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.
Highlights
Cancer has always been one of the most fundamental issues of human health
According to the results of previous studies [14, 15] and the mechanism of the unknown effect of Ziziphora essential oil (ZEO) on HT-29 cells in the present study, our current study is aimed at investigating the expression of apoptotic-related genes in the adjacent culture of colorectal epithelial cancerous cells with ZEO
All biological samples were placed in Rotor-Gene Q 2.3.5 reverse transcription (RT)-PCR, and 95°C cDNA denaturation temperature was performed for 10 minutes; a 40-cycle period including 95°C in 10 seconds, 60°C in 20 seconds, and 72°C in 20 seconds was used for Bax, Bcl-2, caspase 3, and caspase 9 and reading
Summary
Cancer has always been one of the most fundamental issues of human health. Despite a large number of researches and developments in the past decade, cancer remains one of the most important causes of death globally. Ziziphora is a traditional medicinal plant in the family Lamiaceae This plant is a subshrub, its height is between 20 and 50 cm, and the leaves are small, opposite, almost lanceolate, and without petioles. The alcoholic extract of the aerial part of Ziziphora, due to its compounds such as menthol, can cause cytotoxicity in various cancerous cells (e.g., HT-29 and T-47D cell lines). The most important Ziziphora phytochemical compounds are the anticancer ones including pulegone, menthol, and menthone. According to the results of previous studies [14, 15] and the mechanism of the unknown effect of Ziziphora essential oil (ZEO) on HT-29 cells in the present study, our current study is aimed at investigating the expression of apoptotic-related genes in the adjacent culture of colorectal epithelial cancerous cells with ZEO
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