الفهرس 
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Abstract
Cancer has been ranked the second leading cause of death worldwide. Causes of cancer vary from genetic to chemical to physical such as genetic mutations, chemical pollutants and non-ionizing radiation. Colon cancer is the third most occurring with around 10% of all cases of cancer, and the second leading cause of cancer-related deaths worldwide. The average age group that are diagnosed with cancers are in the 50s whereas in Egypt younger population as old as 40 or younger were identified which could be because of an unhealthy lifestyle led by people in this era. Colon cancer can be avoided by following a healthy lifestyle and good dietary habits such as regular exercising, avoiding processed food, quitting smoking and many other healthy habits. Diagnosis usually occurs in advanced stages so regular screening and colonoscopy can increase early diagnosis.
Common cancer therapies such as radio- and chemotherapy, although effective, have adverse effects from weight loss to hair fall to even secondary cancers and diseases. This is why an approach to investigate other treatments with less to no side effects has risen. Natural compounds derived from plants, whether extracts or derived compounds, have shown antioxidant, anti-inflammatory and anticancer effects. Some of which were even used in adjuvant treatments of many cancers and treating cancers resistant to chemotherapy.
Apoptosis is programmed cell death that is controlled by many gene families that maintain the cell homeostasis managing cell survival and death. One of the hallmarks of a cancer is avoiding cell death. In cancerous cells, apoptosis is disrupted by many factors one of which is mutations in apoptotic and antiapoptotic genes responsible for cell death or survival. Many treatments for cancer aim at activating and inducing apoptotic pathways.
Among these treatments are soybean and black seed have been known for their anti-inflammatory, antioxidant, and anticancer effects in many cancers individually. In this study we investigated the effect of different concentrations and a mixture of soybean and black seed ethanol extract on colon cancer cell line (HT-29) through apoptosis. The groups used were negative control (no extracts), positive control (cisplatin-treated cells), soybean-treated cells (IC50 and IC25), black seed-treated cells (IC50 and IC25) and the mixture (soybean IC25 and black seed IC25). The extracts were analyzed by GC-MS and HPLC, and IC50 was determined by cytotoxic test. Apoptosis was tested via flow cytometry, DNA fragmentation (laddering and quantitative), and gene expression of apoptotic genes. The results showed anticancer activity of each extract and a significant anticancer effect of their mixture. Flow cytometry showed 20.65% apoptotic cells with low necrosis percentage of 9.13% in the mixture group compared to negative control unlike Cisplatin which was 9.4% and 7.8% and Ns50 14.93% and 17.7%, respectively. DNA fragmentation of the mixture showed more bands in the gel compared to other treatments and a highly significant DNA fragmentation percentage of 31.82±0.83 compared to 21.93±0.80 and 26.62±0.92 in Cisplatin and Ns50, respectively. Gene expression analysis exhibited an anticancer effect in mixture group with a significant downregulation in anti-apoptotic genes of 0.15-fold change in P53 (p≤0.001), 0.44-fold change in BIRC5 (p≤0.001), 0.48-fold change in BCL2L1 (p≤0.01) and 0.49-fold change in XIAP (p≤0.01) compared to cisplatin 0.34-fold change (p≤0.001), 0.49-fold change (p≤0.001), 0.63-fold change (p≤0.1), and 0.61-fold change (p≤0.01) in P53, BIRC5, BCL2L1, and XIAP, respectively. On the other hand, apoptotic genes were significantly upregulated with a 6.13-fold change in Bax (p≤0.01) compared to 5.19-fold change (p≤0.1) in cisplatin.
Conclusively, soybean and black seed extracts and their mixture have exerted anticancer activity through induction of apoptosis evidenced by the significant DNA fragmentation, early and late apoptotic cell percentage found in our study and by downregulation of anti-apoptotic genes and significant upregulation of Bax proapoptotic gene. The upregulation of proapoptotic and downregulation of antiapoptotic genes of the intrinsic pathway and the significant DNA fragmentation suggest that the mixture of soybean and black seed, and individual extracts induce the activation of the mitochondrial pathway of apoptosis and that it relies on the caspase-dependent pathway.