OBJECTIVE: Huachansu injection (HCSI), a promising anti-cancer Chinese medicine injection, has been reported to have the potential for reducing the toxicity of chemotherapy and improving the quality of life for colorectal cancer (CRC) patients. The objective of this study is to explore the synergistic and detoxifying effects of HCSI when used in combination with irinotecan (CPT-11). METHODS: To investigate the effect of HCSI on anti-CRC efficacy and intestinal toxicity of CPT-11, we measured changes in the biological behavior of LoVo cells in vitro, and anti-tumor effects in LoVo cell xenograft nude mice models in vivo. Meanwhile, the effect of HCSI on intestinal toxicity and the uridine diphosphate-glucuronosyltransferase 1A1 (UGT1A1) expression was investigated in the CPT-11-induced colitis mouse model. Subsequently, we measured the effect of HCSI and its 13 constituent bufadienolides on the expression of UGT1A1 and organic anion transporting polypeptides 1B3 (OATP1B3) in HepG2 cells. RESULTS: The combination index (CI) results showed that the combination of HCSI and CPT-11 exhibited a synergistic effect (CI < 1), which significantly suppressing the LoVo cell migration, enhancing G2/M and S phase arrest, and inhibiting tumor growth in vivo. Additionally, the damage to intestinal tissues was attenuated by HCSI in CPT-11-induced colitis model, while the increased expression of UGT1A1 in HepG2 cells and in mouse was observed. CONCLUSION The co-therapy with HCSI alleviated the intestinal toxicity induced by CPT-11 and exerted an enhanced anti-CRC effect. The detoxifying mechanism may be related to the increased expression of UGT1A1 and OATP1B3 by HCSI and its bufadienolides components. The findings of this study may serve as a theoretical insights and strategies to improve CRC patient outcomes. Please cite this article as: Jiang B, Meng ZY, Hu YJ, Chen JJ, Zong L, Xu LY, Zhang XQ, Zhang JX, Han YL. Huachansu injection enhances anti-colorectal cancer efficacy of irinotecan and alleviates its induced intestinal toxicity through upregulating UGT1A1-OATP1B3 expression in vitro and in vivo. J Integr Med. 2025; 23(5):576-590.
Irinotecan/therapeutic use* ; Animals ; Glucuronosyltransferase/genetics* ; Humans ; Colorectal Neoplasms/metabolism* ; Drugs, Chinese Herbal/therapeutic use* ; Mice, Nude ; Mice ; Up-Regulation/drug effects* ; Male ; Xenograft Model Antitumor Assays ; Mice, Inbred BALB C ; Hep G2 Cells ; Cell Line, Tumor ; Intestines/drug effects* ; Amphibian Venoms

OBJECTIVE: To explore the correlation between chromosome 8 open reading frame 76 (C8orf76) and cyclin-dependent kinase 4 (CDK4) and the potential predictive effect of C8orf76 and CDK4 on the prognosis of colorectal cancer (CRC). METHODS: We constructed a protein-protein interaction network of C8orf76-related genes and analyzed the prognostic signatures of C8orf76 and CDK4. Clinicopathological features of C8orf76 and CDK4 were visualized using a nomogram. RESULTS: C8orf76 and CDK4 levels were positively correlated in two independent human CRC cohorts ( n = 83 and n = 597). A consistent positive correlation was observed between C8orf76 and CDK4 expression in the CRC cell lines. The nomogram included prognostic genes (C8orf76 and CDK4) and pathological N and M stages. The concordance index (C-index) in our cohort was 0.776, which suggests that the ability of the indicators to predict the overall survival of patients with CRC in our cohort was strong. CONCLUSION We found that C8orf76 was positively correlated with CDK4 in both the cohorts as well as in CRC cell lines. Therefore, C8orf76 and CDK4 can be used as potential biomarkers to predict the prognosis of CRC.
Humans ; Colorectal Neoplasms/diagnosis* ; Cyclin-Dependent Kinase 4/metabolism* ; Prognosis ; Male ; Female ; Middle Aged ; Biomarkers, Tumor/genetics* ; Aged ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic

OBJECTIVES: Oxaliplatin (OXA) and 5-fluorouracil (5-FU) are 2 commonly used chemotherapeutic agents for colorectal cancer (CRC). MicroRNAs (miRNAs, miRs) play crucial roles in the development of chemoresistance in various cancers. However, the role and mechanism of miR-224-5p in regulating CRC chemoresistance remain unclear. This study aims to investigate the function of miR-224-5p in chemoresistant CRC cells and the underlying mechanisms. METHODS: CRC datasets GSE28702 and GSE69657 were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed miRNAs between drug-sensitive and resistant groups (OXA or 5-FU) were analyzed, and miR-224-5p was identified as the target miRNA. Chemoresistant cell lines HCT15-OXR, HCT15-5-FU, SW480-OXR, and SW480-5-FU were established. Transient transfections were performed using miR-224-5p mimics, inhibitors, and their respective negative controls (control mimic, control inhibitor) in these cell lines. Cells were treated with different concentrations of OXA or 5-FU post-transfection, and the half-maximal inhibitory concentration (IC₅₀) was determined using the cell counting kit-8 (CCK-8) assay. Cell proliferation was assessed by CCK-8 and colony formation assays. The expression levels of miR-224-5p, LC3, and P62 were measured by real-time polymerase chain reaction (real-time PCR) and/or Western blotting. Autophagic flux was assessed using a tandem fluorescent-tagged LC3 reporter assay. TargetScan 8.0, miRTarBase, miRPathDB, and HADb were used to predict B-cell lymphoma-2 (Bcl-2) as a potential miR-244-5p target, which was further validated by dual-luciferase reporter assays. RESULTS: Chemoresistant CRC cells exhibited down-regulated miR-224-5p expression, whereas up-regulation of miR-224-5p enhanced chemotherapy sensitivity. Exposure to OXA or 5-FU significantly increased autophagic activity in chemoresistant CRC cells, which was reversed by miR-224-5p overexpression. Dual-luciferase assays verified Bcl-2 as a direct target of miR-224-5p. CONCLUSIONS MiR-224-5p regulates chemoresistance in CRC by modulating autophagy through direct targeting of Bcl-2.
Humans ; MicroRNAs/physiology* ; Colorectal Neoplasms/drug therapy* ; Drug Resistance, Neoplasm/genetics* ; Autophagy/drug effects* ; Fluorouracil/pharmacology* ; Oxaliplatin ; Cell Line, Tumor ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Gene Expression Regulation, Neoplastic

OBJECTIVES: To explore the expression of hexokinase 2 (HK2) in colorectal cancer (CRC) and its possible mechanisms for regulating tumor cell behaviors and tumor immune microenvironment. METHODS: We analyzed HK2 expression in CRC and its impact on patient prognosis and tumor immune microenvironment using public databases. HK2 expression was also examined in 8 CRC and paired adjacent tissues using immunohistochemistry, Western blotting and RT-qPCR. In cultured CRC cell lines CT26 and HCT116 with low HK2 expression, the effects of lentivirus-mediated HK2 overexpression and JAK/STAT3 inhibitors on cell proliferation, migration, and invasion were assessed using CCK-8 assay, colony formation assay and Transwell assay and in a subcutaneous tumor-bearing mouse model; the changes were also observed in MC38 and CACO2 cells with high HK2 expressions following treatment with HK2 inhibitor 3-BP. Western blotting was performed to verify the relationship between HK2 and JAK/STAT signaling pathway protein expressions. RESULTS: Informatics analyses suggested that HK2 expression was significantly higher in CRC tissues than in adjacent tissues (P<0.001), and patients with high HK2 expressions had worse prognosis (P=0.09). In the 8 clinical CRC tissues, HK2 expressions were significantly higher in the tumor tissues than in the adjacent tissues (P<0.01). In CT26 and HCT116 cells, HK2 overexpression significantly enhanced cell proliferation, migration and invasion, while in HK2-overexpressing MC38 and CACO2 cells, inhibiting HK2 with 3-BP strongly suppressed these changes. HK2 overexpression promoted STAT3 phosphorylation, and JAK/STAT3 inhibitors effectively suppressed tumor cell proliferation, migration and invasion. TIMER and MCPcounter analyses indicated correlations between HK2 and immune cells, and TCGA and GEO analyses suggested significant positive correlations between HK2 and the immune checkpoints including PDCD1. CONCLUSIONS HK2 is upregulated in CRC to promote tumor cell proliferation, migration and invasion possibly by activating the JAK-STAT signaling pathway and modulating tumor immune microenvironment.
Humans ; Colorectal Neoplasms/metabolism* ; Cell Proliferation ; Hexokinase/genetics* ; Tumor Microenvironment ; Cell Movement ; Signal Transduction ; Animals ; STAT3 Transcription Factor/metabolism* ; Mice ; Neoplasm Invasiveness ; Cell Line, Tumor ; Janus Kinases/metabolism* ; HCT116 Cells ; Caco-2 Cells

Colorectal cancer (CRC), a major global health concern, necessitates innovative treatments. Chimeric antigen receptor (CAR) T cells have shown promises, yet they grapple with challenges. The spotlight pivots to the rising heroes: CAR natural killer (NK) cells, offering advantages such as higher safety profiles, cost-effectiveness, and efficacy against solid tumors. Nevertheless, the specific mechanisms underlying CAR NK cell trafficking and their interplay within the complex tumor microenvironment require further in-depth exploration. Herein, we provide insights into the design and engineering of CAR NK cells, antigen targets in CRC, and success in overcoming resistance mechanisms with an emphasis on the potential for clinical trials.
Colorectal Neoplasms/immunology* ; Humans ; Killer Cells, Natural/metabolism* ; Receptors, Chimeric Antigen/genetics* ; Immunotherapy, Adoptive/methods* ; Tumor Microenvironment/immunology* ; Animals

Humans ; Proto-Oncogene Proteins p21(ras)/genetics* ; Prognosis ; Biomarkers, Tumor/genetics* ; Mutation/genetics* ; Colorectal Neoplasms/genetics* ; Proto-Oncogene Proteins B-raf/metabolism*

This study explored the generation site and regulation mechanism of reactive oxygen species(ROS) in the apoptosis of colorectal cancer cells induced by furanodienone(Fur). RKO cells were treated with 200 μmol·L~(-1) of Fur, and the changes in intracellular nicotinamide adenine dinucleotide phosphate oxidase(NOX) activity were detected by the NOX activity detection method. The control group, Fur group, diphenyleneiodonium(DPI) inhibitor group for general NOX, mitochondrial-targeted antioxidant(MitoTEMPO) group, Fur+DPI group, Fur+MitoTEMPO group, and H_2O_2 positive control group were set up. Intracellular ROS levels were detected by the ROS fluorescent staining method, and NOX1-NOX5 protein expressions were detected by Western blot. The NOX1-specific inhibitor ML171 and NOX4-specific inhibitor(GLX351322) were further introduced, and the cell activity was determined by cell counting kit-8(CCK-8) assay. The effects of ROS level change on the protein expressions of NOX4 and peroxiredoxin 1(PRDX1) were measured by Western blot. BAY11-7082, which is an inhibitor of the inhibitor of nuclear factor κB protein α(IκBα), was used to explore the effect of the expression of phosphorylated nuclear factor κB(p-NF-κB) in the nucleus after the Fur treatment on the NOX4 protein level. The lentiviral plasmid and empty plasmid for PRDX1 gene silencing were constructed to transfect RKO cells, and stably transfected strains were screened. The impact of PRDX1 gene knockout on Fur-induced apoptosis was further analyzed using the flow cytometry assay. The findings demonstrate a considerable increase in mitochondrial ROS level in response to Fur treatment, with an increase in intracellular NOX activity. However, the mitochondrial ROS level is significantly reduced in the Fur+DPI group. The results from Western blot and CCK-8 analysis suggest that intracellular NOX1 and NOX4 protein expressions are elevated by Fur treatment, and GLX351322 effectively reverses the pro-apoptotic effect of Fur, while ML171 has a minimal impact on apoptosis rate. Meanwhile, Fur significantly boosts the level of p-NF-κB in the nucleus, whereas the protein levels of p-NF-κB and NOX4 are reduced after the BAY treatment. The regulation of Fur on NOX4 and PRDX1 protein expressions is negatively correlated. In the stably transfected cell strain with PRDX1 gene knockout, the apoptosis rate is considerably higher than that of the negative control group after Fur treatment. The above results indicate that Fur can induce the apoptosis of colorectal cancer cells by promoting the signal transduction of NF-κB in the nucleus and increasing the generation of mitochondrial ROS derived from NOX4 to inhibit the PRDX1 protein expression.
Humans ; Peroxiredoxins/metabolism* ; Apoptosis/drug effects* ; Reactive Oxygen Species/metabolism* ; NADPH Oxidase 4/metabolism* ; Mitochondria/genetics* ; Cell Line, Tumor ; Colorectal Neoplasms/drug therapy* ; NADPH Oxidases/metabolism* ; Furans/pharmacology*

Objective To investigate the effect of insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) on the proliferation, migration and tumor immune microenvironment of colorectal cancer cells and its possible molecular mechanism. Methods The Cancer Genome Atlas (TCGA) database was used to analyze the expression levels of IGF2BP2 and MYC in colorectal cancer and adjacent tissues. The expression of IGF2BP2 in HCT-116 and SW480 human colorectal cancer cells was silenced by RNA interference (RNAi), and the silencing effect was detected by quantitative real-time PCR. After knocking down IGF2BP2, colony formation assay, CCK-8 assay and 5-ethynyl-2'-deoxyuridine (EdU) assay were employed to detect cell colony formation and proliferation ability. Transwell^TM assay was used to detect cell migration ability. Quantitative real-time PCR was used to detect the mRNA expression of IGF2BP2, MYC, tumor necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β) and interleukin-10 (IL-10). The protein expression of IGF2BP2 and MYC was detected by western blot. The binding ability of IGF2BP2 and MYC in HCT-116 cells was detected by quantitative real-time PCR after RNA immunoprecipitation. Results The results of TCGA database showed that the expression of IGF2BP2 and MYC in colorectal cancer tissues was significantly higher than that in adjacent tissues, and the survival time of colorectal cancer patients with high expression of IGF2BP2 was shorter. After silencing IGF2BP2, the viability, proliferation and migration of HCT-116 and SW480 cells were decreased. The mRNA expression of MYC, TGF-β and IL-10 in IGF2BP2 knockdown group was significantly decreased, while the expression of TNF-α mRNA was increased. The expression of MYC protein and the stability of MYC mRNA were significantly decreased. RIP-qPCR results showed that IGF2BP2 could bind to MYC mRNA. Conclusion Knockdown of IGF2BP2 inhibits colorectal cancer cell proliferation, migration and promotes tumor immunity by down-regulating MYC expression.
Humans ; Cell Line, Tumor ; Cell Movement/genetics* ; Cell Proliferation/genetics* ; Colorectal Neoplasms/metabolism* ; Gene Expression Regulation, Neoplastic ; Interleukin-10/metabolism* ; RNA, Messenger ; RNA-Binding Proteins/metabolism* ; Transforming Growth Factor beta/genetics* ; Tumor Microenvironment/immunology* ; Tumor Necrosis Factor-alpha/metabolism* ; Proto-Oncogene Proteins c-myc/metabolism*

Objective To investigate how the neutrophil extracellular traps (NETs) affect the proliferation and migration of mouse MC38 colorectal cancer cells and its mechanism. Methods Spleen neutrophils were extracted in mouse, followed by collection of NETs after ionomycin stimulation in vitro. The proliferation of MC38 cell was detected by CCK-8 assay, and migration ability were detected by Transwell^TM and cell scratch assay, after co-incubation with MC38 cells. The mRNA expression of cellular matrix metalloproteinase 2 (MMP2) and MMP9 were detected by real-time fluorescence quantitative PCR, and the expression of MMP2, MMP9 and focal adhesion kinase (FAK), phosphorylated FAK protein were detected by Western blot. After silencing MMP9 using small interfering RNA (siRNA), the effect of NETs on the proliferation and migration ability of MC38 cells and the altered expression of related molecules were examined by previous approach. Results NETs promoted the proliferation and migration of MC38 cells and up-regulated the MMP9 expression and FAK phosphorylation. Silencing MMP9 inhibited the promotion of MC38 proliferation and migration by NETs and suppressed FAK phosphorylation. Conclusion NETs up-regulates MMP9 expression in MC38 cells, activates FAK signaling pathway and promotes tumor cell proliferation and migration.
Animals ; Mice ; Focal Adhesion Protein-Tyrosine Kinases/metabolism* ; Matrix Metalloproteinase 2/metabolism* ; Matrix Metalloproteinase 9/metabolism* ; Extracellular Traps/metabolism* ; Cell Movement ; Cell Proliferation ; RNA, Small Interfering/genetics* ; Colorectal Neoplasms/genetics* ; Cell Line, Tumor

OBJECTIVE: To investigate the mechanism by which conditioned medium of colorectal cancer cells promotes the formation of cancer-associated fibroblasts (CAFs). METHODS: Normal human colorectal fibroblasts (CCD-18Co cells) in logarithmic growth phase were treated with the conditioned media of colorectal cancer HCT116 cells (HCT116-CM) or Caco-2 cells (Caco-2-CM) alone or in combination with 300 nmol/L ERK inhibitor SCH772984. The expression levels of CAFs-related molecular markers were detected in the treated cells with real-time quantitative PCR (RT- qPCR) and immunofluorescence assay, and the changes in cell proliferation, colony formation and migration were assessed with RTCA, colony formation and wound healing assays; Western blotting was performed to detect the activated signaling pathways in the fibroblasts and the changes in CAFs formation after blocking of the signaling pathway. RESULTS: HCT116-CM and Caco-2-CM significantly upregulated mRNA expression levels of CAFs markers (including α-SMA, FAP, FN and TGF-β) in CCD-18Co cells, and strongly promoted fibroblast transformation into CAFs (P < 0.05). The two conditioned media also promoted the proliferation, colony formation and migration of CCD-18Co cells (P < 0.05) and significantly increased the levels of α-SMA protein and ERK phosphorylation in the cells (P < 0.05). The ERK inhibitor SCH772984 obviously inhibited the expression of α-SMA and the transformation of CCD-18Co cells into CAFs induced by the conditioned medium of colorectal cancer cells (P < 0.05). CONCLUSION Colorectal cancer cells may induce the formation of colorectal CAFs by activating the ERK pathway in the fibroblasts.
Humans ; Cancer-Associated Fibroblasts/metabolism* ; Culture Media, Conditioned/pharmacology* ; MAP Kinase Signaling System ; Caco-2 Cells ; Fibroblasts ; Signal Transduction ; Cell Proliferation ; Cell Line, Tumor ; Colorectal Neoplasms/genetics* ; Cell Movement