## 4 Discussion
This is the reason why PI can indicate late apoptosis including the efficacy of the extract on cancer cells and the causes of the differences in the effect of the activity of the HRBE between HT-29 (non-metastatic) and SW-620 (metastatic) on the quantity of peptide entering the cells. In apoptotic cells, phosphatidylserine phospholipid is flipped from the inner to the outer leaflet of the plasma membrane to signal for nearby phagocytic cells. Annexin V is a calcium-dependent phospholipid-binding protein with a high affinity for phosphatidylserine [14]. Therefore, the percentage of apoptosis on SW-620 after being treated with HRBE can be detected by Annexin V and flow cytometry. Our result shows that the HRBE at concentrations of 1.25, 2.5, 5 and 10 mg/mL significantly increased the induction of cell apoptosis. This is consistent with previous studies on the extracts of other rice such as the aqueous extract of brewers' rice (WBR) on HT-29, which showed characteristics of apoptosis including early and late apoptosis at 48 h [30] and induced apoptosis by the significant activation of caspase-3 and \(-8\) activities [31]. The methanol extract of the purple rice cultivar exhibited the highest inhibitory growth effect on human hepatocellular carcinoma cells and the greatest ability to induce apoptosis via the mitochondrial pathway with the loss of mitochondrial transmembrane potential (MTP) and the activation of caspase-3 and -9 [32]. Moreover, the extraction of chemical components from the riceberry rice bran induced HL-60 (promyelocytic leukemia cell line) and Caco-2 (colorectal adenocarcinoma) to apoptosis via the p53 protein that increased due to the activation of caspase-3 [3]. p53 is a this tumor-suppressing protein that regulates genes to control the cell cycle in normal cells and induces cell cycle arrest for the repair of DNA, cellular senescence and programmed cell death such as apoptosis to remove irreversibly damaged cells [33]. Therefore, the interesting study of cell cycle arrest (senescence) was also a focus in this particular study. Cellular senescence is the stage of irreversible cell cycle arrest and can be observed by SA-\(\beta\)-gal staining. Our results showed that the cellular senescence inductive effects of the HRBE on HT-29 were higher than those on SW-620. This indicates that HRBE is capable not only of apoptosis induction but also cell senescence induction. Senescence cells could have initially undergone cell cycle arrest that decreased the number of cells in the S and M/G2 phases and increased the number of cells in the G1/G0 phase. For this particular quantitative measurement, flow cytometry can determine the percentage of cells in each cell phase using propidium iodide (PI) and RNase. PI has specific DNA stunning that can indicate the different stages of the cell cycle because the cells in the G1 phase have a constant DNA content (1\(\times\)), while the cells in the G2/M phase have a constant DNA (2\(\times\)) that is twice the intensity of the G1 phase [33]. According to the flow cytometry result in this study, the HRBE increased the number of cells in the G0/G1 phase and decreased the number of cells in the S, G2 and M phases, which also indicates the senescence inductive effect of HRBE on the HT-29 cell line. Since the cyclin-dependent kinases (CDKs) and cyclin are regular proteins controlling the cell cycle, and CDK-cyclin complexes can be regulated by a group of proteins called cyclin-dependent kinase inhibitors (CDKIs) such as p21 that is downregulated by p53, p21 could induce G1 cell cycle arrest by inhibiting the activity of the cyclin-CDK2/4 complexes, E2F and PCNA [34]. This phenomenon correlates with the previous study on HL-60, MCF-7 and Caco-2, in which Cyclin B1 (a complex with Cdk1 controlling the S to G2 phases in the cell cycle) was significantly decreased after being treated with the dichloromethane extract of riceberry bran [3]. Similarly, the cell population at the sub-G0 phase of the HT-29 cells increased after the cells were treated with brewers' rice extract for 48 h [30].