Abstract
BackgroundTransforming growth factor β induced (TGFBI) product, an extracellular matrix (ECM) protein, has been implicated as a putative tumor suppressor in recent studies. Our previous findings revealed that expression of TGFBI gene is down-regulated in a variety of cancer cell lines and clinical tissue samples. In this study, ectopic expression of TGFBI was used to ascertain its role as a tumor suppressor and to determine the underlying mechanism of mesothelioma and breast cancer.MethodsCells were stably transfected with pRc/CMV2-TGFBI and pRc/CMV2-empty vector with Lipofectamine Plus. Ectopic expression of TGFBI was quantified by using quantitative PCR and Western-blotting. Characterization of cell viability was assessed using growth curve, clonogenic survival and soft agar growth. The potential of tumor formation was evaluated by an in vivo mouse model. Cell cycle was analyzed via flow cytometry. Expressions of p21, p53, p16 and p14 were examined using Western-blotting. Senescent cells were sorted by using a Senescence β-Galactosidase Staining Kit. Telomerase activity was measured using quantitative telomerase detection kit.ResultsIn this study, an ectopic expression of TGFBI in two types of cancer cell lines, a mesothelioma cell line NCI-H28 and a breast cancer cell line MDA-MB-231 was found to have reduced the cellular growth, plating efficiency, and anchorage-independent growth. The tumorigenicity of these cancer cell lines as determined by subcutaneous inoculation in nude mice was similarly suppressed by TGFBI expression. Likewise, TGFBI expression reduced the proportion of S-phase while increased the proportion of G1 phase in these cells. The redistribution of cell cycle phase after re-expression of TGFBI was correspondent with transiently elevated expression of p21 and p53. The activities of senescence-associated β-galactosidase and telomerase were enhanced in TGFBI-transfected cells.ConclusionCollectively, these results imply that TGFBI plays a suppressive role in the development of mesothelioma and breast cancer cells, possibly through inhibitions of cell proliferation, delaying of G1-S phase transition, and induction of senescence.
Highlights
Transforming growth factor β induced (TGFBI) product, an extracellular matrix (ECM) protein, has been implicated as a putative tumor suppressor in recent studies
Effects of TGFBI on tumor cell growth in vitro Engineered mesothelioma cell clones (T2804, T2806, and T2807) and breast cancer cell clones (T23108, T23109, and T23113) ectopically expressing TGFBI were generated from their respective parental tumor cell lines, Figure 1 Expression of TGFBI mRNA and protein in TGFBI-transfected, empty vector control, and parental tumor cells. (A) Cells were transfected with pRc/CMV2-TGFBI and pRc/CMV2-empty vector
The results demonstrated that the reintroduction of TGFBI into NCI-H28 and MDA-MB-231 cells dramatically slowed cell growth and prolonged population doubling time 4.38 and 1.16 times (Figure 2A and 2B), respectively
Summary
Transforming growth factor β induced (TGFBI) product, an extracellular matrix (ECM) protein, has been implicated as a putative tumor suppressor in recent studies. The mechanism of how these two types of malignancy undergo malignant transformation remains largely unknown, evidence indicate a multistep process involving both activation of oncogenes and inactivation of tumor suppressor genes exists [21,22] The observation that many latestage tumors are highly resistant to traditional chemotherapy and radiation therapy, highlights the need for innovative therapies based on mechanistic insight of the cancer process. In this regard, the potential role of TGFBI as a tumor suppressor may provide a novel target for manipulation and therapeutic purposes
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