直肠癌,铁死亡 z-bio 2025-07-09 3758

　　Acevaltrate (ACE) of valerian was screened from a series of natural product libraries with anti-tumor activity. In previous studies, ACE has been shown to have antitumor effects on various types of cancer. In addition, acetylvalerate has been shown to induce apoptosis and inhibit proliferation through the HIF-1α, Otub1/c-Maf and USP10/CCND1 axis. Therefore, ACE is considered a promising natural anti-tumor compound. However, its potential antitumor mechanism is not yet known. On July 7, 2025, Professor Zhang Weidong/Liu Sanhong's team from the Institute of Intersectional Sciences of Shanghai University of Traditional Chinese Medicine published an article titled "Acevaltrate targets PCBP1/2 and GPX4 to induce ferroptosis in colorectal cancer" on STTT (IF 52.7). The natural product acevaltrate (ACE) can rapidly and strongly induce iron death in colorectal cancer cells. ACE not only increases Fe2+ levels in colorectal cancer cells by targeting iron chaperone protein PCBP1/2 and reducing its expression, but also destroys the antioxidant system of colorectal cancer cells by targeting GPX4 and inhibiting its enzymatic activity, resulting in its ubiquitin-mediated degradation. This dual effect makes its effect in inducing ferrody death significantly better than the classic ferrody death inducers.

　　Summary

　　Iron death induced by iron ions (Fe2+) and lipid peroxidation accumulation is a novel form of regulatory cell death and has become a hot topic in tumor treatment research. Identifying small molecule drugs that induce ferrode death in tumor cells is a very attractive therapeutic strategy. Here, we screened a natural product acevaltrate (ACE), acevaltrate (ACE), which rapidly and strongly induces iron death in colorectal cancer cells. ACE not only increases Fe2+ levels in colorectal cancer cells by targeting iron chaperone protein PCBP1/2 and reducing its expression, but also destroys the antioxidant system of colorectal cancer cells by targeting GPX4 and inhibiting its enzymatic activity, resulting in its ubiquitin-mediated degradation. This dual effect of ACE makes its effect in inducing ferrody death significantly better than the classic ferrody death inducers. Our animal experiments show that the therapeutic effect of ACE exceeds the existing drugs that induce ferrodysfunction and is superior to first-line clinical drugs such as capecitabine and TAS-102. Importantly, the inhibitory effect of ACE in colorectal tumor organoids is superior to that at the cellular level, emphasizing its potential for clinical application. This study was the first to discover small molecule inhibitors targeting both PCBP1/2 and GPX4, providing a new therapeutic strategy for eliminating cancer cells through ferrous death.

　　1. ACE inhibits the proliferation of colorectal cancer cells and promotes cell death

　　Inspired by the drug resistance, metastasis and side effects of paclitaxel, oxaliplatin and 5-FU, as well as the specificity and broad-spectrum advantages of natural drugs for cancer treatment, we selected RKO cells, a low-differentiated human colon cancer cell line that is sensitive to cell death, screening for anti-tumor activity in a natural compound library. We incubated RKO cells with different compounds (10 μM) for 24 h and tested their viability by the Cell Counting Kit (CCK-8). As shown in Figures 1a and b, 13 compounds were identified as candidate compounds using criteria with an antitumor activity of more than 80%. Furthermore, by measuring the semi-inhibiting concentration of ACE in different tumor cell lines (IC50), we determined that ACE was the best compound with broad-spectrum antitumor effects (Fig. 1c,d). Surprisingly, we found that ACE was more effective against colorectal cancer cells, with an IC50 value ranging from 1.4 to 1.9 μM, which was almost nontoxic to normal intestinal epithelial cells, suggesting that ACE was more suitable for the treatment of colorectal cancer (Figure 1e-i).

　　To determine the anti-proliferative effect of ACE, we conducted colony formation experiments and 5-ethynyl-20-deoxyuracil nucleoside (EdU) experiments, and the results showed that ACE significantly reduced colony formation efficiency and cell growth (Fig. 1j-m). Furthermore, flow cytometry showed that ACE significantly induces G2/M phase block in HCT116 and RKO cells (Fig. 1n,o). To sum up, ACE inhibits cell proliferation, promotes cell cycle arrest, induces cell death, inhibits cell migration and drug resistance.

　　Figure 1 ACE inhibits cell migration and drug resistance　　

　　2. ACE induces specific ferro death in colorectal cancer cells

　　Although ACE significantly inhibits cell proliferation and induces cell death, unlike previous reports, ACE does not activate the classic apoptotic pathway. We further explore its potential non-apoptotic death mechanism. Next, we used multiomics-based strategies (proteomics, transcriptomics, and metabolomics) to investigate possible pathways for ACE-induced cell death. KEGG enrichment analysis of proteomic data revealed that the ferrodynamic pathway was the most enriched pathway, and the ubiquinone biosynthesis and unsaturated fatty acid biosynthesis pathways associated with ferrodynamic were also enriched in the ACE treatment group (Fig. 2a, b). As expected, transcriptome analysis showed that the ferrody death signaling pathway was significantly activated after ACE treatment (Fig. 2c). Based on the important role of ferrodynamic death in overcoming tumor resistance, we preliminarily hypothesized that ACE may induce ferrodynamics in colorectal cancer cells.

　　Since lipid peroxidation is crucial for ferrous death, we next analyzed changes in oxidized fatty acids and redox agent levels in RKO cells in the presence or absence of ACE. First, we observed that ACE treatment resulted in a significant increase in oxidative polyunsaturated fatty acid levels, especially arachidonic acid (AA) and linoleic acid (LA), reflecting the accumulation of lipid peroxidation products in cells (Fig. 2d, e). Furthermore, as shown in Figure 2f-i, the cytotoxicity of ACE can be blocked by ferrodysfunction inhibitors, but not by apoptosis, necrotic apoptosis, or autophagy inhibitors. These results suggest that ACE-induced specific ferrodymortality is a key determinant of colorectal cancer cell death and is largely dependent on Fe2+.

　　Figure 2 ACE can induce ferrous death in colorectal cancer cells

　　3. ACE induces classical and non-classical ferrodymortality

　　Based on the significantly enriched ferrody death pathway, we verified whether ACE induces ferrodymortality through morphological and biochemical characteristics. Unlike other forms of cell death, lipid peroxidation is a key marker of ferrous death. Two fluorescent probes BODIPY-C11 and Liperfluo were used to detect lipid peroxidation levels mediated by angiotensin-converting enzyme. BODIPY-C11 staining showed that ACE promotes lipid peroxidation stronger than ferrodysfunction-induced drugs RSL3 and erastin, which was reversed by the iron chelator DFO and the lipid peroxidation scavenger Lip-1 (Fig. 3a, b). Furthermore, ACE significantly increased intracellular lipid peroxidation levels in a concentration-dependent manner (Fig. 3c). Furthermore, we detected a significant increase in intracellular MDA levels, a cytotoxic product produced by lipid peroxidative metabolism (Fig. 3d). Furthermore, transmission electron microscopy showed a smaller mitochondria, increased membrane density, and decreased cristae, which indicated the morphological characteristics of ferrodemortized cells (Fig. 3e). Furthermore, ACE dose-dependent disruption of mitochondrial function while significantly inhibiting basal and maximal cellular respiration (Fig. 3f). These results suggest that ACE treatment significantly increases the accumulation of lipid peroxides in colorectal cancer, which in turn induces iron death. Taken together, these data suggest that ACE induces classical ferrody death.

　　Fluorescent probes (FerroOrange) and ferrous ion content determination kits were used to detect free iron ions (Fe2+) in cells. As shown in Figure 3g, h, ACE treatment resulted in a significant increase in Fe2+ concentration in RKO and HCT116 cells, and DFO significantly reversed the effect of ACE increasing Fe2+ levels, whereas Lip-1 did not. Surprisingly, ACE significantly increased intracellular Fe2+ levels compared with ferrodymortality-induced factors RSL3 and erastin (Fig. 3g). Furthermore, multiomic results showed that the most significant upregulation gene was HO-1, which was associated with iron metabolism. This finding was subsequently verified by Western blot experiments (Fig. 3i). When free ferrous ions in cells accumulate in the cytoplasm, ROS is generated through the Fenton reaction, resulting in ferrous death in tumor cells. As shown in Figure 3j, ROS levels significantly increased after ACE treatment and resulted in cell death, while ROS inhibitors n-acetyl-l-cysteine (NAC) and GSH reversed ACE-induced cell death (Figure 3k). These results suggest that Fe2+ plays an important role in ACE-induced ferrodymortality (non-classical ferrodymortality). Taken together, these findings suggest that ACE induces ferrodymortality through classical and non-classical pathways.

　　Figure 3 ACE induces ferrodemortem by inactivating GPX4 and accumulating Fe2+

　　4. ACE inhibits the growth of colorectal tumors in the body

　　Although ACE significantly inhibits GPX4 and increases Fe2+ in colorectal cancer cells, thereby inducing ferrous death, the tumor cytotoxicity of this compound in vivo is not known. To verify the potential in vivo antitumor activity of ACE, mice vaccinated with HCT116-luc tumors were treated with oral treatment with corn oil or ACE once daily for 22 days. The tumor fluorescence intensity of the 25 and 50 mg/kg group was 57.5% and 36.95% of the control group, respectively (Fig. 4a). Furthermore, ACE treatment at 25 and 50 mg/kg significantly reduced tumor size, volume, and weight compared to controls (Fig. 4b-f). There was no significant difference in body weight between the groups, and the immunohistochemical results also showed no obvious toxicity to the heart, liver, spleen, lung and kidney (Fig. 4d). These results suggest that ACE treatment significantly inhibited the growth of HCT116 tumors in mice. We then inoculated RKO cells into nude mice and gave ACE when the tumor volume was close to 50 mm3. Similar to its effect at the cellular level, ACE was better in RKO tumor-bearing mice than in HCT116 mice (Fig. 4g-k).

　　Immunoblot analysis of tumor tissues showed that the number of ki67-positive cells in ACE-treated tumors was significantly lower than that in the control group. Furthermore, ACE did not activate caspase-3, indicating that ACE inhibited tumor growth in mice independent of apoptosis (Fig. 4l). To determine whether ACE induces ferrodystrophy in tumor tissue, we measured the levels of lipid peroxidation products MDA and Fe2+ and found that ACE increased the level of MDA and increased the accumulation of Fe2+ in mouse tumor tissue (Fig. 4m, n). It suggests that ACE can induce ferrous death in tumor cells and effectively inhibit the occurrence of colorectal cancer.

　　Figure 4 ACE induces ferrodemortem in vivo

　　5. The downregulation of PCBP1/2 mediates Fe2+ release and induces ferrodystrophy

　　In order to explore the potential mechanism by which ACE improves Fe2+ levels, based on proteomic results, iron metabolic proteins related to ACE were screened through Western blot (Fig. 5a, b). In addition, we used drug affinity responsive target stability (DARTS) technology to detect the binding of small molecule drugs to target proteins and explore the potential target proteins of ACE. DARTS predicted 240 potential target proteins (intensity ratio ≥1.2), of which 4 genes (TF, GCLM, PCBP1, and SLC39A14) were directly associated with ferrody death (Fig. 5c). It is worth noting that PCBP1 or PCBP2, which have similar functions in iron metabolism regulation, were enriched in both assays. Consistent with the multiomic results, PCBP1 and PCBP2 proteins were significantly downregulated in dose and time-dependent (within 1 h) (Fig. 5a, b). Therefore, we speculate that ACE may increase Fe2+ release through PCBP1 and PCBP2.

　　To verify the relationship between elevated Fe2+ levels in ACE and PCBP1/2, we performed immunofluorescence staining and found that PCBP1 and PCBP2 showed a significant negative correlation with intracellular Fe2+ (Fig. 5d). Next, we designed a specific small interfering RNA (siRNA) to simulate the pharmacological inhibitory effect of ACE on PCBP1/2. Interestingly, PCBP1 and PCBP2 knockdown significantly improved Fe2+ and ROS levels (Fig. 5e–g). Furthermore, in cells lacking PCBP1/2, ACE did not significantly increase Fe2+ levels (Fig. 5h, i). Furthermore, we observed very poor cell status after knockdown, and flow cytometry results indicated increased cell death in colorectal cancer cells (Fig. 5j). These results suggest that ACE may induce ferrodymortality by increasing Fe2+ and ROS by PCBP1 and PCBP2. As expected, overexpression of PCBP1 and PCBP2 significantly inhibited the ability of ACE to increase lipid peroxidation and Fe2+, which further confirmed our speculation (Fig. 5k-m). Furthermore, overexpression of PCBP1 and PCBP2 in RKO cells significantly promoted cell proliferation and significantly inhibited the cytotoxicity of ACE (Fig. 5n,o). These results suggest that ACE induces ferrous death in colorectal cancer through PCBP1 and PCBP2.

　　Figure 5 PCBP1/2 mediates ace-induced Fe2+ accumulation and ferrodemortem

　　6. ACE binding to PCBP1/2 induces ferrodystrophy

　　To study whether ACE directly binds to PCBP1 and PCBP2, we used Cell Thermal Shift Assay (CETSA) to detect whether the thermal stability of proteins changed after ACE treatment. As shown in Figure 6a and b, the thermal stability of PCBP1 and PCBP2 proteins decreased after ACE treatment, which is consistent with the results of DARTS. Furthermore, we determined the affinity between PCBP1 and ACE by surface plasmon resonance (SPR), and the results showed that ACE directly binds to PCBP1 with a KD value of 0.8464 μM (Fig. 6c). Furthermore, ACE slowly dissociated from the PCBP1 protein, indicating that ACE binds to the PCBP protein in a more stable manner (Fig. 6c). Furthermore, DTT, a cysteine-rich thiol donor, can compete for binding to cysteine-dependent ACE of PCBP1/2. As shown in Figure 6d,e, the downregulation of PCBP1/2 and the increase in cytotoxicity caused by ACE are partially reversed by the addition of excess DTT, indicating that ACE directly binds to the cysteine residues of PCBP. We then simulated ACE covalent binding to PCBP1/2 through its cysteine residues using Molecular Operating Environment (MOE) software. The binding mode of ACE to each cysteine residue site of PCBP1 (AlphaFold ID AF-Q15365-F1) and PCBP2 (AlphaFold ID AF-Q15366-F1) was explored through docking simulation. As shown in Figure 6f, a stable hydrogen bond formed between Cys54 and the ACE backbone, indicating that Cys54 may be the binding site of PCBP1 and PCBP2. Furthermore, Cys293 may be another binding site for PCBP1. To further confirm the direct binding of ACE to PCBP1/2, we overexpressed wild-type PCBP1/2, Cys54 mutant PCBP1, Cys293 mutant PCBP1 and GFP-tagged Cys54 mutant PCBP2, and then analyzed the direct binding of ACE to PCBP1/2 by microcalorimetry (MST). As expected, ACE is strongly combined with PCBP1/2, with estimated Kd values of 4.64 nM and 204 nM. When Cys293 mutates to alanine, its Kd is 325 times that of wild-type PCBP1, while when Cys54 mutates to alanine, neither ACE binding was detected by PCBP1 and PCBP2 (Fig. 6h, i). We consistently found that the cytotoxicity of ACE was reduced in RKO cells overexpressing PCBP1/2 C54A (Fig. 6j). These results show that ACE increases Fe2+ levels by directly binding to PCBP1 and PCBP2, thereby inducing iron death in colorectal cancer.

　　Figure 6 Direct combination of ACE with PCBP1/2

　　7.ACE is a natural ferrodysfunction inducer

　　Since upregulation of a series of antioxidant genes and factors was observed in multiomic data, we detected changes in expression of some antioxidant genes. Consistent with the multiomic results, ACE significantly upregulated the expression of nuclear factor erythrocyte-derived 2-like protein 2 (Nrf2) and its downstream target genes (such as SLC3A2, SLC7A11 and GCLM), ferrodysfunction inhibitor protein 1 (FSP1), and dihydroorotate dehydrogenase (DHODH). Furthermore, we found that expression of the core protein GPX4, which is a ferrodemortem defense mechanism, was significantly downregulated (Fig. 7a,b). Inactivation of GPX4 can occur through two processes: intracellular glutathione (GSH) depletion or direct targeting of GPX4. As shown in Figure 7c, ACE treatment increased the levels of GSH and cystine in RKO cells. Furthermore, ACE rapidly increased ROS levels, inhibiting GPX enzyme activity, suggesting that ACE may bind directly to GPX4 like RSL3 (Fig. 7d). As expected, the lack of GPX4 significantly promoted lipid peroxidation accumulation and cell death (Fig. 7e,f). However, GPX4 overexpression increased the IC50 value of ACE in RKO cells (Fig. 7g). Excess DTT reversed the downregulation of GPX4, confirming the binding of ACE to the cysteine residues of GPX4 (Fig. 7h). As shown in Figure 7i, we performed a similar molecular docking simulation and found that the active site selenocysteine (U46) is the most favorable site for covalent binding of ACE (Protein Data Bank ID 6NH3) to GPX4. Furthermore, the thermal stability of GPX4 protein increased after ACE and RSL3 (positive control) treatment, indicating that ACE can bind and stabilize GPX4 protein (Fig. 7j). Finally, we performed MST analysis of gfp-tagged GPX4 in wild-type and destructive mutant U46. The Kd value of ACE-bound GPX4 protein was estimated to be 196 nM (Fig. 7i). However, once U46 is mutated to alanine, the binding between GPX4 and ACE disappears, which further reduces the cytotoxicity of ACE (Fig. 7i). These results suggest that ACE inactivates GPX4 by directly binding to U46 in GPX4, triggering lipid peroxidation and subsequent ferrous death.

　　Next, we studied the mechanism by which ACE triggers the degradation of GPX4 protein. Western blot and real-time PCR results showed that ACE not only downregulates GPX4 expression, but also downregulates its mRNA levels (Fig. 7a, b). In this study, we mainly investigated post-translational regulation of GPX4 because the protein levels of GPX4 drop faster than their RNA levels (Fig. 7b). As shown in Figure 7k,l, GPX4 half-life in ACE-treated cells was faster than that in untreated cells when combined with the protein translation inhibitor cycloheximide (CHX), indicating that ACE mainly degrades GPX4 at the protein level. In addition, Western blot experiments showed that the proteasome inhibitor MG132 reversed the degradation of GPX4 by ACE, while CQ had no reversal effect on the degradation of GPX4 (Fig. 7m). Furthermore, IP experiments showed that ACE increased ubiquitination of GPX4 (Fig. 7n). ACE mediates GPX4 degradation through the ubiquitin-proteasome pathway, inducing ferrous death in colorectal cancer cells.

　　Finally, the cytotoxicity of RSL3 disappeared, and when GPX4 was knocked down, the cytotoxicity of ACE was partially reduced because RSL3 did not increase Fe2+ (Fig. 7o, p). As shown in Figure 41, the levels of GPX4, PCBP1 and PCBP2 proteins in tumor tissues showed a dose-dependent reduction, which is consistent with the results of in vitro experiments. These results underline the effectiveness of the dual mechanisms of ACE inducing ferrody death in colorectal cancer cells, namely inactivate GPX4 and downregulate PCBP1/2 release of Fe2+.

　　Figure 7 ACE-induced GPX4 depletion leads to ferrodystrophy

　　8. Protein expression levels of PCBP1/2 and GPX4 in human tumor tissues

　　To verify the potential of the above model for clinical tumor treatment, we compared the tumor suppression effects of ACE with clinical first-line drugs and ferrodyphobic drugs in the xenograft model. We found that the tumor suppressor effect of ACE was significantly greater than that of the ferrodymortality-positive drugs sorafenib and artemisinin, and even better than the clinical first-line drugs capecitabine and TAS-102, because ACE had significantly better effects on elevating Fe2+ and lowering PCBP2 and GPX4 than other treatments (Figure 8a–d). Although ACE induces ferrodystrophy in tumor cells by targeting PCBP1/2 and GPX4, studies have shown that ferrodystrophy can cause acute liver and acute renal injury. To evaluate the safety of ACE in treating tumors, we further performed acute toxicology experiments, administering ACE (50 mg/kg) orally in mice for 10 days per day. As shown in Supplementary Table 1, there were no significant changes in the blood parameters of mice such as red blood cells, white blood cell count, and platelet count; liver and kidney injury indexes ALT, AST, BUN, Cr, etc. were all within the normal range (Fig. 8e). Furthermore, we examined Fe2+ levels in the heart, liver, and kidney tissue of mice and found no significant differences between heart, liver, and kidney tissues of the ace-treated mice and control groups (Fig. 8f). These results indicate that there is no obvious risk of inducing ferrody death in normal tissues at therapeutic doses of ACE, and the clinical application of ACE provides a solid foundation for translational medicine.

　　Construct a colorectal carcinoma organoid model to evaluate the sensitivity of ACE. After 6 days of culture with different concentrations of ACE (0.01, 0.1, 1, 5, 10, 25, 50 μM), the size of living cells represented by green fluorescence was significantly reduced, and the number of dead cells represented by red fluorescence was significantly increased, indicating that the growth of organoids was significantly inhibited, as shown in Figure 8g. The sensitivity of organoids was measured by the area method, and the IC50 values of ACE for the three organoid models were 58, 148 and 340 nM, respectively (Fig. 8h). Overall survival analysis of patients with colorectal cancer showed that overall survival was significantly worse in patients with high levels of GPX4, PCBP1 and PCBP2 (Fig. 8i). Furthermore, we studied the protein expression of PCBP1, PCBP2 and GPX4 in colon cancer patients by immunofluorescence staining (Fig. 8j). These findings suggest that PCBP1 and PCBP2 have potential clinical significance as biomarkers for tumor diagnosis.

　　Figure 8 Targeting PCBP1/2 as a potential tumor suppression strategy

　　in conclusion

　　To sum up, our study shows that ACE induces ferrody death in tumor cells through dual mechanisms: on the one hand, ACE enhances Fe2+-induced ferrodymortality in cells by directly binding to and degrading PCBP 1/2, and on the other hand, ACE induces ubiquitination and degradation of GPX4 by directly binding to GPX4. In addition, ACE showed strong anti-tumor effects in mouse colon cancer models and even human colon cancer organoid models. The dual-target mechanism of ACE not only avoids the compensatory drug resistance of single-target inducers, but also achieves efficient and low-toxic tumor selective killing through the multi-target characteristics of natural products. We look forward to this dual mechanism providing new strategies for the clinical management of colorectal cancer.