Connective Tissue Growth Factor Induces Apoptosis in Human Breast Cancer Cell Line MCF-7

Abstract

Connective tissue growth factor (CTGF) is a member of an emerging CCN gene family that is implicated in various diseases associated with fibro-proliferative disorder including scleroderma and atherosclerosis. The function of CTGF in human cancer is largely unknown. We now show that CTGF induces apoptosis in the human breast cancer cell line MCF-7. CTGF mRNA was completely absent in MCF-7 but strongly induced by treatment with transforming growth factor ␤ (TGF-␤). TGF-␤ by itself induced apoptosis in MCF-7, and this effect was reversed by co-treatment with CTGF antisense oligonucleotide. Overexpression of CTGF gene in transiently transfected MCF-7 cells significantly augmented apoptosis. Moreover, recombinant CTGF protein significantly enhanced apoptosis in MCF-7 cells as evaluated by DNA fragmentation, Tdt-mediated dUTP biotin nick end-labeling staining, flow cytometry analysis, and nuclear staining using Hoechst 33258. Finally, recombinant CTGF showed no effect on Bax protein expression but significantly reduced Bcl2 protein expression. Taken together, these results suggest that CTGF is a major inducer of apoptosis in the human breast cancer cell line MCF-7 and that TGF-␤-induced apoptosis in MCF-7 cells is mediated, in part, by CTGF. Connective tissue growth factor (CTGF)1 is a member of an emerging gene family known as the CCN family (1), which includes CTGF (also known as fisp-12), CYR61(cysteine-rich 61/CEF10), Nov (nephroblastoma overexpressed) and the newly discovered WISP1/elm1, WISP2/rCop1, and WISP3 (2-5). CTGF was originally identified in conditioned medium of human umbilical vein endothelial cells (6) and plays a role in various human diseases including systemic scleroderma (7), atherosclerosis (8), renal diseases (9), hepatic fibrosis in biliary atresia (10), and malignant melanoma (11). A common finding in most of these diseases was a high level expression of CTGF in fibro-proliferative areas of affected tissues. In most cases, a direct correlation between high level CTGF expression and high levels of transforming growth factor-␤ (TGF-␤) expression could be established. In fact, CTGF gene expression has been shown to be regulated by TGF-␤ (2).TGF-␤ is a pleiotropic growth modulator with a wide variety of activities on different cell types. TGF-␤ stimulates proliferation of mesenchymal cells but inhibits that of endothelial (12) and epithelial cells (13). TGF-␤ is also known to inhibit the proliferation of a variety of cancer cell lines including the human breast cancer cell line 15). In fact, the anticancer effect of tamoxifen has been attributed to indirect activation of TGF-␤ pathway and induction of apoptosis by TGF-␤ (16). Because CTGF is thought to be a mediator of TGF-␤ action, it is conceivable that CTGF may also inhibit cancer cell proliferation by inducing apoptosis in these cells. In the present study, we utilized antisense technology, transient overexpression techniques, and recombinant CTGF protein to gain insight into the biological function of CTGF in the MCF-7 breast cancer cell line...

Identification of an RNA element that confers post-transcriptional repression of connective tissue growth factor/hypertrophic chondrocyte specific 24 (ctgf/hcs24) gene: Similarities to retroviral RNA-protein interactions

“…In addition, the fact that high levels of CTGF expression is limited to particular types of cells, such as hypertrophic chondrocytes, suggests that expression of the ctgf gene may be under repressed conditions generally in vivo. Otherwise, even apoptosis might be induced by over-expressed CTGF, as reported in a particular cell line (Hishikawa et al, 1999). In concordance with these data, we had previously found that the 3'-untranslated region (3'-UTR) of the CTGF gene exerts repressive e ects on gene expression in cis (Kubota et al, 1999b).…”

Suppression of Cell Proliferation and Signaling Transduction by Connective Tissue Growth Factor in Non–Small Cell Lung Cancer Cells

“…In addition, breast cancer patients with a poor prognosis and mortality had significantly lower CTFG levels compared with their normal breast tissues (13). Forced expression of CTGF in MCF-7 breast cancer cells increased cell apoptosis (15). In addition, patients with lung adenocarcinoma with low CTGF expression more often had an advanced stage and a poorer prognosis (18).…”

“…4A, right). MCF-7 cells were used as control because recombinant CTGF protein has been shown to cause growth inhibition in MCF-7 cells (15). Heparin affinity chromatography has been used in CTGF purification (21).…”

“…Similarly, the metastatic breast cancer cell line, MDA-MB-231, expresses lower levels of CTGF than the less invasive MCF-7 cells (13). Forced expression of CTGF in MCF-7 cells induces apoptosis (15). In the progression of colorectal cancer, patients with high CTGF expression in their tumors have higher survival rates compared with patients with low CTGF expression in their tumors (16).…”

Apoptotic mechanisms in T47D and MCF-7 human breast cancer cells

“…Therefore, we suggest that in MCF-7 cells another mechanism exists, which is a caspase-3 independent pathway causing the DNA fragmentation observed in this study. Many studies have utilised MCF-7 cells to study apoptosis and have demonstrated DNA fragmentation in response to many stimuli (Saunders et al, 1997;Hishikawa et al, 1999;Tudor et al, 2000). For example, nitric oxide induced apoptosis in MCF-7 cells with a lack of DEVDase activity substantiating the suggestion for an alternative mechanism in MCF-7 and possibly other cells that do not express caspase-3 (Umansky et al, 2000).…”