Apoptosis Induced by Ziziphora tenuior Essential Oil in Human Colorectal Cancer Cells

Abstract

Ziziphora (Cacotti in Persian) belongs to the Lamiaceae family (mint group) and is vastly found in Iran and Asia. This traditional medicinal plant is normally used as analgesic and for treatment of particular gastrointestinal diseases. Since colorectal cancer is one of the most common causes of death in the world and the second leading cause of cancer death among adults, there is a pressing need to inhibit this malignancy by using methods with minimal side effects. One of these methods is the use of natural resources such as medical plants. This study is aimed at investigating the expression of apoptosis-related genes in the adjacent culture of colorectal cancer epithelial cells (HT-29) with Ziziphora essential oil (ZEO). The essential oil was extracted from Ziziphora leaves, and its compounds were determined and then added to the HT-29 culture medium at different concentrations. After 24 hours, the HT-29 cells were harvested from the medium and cytotoxicity was analyzed by MTT assay. After MTT assay and determination of the percentage of apoptosis by flow cytometry, RNA extraction was performed and the expression levels of Bax, Bcl-2, caspase 3 (C3), and caspase 9 (C9) were analyzed using newly designed primers by reverse transcription (RT) qPCR method and GeniX6 software. Also, specific antibodies were used for western blot analyses of those molecules. GC analysis revealed 42 different compounds in the ZEO, including pulegone (26.65%), menthone (5.74%), thymol (5.51%), and menthol (1.02%). MTT assay showed that the concentration of 200 μg/ml of ZEO had the highest HT-29 cell death during 24 hours. After incubation with the concentration of 50 μg/ml of ZEO for 24 and 48 hours, caspase 3 and 9 gene expressions in the treated group increased compared to those in the control group (P < 0.001), while the Bcl-2 expression decreased. The results showed that having anticancer compounds, ZEO can increase C3 and C9 and decrease Bcl-2 expressions, causing apoptosis in HT-29 cells in vitro. This can lead to the use of ZEO as a factor for colorectal cancer treatment.

Figure 1

The existence of pulegone in ZEO GC/MS. (a) Chromatogram of Ziziphora essential oil (ZEO). (b) Chemical structure of pulegone in ZEO.

Figure 2

Percentage of the viability of treated HT-29 cells with different concentrations of Ziziphora essential oil (ZEO).

Figure 3

Effect of concentration of 50 μg/ml of Ziziphora essential oil (ZEO) on apoptosis in HT-29 cells. (a) Flow cytometry figure (annexin v/PI), Q4 healthy cells, Q3 early apoptosis, Q2 late apoptosis, and Q1 necrosis. (b) Percentage of early apoptotic cells after 24- and 48-hour incubation of HT-29 cells with ZEO.

Figure 4

Ziziphora essential oil- (ZEO-) induced apoptosis includes an increase in caspase 3 and 9 and a decrease in Bcl-2 and Bax expressions in HT-29 cells. (a) Light microscopic pictures (×100 magnification) with arrows represent the morphological changes caused by apoptosis in the examined HT-29 cells. (b) Bax, Bcl-2, C3, and C9 expression results obtained by western blot in HT-29 cells. (c) mRNA, Bax, Bcl-2, C3, and C9 results obtained by RT-PCR.

Figure 5

Schematic view of the effect of Ziziphora essential oil (ZEO) on apoptosis through pulegone and menthol. On the other hand, menthol activates caspase 10, followed by activation of the Bid pathway, and caspases 3 and 7 cause apoptosis.