Nimbolide, a limonoid triterpene, inhibits growth of human colorectal cancer xenografts by suppressing the proinflammatory microenvironment

Abstract

Purpose: Extensive research over the past decade has revealed that the proinflammatory microenvironment plays a critical role in the development of colorectal cancer. Whether nimbolide, a limonoid triterpene, can inhibit the growth of colorectal cancer was investigated in the present study.

Experimental design: The effect of nimbolide on proliferation of colorectal cancer cell lines was examined by MTT assay, apoptosis by caspase activation and poly-ADP ribose polymerase cleavage, NF-κB activation by DNA-binding assay, and protein expression by Western blotting. The effect of nimbolide on the tumor growth in vivo was examined in colorectal cancer xenografts in a nude mouse model.

Results: Nimbolide inhibited proliferation, induced apoptosis, and suppressed NF-κB activation and NF-κB-regulated tumorigenic proteins in colorectal cancer cells. The suppression of NF-κB activation by nimbolide was caused by sequential inhibition of IκB kinase (IKK) activation, IκBα phosphorylation, and p65 nuclear translocation. Furthermore, the effect of nimbolide on IKK activity was found to be direct. In vivo, nimbolide (at 5 and 20 mg/kg body weight), injected intraperitoneally after tumor inoculation, significantly decreased the volume of colorectal cancer xenografts. The limonoid-treated xenografts exhibited significant downregulation in the expression of proteins involved in tumor cell survival (Bcl-2, Bcl-xL, c-IAP-1, survivin, and Mcl-1), proliferation (c-Myc and cyclin D1), invasion (MMP-9, ICAM-1), metastasis (CXCR4), and angiogenesis (VEGF). The limonoid was found to be bioavailable in the blood plasma and tumor tissues of treated mice.

Conclusions: Our studies provide evidence that nimbolide can suppress the growth of human colorectal cancer through modulation of the proinflammatory microenvironment.

FIGURE 1. Nimbolide inhibits growth and induces apoptosis in colorectal cancer cells

A, Molecular structure of nimbolide. B, Cells were treated with indicated concentrations of nimbolide, and cell growth was analyzed on days 0, 1, 3, and 5 by MTT assay. C, Cytotoxicity was measured by the live/dead assay. D, Nimbolide induces caspase activation and PARP cleavage in CRC cells. Whole-cell extracts from treated cells were analyzed by Western blotting by using indicated antibodies. *-indicates the significance of difference compared with control; P< 0.05. NL, nimbolide.

FIGURE 2. Nimbolide inhibits the expression of tumorigenic proteins in CRC cells

A, Whole-cell extracts from treated cells were analyzed by Western blotting by using indicated antibodies. B, Nimbolide suppresses mRNA levels of tumorigenic proteins. HCT-116 cells were treated with indicated concentrations of nimbolide for 12 h, total RNA was extracted and examined for mRNA levels by qRT-PCR after normalization to GAPDH data. C, Nimbolide reduces colony formation by colon cancer cells. HCT-116 cells were treated with indicated concentrations of nimbolide for 12 hours. Cells were then washed, allowed to form colonies for 10 days, stained with crystal violet, and then counted. NL, nimbolide.

FIGURE 3. Nimbolide down-regulates constitutive NF-κB activation in colon cancer cells through inhibition of IKK activity

A (left), HCT-116 cells were treated with 10 μM nimbolide for the times indicated. Nuclear extracts were prepared and assayed for NF-κB activation by using EMSA. A (right), Nimbolide neither inhibits STAT3 phsphorylation nor total STAT3 in HCT 116 cells. The whole cell extract from normal and treated cells were analyzed by Western blotting for pSTAT3 and STAT3. B (left), Nimbolide inhibits phosphorylation of IκBα in HCT-116 cells. Cells were treated with 10 μM nimbolide for the times indicated. Cytoplasmic extracts were analyzed by Western blotting by using a phosphospecific IκBα antibody (Ser^32/36). B (right), Nimbolide inhibits IKK activation in HCT-116 cells. Cells were incubated with 10 μM nimbolide for the times indicated. Whole-cell extracts were immunoprecipitated with an antibody against IKK-β and analyzed with use of an immune complex kinase assay. The effect of nimbolide on IKK protein expression was determined by Western blot analysis by using IKK-α and IKK-β antibodies. C (left), Nimbolide directly inhibits IKK activation in HCT-116 cells. Whole-cell extracts were immunoprecipitated with an IKK-β antibody. The immunoprecipitated complex was incubated with the indicated concentrations of nimbolide, and an immunocomplex kinase assay was performed. C (right), D, Nimbolide induces redistribution of p65 and inhibits p65 phosphorylation in HCT-116 cells. C (right), Cells were incubated with 10 μM nimbolide for 4 hours and analyzed for p65 localization by immunocytochemical analysis with use of a confocal microscope. D, Cells were incubated with 10 μM nimbolide for the times indicated, and the nuclear extract was analyzed for p65 and p-p65. PARP was used as a loading control. NL, nimbolide.

FIGURE 4. Nimbolide inhibits the growth of colorectal tumors in nude mice

A, Outline of the experimental protocol. Group I was given DMSO (50 μL, i.p. daily) for 10 days. Groups II and III were given nimbolide at 5 mg/kg and 20 mg/kg of body weight (i.p. daily), respectively, for 10 days. B, Bioluminescence imaging of live anesthetized mice implanted with CRC tumors (left); measurements of tumor volume on the 10th day with use of bioluminescence imaging (right, n = 4). C, Measurements of tumor volume with use of Vernier calipers and the formula 1/2× L × W², where L and W represent the length and width of tumors, respectively, on various days. Data represent mean ± SD of tumor volume of 4 mice. D, Effect of nimbolide on the body weight of mice over the treatment time.

FIGURE 5. Nimbolide inhibits NF-κB activation and down-regulates NF-κB–regulated gene products

A–B, Protein extracts prepared from colorectal tumor tissues of mice were analyzed by Western blotting by using antibodies against (A) antiapoptotic, (B, top) proliferative, (B, bottom) invasive, and angiogenic proteins. β-actin was used as an internal control. C, Nimbolide inhibits NF-κB activation in colorectal tumor tissues. Nuclear extracts prepared from tumor tissues were assayed for NF-κB activation by EMSA. D, Nimbolide induces DR5 but does not affect STAT3 phsphorylation or total STAT3 in tumor tissues. The whole cell extract from tumor tissues were analyzed by Western blotting for pSTAT3, STAT3 and DR5. NL, nimbolide.

FIGURE 6. Nimbolide inhibits the expression of cyclin D1, MMP-9, CXCR4, VEGF, and Ki67 in xenograft tumors from mice

The sections were stained and analyzed as described in the Materials and Methods section. Values across each photomicrograph represent percent mean ± SD of positive cells.