Chemopreventive potential of beta-Sitosterol in experimental colon cancer model--an in vitro and In vivo study

Abstract

Background: Asclepias curassavica Linn. is a traditional medicinal plant used by tribal people in the western ghats, India, to treat piles, gonorrhoea, roundworm infestation and abdominal tumours. We have determined the protective effect of beta-sitosterol isolated from A. curassavica in colon cancer, using in vitro and in vivo models.

Methods: The active molecule was isolated, based upon bioassay guided fractionation, and identified as beta-sitosterol on spectral evidence. The ability to induce apoptosis was determined by its in vitro antiradical activity, cytotoxic studies using human colon adenocarcinoma and normal monkey kidney cell lines, and the expression of beta-catenin and proliferating cell nuclear antigen (PCNA) in human colon cancer cell lines (COLO 320 DM). The chemopreventive potential of beta-sitosterol in colon carcinogenesis was assessed by injecting 1,2-dimethylhydrazine (DMH, 20 mg/kg b.w.) into male Wistar rats and supplementing this with beta-sitosterol throughout the experimental period of 16 weeks at 5, 10, and 20 mg/kg b.w.

Results: beta-sitosterol induced significant dose-dependent growth inhibition of COLO 320 DM cells (IC50 266.2 microM), induced apoptosis by scavenging reactive oxygen species, and suppressed the expression of beta-catenin and PCNA antigens in human colon cancer cells. beta-sitosterol supplementation reduced the number of aberrant crypt and crypt multiplicity in DMH-initiated rats in a dose-dependent manner with no toxic effects.

Conclusion: We found doses of 10-20 mg/kg b.w. beta-sitosterol to be effective for future in vivo studies. beta-sitosterol had chemopreventive potential by virtue of its radical quenching ability in vitro, with minimal toxicity to normal cells. It also attenuated beta-catenin and PCNA expression, making it a potential anticancer drug for colon carcinogenesis.

Figure 1

Structure of β-sitosterol isolated from A. curassavica. The compound was identified based on the following evidence: IR spectrum showed hydroxyl (3430 cm^-1) and tris-substituted double-bond (1642 and 80.1 cm^-1). The EI/MS mass spectrum showed m+ at m/z 414, corresponding to the molecular formula (C₂₉H₅₀O) for β-sitosterol. The other characteristic peaks were at m/z 273 [m-sidechain]⁺ 255 [m-side chain - H₂O]⁺ 231 [m-side + +1chain-ring D], 213 [231-H₂O] and 300. The H-NMR spectrum showed 2 tertiary methyl groups at δ 0.68 and 1.02, corresponding to H-18 and H-19. Three secondary methyls appeared at δ 0.92, 0.82 and 0.84, corresponding to H-21, H-26 and H-27, respectively. (J = 6.5 Hz). H-29 appeared as triplet at δ 0.85 (J = 7.0 Hz). The C-NMR spectrum indicated 29 carbon atoms, 10 primary, 10 secondary and 3 tertiary carbon; 6 methyl groups being present. C-5 and C-6 were olefinic carbon atoms appearing at δ 121.70 and 140.74.

Figure 2

Anti-oxidant activity of β-sitosterol. The free radical scavenging ability of β-sitosterol was determined by DPPH and nitric oxide scavenging assays compared with ascorbic acid as a standard at various concentrations.

Figure 3

Induction of apoptosis in COLO 320 DM. Human colon cancer cell lines (COLO 320 DM) cells were treated 120 or 240 μM β-sitosterol. The anti-proliferative effect is shown in (a), flow cytometric analysis for the determination of pro-apoptotic/apoptotic/necrotic cells is given in (b), and fluorescent staining of nuclei by Hoechst 33258 at 20× is seen in (c).

Figure 4

ROS Scavenging ability. β-sitosterol was treated with COLO 320 DM cells for 24 h at 120 or 240 μM and the ROS scavenging ability determined using DCF staining and confocal microscopy. (A & B) COLO 320 DM cells untreated, (C) COLO 320 DM cells treated with β-sitosterol at 120 μM, (D) COLO 320 DM cells treated with β-sitosterol at 240 μM.

Figure 5

Expression of β catenin (A) and PCNA (B) in COLO 320 DM cells. Human colon cancer (COLO 320 DM) cells were treated with 15, 30, 60, or 120 μM/ml β-sitosterol for 24 h and 50 μg of extracted protein were loaded.

Figure 6

Topographical view of ACF. Topographical views of (A & B) normal crypt (40×), (C) ACF (arrows) with multiple crypts in the colon from a rat treated with DMH (40×), (D) ACF (arrow) with 2 and 3 crypts in the colon from a rat treated with DMH+ β-sitosterol (5 mg/kg), (E) ACF (arrow) with multiple 2 crypts in the colon from a rat treated with DMH+ β-sitosterol (10 mg/kg), and (F) ACF (arrow) with single 2 crypts in the colon from a rat treated with DMH+ β-sitosterol (20 mg/kg).