Caesalpinia sappan L. has exhibited various pharmacological effects, yet its anticancer activities against colorectal cancer (CRC) and underlying molecular mechanisms remain unclear. This study investigated the anticancer properties of an ethanol extract of C. sappan L. (CSE) against CRC cells, focusing on the identification of bioactive compounds and their mechanisms of action. A network pharmacology analysis was conducted to identify potential CRC targets and bioactive compounds of CSE, using LC-MS for compound identification. The anticancer effects of CSE were then validated through in vitro and in vivo models of CRC. The network pharmacological approach identified 87 overlapping genes between CSE targets and CRC-related genes, with protein–protein interaction analysis highlighting 33 key target genes. CSE inhibited cell proliferation in human CRC cell lines, including HCT 116, KM12SM, HT-29, and COLO 205, and induced apoptosis via caspase 3/7 activation. Western blot analyses confirmed the modulation of critical signaling pathways, including STAT3, AKT, and mitogen-activated protein kinases. Furthermore, CSE significantly suppressed tumor growth in MC38 CRC-bearing mice. These findings suggest that CSE possesses substantial potential as a natural anticancer agent for CRC treatment, highlighting the need for further exploration in therapeutic development.

Bojungikki-Tang (BJIKT) is traditionally used to enhance digestive function and immunity. It has gained attention as a supplement to chemotherapy or targeted therapy owing to its immune-boosting properties. This study aimed to evaluate the synergistic anti-tumor effects of BJIKT in combination with pembrolizumab in a preclinical model. MHC I/II double knockout NSG mice were humanized with peripheral blood mononuclear cells (PBMCs) and injected subcutaneously with H460 lung tumor cells to establish a humanized tumor model. Both agents were administered to evaluate their impact on tumor growth and immune cell behavior. Immunohistochemistry showed decreased exhaustion markers in CD8(+) and CD4(+) T cells within the tumor, indicating enhanced T cell activity. Additionally, RNA sequencing, transcriptome analysis, and quantitative PCR analysis were performed on tumor tissues to investigate the molecular mechanisms underlying the observed effects. The results confirmed that BJIKT improved T cell function and tumor necrosis factor signaling while suppressing transforming growth factor-β signaling. This modulation led to cell cycle arrest and apoptosis. These findings demonstrate that BJIKT, when combined with pembrolizumab, produces significant anti-tumor effects by altering immune pathways and enhancing the anti-tumor immune response. This study provides valuable insights into the role of BJIKT in the tumor microenvironment and its potential to improve therapeutic outcomes.

Immunogenic cell death (ICD) converts dying tumor cells into an anticancer vaccine, yet whether protection depends on how those cells are prepared and when they are given has rarely been examined. Using mitoxantrone (MTX), a clinically approved small molecule classified as an ICD inducer, we examined both variables in the murine CT-26 colon carcinoma model. MTX reduced viability in a concentration-dependent manner and induced extracellular ATP release. The total extent of cell death, however, was similar across concentrations, indicating that the concentration-dependent loss of viability was not accompanied by a proportional increase in cell death. What differed was the composition of the dying population, which shifted from early to late apoptotic cells as the concentration increased. Prophylactic vaccination with MTX-treated cells protected BALB/c mice, leaving 75.0–87.5% of animals tumor-free after vaccination at 1 μM and 37.5–62.5% at 3 μM across independent experiments. Protection increased with the interval between vaccination and challenge, with 0%, 50.0%, and 87.5% of animals tumor-free after simultaneous injection, a 3-day interval, and a 7-day interval, respectively. Cells killed completely by freeze–thaw were inactive as a vaccine, and cisplatin-treated cells conferred only a marginal delay, whereas responders to MTX-treated cells resisted rechallenge for the remainder of the observation period. Taken together, these results show that use of an ICD-inducing drug alone is not sufficient for effective dying-cell vaccination and that protective efficacy is influenced by vaccine preparation and by the interval between vaccination and tumor challenge, rather than by the extent of cell death alone.

Plant-derived saponins have attracted significant interest for their potential to promote apoptotic cell death and enhance antitumor immune responses through immunogenic cell death (ICD). Akebia quinata, a saponin-rich medicinal plant, exhibits diverse pharmacological properties; however, studies on its seeds are limited, and their immunomodulatory activity in cancer remains largely unexplored. In this study, A. quinata seeds were extracted using 70% ethanol, and the phytochemical profile was characterized using UHPLC–QTOF MS/MS. We investigated the anticancer properties of A. quinata seed extract (AQSE), focusing on its role in inducing apoptosis and ICD in non-small cell lung cancer (NSCLC). In human NSCLC cell lines (A549 and H460), AQSE exhibited potent cytotoxic effects in a dose-dependent manner. Flow cytometric analysis confirmed the induction of apoptosis, evidenced by a significant increase in Annexin V-positive cells and an elevated sub-G1 population. Mechanistically, AQSE treatment induced cell death by simultaneously inhibiting the survival-promoting MEK/ERK/CREB axis and activating the stress-responsive JNK pathway. Furthermore, AQSE triggered hallmark features of ICD, characterized by surface exposure of calreticulin and the release of extracellular HMGB1 and ATP. Most importantly, an in vivo vaccination assay using a syngeneic mouse model demonstrated that immunization with AQSE-treated dying cells significantly suppressed tumor growth upon rechallenge, confirming the establishment of antitumor immunological memory. Additionally, bioassay-guided fractionation revealed that the anticancer activity was primarily concentrated in the ethyl acetate fraction. These findings suggest that AQSE exerts anticancer effects via the induction of apoptosis and ICD, highlighting its potential as a promising natural candidate for the development of novel therapeutic strategies against NSCLC.