Purpose: 1,1-Bis(3′-indolyl)-1-(p-substitutedphenyl)methanes [methylene-substituted diindolylmethanes (C-DIM)] containing p-trifluoromethyl, p-t-butyl, and p-phenyl substituents activate peroxisome proliferator-activated receptor γ (PPARγ) and inhibit growth of several different cancer cell lines through receptor-dependent and receptor-independent pathways. The purpose of this study is to investigate the anticancer activity of these compounds in renal cell carcinoma.
Experimental Design: The anticancer activity of the p-t-butyl–substituted C-DIM compound (DIM-C-pPhtBu) was investigated in ACHN and 786-0 renal cell carcinoma cell lines and in an orthotopic model for renal carcinogenesis using ACHN cells injected directly into the kidney.
Results: PPARγ is overexpressed in ACHN cells and barely detectable in 786-0 cells, and treatment with DIM-C-pPhtBu induces proteasome-dependent degradation of cyclin D1 and variable effects on p21 and p27 expression in both cell lines. DIM-C-pPhtBu also induced several common proapoptotic responses in ACHN and 786-0 cells, including increased expression of nonsteroidal anti-inflammatory drug-activated gene-1 and endoplasmic reticulum stress, which activates death receptor 5 and the extrinsic pathway of apoptosis. Activation of these responses was PPARγ independent. In addition, DIM-C-pPhtBu (40 mg/kg/d) also inhibited tumor growth in an orthotopic mouse model for renal carcinogenesis, and this was accompanied by induction of apoptosis in renal tumors treated with DIM-C-pPhtBu but not in tumors treated with the corn oil vehicle (control).
Conclusions: DIM-C-pPhtBu and related compounds are cytotoxic to renal cancer cells and activate multiple proapoptotic and growth-inhibitory pathways. The results coupled with in vivo anticancer activity show the potential of DIM-C-pPhtBu and related C-DIMs for clinical treatment of renal adenocarcinoma.
Peroxisome proliferator-activated receptor γ (PPARγ) is a member of the nuclear receptor superfamily of ligand-activated transcription factors (1, 2). PPARγ also plays a role in metabolic diseases, atherosclerosis, and cancer (3–9). PPARγ agonists, such as the thiazolidinediones rosiglitazone and pioglitazone, have been developed for treatment of type II diabetes due to their activity as insulin-sensitizing agents (10, 11). PPARγ is highly expressed in many tumor samples and cancer cell lines derived from hematopoietic and nonhematopoietic tumors (12), and PPARγ agonists have been extensively investigated as potential antitumor drugs (13–23). Thiazolidinediones, such as rosiglitazone, troglitazone, and 15-deoxy-Δ12,14prostaglandin J2 (PGJ2), typically inhibit cancer cell growth. This is accompanied by inhibition of G0-G1 to S phase progression, down-regulation of cyclin D1, and induction of p21 and/or p27. In addition, these compounds and other classes of PPARγ agonists, such as 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid and related triterpenoids and 1,1-bis(3′-indolyl)-1-(p-substitutedphenyl)methanes [methylene-substituted diindolylmethanes (C-DIM)], also induce apoptotic pathways in cancer cell lines and inhibit tumor growth in in vivo models (17, 24–38). However, most of the growth-inhibitory responses induced by these compounds are receptor independent.
Research in this laboratory has focused on PPARγ-active C-DIM compounds containing p-substituted trifluoromethyl (DIM-C-pPhCF3), t-butyl (DIM-C-pPhtBu), and phenyl (DIM-C-pPhC6H5) groups (28–37). These compounds induce PPARγ-dependent p21 and caveolin-1 expression in pancreatic and colon cancer cell lines, respectively. However, many of the induced responses are PPARγ independent. For example, C-DIMs induce endoplasmic reticulum (ER) stress in pancreatic and ovarian cancer cells, leading to constitutive activation of death receptor 5 (DR5) and caspase-8–mediated apoptosis (34, 36). In addition, C-DIMs induce proteasome-dependent down-regulation of cyclin D1 and increased expression of nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1) and activating transcription factor 3 (ATF3; ref. 33). Induction of NAG-1 by C-DIMs was associated with kinase-dependent induction of early growth response-1 gene.
It has previously been reported that PGJ2 and thiazolidinediones induce cell cycle arrest and apoptosis in human renal cell carcinoma (RCC) cell lines (38–40), and this study investigates the effects of C-DIMs in ACHN and 786-0 RCC cells. The results show that these compounds inhibit growth of both cell lines with IC50 values ≤1 to 5 μmol/L. C-DIMs activate a PPARγ-GAL4 chimera in both cell lines, although PPARγ is expressed in ACHN cells and only barely detectable in 786-0 cells. C-DIMs induced differential expression of cyclin-dependent kinase inhibitors p21 and p27 in ACHN (high) and 786-0 (low to nondetectable) cells but induced a similar pattern of other growth-inhibitory and proapoptotic responses in both cell lines. For example, DIM-C-pPhtBu induced NAG-1, ATF3, and ER stress responses, including activation of DR5 and the extrinsic apoptosis pathway in ACHN and 786-0 cells. Moreover, in an orthotopic model of renal adenocarcinoma, DIM-C-pPhtBu inhibited tumor growth and induced extensive apoptosis in the tumors.
Materials and Methods
Cell culture. Human renal clear cell carcinoma cell lines ACHN and 786-0 were obtained from the American Type Culture Collection. ACHN cells were maintained in MEM (Sigma) supplemented with 10% fetal bovine serum and 10 mL/L of 100× antibiotic/antimycotic solution (Sigma-Aldrich). 786-0 cells were maintained in RPMI 1640 (Sigma) supplemented with 10% fetal bovine serum and 10 mL/L of 100× antibiotic/antimycotic solution. Cells were maintained at 37°C in the presence of 5% CO2.
Reagents. Antibodies for DR5, cleaved poly(ADP-ribose) polymerase (PARP), cleaved caspase-3, and caspase-8 were purchased from Cell Signaling. Antibodies CHOP, GRP78, PPARγ, cyclin D1, p27, caveolin, and β-tubulin were purchased from Santa Cruz Biotechnology, and NAG-1 antibody was obtained from Upstate. p21 antibody was obtained from BD PharMingen, and monoclonal β-actin was obtained from Sigma-Aldrich. Reporter lysis buffer and luciferase reagent for luciferase studies were supplied by Promega. β-Galactosidase (β-Gal) reagent was obtained from Tropix, and Lipofectamine 2000 reagent was purchased from Invitrogen. Western Lightning Chemiluminescence reagent was from Perkin-Elmer Life and Analytical Sciences. MG132 was obtained from Sigma. Z-VAD-FMK and Z-IETD-FMK were obtained from BD Biosciences, and the C-substituted DIMs (C-DIMs) and T007 were prepared in this laboratory as described previously (28–30).
Plasmids. The GAL4 reporter containing 5× GAL4 response elements (pGAL4) was kindly provided by Dr. Marty Mayo (University of North Carolina, Chapel Hill, NC). GAL4DBD-PPARγ construct (gPPARγ) was a gift from Jennifer L. Oberfield (GlaxoSmithKline Research and Development, Research Triangle Park, NC). The GRP78 promoter-luciferase construct contains 374 bp from the promoter and was provided by Dr. K. Park (Center for Molecular Medicine, Sungkyunkwan University, Seoul, Korea). Human CHOP promoter constructs were provided by Dr. Pierre Fafournoux (Saint Genes, Champarelle, France).
Cell proliferation and fluorescence-activated cell sorting assays. ACHN (3 × 104 cells/mL) and 786-0 (1.5 × 104 cells/mL) were plated in 12-well plates. After cell attachment for 24 h, the medium was changed to DMEM/Ham's F12 containing 2.5% charcoal-stripped fetal bovine serum and either vehicle (DMSO) or the indicated compound. Fresh medium and compound were added every 48 h, and representative samples were trypsinized and counted using the Coulter Z1 cell counter (Beckman Coulter). Each experiment was done in triplicate and results are expressed as mean ± SE for each determination. Cells were analyzed on a FACSVantage SE DiVa (Becton Dickinson) using BD FACSDiva software version 4.1.1. Propidium iodide 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 propidium iodide width versus propidium iodide area. Data analysis was done in BD FACSDiva software version 4.1.1 using propidium iodide width versus propidium iodide area to exclude cell aggregates.
Western blot analysis. Cell lysates were prepared using lysis buffer [50 mmol/L HEPES, 0.5 mol/L NaCl, 1.5 mmol/L MgCl2, 1 mmol/L EGTA, 10% (v/v) glycerol, 1% (v/v) Triton X-100, 5 μL/mL protease inhibitor cocktail (Sigma Aldrich)]. The lysates were incubated on ice for 1 h with intermittent vortexing followed by centrifugation at 20,000 × g for 10 min at 4°C. Protein samples (60-100 μg) were size separated by electrophoresis on SDS-polyacrylamide gels under nonreducing conditions. Separated proteins were electroblotted onto polyvinylidene membranes (polyvinylidene diflouride; Bio-Rad). The blot was blocked by incubating in blocking buffer [5% skim milk, 10 mmol/L Tris-HCl, 150 mmol/L NaCl (pH 8), 0.1% Tween 20] for 1 h at 20°C and then incubated with the primary antibody overnight at 4°C. Incubation with a horseradish peroxidase–conjugated anti-mouse or rabbit secondary antibody was then carried out at 37°C for 1 h, and antibody-bound proteins were detected by the enhanced chemiluminescence Western blotting analysis system.
Transfection and luciferase assay. Cells were cultured in 12-well plates (DMEM/Ham's F12 containing 2.5% charcoal-stripped fetal bovine serum). After 16 to 20 h, when cells were 50% to 60% confluent, reporter gene constructs were transfected using Lipofectamine 2000 (Invitrogen). The effects of different treatments on transactivation were investigated on ACHN and 786-0 cells. Cells were transfected with either 500 ng of pGRP78, pCHOP, GAL4Luc, and/or PPARγ-GAL4 constructs in the presence of 40 ng β-Gal according to the manufacturer's protocol. Five hours after transfection, the transfection mix was replaced with complete medium containing either vehicle (DMSO) or the indicated ligand for 24 h. Cells were then lysed with 100 μL of 1× reporter lysis buffer, and 30 μL of cell extract were used for luciferase and β-Gal assays. A LumiCount luminometer (Perkin-Elmer Life and Analytical Sciences) was used to quantitate luciferase and β-Gal activities. Luciferase activities were then normalized to β-Gal activity.
Animals and orthotopic implantation of tumor cells. Male athymic nude mice (NCI-nu) were purchased from the Animal Production Area of the National Cancer Institute Frederick Cancer Research and Development Center (Frederick, MD). The mice were housed and maintained under specific pathogen-free conditions in facilities approved by the American Association for Accreditation of Laboratory Animal Care and in accordance with current regulations and standards of the U.S. Department of Agriculture, U.S. Department of Health and Human Services, and the NIH. The mice were used in accordance with institutional guidelines when they were 8 to 12 weeks old.
To produce tumors, ACHN cells were harvested from subconfluent cultures by a brief exposure to 0.25% trypsin and 0.02% EDTA. Trypsinization was stopped with medium containing 10% fetal bovine serum, and the cells were washed once in serum-free medium and resuspended in HBSS. Only suspensions consisting of single cells with >90% viability were used for the injections. Injection of cells into the kidney subcapsule was done. Briefly, mice (six per treatment group) were anesthetized and placed in the left lateral decubitus position. A vertical incision was made in the right flank through the skin and peritoneum, exposing the lateral aspect of the kidney. The kidney was lifted gently and stabilized. A 27-gauge needle was inserted into the renal parenchyma from the lower pole of the kidney and advanced until its point reached just below the renal capsule. At this time, the mice were injected with 1 million viable tumor cells in 50 μL HBSS. After injection, the kidney was returned to the abdominal cavity, and the wound was closed in one layer with wound clips.
The mice were killed 5 weeks after injection, and normal and the injected kidneys were removed and fixed in 10% buffered formalin solution after measuring the weight. All animals injected with kidney cancer cells developed kidney tumors. Tumors usually grow in kidney subcapsules and invade into the cortex and medulla of the kidney.
Terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling assay and histologic studies. For H&E staining and terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) assay, tumor tissue was fixed in formalin and embedded in paraffin, one section was processed for H&E staining, and the others were used for TUNEL assay. TUNEL staining was carried out using DeadEnd Colorimetric TUNEL System (Promega). Paraffin-embedded sections (4-6 μm thick) were processed per manufacturer's protocol. Briefly, sections were deparaffinized in xylene and then treated with a graded series of alcohol [100%, 95%, 85%, 70%, and 50% ethanol (v/v) in double-distilled water] and rehydrated in PBS (pH 7.5). Tissues were then treated with proteinase K solution for permeabilization and then refixed with 4% paraformaldehyde solution. Slides were then treated with recombinant terminal deoxynucleotidyl transferase reaction mix and incubated at 37°C for 1 h. Reaction was terminated by immersing the slides in 2× SSC solutions for 15 min at room temperature. After blocking the endogenous peroxidases activity (by 0.3% hydrogen peroxide), slides were washed with PBS and then incubated with streptavidin horseradish peroxidase solution for 30 min at room temperature. After washing, slides were incubated with 3,3′-diaminobenzidine (substrate) solution until a light brown background appears (10 min) and then rinsed several times in deionized water. After mounting, slides were observed by light microscope. The percentage of TUNEL-positive cells was quantified by a point counting procedure in seven randomly selected zones in slides from mice treated with corn oil (control) or DIM-C-pPhtBu. Results are expressed as mean ± SD.
Statistical analysis. Statistical significance was assessed using Student's t test. A value of P < 0.05 compared with solvent control was considered statistically significant.
The growth-inhibitory effects of C-DIMs were investigated in both 786-0 and ACHN renal carcinoma cell lines (Fig. 1). IC50 values for growth inhibition by DIM-C-pPhCF3, DIM-C-pPhtBu, and DIM-C-pPhC6H5 were in the 1 to 5 μmol/L range in both cell lines. ACHN cells seemed to be slightly more sensitive to the C-DIM compounds than 786-0 cells because significant growth inhibition after 6 days was observed in the former cell line after treatment with 1.0 μmol/L concentration. These results indicated that C-DIMs were more potent than pioglitazone or PGJ2, which exhibited growth inhibition IC50 values ranging from 10 to 20 μmol/L in ACHN and Cak-1 cell lines (38–40). ACHN and 786-0 cells were also treated with 5 or 10 μmol/L of DIM-C-pPhtBu for 24 h and the percent distribution of cells in G0-G1, S, and G2-M phases was determined (Fig. 1D). The effects of DIM-C-pPhtBu on 786-0 cells were minimal with only a small but significant decrease in the percentage of cells in G2-M and an increase in G0-G1 (not significant) after treatment with 10 μmol/L DIM-C-pPhtBu. A similar but more dramatic pattern of effects was observed in ACHN cells where DIM-C-pPhtBu induced a concentration-dependent percentage increase in G0-G1 and decrease in G2-M phases of the cell cycle. In addition, although the overall percentage of apoptotic cells was low after treatment for 24 h, 10 μmol/L DIM-C-pPhtBu induced a significant 2.2- and 2.9-fold increase in apoptosis in ACHN and 786-0 cells, respectively.
Results in Fig. 2A illustrated the effects of DIM-C-pPhtBu on activation of luciferase activity in ACHN and 786-0 cells transfected with the PPARγ-GAL4 chimera and a reporter gene construct (pGAL4) containing five tandem yeast GAL4 response elements linked to a luciferase reporter gene. The PPARγ-GAL4 chimera contains the ligand-binding domain of PPARγ linked to the DNA-binding domain of the yeast GAL4 protein. The results show that DIM-C-pPhtBu induced luciferase activity in both cell lines and induction was also observed for DIM-C-pPhCF3 and DIM-C-pPhC6H5 (data not shown). In contrast, Western blot analysis of whole-cell lysates from 786-0 and ACHN cells shows that PPARγ is highly expressed in the latter cell line, whereas low to nondetectable levels of PPARγ protein were detected in 786-0 cells (Fig. 2A). PPARγ protein expression was not affected by treatment with 10 μmol/L DIM-C-pPhtBu. These data are consistent with a previous study showing highly variable expression of PPARγ in kidney cancer cell lines and tumors, and renal carcinoma is an example of a tumor type where decreased PPARγ expression in tumor versus nontumor tissue has been observed (41).
PPARγ agonists typically affect cell cycle proteins, such as cyclin D1, p27, and p21, and results in Fig. 2B and C summarize the concentration- and time-dependent effects of DIM-C-pPhtBu on these same proteins in ACHN and 786-0 cells, respectively. p27 is uniformly expressed after 12 or 24 h in ACHN cells and is unaffected by treatment with DIM-C-pPhtBu. In contrast, p21 expression is low after 12 h and enhanced after 24 h. Some induction of p27 is observed only at the highest concentration (12.5 μmol/L) of DIM-C-pPhtBu. p21 is expressed in both cell lines and DIM-C-pPhtBu decreases expression of p21 in ACHN but not 786-0 cells. In contrast, DIM-C-pPhtBu induced a significant decrease in the expression of cyclin D1 protein as previously observed in other cancer cell lines treated with C-DIMs (28, 36, 37), and this response was reversed after cotreatment with the proteasome inhibitor MG132 (Fig. 2D).
Previous studies show that caveolin-1 is induced by C-DIMs in colon but decreased in prostate cancer cells (24, 36). DIM-C-pPhtBu decreased caveolin-1 in 786-0 cells but did not affect expression in ACHN cells (Fig. 3A). Previous studies have also shown that the proapoptotic NAG-1 gene and ATF3 are induced by C-DIMs. Results in Fig. 3B show that both proteins were induced by DIM-C-pPhtBu in ACHN and 786-0 cells, and up to a 4-fold induction response was observed (Fig. 3C).
It has also been reported that C-DIM compounds activate receptor-independent ER stress and apoptosis in pancreatic and ovarian cancer cells (34, 35). Results in Fig. 4A summarize the effects of DIM-C-pPhtBu on ER stress pathways in ACHN and 786-0 cells. Minimal induction of ER stress responses for GRP78 and CHOP was observed after treatment of both cell lines for 6 h (data not shown), whereas both markers of ER stress were elevated after treatment with DIM-C-pPhtBu for 12 or 18 h and this persisted for up to 24 h. Similar induction responses for GRP78 were also observed for DIM-C-pPhCF3 and DIM-C-pPhC6H5 (data not shown). In addition, a similar time course was observed for induction of DR5 and cleaved caspase-8, which is indicative of activation of the extrinsic apoptotic pathway associated with induction of DR5. There were minimal differences in constitutive and inducible GRP78, CHOP, DR5, and caspase-3 cleavage in ACHN and 786-0 cells, except that higher constitutive levels of DR5 were expressed in the former cell line. Figure 4B directly compares and quantitates induction of GRP78 and CHOP proteins in ACHN and 786-0 cells after treatment with 5 to 12.5 μmol/L of DIM-C-pPhtBu for 18 h. Significant induction was observed in both cell lines using 10 μmol/L DIM-C-pPhtBu. We also showed that 10 μmol/L DIM-C-pPhtBu induced transactivation in ACHN and 786-0 cells transfected with pGRP78, a construct containing the −374 to +1 region of the GRP78 gene promoter that has an ER stress response element (Fig. 4C). Similar induction of luciferase activity by DIM-C-pPhtBu was observed in cells transfected with pCHOP, which contains the −954 to +1 region of the CHOP gene promoter.
Activation of ER stress pathways and the subsequent induction of DR5 and cleaved caspase-8 (Fig. 4A) is consistent with the induction of RCC cell death observed after treatment with the C-DIM compounds (Fig. 1). Results in Fig. 5A confirm that DIM-C-pPhtBu induced a concentration- and time-dependent induction of cleaved (activated) caspase-3 and caspase-dependent PARP cleavage in ACHN and 786-0 cells at concentrations of 5 and 7.5 μmol/L with the maximal effects observed after 72 h. The role of PPARγ in mediating DIM-C-pPhtBu–induced PARP cleavage, cyclin D1 down-regulation, and induction of NAG-1 and GRP78 was investigated in ACHN and 786-0 cells treated with the C-DIM compound alone or in combination with the PPARγ antagonist T007 (Fig. 5B). The results show that T007 did not affect any of the DIM-C-pPhtBu–induced responses, suggesting that these effects were PPARγ independent. Similar results were observed in previous studies with C-DIMs in other cancer cell lines (29–37). Results in Fig. 5C and D also show (quantitatively) that DIM-C-pPhtBu induced PARP cleavage in 786-0 and ACHN cell lines, respectively. These responses were blocked after cotreatment with both a caspase-8 (Z-IETD-FMK) and a pancaspase (Z-VAD-FMK) inhibitor. These results confirm that DIM-C-pPhtBu induces caspase-8–dependent and caspase-3–dependent apoptosis in both ACHN and 786-0 cells, and this is consistent with activation of ER stress (Fig. 4) and other proapoptotic genes, such as NAG-1 and ATF3 (Fig. 3), by DIM-C-pPhtBu in the RCC cell lines.
We also investigated the effects of DIM-C-pPhtBu on renal adenocarcinoma development in an orthotopic model for kidney cancer in which ACHN and 786-0 cells were directly injected into the kidney. Preliminary studies showed that ACHN but not 786-0 cells formed tumors in this model. Figure 6 illustrates the effects of DIM-C-pPhtBu (40 mg/kg/d) and corn oil vehicle control on normal kidney weights and kidney weights in tumor-bearing animals. Normal kidney weights in animals administered corn oil or DIM-C-pPhtBu were 0.262 ± 0.047 and 0.274 ± 0.03 g, respectively, and kidney weights in tumor-bearing animals were 0.752 ± 0.08 and 0.444 ± 0.07 g, respectively (Fig. 6A and B). Thus, DIM-C-pPhtBu had no effect on kidney weights but significantly decreased kidney tumor weights in animals bearing ACHN cells. H&E staining of kidney tumors and nontumor tissue showed distinct differences. In addition, there were no significant differences in body or organ (heart and liver) weights in treated versus nontreated animals or any evidence of toxicity by histopathologic analysis. The normal kidney (Fig. 6C, top) consists of low numbers of glomerulas surrounded by normal-appearing tubular epithelium, whereas the appearance of tumor tissue contained neoplastic cells with atypical epithelium with variable malignant features (Fig. 6C, bottom). In addition, using the colorimetric TUNEL assay, it was apparent that DIM-C-pPhtBu induced massive staining in the tumors compared with corn oil (control) tumors, showing that apoptosis was induced in both ACHN tumors (Fig. 6D) and cells. Quantitation of the TUNEL assay results illustrated the significantly increased apoptosis in the tumors from C-DIM–treated mice. This was a major pathway for the anticarcinogenic activity of DIM-C-pPhtBu in this kidney cancer model and is consistent with the PPARγ-independent activation of apoptosis by C-DIMs in RCC cells (Figs. 4 and 5).
RCC is a complex disease and the most predominant form is clear cell carcinoma, which is highly metastatic and resistant to many chemotherapies. IFN and interleukin-2 have been used for treatment of metastatic RCC; however, only 15% to 20% of patients benefit from these treatments (41). RCC typically exhibits high expression of hypoxia-inducible factor-1α and up-regulation of vascular endothelial growth factor expression, and newer chemotherapies for treatment of RCC are targeting vascular endothelial growth factor/vascular endothelial growth factor receptor signaling using antibodies and kinase inhibitors (42).
PPARγ is an orphan nuclear receptor that is overexpressed in many tumor types (12), and different structural classes of PPARγ agonists show some promise for cancer chemotherapy (8). For example, both thiazolidinediones and PGJ2 inhibit growth of RCC cells and typically affect cell cycle genes/proteins associated with G0-G1 to S phase progression (38, 39). However, unlike many cancer cell lines and tumors, there is some suggestion that the overexpression of PPARγ in RCC is somewhat variable (40). For example, in a study of six RCC cell lines, the expression of PPARγ was lower in five of these cell lines compared with normal kidney tissue. Only ACHN cells expressed PPARγ mRNA levels higher than observed in normal kidney cells. Similar results were observed in the comparison of PPARγ mRNA in RCC tumors versus normal kidney tissue where the tumor samples frequently exhibited lower levels of this transcript.
Studies in our laboratory have identified a novel class of PPARγ agonists derived from DIM, and like many other ligands for PPARγ, the C-DIM compounds inhibit cancer cell/tumor growth through receptor-dependent and receptor-independent pathways (28–37). Results of growth-inhibitory studies in ACHN and 786-0 RCC cells showed that IC50 values for PPARγ-active C-DIMs were between 1 and 5 μmol/L in both cell lines, and this was much lower than observed for pioglitazone or PGJ2 in other studies (40). The growth inhibition studies also showed that the effects of C-DIMs were both structure- and time-dependent in 786-0 cells. After treatment of these cells for up to 4 days with 5 μmol/L DIM-C-pPhtBu or DIM-C-pPhC6H5, growth inhibition was not observed, whereas significant inhibition was observed after 6 days. In contrast, 5 μmol/L DIM-C-pPhCF3 was cytotoxic to 786-0 cells after 4 and 6 days. The effects of 5 or 10 μmol/L DIM-C-pPhtBu on the percent distribution of ACHN and 786-0 in G0-G1, S, and G2-M phases of the cell cycle showed that 786-0 cells were resistant to change compared with ACHN cells (Fig. 1D). In this cell line, 10 μmol/L DIM-C-pPhtBu caused a 16.3% increase in cells in G0-G1, which is consistent with the sensitivity of this cell line to DIM-C-pPhtBu–dependent down-regulation of cyclin D1 (Fig. 2B). However, increased G0-G1 was not accompanied by a decrease in the percentage of cells in S phase, but instead, there was an 18.3% decrease of ACHN cells in G2-M. This suggests that, in ACHN cells, other cell cycle regulators may be affected by DIM-C-pPhtBu and the identity of these genes/proteins is currently being investigated. These results suggest that C-DIM compounds are potentially effective drugs for treatment of RCC. Moreover, due to the differential expression of PPARγ protein in ACHN (high) versus 786-0 (low to nondetectable; Fig. 2A), these cell lines are ideal for investigating activation of receptor-dependent and receptor-independent growth-inhibitory/proapoptotic pathways by C-DIMs.
In transactivation studies using a PPARγ-GAL4 chimera, we observed induction responses by DIM-C-pPhtBu (Fig. 2) in ACHN and 786-0 cell lines, suggesting that the appropriate cofactors required for this response are expressed in both cell lines. The effects of PPARγ-active C-DIMs on p27 and p21 were highly variable in ACHN and 786-0 cells (Fig. 2B and C). However, DIM-C-pPhtBu induced proteasome-dependent degradation of cyclin D1 (Fig. 2), which was not inhibited after cotreatment with a PPARγ antagonist (Fig. 5B). These results are consistent with previous reports showing that C-DIMs induce receptor-independent degradation of cyclin D1 and other cancer cell lines (28, 29, 36, 37). PPARγ-active C-DIMs induce receptor-dependent expression of caveolin-1 in colon cancer cells (29, 37), where this gene may exhibit tumor suppressor activity (43), but decrease caveolin-1 in prostate cancer cells (36), where it may have tumor-enhancing activity (44, 45). Caveolin-1 overexpression in RCC patients predicts poor disease-free survival (46); however, the functional role of caveolin-1 in RCC has not been explored. In our studies, 7.5 μmol/L DIM-C-pPhtBu decreased caveolin-1 expression in 786-0 but not in ACHN (no change) cells (Fig. 3A) and resembled the responses observed in prostate cancer cells where PPARγ-active C-DIMs decrease levels of caveolin-1 protein (36). The function of this effect of DIM-C-pPhtBu (i.e., decreased caveolin-1 expression) in RCC cells is now being further investigated.
PPARγ-active C-DIMs also induce NAG-1 and ER stress in colon and pancreatic cancer cells. These receptor-independent responses lead to apoptosis (33–35, 37). Results summarized in Figs. 3 to 5 show that DIM-C-pPhtBu also induced NAG-1 and ER stress proteins and DR5, and these responses are coupled with activation of caspase-dependent PARP cleavage and fluorescence-activated cell sorting analysis also confirmed that DIM-C-pPhtBu induced apoptosis in 786-0 and ACHN cells (data not shown). This suggests that DIM-C-pPhtBu–induced apoptosis in RCC cells is similar to that observed in colon and pancreatic cells (33–36), and these responses are PPARγ independent (Fig. 5B). The concentrations of DIM-C-pPhtBu required to induce PARP cleavage, NAG-1, or ER stress after treatment for up to 24 h (Figs. 3B and C, 4A and B, and 5B) were ∼10 μmol/L; however, lower concentrations (5 μmol/L) induced PARP cleavage after treatment for 72 h (Fig. 5A). These time-dependent effects of DIM-C-pPhtBu were similar to the temporal effects of this compound on cell proliferation (Fig. 1).
We also used an orthotopic model of RCC and directly injected human ACHN cells into the kidney of each athymic mouse. The results in Fig. 6A and B show that treatment with DIM-C-pPhtBu (40 mg/kg/d) did not affect normal kidney weight but significantly decreased the weight of the tumor-bearing kidney. H&E staining of normal kidney and kidney tumors exhibited the expected differences in staining, and the TUNEL assay showed extensive staining for apoptosis in the treated animals but not in the controls (Fig. 6C). These results show a parallel mode of action between the in vivo and in vitro studies where DIM-C-pPhtBu inhibits both RCC cell and tumor growth, and this is due, in part, through activation of apoptosis. Thus, DIM-C-pPhtBu, a prototypical C-DIM compound, represents a novel class of drugs for treatment of renal tumors through PPARγ-independent activation of proapoptotic proteins (NAG-1) and pathways (ER stress). We are now investigating the role of C-DIM–induced kinases in the induction of NAG-1 and also the mechanisms of ER stress activation that may involve direct effects on mitochondria (data not shown). Current studies are also focused on identifying the most active proapoptotic C-DIM analogues (among >100 compounds) for potential clinical applications in treating RCC.
Grant support: NIH grants ES09106 and CA112337 and Texas Agricultural Experiment Station.
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