pubmed.ncbi.nlm.nih.gov

Antitumor effect of retinoic acid receptor-beta2 associated with suppression of cyclooxygenase-2 - PubMed

Antitumor effect of retinoic acid receptor-beta2 associated with suppression of cyclooxygenase-2

Shumei Song et al. Cancer Prev Res (Phila). 2009 Mar.

Abstract

Retinoic acid receptor-beta2 (RAR-beta2) is a putative tumor suppressor gene in various cancers. To determine the underlying molecular mechanisms, we transfected RAR-beta2 cDNA into esophageal cancer TE-1 and TE-8 cells and found that RAR-beta2 suppressed tumor cell growth in vitro and tumor formation in nude mice in TE-8 cells, whereas the stable transfection of RAR-beta2 did not restore retinoid sensitivity or inhibit tumor formation in nude mouse in TE-1 cells. Molecularly, we revealed that RAR-beta2 antitumor activity was associated with expression and suppression of cyclooxygenase-2 (COX-2) in these tumor cell lines. Moreover, antisense RAR-beta2 cDNA induced COX-2 expression in TE-3 cells. Furthermore, when COX-2 expression is first blocked by using antisense COX-2 expression vector, the effect of RAR-beta2 is diminished in these tumor cells. In addition, we analyzed expression of RAR-beta2 and COX-2 mRNA in tissue specimens and found that RAR-beta2 expression is associated with low levels of COX-2 expression in esophageal cancer tissues. Induction of RAR-beta2 expression in oral leukoplakia tissues after the patients treated with 13-cis RA correlated with a reduction in COX-2 expression and clinical response. Our findings indicate that some of RAR-beta2 antitumor activities are mediated by suppression of COX-2 expression in some of these esophageal cancer cells. After correlating antitumor effect of RAR-beta2 with COX-2 expression in the published studies, we also found the association. Thus, further studies will determine whether manipulation of COX-2 expression in different cancers can antagonize RAR-beta2 activity.

PubMed Disclaimer

Conflict of interest statement

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Figures

Fig. 1
Fig. 1

Characterization of RAR-β2–transfected esophageal cancer cells. TE-1V1 is the vector only–transfected subline and TE-1S4 and TE-1S16 are RAR-β2–transfected sublines. A, Western blotting analysis of RAR-β2 expression in RAR-β2–transfected TE-1 cells. PC, positive control; NS, nonspecific band. B, cell viability assay. The cells were treated with or without ATRA for 5 d, and cell viability was measured by a sulforhodamine B assay and then summarized as percentage of control (see Materials and Methods for details). C, colony formation assay. The cells were grown in soft agar and treated with or without ATRA for 21 d, after which colonies were counted and summarized as the mean ± SD. These experiments were repeated at least once.

Fig. 2
Fig. 2

Nude mouse xenograft assay. A, growth curve of tumor masses in nude mice injected with RAR-β2–transfected TE-1S4 and TE-1S16 esophageal cancer cells or with TE-1V1 vector control cells. B, growth curve of tumor masses in nude mice injected with RAR-β2–transfected TE-8S20 and TE-8S22 esophageal cancer cells or with TE-8V1 vector only–transfected cells. The results showed that the role of RAR-β2 in suppressing tumor formation is dependent on COX-2 expression.

Fig. 3
Fig. 3

Western blot analysis of gene expression in RAR-β2–transfected esophageal cancer cells. TE-1V1, TE-8V1, and TE-3V1 are stable vector only–transfected sublines, whereas TE-1S4, TE-1S16, TE-8S20, and TE-8S22 are RAR-β2–transfected sublines and TE-3A3 and TE-3A5 are antisense RAR-β2–transfected sublines of TE-1, TE-3, and TE-8, respectively. The cells were maintained in 200 µg/mL G418, and total cellular protein was extracted and subjected to Western blot analysis of COX-2 expression.

Fig. 4
Fig. 4

Reduced Ki67 positivity by transient transfection of RAR-β2 and association of COX-2 expression. A, cell proliferation assay. Esophageal cancer cell lines TE-1, TE-8, HCE-4, and SKGT-4 were transiently transfected with either pCMS/EGFP plus pRC/CMV vector or pCMS/EGFP plus pRC/CMV/ RAR-β2 for 2 d. The cells were then treated with 400 µg/mL G418 for additional 24 h and stained for Ki67 immunohistochemically. After that, more than 200 cells in 10 fields of 20× objective were counted for positive staining of GFP (green) as well as for positive or negative Ki67 (red) staining in these cells. The percentage of control of cell proliferation was calculated from the following equation: % control = NT/NV × 100, where NT and NV are the numbers of Ki67-positive cells in GFP-positive cells of RAR-β2–transfected and vector control cultures, respectively. B, Western blot analysis of COX-2 expression. The cells were grown in monolayer for 5 d and total cellular protein was extracted and subjected to Western blot analysis.

Fig. 5
Fig. 5

Antisense COX-2–antagonized RAR-β2 effects in esophageal cancer cell lines. Esophageal cancer cell lines TE-1, TE-8, HCE-4, and SKGT-4 were first transfected with pCMS/EGFP vector plus pcDNA3.1 vector or pCMS/EGFP plus pcDNA3.1/COX2-AS. Two days later, GFP was expressed in these cells. We then transfected RAR-β2 expression vector into these tumor cells for 48 h. After that, the cells were stained for Ki67 immunohistochemically. More than 200 cells in 10 fields of 20× objective were counted for positive staining of GFP (green) as well as for positive or negative Ki67 (red) staining in these cells. The percentage of control of cell proliferation was calculated from the following equation: %control = NT/NV × 100, where NT and NV are the numbers of Ki67-positive cells in GFP-positive cells of COX-2 antisense–transfected or RAR-β2–transfected and control cultures, respectively.

Fig. 6
Fig. 6

In situ detection of RAR-β2 and COX-2 mRNA expression. Paraffinembedded sections were from patients with oral premalignant lesions treated with 13-cis RA for 3 mo. Biopsy specimens were collected both before and after treatment and processed for in situ hybridization analysis of RAR-β2 and COX-2 mRNA expression by using digoxigenin-labeled probes.

Fig. 7
Fig. 7

Cell viability assay. Stable RAR-β2–transfected TE-1 and TE-8 sublines were grown in monolayer and treated with ATRA (1 µmol/L), COX-2 inhibitor NS398 (12.5 µmol/L), or their combination for 5 d. Cell viability assay was then done (see Materials and Methods). The experiments were repeated thrice with similar results.

Similar articles

Cited by

References

    1. Soprano DR, Qin P, Soprano KJ. Retinoic acid receptors and cancers. Annu Rev Nutr. 2004;24:201–221. - PubMed
    1. Xu X-C. Tumor-suppressive activity of retinoic acid receptor-β in cancer. Cancer Lett. 2007;253:14–24. - PMC - PubMed
    1. Cheung B, Hocker JE, Smith SA, Norris MD, Haber M, Marshall GM. Favorable prognostic significance of high-level retinoic acid receptor β expression in neuroblastoma mediated by effects on cell cycle regulation. Oncogene. 1998;17:751–759. - PubMed
    1. Wang XD, Liu C, Bronson RT, Smith DE, Krinsky NI, Russell M. Retinoid signaling and activator protein-1 expression in ferrets given β-carotene supplements and exposed to tobacco smoke. J Natl Cancer Inst. 1999;91:60–66. - PubMed
    1. Vuillemenot BR, Pulling LC, Palmisano WA, Hutt JA, Belinsky SA. Carcinogen exposure differentially modulates RAR-β promoter hypermethylation, an early and frequent event in mouse lung carcinogenesis. Carcinogenesis. 2004;25:623–629. - PubMed

MeSH terms

Substances