Boswellic acid inhibits growth and metastasis of human colorectal cancer in orthotopic mouse model by downregulating inflammatory, proliferative, invasive and angiogenic biomarkers

Abstract

Numerous cancer therapeutics were originally identified from natural products used in traditional medicine. One such agent is acetyl-11-keto-beta-boswellic acid (AKBA), derived from the gum resin of the Boswellia serrata known as Salai guggal or Indian frankincense. Traditionally, it has been used in Ayurvedic medicine to treat proinflammatory conditions. In this report, we hypothesized that AKBA can affect the growth and metastasis of colorectal cancer (CRC) in orthotopically implanted tumors in nude mice. We found that the oral administration of AKBA (50-200 mg/kg) dose-dependently inhibited the growth of CRC tumors in mice, resulting in decrease in tumor volumes than those seen in vehicle-treated mice without significant decreases in body weight. In addition, we observed that AKBA was highly effective in suppressing ascites and distant metastasis to the liver, lungs and spleen in orthotopically implanted tumors in nude mice. When examined for the mechanism, we found that markers of tumor proliferation index Ki-67 and the microvessel density cluster of differentiation (CD31) were significantly downregulated by AKBA treatment. We also found that AKBA significantly suppressed nuclear factor-κB (NF-κB) activation in the tumor tissue and expression of proinflammatory (cyclooxygenase-2), tumor survival (bcl-2, bcl-xL, inhibitor of apoptosis (IAP-1) and survivin), proliferative (cyclin D1), invasive (intercellular adhesion molecule 1 and matrix metalloproteinase-9) and angiogenic C-X-C (CXC) receptor 4 and vascular endothelial growth factor) biomarkers. When examined for serum and tissue levels of AKBA, a dose-dependent increase in the levels of the drug was detected, indicating its bioavailability. Thus, our findings suggest that this boswellic acid analog can inhibit the growth and metastasis of human CRC in vivo through downregulation of cancer-associated biomarkers.

Figure 1

AKBA inhibits the growth of orthotopically implanted CRC tumors in nude mice. (A) Schematic of the experimental protocol described in Materials and Methods. Group I was given corn oil (100 μL orally, daily); Group II was given AKBA (50 mg/kg orally, daily); Group III was given AKBA (100 mg/kg orally, daily); and Group IV was given AKBA (200 mg/kg orally, daily). (B) Bioluminescence imaging of orthotopically implanted CRC in live, anesthetized mice (left panel); measurements (photons/sec) of tumor volume (mean ± standard error) at various time points using live bioluminescence imaging at the indicated times (n = 6) (right panel). (C) Necropsy photographs of mice with orthotopically implanted CRC (left panel); tumor volumes (mean ± standard error; P < 0.001, control vs. 200-mg/kg AKBA and P < 0.034, control vs. 100-mg/kg AKBA) in mice measured on the last day of the experiment at autopsy using Vernier calipers and calculated using the formula V = 2/3 πr³ (n = 6). (D) As shown by the body weight change in mice, AKBA had no toxicity in the amount tested. There was no significant difference in body weight between the treated group and the control group.

Figure 2

AKBA inhibits ascites and distant organ metastasis in an orthotopic CRC murine model. (A) AKBA inhibited the development of ascites in CRC. The number of animals with ascites was counted 40 days after tumor cell implantation, and the percentages of animals with ascites were plotted. V, vehicle; L, low (AKBA 50 mg/kg); M, medium (100 mg/kg); and H, high (200 mg/kg). (B) AKBA inhibited metastasis to the lungs, liver, and spleen. Mice were killed and their abdomens were surgically opened; the number of metastatic foci in each organ was counted. Values are the mean ± standard deviation, n = 6. V, vehicle; L, low (AKBA 50 mg/kg); M, medium (100 mg/kg); and H, high (200 mg/kg).

Figure 3

AKBA inhibits tumor cell proliferation and angiogenesis in CRC. (A) The results of an IHC analysis of proliferation marker Ki-67 indicated that CRC cell proliferation was inhibited in mice treated with AKBA at different dose concentrations: V, vehicle; L, low (AKBA 50 mg/kg); M, medium (100 mg/kg); and H, high (200 mg/kg) (Left panel). Quantification of Ki-67 cells, as described in Materials and Methods. Values are represented as the mean ± standard error of triplicate (Right panel). (B) The results of an IHC analysis of CD31 for microvessel density indicated that angiogenesis was inhibited by AKBA at different dose concentrations. V, vehicle; L, low (AKBA 50 mg/kg); M, medium (100 mg/kg); and H, high (200 mg/kg) (Left panel). Quantification of CD31-positive microvessel density, as described in Materials and Methods. Values are the mean ± standard error of triplicate.

Figure 4

AKBA inhibits the expression of NF-κB and NF-κB-regulated gene products in CRC samples. (A) Electrophoretic mobility shift assay analysis showed the inhibition of NF-κB by AKBA in nuclear extracts from animal tissue. (B) Western blot showing that AKBA inhibited the expression of NF-κB-dependent gene products that regulate proliferation (cyclin D1 and COX-2), invasion (ICAM-1 and MMP-9), metastasis (CXCR4), and angiogenesis (VEGF). AKBA inhibits the expression of antiapoptotic gene products such as IAP-1, Bcl-xL, Bcl-2, and survivin in CRC tissues. Samples from three animals in each group were analyzed, and representative data are shown. V, vehicle; L, low (AKBA 50 mg/kg); M, medium (100 mg/kg); and H, high (200 mg/kg).

Figure 5

(A) AKBA inhibits the expression of COX-2, cyclin D1, MMP-9, and VEGF in CRC tissues from mice. Percentages indicate positive staining for the given biomarker. Samples from three animals in each group were analyzed, and representative data are shown. V, vehicle; L, low (AKBA 50 mg/kg); M, medium (100 mg/kg); and H, high (200 mg/kg). (B) Schematic representation of mechanism by which AKBA inhibited growth/ Metastasis of orthotopically implanted CRC.

Figure 6

HPLC chromatogram of AKBA in plasma and CRC tissue in nude mice. (A) HPLC chromatogram of pure AKBA. (B) HPLC chromatogram of AKBA in mice plasma from dosed animal, n = 6. (C) HPLC chromatogram of AKBA in mice CRC tissue from dosed animal, n = 6.