Colorectal cancer is one of the leading causes of cancer-related death in Thailand and many other countries. The standard practice for curing this cancer is surgery with an adjuvant chemotherapy treatment. However, the unfavorable side effects of chemotherapeutic drugs are undeniable. Recently, protein hydrolysates and anticancer peptides have become popular alternative options for colon cancer treatment. Therefore, we aimed to screen and select the anticancer peptide candidates from the in silico pepsin hydrolysate of a Cordyceps militaris (CM) proteome using machine-learning-based prediction servers for anticancer prediction, i.e., AntiCP, iACP, and MLACP. The selected CM-anticancer peptide candidates could be an alternative treatment or co-treatment agent for colorectal cancer, reducing the use of chemotherapeutic drugs. To ensure the anticancer properties, an in vitro assay was performed with “CM-biomimetic peptides” on the non-metastatic colon cancer cell line (HT-29). According to the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay results from peptide candidate treatments at 0–400 µM, the IC₅₀ doses of the CM-biomimetic peptide with no toxic and cancer-cell-penetrating ability, original C. militaris biomimetic peptide (C-ori), against the HT-29 cell line were 114.9 µM at 72 hours. The effects of C-ori compared to the doxorubicin, a conventional chemotherapeutic drug for colon cancer treatment, and the combination effects of both the CM-anticancer peptide and doxorubicin were observed. The results showed that C-ori increased the overall efficiency in the combination treatment with doxorubicin. According to the acridine orange/propidium iodine (AO/PI) staining assay, C-ori can induce apoptosis in HT-29 cells significantly, confirmed by chromatin condensation, membrane blebbing, apoptotic bodies, and late apoptosis which were observed under a fluorescence microscope.

Riceberry rice bran is the part of rice that has been scrubbed out during the coloring process. There are various health benefits to be gained from foods with a high protein content. This work aimed to study the effect of hydrolyzed riceberry rice bran extracts on colon cancer cell lines compared to normal cells. The MTT assay result showed that our extract has lower cytotoxicity effects on normal cells (PCS-201-010, IC₅₀ = 6745 µg/mL) compared to colon cancer cell lines and has a greater effect on metastatic cancer cell lines (SW-620, IC₅₀ = 5468 µg/mL) than non-metastatic cancer cell lines (HT-29, IC₅₀ = 6054 µg/mL). The apoptotic inductive effects of HRBE on SW-620 were observed after 72 h at a maximum rate of 76% and at maximum concentration. According to the result of the cell senescence analysis after 24 h of HRBE treatment, the percentage of HT-29 (86%) expressing SA-β-gal was much higher than that of SW-620 (32%). Consequently, the decrease in the cell population in the S and M/G2 phases indicated cell cycle arrest in HT-29 cells after being treated with HRBE. Focusing on the peptide fraction size of HRBE, the largest (>50 kDa) fraction showed the highest anticancer activity compared to other fractions. In conclusion, the hydrolyzed riceberry rice bran extract induced the apoptosis process in the metastatic cancer cells and induced the senescence process in the non-metastatic cancer cells. This observed information will be useful and applicable for medical research and colon cancer treatment in the future.