Effects of Hexane Root Extract of Ferula hermonis Boiss. on Human Breast and Colon Cancer Cells: An In Vitro and In Vivo Study

Abstract

Breast and colon cancers are leading causes of cancer-related deaths globally. Plants are a potential source of natural products that may be used for the treatment of cancer. Ferula hermonis (FH) is reported to have diverse therapeutic effects. However, there are few reports on the in vitro anticancer potential of FH extract. Our results showed that the Ferula hermonis root hexane extract (FHRH) can induce dose-dependent cytotoxic effects in breast and colon cancer cells with MTT IC₅₀ values of 18.2 and 25 μg/ml, respectively. The FHRH extract induced apoptosis in both breast and colon cancer cells; this was confirmed by light and nuclear staining, q-PCR, and caspase 3/7 activation. This study also demonstrated the antitumor activity of FHRH in 9,10-dimethylbenz[α]anthracene DMBA-induced rodent mammary tumor model. The GC/MS analysis revealed the presence of 3,5-Dimethylbenzenemethanol, Alpha-Bisabolol, Alpha-pinene, Beta-pinene, and Baccatin III that have various pharmacological potentials. Overall, the present study suggests that FHRH extract possesses anticancer potential which is mediated through apoptotic effects in MDA-MB-231 and LoVo cells. The present study also considered a basis for further investigations into the potential use of FHRH extract as an anticancer therapy for breast and colon cancers.

Figure 1

The effect of FHRH extracts in MDA-MB-231 and LoVo cells using MTT and LDH assays. (a) The absorbance of the MTT formazan was determined at 540 nm in an ELISA reader. (b) MDA-MB-231 (18.2 μg/ml) and LoVo (25 μg/ml) cells were treated with the FHRH extract for 48 h. LDH activity was determined at 490 nm in an ELISA reader. Data represent the mean ±SD (∗P< 0.05, ∗∗P< 0.01, and ∗∗∗P< 0.001 were considered significant compared to control) of three independent experiments carried out in triplicate. FH: Ferula hermonis, R: root, H: hexane.

Figure 2

Nuclear morphology following FHRH treatment. Cells were stained using Hoechst 33258 to observe nuclear changes and photographed with fluorescence microscopy. Arrows indicate the cells with DNA fragmentation (magnification: 200 x).

Figure 3

Effect of the FHRH extract on the mRNA expression of apoptosis-associated genes. Fold change of p53, Bax, and caspases 3, 8, 9 genes in MDA-MB-231 cancer cells treated with IC₅₀ concentrations (18.2 μg/ml) for 48 h. ∗P<0.05, ^∗∗P<0.005, and ∗∗∗P<0.0005 compared to the control cells (DMSO vehicle). Data are presented as the mean ± standard deviation in the three independent experiments carried out in triplicate.

Figure 4

Detection of caspase 3 and 7 activity after MDA-MB-231 (18.2 μg/ml) and LoVo (25 μg/ml) cells treatment with FHRH extract for 48h. Caspase-3/7 activities were assayed by the CellEvent™ caspase-3′7 green detection reagent. FH: Ferula hermonis, R: root, H: hexane. (∗P< 0.05, ∗∗P< 0.01, and ∗∗∗P< 0.001 compared with the control (untreated cells)). Data are presented as the mean ± standard deviation of three independent experiments carried out in duplicate.

Figure 5

The effect of the FHRH extract on MDA-MB-231 cell migration. Cells were scratched with a pipette tip and then incubated with 9 μg/ml of the FHRH extract for 48 h. Migrating cells were photographed under a phase-contrast microscope. FH: Ferula hermonis, R: root, H: hexane. ∗P<0.05 compared with the control (untreated cells). Data are presented as the mean ± standard deviation of three independent experiments carried out in duplicate.

Figure 6

Light micrographs of tumor growths induced by DMBA (16 weeks postinjection) in mammary glandular tissue of female rats in comparison to rats injected with DMBA and then treated for 10 weeks with FHRH extract. (a) Proliferating neoplastic cells in the epithelial lining of mammary glandular ducts (small arrows). Neoplastic cells are also seen infiltrating the interstitial tissue. DMBA-injected animal. HE: 100X. (b) Neoplastic cell growth in the epithelial lining of mammary glandular ducts and presence of carcinomatous cells in the ductal lumina (arrowhead). DMBA-injected animal. HE: 200X. (c) Neoplastic cells in the ductal epithelial lining (arrows) and also in the ductal lumina (arrowheads). Note the nuclear pleomorphism of neoplastic cells. DMBA-injected animal. HE: 400X. (d) Area of necrosis (∗) in the mammary glandular tissue. Note the cytoplasmic and nuclear cell debris (arrows). DMBA-injected animal. HE: 100X. (e) Restriction of the proliferating neoplastic cells to a relatively limited number of mammary glandular ducts (arrows). Note absence of distinct neoplastic cell infiltration and also absence of remarkable areas of necrosis in interstitial tissue (∗). FHRH extract-treated animal. HE: 400X. (f) Proliferating neoplastic cells constituting only a few layers in the epithelial lining of mammary glandular ducts (arrows). No distinct areas of necrosis in the interstitial tissue (∗). (g-h) Normal structure of rat mammary gland tissue. Arrow indicates the ducts and epithelial lining cells. The adipose tissue (∗) surrounded the ducts (HE: 200x and 100x, respectively).

Figure 7

Images represent the breast cancer tissue of in vivo experiment in rats. Solid tumor was induced in Wistar albino rats (Rattus norvegicus) with a single subcutaneous injection by injecting 9,10-dimethylbenz[α]anthracene (DMBA). After 8 weeks postinjection, group I remained untreated, while group II were injected with FHRH extract at a dose of 100 mg/kg into the tumor itself. (a) Tumor size in DMBA group and after treatment with FHRH extract. (b) Tumor volume was calculated as described in materials and methods.

Figure 8

GC-MS analysis of the hexane extract of F. hermonis.