Background
Colorectal cancer is a leading cause of death in most developed countries including the United States, and in 2010 it is estimated that over 102,700 new cases will be diagnosed and 51,370 deaths will occur in the United States [
1]. Genetic susceptibility accounts for 15 - 25% of colon cancer cases, and genetic markers provide important insights on factors important for the molecular and genetic changes that result in development of this disease [
2]. Familial adenomatous polyposis syndromes [
3,
4], hereditary non-polyposis colorectal cancer [
5‐
8], and other polyposis syndromes which increase the incidence of colorectal cancer including Peutz Jegher's syndrome, familial juvenile polyposis, and hereditary mixed polyposis syndrome, are linked to mutations in LKB1, STK11, SMAD4, PTEN, E-cadherin, cyclin D1, and transforming growth factor β receptors [
2].
The incidence rates of sporadic colon cancer are highly variable among different regions of the world and the changes in incidence of this disease in migrants suggests that environmental factors related to diet contribute to development of colon cancer [
9,
10]. Fruits, nuts and vegetables contain diverse anticarcinogenic phytochemicals; however, epidemiological studies give variable results with respect to their chemopreventive effects and similar variability among studies has been reported for the protective effects of dietary folate [
11‐
14]. Most colon cancer patients present with localized disease which is treated with curative surgery; however, disease relapse is experienced by up to 40% of patients [
15‐
17]. Cytotoxic drugs are primarily used for colon cancer chemotherapy and there is a increasing need to develop mechanism-based drugs for treating this disease.
Specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 are overexpressed in colon and other cancer cell lines [
18‐
23], and Sp1 is a negative prognostic factor for survival of pancreatic and gastric cancer patients [
24,
25]. The potential importance of Sp transcription factors as drug targets is due not only to their overexpression in multiple tumor types but also to their relatively low expression in non-tumor rodent and human tissues, and this is consistent with the reported decrease of Sp1 expression with increasing age [
26‐
28]. RNA interference studies which knockdown Sp1, Sp3 and Sp4 (individually or combined) have identified several Sp-regulated gene-products that are themselves individual targets for new mechanism-based drugs. Sp-regulated genes include several that are important for cancer cell proliferation [cyclin D1, epidermal growth factor receptor (EGFR), hepatocyte growth factor receptor (c-MET)], survival (bcl-2 and survivin), angiogenesis [vascular endothelial growth factor (VEGF) and its receptors (VEGFR1/R2) and pituitary tumor-transforming gene 1 (PTTG-1)], and inflammation (p65 subunit of NFκB) [
23,
29‐
38].
Betulinic acid (BA) is a naturally occurring triterpenoid which inhibits growth of multiple tumors [
39,
40]. Studies in this laboratory show that BA inhibits prostate cancer cell and tumor (xenograft) growth and this is due, in part, to proteasome-dependent downregulation of Sp1, Sp3, Sp4 and several Sp-regulated genes [
20]. In this study, we show that BA inhibits growth of colon cancer cells and tumors and downregulates Sp transcription factors through activation of proteasome-dependent (SW480 cells) and proteasome-independent (RKO cells) pathways.
Methods
Cell proliferation and cell cycle progression assays
The RKO and SW480 colon cancer cell lines were previously characterized at the M.D. Anderson Cancer Center (Houston, TX) and kindly provided by Dr. Stanley Hamilton. RKO and SW480 colon cancer cells (2 × 104 per well) were plated in 12-well plates and allowed to attach for 24 h. The medium was then changed to DMEM/Ham's F-12 medium containing 2.5% charcoal-stripped FBS, and either vehicle [dimethyl sulfoxide (DMSO)] or different concentrations of the compound were added. Fresh medium and compounds were added every 48 h, and cells were then trypsinized and counted after 48 and 96 h using a Coulter Z1 cell counter. Results are expressed as means ± SE for at least 3 replicate determinations for each treatment group. RKO and SW480 cells were treated with either the vehicle (DMSO) or BA for 24 h. Cells were trypsinized, centrifuged and resuspended in staining solution containing 50 μg/ml propidium iodide, 4 mmol/L sodium citrate, and 30 units/ml RNase. After incubation at room temperature for 1 h, cells were analyzed on a FACS Vantage SE DiVa made by Becton Dickinson, using FACSDiva Software V4.1.1. Propidium iodide (PI) fluorescence was collected through a 610SP bandpass filter, and list mode data were acquired on a minimum of 50,000 single cells defined by a dot plot of PI width vs. PI area. Data analysis was performed in BD FACSDiva Software V4.1.1 using PI width vs. PI area to exclude cell aggregates.
Plasmids, transfection assay and antibodies
Sp1 and Sp3 promoter constructs were kindly provided by Drs. Carlos Cuidad and Veronique Noe (University of Barcelona, Barcelona, Spain). The pVEGF-2068 construct contains a VEGF promoter insert (positions -2068 to +54) linked to luciferase reporter gene. The pSurvivin-269 was kindly provided by Dr. M. Zhou (Emory University, Atlanta, GA). The PTTG-1-luc construct containing the -1373 to +3 region of the PTTG-1 promoter was provided by Dr. Kakar (University of Louisville, Louisville, KY). Colon cancer cells (1.5 × 10
5) were seeded in 12-well plates using DMEM:Ham's F-12 media containing 2.5% charcoal stripped serum. After 24 h, cells were transfected with 0.4 μg of reporter gene constructs and 0.04 μg of β-Gal using Lipofectamine 2000 according to manufacturer's protocol. Reporter lysis buffer and luciferase reagent for luciferase studies were supplied by Promega (Madison, WI). Five h after transfection, cells were treated with control or BA for 22-24 h and luciferase activity (normalized to β-galactosidase) was determined using Lumicount luminometer (PerkinElmer Life and Analytical Sciences). For RNA interference assays with iSp, a mixture of oligonucleotides containing siRNAs against Sp1, Sp3 and Sp4 (combined) was used as previously described [
20,
21,
34]. Antibodies for Sp1, Sp3, Sp4, cyclin D1, EGFR, NFκB (p65), VEGF and VEGFR1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). c-PARP and survivin antibodies were purchased from Cell Signaling Technology (Danvers, MA). Monoclonal β-actin antibody was purchased from Sigma-Aldrich. Western blots were determined with whole cell lysates essentially as described [
20‐
23].
Northern blot analysis
For miRNA analysis, 20 μg total RNA per lane was electrophoresed on 15% TBE urea polyacrylaminde gel (Invitrogen), electrophoretically transferred in 0.5 × TBE at 300 mÅ for 45 min to GeneScreen Plus membrane (PerkinElmer, Boston, MA), UV cross-linked and hybridized in ULTRAhyb-Oligo hybridization buffer (Ambion, Austin, TX) at 42°C with 32P end-labeled DNA oligonucleotides complementary to miR-27a. Blots were washed at 42°C in 2X SSC and 0.5% SDS for 30 min with gentle agitation.
Semiquantitative reverse transcription and real time PCR
RKO and SW480 colon cancer cells were treated with BA at different concentrations for 24 h. Total RNA was extracted using RNeasy Mini Kit (Qiagen), and 2 μg of RNA was used to synthesize cDNA using Reverse Transcription System (Promega). Primers were obtained from IDT and used for amplification were as follows: ZBTB10 (sense 5'-GCT GGA TAG TAG TTA TGT TGC-3'; antisense 5'-CTG AGT GGT TTG ATG GAC AGA G-3'). PCR products were electrophoresed on 1% agarose gels containing ethidium bromide and visualized under UV transillumination. Real time PCR for determining miR-27a, ZBTB10 and Myt-1 RNA levels were determined essentially as described [
20‐
23].
Reactive oxygen species (ROS) and mitochondrial membrane potential assays
Cellular ROS levels were evaluated with the cell permeant probe CM-H2DCFDA (5-(and-6)-chloromethyl-2'7'-dichlorodihydrofluorescein diacetate acetyl ester) from Invitrogen. Following 36 h treatment, cells plated on a 6-well cell culture plate were loaded with 10 μM CM-H2DCFDA for 1 h, washed once with serum-free medium, and analyzed for ROS levels using Beckman Coulter XL four-color cytometer. Each experiment was done in triplicate and results are expressed as mean ± S.E. for each treatment group. Mitochondrial membrane potential (MMP) was measured with Mitochondrial Membrane Potential Detection Kit (Stratagene) according to the manufacturer's protocol using JC-1 dye, and mitochondrial membrane potential shift was measured using FACS Calibur flow cytometer using CellQuest acquisition software (BD Biosciences). J-aggregates were detected as red fluorescence and J-monomers are detected as green fluorescence.
Xenograft studies in athymic mice
Female athymic nude mice were purchased from Harlan Laboratories (Indianapolis, IN) and were cared for and used in accordance with institutional guidelines. To produce tumors, RKO cells (5 × 106; ≥ 90% viable) were subcutaneously injected into the flanks of individual mice. Tumors were allowed to grow for 6 days until palpable and mice were then randomized into two groups (6 mice/group) and dosed by oral gavage with corn oil or BA (25 mg/kg/day) every second day for 22 days. The mice were weighed, and tumor size was measured every second day with calipers to permit calculation of tumor volumes: V = LW2/2, where L and W were length and width, respectively. After BA treatment, the animals were sacrificed; final body and tumor weights were determined, and major visceral organs were collected and lysates were used for western blot analysis of Sp proteins.
Discussion
The anticancer activity of BA initially showed high potency against melanoma in cell culture and animal models, and subsequent studies show the effectiveness of this compound against multiple tumor types [
39,
40,
42]. The low
in vivo toxicity of BA coupled with supporting
in vitro and
in vivo results suggest that this compound or some derivative has potential for clinical applications in cancer chemotherapy. However, BA is a highly lipophilic molecule with limited water solubility and this may decrease
in vivo uptake of this compound; therefore, development of specialized formulations/carriers such as liposomes may help to enhance the
in vivo efficacy of BA as an anticancer agent [
43]. Previous studies in this laboratory showed that BA inhibits prostate cancer cell and tumor growth and this is accompanied by proteasome-dependent degradation of Sp1, Sp3 and Sp4 and several Sp-regulated pro-oncogenic gene products [
20]. Several other anticancer agents including tolfenamic acid, curcumin, arsenic trioxide, a nitro-NSAID (GT-094), and two synthetic triterpenoid derivatives, CDDO-Me and CDODA-Me, also induce Sp downregulation in various cancer cell lines via proteasome-dependent and -independent pathways [
19,
21,
33‐
38].
BA inhibits colon cancer cell growth and induces caspase-dependent PARP cleavage in RKO and SW480 colon cancer cells (Figure
1) and these results are consistent with other reports on the effects of BA on colon cancer cell lines [
39,
40,
44‐
46]. Moreover, BA also inhibited tumor growth in athymic nude mice bearing RKO cells as xenografts (Figure
6). We observed that BA decreased expression of Sp1, Sp3 and Sp4 proteins in both RKO and SW480 colon cancer cells and tumors (Figures
2A and
6C) and this was accompanied by parallel decreases in survivin and VEGF (Figures
2A and
2B), and these results are comparable to those observed in LNCaP prostate and KU7 bladder cancer cells treated with BA [
20,
32]. Recent RNA interference studies show that p65 (NFκB subunit), EGFR, cyclin D1, and pituitary tumor transforming gene-1 (PTTG-1) are also Sp-regulated genes [
32‐
35], and results in Figure
3C demonstrate that BA decreased expression of these gene products in RKO and SW480 cells. Moreover, knockdown of Sp1, Sp3 and Sp4 (in combination) in RKO colon cancer cells also decreased expression of EGFR, cyclin D1, p65 and PTTG-1, confirming the role of Sp transcription factors in regulating expression of these genes. These results are consistent with the induction of apoptosis by BA since many of these Sp-regulated genes are important for survival pathways.
Previous studies showed that BA-induced downregulation of Sp1, Sp3 and Sp4 was proteasome-dependent in LNCaP cells but proteasome-independent in KU7 bladder cancer cells [
20,
32]. Similar variability was observed in RKO and SW480 colon cancer cells (Figure
2D) where BA-induced downregulation of Sp proteins was proteasome-independent and -dependent, respectively. This demonstrates that, for BA and possibly other drugs that downregulate Sp1, Sp3, Sp4 and Sp-regulated genes, the pathways required for this response are variable and dependent not only on tumor type but also cell context within the same tumor. At least two of these pathways, namely induction of proteasome- and caspase-dependent degradation of Sp proteins, involve activation of post-transcriptional processes [
20,
21,
37]; however, their mechanisms have not been determined and are currently being investigated in this laboratory.
We have previously reported that the synthetic triterpenoid CDODA-Me and the NO-NSAID GT-094 decrease Sp protein expression in SW480 and RKO colon cancer cells through a transcriptional repression pathway in which miR-27a is decreased and this results in the induction of ZBTB10, a transcriptional repressor [
36,
38]. BA decreased luciferase activity in RKO cells transfected with constructs containing several GC-rich promoter inserts (Figures
3B-D) and also decreased expression of miR-27a and induced expression of ZBTB10 in RKO cells (Figures
5A-C). Since overexpression of ZBTB10 and antisense-miR-27a also decreases expression of Sp1, Sp3, Sp4 and Sp-regulated genes in colon cancer cells [
36], the mechanism of action of BA in RKO cells is linked to disruption of miR-27a:ZBTB10 as previously reported for CDODA-Me and GT-094 in colon cancer cells [
36,
38].
BA is known to be a mitochondriotoxic drug and decreases the mitochondrial membrane potential in several different cancer cell lines leading to induction of apoptosis [
39,
40,
44] and BA also decreased MMP in RKO cells (Figure
4C). Previous studies have demonstrated that at least four agents that are mitochondriotoxic and induce ROS also downregulate Sp proteins; this effect is ROS-dependent and reversible with antioxidants or catalase, and compounds activating this pathway include arsenic trioxide (bladder), curcumin and CDDO-Me (pancreatic), and GT-094 (colon) [
33,
37,
38]. Moreover, for GT-094 and CDDO-Me, the mechanism of ROS-dependent downregulation of Sp1, Sp3, and Sp4 involves disruption of miR-27a:ZBTB10 [
33,
38]. Results of this study show that BA also induced ROS-downregulated Sp1, Sp3, Sp4 and miR-27a and induced ZBTB10 in RKO cells, and all of these responses were significantly attenuated in cells cotreated with BA plus catalase (Figure
4). Moreover, catalase also reversed the growth inhibitory effects of BA (Figure
4C), further demonstrating the importance of ROS activation for the anticancer activity of this compound in RKO cells. In contrast to previous studies showing that CDODA-Me and GT-094 activated transcriptional repression of Sp proteins in both RKO and SW480 cells [
33,
36], BA induced transcriptional repression in RKO cells but activated the proteasome pathway for degradation of Sp proteins in SW480 cells. The mitochondrial or extra-mitochondrial origins of ROS in cancer cells treated with BA and other agents that downregulate Sp transcription factors is currently being investigated.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
SC carried out and supervised the in vitro studies on BA-induced downregulation of Sp proteins and Sp-regulated genes and also the RNA interference studies. SP carried out the in vitro studies on downregulation of Sp1, Sp3 and Sp4 and Sp-regulated genes. PL carried out some of the in vitro experiments including the studies on miR-27a:ZBTB10. SP carried out the in vivo study and analyzed the tumor tissue. SS carried out the experimental design and drafted the manuscript. All authors have read and approved the final manuscript.