## 4 Discussion  
Colon cancer is the third most common cancer and the second leading cause of cancer-related death worldwide [16]. There have been several studies on natural substances to replace or use in conjunction with chemotherapy drugs [17; 18; 19]. Moreover, it has been reported that proteins from nutrients are an efficient source for bioactive peptides with selectively anticancer properties [20; 21; 22; 23]. In the present study, cytotoxicity tests of hydrolyzed riceberry rice bran extracts (HRBE) by MTT assay were performed on human normal fibroblast cell lines (PCS-201-010), non-metastatic colon cancer cell lines (HT-29) and metastatic colon cancer cell lines (SW-620). The percentage of cell viability after treatment decreased when increasing the concentration of the extract (dose-dependent), and considering the IC\({}_{50}\) value was effective when cells were treated with HRBE at 48 and 72 h (time-dependent). A similar observation by [PERSON] et al. (2009) [24] was comparable with our results when the cytotoxicity of purple glutinous rice extract was tested on human hepatocellular carcinoma (HepG2) and prostate cancer (LNCaP) and was found to be more effective at 48 h than 24 h. The efficacy of rice bran extract on colon cancer cells (HCT-116) and breast cancer (HTB-26) was also revealed when the cytotoxicity rate of both cancer cells increased dependent on the time [25]. Focusing on the cell-specific mechanisms, the inhibitory effects on the cell proliferation of the three cell lines were significantly different when treated with HRBE. The IC\({}_{50}\) values of the HRBE treatment when used on HT-29 were significantly higher than those of SW-620. This phenomenon could be explained based on the morphology  
Figure 9: Fluorescence microscope AO/PI double staining of HRBE fractions on SW-620 cells after being treated with 0 mg/mL, considered as negative control (**A**), 2.5 mg/mL (**B**), 5 mg/mL (**C**) and 10 mg/mL (**D**) for 72 h compared to positive control groups treated with 0.05 mg/mL mitomycin C treatment (**E**) and doxorubicin 0.116 mg/mL (**F**). VI: viable cell; blebbing of the cell membrane; CC: chromatin condensation; LA: late apoptosis. of non-metastatic cancer cells and the fact that they have denser adhesion between cells (cell-cell junction) than metastatic cancer so that the loss of the cell junction changes the morphology and leads to the cancer spreading to other organs [26]. This may correlate with our results that show that the HRBE had higher cytotoxic efficiency on SW-620 (metastatic colon cancer cells) than HT-29 (non-metastatic colon cancer cells) and had no effect on PCS-201-010 (normal fibroblast cells). According to the result of the MTT assay, we selected SW-620 cells for the investigation of the apoptotic inductive effect of HRBE confirmed by the pattern of cellular apoptotic morphology using the fluorescence microscope AO/PI double staining test. The apoptosis cell morphology such as membrane blebbing and chromatin condensation in early apoptosis was observed after being treated with HRBE. Notably, the late apoptosis feature was indicated in the red color from PI that binds specifically to DNA but cannot passively traverse into cells that possess an intact plasma membrane because of the differences between the cell membranes of normal and cancer cells [27; 28]. A normal cell membrane contains zwitterion phosphatidylcholine and sphingomyelin in an outer leaflet, and anionic phosphatidylserine and phosphatidylethanolamine in the PCSinner leaflet with asymmetric distribution. In contrast, a cancer cell loses asymmetric distribution, contains more negative charges and pH is altered in the cell membrane [26], the physical changes of which are caused by mutations in the proto-oncogenes and tumor suppressor genes [29].