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Colorectal cancer (CRC) is a very common and deadly cancer worldwide, and oxaliplatin is used as first‐line chemotherapy. However, resistance usually develops, limiting treatment. Echinatin (Ech) is the main component of licorice and exhibits various therapeutic effects on inflammation‐mediated diseases and cancer, ischemia/reperfusion, and liver injuries. The present study elucidated the underlying molecular mechanism of Ech‐induced apoptosis in both oxaliplatin‐sensitive (HT116 and HT29) and ‐resistant (HCT116‐OxR and HT29‐OxR) CRC cells. To evaluate the antiproliferative activities of Ech, we performed MTT and soft agar assays. Ech reduced viability, colony size, and numbers of CRC cells. The underlying molecular mechanisms were explored by various flow cytometry analyses. Ech‐induced annexin‐V stained cells, reactive oxygen species (ROS) generation, cell cycle arrest, JNK/p38 MAPK activation, endoplasmic reticulum (ER) stress, mitochondrial membrane potential depolarization, and multi‐caspase activity. In addition apoptosis‐, cell cycle‐, and ER stress‐related protein levels were confirmed by western blotting. Moreover, we verified ROS‐mediated cell death by treatment with inhibitors such as N‐acetyl‐L‐cysteine, SP600125, and SB203580. Taken together, Ech exhibits anticancer activity in oxaliplatin‐sensitive and ‐resistant CRCs by inducing ROS‐mediated apoptosis through the JNK/p38 MAPK signaling pathway. This is the first study to show that Ech has the potential to treat drug‐resistant CRC, providing new directions for therapeutic strategies targeting drug‐resistant CRC.

Podophyllotoxin (PT), a lignan compound from the roots and rhizomes of Podophyllum peltatum, has diverse pharmacological activities including anticancer effect in several types of cancer. The molecular mechanism of the anticancer effects of PT on colorectal cancer cells has not been reported yet. In this study, we sought to evaluate the anticancer effect of PT on human colorectal cancer HCT116 cells and identify the detailed molecular mechanism. PT inhibited the growth of cells and colony formation in a concentration-dependent manner and induced apoptosis as determined by the annexin V/7-aminoactinomycin D double staining assay. PT-induced apoptosis was accompanied by cell cycle arrest in the G2/M phase and an increase in the generation of reactive oxygen species (ROS). The effects of PT on the induction of ROS and apoptosis were prevented by pretreatment with N-acetyl-L-cysteine (NAC), indicating that an increase in ROS generation mediates the apoptosis of HCT116 cells induced by PT. Furthermore, Western blot analysis showed that PT upregulated the level of phospho (p)-p38 mitogen-activated protein kinase (MAPK). The treatment of SB203580, a p38 inhibitor, strongly prevented the apoptosis induced by PT, suggesting that PT-induced apoptosis involved the p38 MAPK signaling pathway. In addition, PT induced the loss of mitochondrial membrane potential and multi-caspase activation. The results suggested that PT induced cell cycle arrest in the G2/M phase and apoptosis through the p38 MAPK signaling pathway by upregulating ROS in HCT116 cells.

Picropodophyllotoxin (PPT), an epimer of podophyllotoxin, is derived from the roots of Podophyllum hexandrum and exerts various biological effects, including anti-proliferation activity. However, the effect of PPT on colorectal cancer cells and the associated cellular mechanisms have not been studied. In the present study, we explored the anticancer activity of PPT and its underlying mechanisms in HCT116 cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to monitor cell viability. Flow cytometry was used to evaluate cell cycle distribution, the induction of apoptosis, the level of reactive oxygen species (ROS), assess the mitochondrial membrane potential (Δψm), and multi-caspase activity. Western blot assays were performed to detect the expression of cell cycle regulatory proteins, apoptosis-related proteins, and p38 MAPK (mitogen-activated protein kinase). We found that PPT induced apoptosis, cell cycle arrest at the G1 phase, and ROS in the HCT116 cell line. In addition, PPT enhanced the phosphorylation of p38 MAPK, which regulates apoptosis and PPT-induced apoptosis. The phosphorylation of p38 MAPK was inhibited by an antioxidant agent (N-acetyl-L-cysteine, NAC) and a p38 inhibitor (SB203580). PPT induced depolarization of the mitochondrial inner membrane and caspase-dependent apoptosis, which was attenuated by exposure to Z-VAD-FMK. Overall, these data indicate that PPT induced G1 arrest and apoptosis via ROS generation and activation of the p38 MAPK signaling pathway.

Picropodophyllotoxin (PPT) is an epimer of podophyllotoxin (PT), which is a bitter-tasting aryltetralin-type lignan and was the main component in the alcohol-soluble fraction of Podophyllum species (Canel et al., 2000;Gordaliza et al., 2004;Shah et al., 2021). PT targets microtubule assembly and exhibits potent anticancer activity but has severe systemic toxicity in normal cells (Gordaliza et al., 2004;Zhao et al., 2021). Thus, PPT with a low toxicity, 50% lethal dose (LD50) of more than 500 mg/kg in rodents was developed (Girnita et al., 2004;Vasilcanu et al., 2004). Interestingly, despite its high structural

Licochalcone B (LCB) exhibits anticancer activity in oral cancer, lung cancer, and hepatocellular carcinoma cells. However, little is known about its antitumor mechanisms in human oxaliplatin-sensitive and -resistant colorectal cancer (CRC) cells. The purpose of the present study was to investigate the antitumor potential of LCB against human colorectal cancer in vitro and analyze its molecular mechanism of action. The viability of CRC cell lines was evaluated using the MTT assay. Flow cytometric analyses were performed to investigate the effects of LCB on apoptosis, cell cycle distribution, reactive oxygen species (ROS), mitochondrial membrane potential (MMP) dysfunction, and multi-caspase activity in CRC cells. The results demonstrated that LCB induced a reduction in cell viability, apoptosis, G2/M cell cycle arrest, ROS generation, MMP depolarization, activation of multi-caspase, and JNK/p38 MAPK. However, p38 (SB203580) and JNK (SP600125) inhibitors prevented the LCB-induced reduction in cell viability. The ROS scavenger N-acetylcysteine (NAC) inhibited LCB-induced reduction in cell viability, apoptosis, cell cycle arrest, ROS generation, MMP depolarization, and multi-caspase and JNK/p38 MAPK activities. Taken together, LCB has a potential therapeutic effect against CRC cells through the ROS-mediated JNK/p38 MAPK signaling pathway. Therefore, we expect LCB to have promising potential as an anticancer therapeutic and prophylactic agent.

The mechanistic functions of 3-deoxysappanchalcone (3-DSC), a chalcone compound known to have many pharmacological effects on lung cancer, have not yet been elucidated. In this study, we identified the comprehensive anti-cancer mechanism of 3-DSC, which targets EGFR and MET kinase in drug-resistant lung cancer cells. 3-DSC directly targets both EGFR and MET, thereby inhibiting the growth of drug-resistant lung cancer cells. Mechanistically, 3-DSC induced cell cycle arrest by modulating cell cycle regulatory proteins, including cyclin B1, cdc2, and p27. In addition, concomitant EGFR downstream signaling proteins such as MET, AKT, and ERK were affected by 3-DSC and contributed to the inhibition of cancer cell growth. Furthermore, our results show that 3-DSC increased redox homeostasis disruption, ER stress, mitochondrial depolarization, and caspase activation in gefitinib-resistant lung cancer cells, thereby abrogating cancer cell growth. 3-DSC induced apoptotic cell death which is regulated by Mcl-1, Bax, Apaf-1, and PARP in gefitinib-resistant lung cancer cells. 3-DSC also initiated the activation of caspases, and the pan-caspase inhibitor, Z-VAD-FMK, abrogated 3-DSC induced-apoptosis in lung cancer cells. These data imply that 3-DSC mainly increased mitochondria-associated intrinsic apoptosis in lung cancer cells to reduce lung cancer cell growth. Overall, 3-DSC inhibited the growth of drug-resistant lung cancer cells by simultaneously targeting EGFR and MET, which exerted anti-cancer effects through cell cycle arrest, mitochondrial homeostasis collapse, and increased ROS generation, eventually triggering anti-cancer mechanisms. 3-DSC could potentially be used as an effective anti-cancer strategy to overcome EGFR and MET target drug-resistant lung cancer.

Licochalcone H (LCH), a regioisomer of licochalcone C derived from the root of Glycyrrhiza inflata. CRC treatment has always been challenged by the development of resistance. We investigated the antiproliferative activity of LCH in oxaliplatin (Ox)-sensitive and -resistant CRC cells. LCH significantly inhibited cell viability and colony growth in both Ox-sensitive and Ox-resistant CRC cells. We found that LCH decreased epidermal growth factor receptor (EGFR) and AKT kinase activities and related activating signaling proteins including phospho (p)-EGFR and pAKT. A computational docking model indicated that LCH may interact with EGFR, AKT1, and AKT2 at the ATP-binding sites. LCH induced ROS generation, as verified by N-acetyl-L-cysteine (NAC) treatment, and increased the expression of the ER stress markers. LCH treatment of CRC cells induced depolarization of MMP and increased. Multi-caspase activity was induced by LCH treatment and confirmed by Z-VAD-FMK treatment. LCH increased the number of subG1 cells and arrested the cell cycle at the G1 phase. LCH inhibits the growth of Ox-sensitive and Ox-resistant CRC cells by targeting EGFR and AKT, and inducing ROS generation and ER stress-mediated apoptosis. Therefore, LCH could be a potential therapeutic agent for improving not only Ox-sensitive but also Ox-resistant CRC treatment.