Overexpression of caudal-related homeobox transcription factor 2 inhibits the growth of transplanted colorectal tumors in nude mice

Abstract

Caudal‑related homeobox transcription factor 2 (CDX2) is a transcription factor, which is specifically expressed in the adult intestine. It is essential for the development and homeostasis of the intestinal epithelium and its functions as a tumor suppressor have been demonstrated in the adult colon. The present study aimed to examine the inhibitory effects of the overexpression of CDX2 on subcutaneously‑transplanted tumors, derived from LoVo colon cancer cells, in nude mice, and to provide experimental evidence for the biotherapy of colon cancer. A pEGFP‑C1‑CDX2 eukaryotic expression vector was transfected into the LoVo cells via lipofection, and LoVo cells stably‑expressing CDX2 (pEGFP‑C1‑CDX2 cells) were obtained using G418 selection. A nude mouse subcutaneously‑transplanted tumor model was established by inoculating the nude mice with the pEGFP‑C1‑CDX2 cells, and the effects of overexpression of CDX2 on transplanted tumor growth in the LoVo cells were observed. Western blotting results demonstrated that the protein expression of CDX2 in the LoVo cells was higher in the pEGFP‑C1‑CDX2 cell group, compared with that in the pEGFP‑C1 cell group and the untreated cell group. At 20 days post‑inoculation with either pEGFP‑C1‑CDX2 or pEGFP‑C1, the transplanted tumor masses were significantly lower in the pEGFP‑C1‑CDX2 group, compared with those in the pEGFP‑C1 and untreated groups. Immunohistochemistry revealed that the expression levels of CDX2 and matrix metalloproteinase‑2 (MMP‑2) were detected in each group, and the protein expression of CDX2 was increased in the tumor tissues from the nude mice in the pEGFP‑C1‑CDX2 group. However the expression of MMP‑2 was downregulated in the tumor tissues of the nude mice in the pEGFP‑C1‑CDX2 group. Taken together, these data suggested that pEGFP‑C1‑CDX2 cells exhibited suppressed tumor growth in vivo. Overexpression of CDX2 was observed in transplanted tumors in the pEGFP‑C1‑CDX2 group, and the gene expression of MMP‑2 was reduced. These results indicate that CDX2 inhibited the growth of colorectal tumor cells, possibly by downregulating the gene expression.

Figure 1

Identification of the pEGFP-C1-CDX2 eukaryotic expression vector and overexpression of CDX2 in the LoVo cells. (A) pEGFP-C1-CDX2 was identified using a double enzyme digestion assay with HindII/BamHI. The DNA fragments of 1,055 bp (CDX2) and 4.7 kb (pEGFP-C1) were amplified. (B) Monoclones of the pEGFP-C1-CDX2 cells were visualized using light microscopy (left) and fluorescence microscopy (right). Magnification, ×100. (C) Protein expression of CDX2 was detected using western blotting. The expression of β-actin was used as internal control. M, marker; 1, double enzyme digestion; CDX2, caudal-related homeobox transcription factor 2.

Figure 2

Overexpression of the CDX2 gene inhibits tumour growth of LoVo cells in vivo. (A) Representative images of the tumors isolated from three mice 3 weeks after inoculation with either untreated cells, pEGFP-C1 cells or pEGFP-C1-CDX2 cells (n=3/group). (B) Overexpression of CDX2 inhibited tumour growth of the LoVo cells in vivo (n=6/group). Data are expressed as the mean ± standard error of the mean. ^*P<0.05 and ^**P<0.01 vs. untreated and pEGFP-C1 cell groups. CDX2, caudal-related homeobox transcription factor 2.

Figure 3

Immunohistochemical analysis of MMP-2 and CDX2 proteins in LoVo xenograft tumors. The sections were immunostained with 3,3′-diaminobenzidine and counterstained with hematoxylin for the expression of MMP-2 and CDX2. Representative samples are shown (A–C) magnification, ×400; (D–F) magnification, ×200. It was observed that MMP-2 was expressed in the cytoplasm in LoVo xenograft tumors, and that CDX2 was expressed in the nucleus. MMP2, matrix metalloproteinase-2; CDX2, caudal-related homeobox transcription factor 2.