1,1-Bis(3'-indolyl)-1-(p-biphenyl)methane inhibits basal-like breast cancer growth in athymic nude mice

Abstract

Introduction: 1,1-Bis (3'-indolyl)-1-(p-biphenyl) methane (CDIM9) has been identified as a new peroxisome proliferator-activated receptor (PPAR)-gamma agonist that exhibits both receptor dependent and independent antitumor activities. CDIM9 has not previously been studied with respect to its effects against basal-like breast cancer. Our goal in the present study was to investigate the anti-basal-like breast tumor activity of CDIM9 in vitro and in vivo.

Methods: The effects of CDIM9 on cell protein and DNA syntheses were determined in basal-like breast cancer MDA-MB231 and BT549 cells in vitro. Maximum tolerated dose and dose-limited toxicity were determined in BalB/c mice, and antitumor growth activities were assessed in MDA-MB231 basal-like breast tumor xenografts in athymic nude mice.

Results: CDIM9 exhibited selective cell cytotoxicity and anti-proliferation effects on basal-like breast cancer lines. In MDA-MB231 cell, CDIM9 induced caveolin-1 and p27 expression, which was significantly downregulated by co-treatment with the PPAR-gamma antagonist GW9662. Nonsteroidal anti-inflammatory drug-activated gene-1 and activating transcription factor-3 were upregulated by CDIM9 through a PPAR-gamma independent pathway. CDIM9 (40 mg/kg daily, intraperitoneally, for 35 days) inhibited the growth of subcutaneous MDA-MB231 tumor xenografts by 87%, and produced a corresponding decrease in proliferation index. Nearly half of the treated mice (46%) had complete durable remissions, confirmed by histology. The growth of an established tumor was inhibited by CDIM9 treatment (64 mg/kg daily, intraperitoneally, for 10 days), with a mean tumor growth inhibition of 67% as compared with controls. CDIM9 induced increases in tumor caveolin-1 and p27 in vivo, which may contribute to its antitumor activity in basal-like breast cancer.

Conclusion: CDIM9 showed potent antiproliferative effects on basal-like breast cancer cell in tissue culture and dramatic growth inhibition in animal models at safe doses. These findings justify further development of this drug for treatment of basal-like breast cancer.

Figure 1

The cytotoxic and growth inhibitory effects of CDIM9 in MD-MB231 and BT549 cells. (a) MDA-MB231 cells, SKMCs, BT549 cells, and HSMMs were treated with serial diluted CDIM9 at between 1 × 10^-3 and 1.9 × 10^-6 mol/l for 48 hours. The EC₅₀ values were determined using GraphPad Prism software. (b) Cell growth inhibition assay. MD-MB231 and BT549 cells were treated with 1 to 10 μmol/l CDIM9 for 6 days, and cell numbers were determined as described in Materials and methods. Results are expressed as means ± standard error for three separate determinations at each time point. CDIM9, 1,1-bis (3'-indolyl)-1-(p-biphenyl) methane; EC₅₀, concentration producing 50% of the maximum possible response; HSMM, human muscle myoblast; SKMC, human skeletal muscle cell.

Figure 2

Induction of cell cycle proteins by CDIM9. (a) MDA-MB231 and (b) BT549 cells were treated with Me₂SO or 5, 10, and 20 μmol/l CDIM9 for 24 hours. Whole cell lysates were analyzed for caveolin-1, p27, and p21 by Western blot assays, as described in Materials and methods. β-Actin served as loading control. CDIM9, 1,1-bis (3'-indolyl)-1-(p-biphenyl) methane.

Figure 3

Effect of CDIM9 on apoptosis and induction of NAG-1 and ATF3. (a) MDA-MB231 cells were treated with Me₂SO and 20 μmol/l CDIM9 for 24 hours and apoptotic cells were detected using TUNEL staining. The arrows indicated infrequent apoptotic cells. (b) MDA-MB231 and BT549 cells were treated with Me₂SO or 5, 10, and 20 μmol/l CDIM9 for 24 hours. Whole cell lysates were analyzed for NAG-1 and ATF3 by Western blot assays. β-Actin served as loading control. ATF, activating transcription factor; CDIM9, 1,1-bis (3'-indolyl)-1-(p-biphenyl) methane; NAG, nonsteroidal anti-inflammatory drug-activated gene; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling.

Figure 4

Maximum tolerated dose and dose limiting toxicity of CDIM9. (a) Kaplan-Meier curves for 10 BalB/c mice (age 4 to 6 weeks) injected intraperitoneally (i.p.) with CDIM9 daily for a total of 35 doses. (b) Hematoxylin and eosin staining of kidney and liver harvested from animals treated with 160 mg/kg per day CDIM9 for 12 days and after animals had recovered from treatment. Arrows indicated the tubular vacuolization (kidney) and steatosis (liver). CDIM9, 1,1-bis (3'-indolyl)-1-(p-biphenyl) methane.

Figure 5

Inhibition of basal-like breast tumor growth by CDIM9. Shown are tumor sizes (following inoculation of MDA-MB231 basal-like breast cancer cells subcutaneously) tumor size in athymic nude mice treated daily with CDIM9 (40 mg/kg in 50 μl placebo, intraperitoneally), placebo, or PBS starting on day 4 after tumor inoculation. Values are expressed as mean ± standard error. CDIM9, 1,1-bis (3'-indolyl)-1-(p-biphenyl) methane; PBS, phosphate-buffered saline.

Figure 6

The growth inhibitory effects of CDIM9 on established MDA-MB231 tumor. (a) Athymic nude mice bearing established MDA-MB231 tumors were injected intraperitoneally with 64 mg/kg CDIM9 in 100 μl placebo or 100 μl saline for 10 total doses. The tumor size was measured on days 0 and 10. (b) Tumors collected from CDIM9 (64 mg/kg per day, intraperitoneally) or PBS treated mice were tested by Ki-67 immunohistochemistry staining and H&E staining. (c) The in vivo gene modulation activity of CDIM9 was investigated by immunoblotting analyses of MDA-MB231 tumor lysates after treatment with 64 mg/kg CDIM9 by intraperitoneal injection every day for seven total doses. CDIM9, 1,1-bis (3'-indolyl)-1-(p-biphenyl) methane; H&E, hematoxylin and eosin; PBS, phosphate-buffered saline.