Betulinic acid inhibits colon cancer cell and tumor growth and induces proteasome-dependent and -independent downregulation of specificity proteins (Sp) transcription factors

Abstract

Background: Betulinic acid (BA) inhibits growth of several cancer cell lines and tumors and the effects of BA have been attributed to its mitochondriotoxicity and inhibition of multiple pro-oncogenic factors. Previous studies show that BA induces proteasome-dependent degradation of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 in prostate cancer cells and this study focused on the mechanism of action of BA in colon cancer cells.

Methods: The effects of BA on colon cancer cell proliferation and apoptosis and tumor growth in vivo were determined using standardized assays. The effects of BA on Sp proteins and Sp-regulated gene products were analyzed by western blots, and real time PCR was used to determine microRNA-27a (miR-27a) and ZBTB10 mRNA expression.

Results: BA inhibited growth and induced apoptosis in RKO and SW480 colon cancer cells and inhibited tumor growth in athymic nude mice bearing RKO cells as xenograft. BA also decreased expression of Sp1, Sp3 and Sp4 transcription factors which are overexpressed in colon cancer cells and decreased levels of several Sp-regulated genes including survivin, vascular endothelial growth factor, p65 sub-unit of NFκB, epidermal growth factor receptor, cyclin D1, and pituitary tumor transforming gene-1. The mechanism of action of BA was dependent on cell context, since BA induced proteasome-dependent and proteasome-independent downregulation of Sp1, Sp3 and Sp4 in SW480 and RKO cells, respectively. In RKO cells, the mechanism of BA-induced repression of Sp1, Sp3 and Sp4 was due to induction of reactive oxygen species (ROS), ROS-mediated repression of microRNA-27a, and induction of the Sp repressor gene ZBTB10.

Conclusions: These results suggest that the anticancer activity of BA in colon cancer cells is due, in part, to downregulation of Sp1, Sp3 and Sp4 transcription factors; however, the mechanism of this response is cell context-dependent.

Figure 1

BA inhibits growth and induces apoptosis in colon cancer cells. (A) Inhibition of cell proliferation. Cells were treated with BA for 48 or 96 h and then counted as described in the Materials and Methods. Cell cycle progression in RKO (B) and SW480 (C) cells. Cells were treated with DMSO (0), 10 or 15 μM BA for 24 h and analyzed by FACS analysis as described in the Materials and Methods. (D) Induction of PARP cleavage. RKO and SW480 cells were treated with BA for 24 h and whole cell lysates were analyzed for cleaved PARP as described in the Materials and Methods. Results in (A) - (C) are expressed as means ± SE for at least 3 replicate experiments and significant (p < 0.05) differences from controls (0, DMSO) are indicated (*).

Figure 2

BA decreases Sp proteins and Sp-regulated genes. BA decreases Sp proteins in RKO and SW480 cells (A) and Sp-regulated genes (B). Cells were treated with DMSO (0) or BA for the indicated times and whole cell lysates were analyzed by western blots as described in the Materials and Methods. (C) BA and Sp1/Sp3/Sp4 (iSp) knockdown decrease Sp-regulated genes. Cells were either treated with BA or transfected with iSp and whole cell lysates were analyzed by western blots as described in the Materials and Methods. (D) Effects of proteasome inhibitors. Cells were treated with BA ± proteasome inhibitors MG132 or lactacystin for 24 h and whole cell lysates were analyzed by western blots as described in the Materials and Methods.

Figure 3

BA inhibits luciferase activity in RKO cells transfected with GC-rich constructs. Transfection with constructs containing Sp1 (A), Sp3 (B) and Sp-regulated (C) gene promoter constructs. RKO cells were transfected with the indicated constructs, treated with DMSO or BA, and luciferase activity (normalized to β-galactosidase) was determined as described in the Materials and Methods. Results are expressed as means ± SE for at least 3 replicated determinations as significant (p < 0.05) is indicated (*).

Figure 4

BA decreases MMP and induces ROS in RKO cells. (A) Induction of ROS. RKO cells were treated with 15 μM BA, catalase or BA plus catalase for 36 h and ROS production was determined using the fluorescent probe H2DCFDA as described in the Materials and Methods. Role of ROS in BA-induced Sp downregulation (B) and growth inhibition and BA effects on MMP (C). Cells were treated with DMSO (control), BA, catalase or BA plus catalase and Sp proteins (in whole cell lysates), MMP and cell growth were determined as described in the Materials and Methods. Results (C) are expressed as means ± SE for 3 replicate experiments and significant (p < 0.05) growth inhibition by BA (*) and rescue by catalase (**) are indicated. BA-induced inhibition of MMP was not affected by cotreatment with catalase (data not shown).

Figure 5

Role of miR-27a in regulation of BA-mediated responses. (A) Repression of miR-27a and induction of ZBTB10. RKO cells were treated with DMSO or BA and miR-27a and ZBTB10 expression were determined by Northern blot and semi-quantitative RT-PCR, respectively, as described in the Materials and Methods. (B) BA decreases miR-27a promoter activity. RKO cells were transfected with pMiR-27a(-639/+36)-luc, treated with DMSO or BA and luciferase activity determined as described in the Materials and Methods. Role of BA-induced ROS on expression of miR-27a and ZBTB10 (C) and Myt-1 (D). RKO cells were treated with DMSO, BA, catalase or BA plus catalase for 36 h and miR-27a, ZBTB10 and Myt1 mRNA levels were determined by real time PCR as described in the Materials and Methods. Results in (B) - (D) are expressed as means ± SE for at least 3 replicate determinations and significant (p < 0.05) effects by BA (*) and reversal by catalase (**) are indicated.

Figure 6

BA inhibits colon tumor growth in vivo. Inhibition of tumor growth (A) and weights (B). BA (25 mg/kg/d) was administered (orally) to athymic nude mice every second day and tumor volumes and weights were determined as described in the Materials and Methods. (C) Downregulation of Sp1, Sp3 and Sp4. Tumor lysates from individual mice were analyzed by western blots as described in the Materials and Methods and Sp1, Sp3 and Sp4 protein levels were normalized to β-actin. Results in (A) - (C) are means ± SE for at least 8 mice in the control (corn oil) and BA-treated groups and significant (p < 0.05) inhibition by BA is indicated (*).