells in a matrigel invasion assay. Similarly, ectopic expression of the anti-apoptotic BCL-x_L protein leads to enhanced matrigel invasion by LN-18 and LN-229 glioma cells. We outline the possible interrelations of TGF-, proteins of the BCL-2 family, integrins and metalloprotease activity. By virtue of its promotion of glioma invasion and its growth regulatory and immunomodulatory properties, TGF- continues to be one of the most promising targets for the experimental therapy of human malignant glioma.     

IL-8 is a mediator of NF-κB induced invasion by gliomas

Glioblastoma (GBM) is the most common and deadly form of primary brain tumor with a median survival of eleven months, despite use of extensive chemotherapy, radiotherapy and surgery. We have previously shown that nuclear factor-kappa B (NF-κB) is aberrantly expressed in GBM tumors and primary cell lines derived from tumor tissue. Here we show that IL-8, a chemokine is also aberrantly expressed by GBM cell lines and expression of IL-8 is in large part, attributable to the aberrant activation of NF-κB. We hypothesized that invasiveness of GBM cells is driven at least in part by aberrantly expressed IL-8. In support of the hypothesis we found that treatment of glioma cells with an IL-8 neutralizing antibody markedly decreased their invasiveness compared to cells treated with control IgG or left untreated. Furthermore, downregulation of IL-8 protein production with use of IL-8 targeted siRNA also resulted in decreased invasion in matrigel. We next investigated the presence of IL-8 receptors by FACS analysis and found that GBM cells (U87, U251, D54 and LN229) only express CXCR1 but not CXCR2. Treatment of U87 cells with a blocking CXCR1 antibody reduced their invasion through matrigel. Finally, we found that addition of exogenous IL-8, following downregulation of NF-κB which results in loss of endogenous IL-8 production, incompletely restored tumor cell invasion. Our data indicate that IL-8 is necessary but not solely responsible for glioma cell invasion and mediates its effect in an autocrine manner.     

Sensitization of interferon-γ induced apoptosis in human osteosarcoma cells by extracellular S100A4

Background

S100A4 is a small Ca²⁺-binding protein of the S100 family with metastasis-promoting properties. Recently, secreted S100A4 protein has been shown to possess a number of functions, including induction of angiogenesis, stimulation of cell motility and neurite extension.

Methods

Cell cultures from two human osteosarcoma cell lines, OHS and its anti-S100A4 ribozyme transfected counterpart II-11b, was treated with IFN-γ and recombinant S100A4 in order to study the sensitizing effects of extracellular S100A4 on IFN-γ mediated apoptosis. Induction of apoptosis was demonstrated by DNA fragmentation, cleavage of poly (ADP-ribose) polymerase and Lamin B.

Results

In the present work, we found that the S100A4-expressing human osteosarcoma cell line OHS was more sensitive to IFN-γ-mediated apoptosis than the II-11b cells. S100A4 protein was detected in conditioned medium from OHS cells, but not from II-11b cells, and addition of recombinant S100A4 to the cell medium sensitized II-11b cells to apoptosis induced by IFN-γ. The S100A4/IFN-γ-mediated induction of apoptosis was shown to be independent of caspase activation, but dependent on the formation of reactive oxygen species. Furthermore, addition of extracellular S100A4 was demonstrated to activate nuclear factor-κB (NF-κB).

Conclusion

In conclusion, we have shown that S100A4 sensitizes osteosarcoma cells to IFN-γ-mediated induction of apoptosis. Additionally, extracellular S100A4 activates NF-κB, but whether these events are causally related remains unknown.

     

LncRNA PlncRNA-1 overexpression inhibits the growth of breast cancer by upregulating TGF-β1 and downregulating PHGDH

Objective

To investigate the role of lncRNA PlncRNA-1 in the pathogenesis of breast cancer.

Methods

A total of 78 patients with breast cancer as well as 48 healthy females were included in this study. Expression in tumor tissues and adjacent healthy tissues of breast cancer patients, as well as in breast tissues and serum of both patients and healthy control was detected by qRT-PCR. Cell proliferation was detected by CCK-8 assay, and cell apoptosis was tested by MTT assay. PlncRNA-1 overexpression cell lines were constructed and the effects on TGF-β1 as well as phosphoglycerate dehydrogenase (PHGDH) were explored by western blot.

Results

Expression levels of PlncRNA-1 were significantly lower in tumor tissues than those in adjacent healthy tissues. Significantly lower expression levels of PlncRNA-1 were also found in breast cancer patients than those in healthy controls in both breast tissue and serum. Upregulation of PlncRNA-1 promoted the expression of TGF-β1, but inhibited the expression of PHGDH. LncRNA PlncRNA-1 overexpression reduced the proliferation rate, but increased the apoptosis rate of breast cancer cells, while treatment with TGF-β inhibitor reduced those effects of PlncRNA-1 overexpression.

Conclusion

LncRNA PlncRNA-1 overexpression inhibits the growth of breast cancer by upregulating TGF-β1 and downregulating PHGDH.

     

Temozolomide decreases invasion of glioma stem cells by down-regulating TGF-β2

Gliomas are characterized by excessive proliferation, diffuse infiltration and immunosuppression. Recent studies implicate a key role for a restricted population of glioma stem cells (GSCs) in glioma invasive growth and recurrence. Transforming growth factor (TGF)-β2 is a mediator of immunosuppression associated with malignant glioma and also influences pro-invasive functions. Temozolomide (TMZ), is a new alkylating agent with promising antitumour efficacy for malignant gliomas, and the effect of TMZ on GSCs invasion has not been known. To address this issue, we developed studies aimed at neurospheres from primary cultured glioma cells, due to the fact that since neurospheres can be enriched in GSCs, we could examine whether TMZ inhibits the invasion of GSCs. TMZ reduced the TGF-β2-mediated invasion, and down-regulated TGF-β2 expression at the mRNA and protein levels. Thus, these results indicate that TMZ, as a chemotherapeutic agent, can reduce the invasion of GSCs and their immunosuppressive activity. TMZ may be used as an immunomodulating agent for glioma therapy.