Antisense Tcf inhibits the neoplastic growth of liver cancer cells

Abstract

Purpose: T cell transcription factors are nuclear effectors of the Wnt signaling transduction pathway and play crucial roles in embryonic and malignant development. Our previous study showed increased expression level of Tcf mRNA in liver cancer. In the present paper, antisense Tcf RNA was used to explore the possible therapeutic effect on liver cancer cells by interrupting the abnormal Wnt pathway.

Methods: Antisense expression vectors containing the conserved sequence of Tcf cDNA were constructed and transfected into a human liver cancer cell line SMMC-7721. Tumorigenic potential was determined by cellular growth assay and tumor growth in nude mice.

Results: The stable transfection of anti-sense Tcf in SMMC-7721 cells significantly reduced Tcf expression at both mRNA and protein levels compared with parental and mock-transfected cells. Antisense-mediated suppression of Tcf inhibited the in vitro proliferation and in vivo tumor formation ability. Furthermore, the apoptosis rate of antisense transfected cells was significantly higher than that of control, indicating that antisense RNA suppressed malignant growth by induction of apoptosis.

Conclusion: Our studies demonstrate the critical role of Wnt signaling pathway in the neoplastic growth of liver cancer cells and suggest that inhibition of Tcf activity with antisense Tcf RNA may be a potential new gene therapy method for liver cancer.

Fig. 1A, B

The expression of Tcf mRNA and protein is decreased in Tcf antisense RNA-expressing clone. A RT-PCR analysis showing the expression of Tcf mRNA in parental SMMC-7721 (lane A1), mock-7721 (lane A2), and pTas-7721 (lane A3). Expression of the housekeeping gene β-actin was used as internal control. Molecular weight marker (lanes Am) was electrophoresed in parallel. Lower expression level of Tcf mRNA was observed in pTas-7721 cells. There were no changes in the level of SMMC-7721 and mock-7721 transcripts. Expression levels shown are the representative results of four independent experiments with at least three different cell clones; B Western Blot showing expression of Tcf protein in SMMC-7721(lane B1), mock-7721(lane B2), and pTas-7721(lane B3). Fifty-microgram protein extracts were loaded in each lane, and the blot was probed with the human anti-Tcf-4 antibody. Antisense RNA transfection suppressed the expression of Tcf protein

Fig. 2

Proliferation assay. 5×10³ cells were seeded in 96-well culture plate. At the indicated time point, the numbers of viable cells were assessed by the MTT conversion. The mean values from three separate experiments are shown

Fig. 3A–C

Suppression of tumorigenesis of antisense-transfected cells in vivo. 1×10⁷ cells were injected s.c. in the right flank of nude mice. Tumor volumes were estimated once a week for up to 8 weeks with calipers, and data are shown as the mean of each group (n=8). A mice were injected with pTas-7721(left), SMMC-7721(middle), and mock-7721(right). Photographs of the animals bearing tumors were taken on day 28; B tumors were excised from nude mice on day 56 after injection. The pTas-7721 cells(bottom) generate small tumors as compared with the tumors derived from SMMC-7721(middle) and mock-7721 cells(top); C growth curves of tumors in nude mice

Fig. 4

Antisense Tcf RNA induce apoptosis. After control cells (SMMC-7721 or mock-7721) or antisense-transfected cells (pTas-7721) were incubated in RPMI1640 with 10% FCS for 72 h, 5×10⁵ cells were collected, washed twice, counterstained with PI, and annexin V conjugated with FITC, and then analyzed using FACS. Annexin V-FITC−/PI− (LL) represent live cells, Annexin V-FITC+/PI−(LR) represent apoptotic cells, Annexin V-FITC+/PI+(UR) represent necrotic cells, and Annexin V-FITC−/PI+(UL) represent cell debris