Cytotoxic activity of Piper cubeba extract in breast cancer cell lines

Abstract

This study aimed to evaluate the cytotoxicity of a crude extract of Piper cubeba against normal and breast cancer cell lines. To prepare the extract, P. cubeba seeds were ground, soaked in methanol and dichloromethane and isolated by column chromatography. Fractions were tested for cytotoxicity effects on normal fibroblast (L929), normal breast (MCF-12A) and breast cancer cell lines (MCF-7, MDA-MB-468 and MDA-MB-231). The most effective fraction was selected for DNA fragmentation assay to detect apoptotic activity. The results showed that the methanolic crude extract had a higher cytotoxic activity against MDA-MB-468 and MCF-7 than a dichloromethane crude extract. Then, the methanolic crude extract was separated into six fractions, designated A to F. Fraction C was highly active against breast cancer cell lines with an IC50 value less than 4 μg/mL. Therefore, Fraction C was further separated into seven fractions, CA to CG. The 1H-NMR profile showed that Fraction CE was long chain hydrocarbons. Moreover, Fraction CE demonstrated the highest activity against MCF-7 cells with an IC50 value of 2.69 ± 0.09 μg/mL and lower cytotoxicity against normal fibroblast L929 cells with an IC50 value of 4.17 ± 0.77 μg/mL. Finally, DNA fragmentation with a ladder pattern characteristic of apoptosis was observed in MCF-7, MDA-MB-468, MDA-MB-231 and L929 cells, but not in MCF-12A cells.

Figure 1

The ¹H-NMR spectra of (A) Fraction C and (B) Fraction CE.

Figure 2

The effect of 5 μg/mL Fraction CE on MCF-7, MDA-MB-468, MDA-MB-231, MCF-12A and L929 cells after an exposure time of 72 h. The values are expressed as the means ± S.D. (n = 3). * p < 0.05 versus normal L929 cells.

Figure 3

Analysis of DNA fragmentation induced by Fraction CE of P. cubeba in five cell lines. Cells were treated for seven days with Fraction CE, and DNA fragmentation was assessed by 1.5% agarose gel electrophoresis and ethidium bromide staining. Fraction CE was used to treat (A) MCF-7; (B) MDA-MB-468 (C) MDA-MB-231; (D) MCF-12A and (E) L929 with 2.69 μg/mL, 2.97 μg/mL, 3.98 μg/mL, 2.91 μg/mL and 4.17 μg/mL, respectively. The data are representative of three independent experiments carried out under the same conditions.