Proliferation Of Colon Cancer Cells Research Articles

Negative regulation of HDAC3 transcription by histone acetyltransferase TIP60 in colon cancer.

Colon cancer is the third most common cancer globally. The expression of histone deacetylase 3 (HDAC3) is upregulated, whereas the expression of tat interactive protein, 60kDa (TIP60) is downregulated in colon cancer. However, the relationship between HDAC3 and TIP60 in colon cancer has not been clearly elucidated. We investigated whether TIP60 could regulate the expression of HDAC3 and suppress colon cancer cell proliferation. RNA sequencing data (GSE108834) showed that HDAC3 expression was regulated by TIP60. Subsequently, we generated TIP60-knockdown HCT116 cells and examined the expression of HDAC3 by western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). We examined the expression pattern of HDAC3 in various cancers using publicly available datasets. The promoter activity of HDAC3 was validated using a dual-luciferase assay, and transcription factors binding to HDAC3 were identified using GeneCards and Promo databases, followed by validation using chromatin immunoprecipitation-quantitative polymerase chain reaction. Cell proliferation and apoptosis were assessed using colony formation assays and fluorescence-activated cell sorting analysis of HCT116 cell lines. In response to TIP60 knockdown, the expression level and promoter activity of HDAC3 increased. Conversely, when HDAC3 was downregulated by overexpression of TIP60, proliferation of HCT116 cells was inhibited and apoptosis was promoted. TIP60 plays a crucial role in the regulation of HDAC3 transcription, thereby influencing cell proliferation and apoptosis in colon cancer. Consequently, TIP60 may function as a tumor suppressor by inhibiting HDAC3 expression in colon cancer cells.

Novel miR-490-3p/hnRNPA1-b/PKM2 axis mediates the Warburg effect and proliferation of colon cancer cells via the PI3K/AKT pathway.

Heterogeneous ribonucleoprotein A1 (hnRNPA1) has been reported to enhance the Warburg effect and promote colon cancer (CC) cell proliferation, but the role and mechanism of the miR-490-3p/hnRNPA1-b/PKM2 axis in CC have not yet been elucidated. To investigate the role and mechanism of a novel miR-490-3p/hnRNPA1-b/PKM2 axis in enhancing the Warburg effect and promoting CC cell proliferation through the PI3K/AKT pathway. Paraffin-embedded pathological sections from 220 CC patients were collected and subjected to immunohistochemical analysis to determine the expression of hnRNPA1-b. The relationship between the expression values and the clinicopathological features of the patients was investigated. Differences in mRNA expression were analyzed using quantitative real-time polymerase chain reaction, while differences in protein expression were analyzed using western blot. Cell proliferation was evaluated using the cell counting kit-8 and 5-ethynyl-2'-deoxyuridine assays, and cell cycle and apoptosis were detected using flow cytometric assays. The targeted binding of miR-490-3p to hnRNPA1-b was validated using a dual luciferase reporter assay. The Warburg effect was evaluated by glucose uptake and lactic acid production assays. The expression of hnRNPA1-b was significantly increased in CC tissues and cells compared to normal controls (P < 0.05). Immunohistochemical results demonstrated significant variations in the expression of the hnRNPA1-b antigen in different stages of CC, including stage I, II-III, and IV. Furthermore, the clinicopathologic characterization revealed a significant correlation between hnRNPA1-b expression and clinical stage as well as T classification. HnRNPA1-b was found to enhance the Warburg effect through the PI3K/AKT pathway, thereby promoting proliferation of HCT116 and SW620 cells. However, the proliferation of HCT116 and SW620 cells was inhibited when miR-490-3p targeted and bound to hnRNPA1-b, effectively blocking the Warburg effect. These findings suggest that the novel miR-490-3p/hnRNPA1-b/PKM2 axis could provide a new strategy for the diagnosis and treatment of CC.

Apoptotic effect of 5-fluorouracil-doxorubicin combination on colorectal cancer cell monolayers and spheroids.

Drug combination studies help to improve new treatment approaches for colon cancer. Tumor spheroids (3D) are better models than traditional 2-dimensional cultures (2D) to evaluate cellular responses to chemotherapy drugs. The cultivation of cancer cells in 2D and 3D cultures affects the apoptotic process, which is a major factor influencing the response of cancer cells to chemotherapeutic drugs. In this study, the antiproliferative effects of 5-fluorouracil (5-FU) and doxorubicin (DOX) were investigated separately and in combination using 2D and 3D cell culture models on two different colon cancer cell lines, HT-29 (apoptosis-resistant cells) and Caco-2 2 (apoptosis-susceptible cells). The effect of the drugs on the proliferation of both colon cancer cells was determined by performing an MTT assay in 2D culture. The apoptotic effect of 5-FU and DOX, both as single agents and in combination, was assessed in 2D and 3D cultures through quantitative real-time polymerase chain reaction analysis. The expression of apoptotic genes, such as caspases, p53, Bax, and Bcl-2, was quantified. It was found that the mRNA expression of proapoptotic genes was significantly upregulated, whereas the mRNA expression of the antiapoptotic Bcl-2 gene was significantly downregulated in both colon cancer models treated with 5-FU, DOX, and 5-FU + DOX. The results indicated that the 5-FU + DOX combination therapy induces apoptosis and renders 5-FU and DOX more effective at lower concentrations compared to their alone use. This study reveals promising results in reducing the potential side effects of treatment by enabling the use of lower drug doses.

SP1-induced circ_0017552 modulates colon cancer cell proliferation and apoptosis via up-regulation of NET1

Colon cancer (CC) is a common malignancy over the world and its morbidity and mortality significantly went up in China in recent years. Molecular functions in cancers have gradually been the pivot subject in cancer research. Neuroepithelial cell transforming 1 (NET1) was reported to contribute to prostate cancer and gastric cancer. Our study figured out that NET1 was overexpressed in CC cells. Then, loss-of-function assays revealed that NET1 facilitated CC cell proliferation and repressed CC cell apoptosis. Next, miR-338-3p was confirmed to target NET1. After that, we verified that circ_0017552 which originates from NET1 could positively modulate NET1 expression. Besides, circ_0017552 was a sponge of miR-338-3p. Rescue assays’ results demonstrated that circ_0017552 could regulate CC cell proliferation and apoptosis through up-regulation of NET1. A transcription factor named Sp1 (SP1) was found to be present in circ_0017552. SP1 induced transcription of circ_0017552 to facilitate CC cell proliferation and inhibit CC cell apoptosis. In a word, SP1-induced circ_0017552 regulated CC cell proliferation and apoptosis through miR-338-3p/NET1 axis.

Evaluating the Anti-proliferative and Apoptotic Role of Atrial Natriuretic Peptide in Colon Cancer Cell Lines

The use of small peptides and conventional anticancer drugs is gaining importance in oncology as small peptides may help increase chemo or radiation sensitivity. The present study aimed to study the impact of atrial natriuretic peptide (ANP) in reducing colon cancer cell proliferation in primary and metastatic colon cancer cell lines. The proliferation of colon cancer cell lines (SW480 and SW620) was analysed by CCK-8 assay and cell damage was analyzed by lactate dehydrogenase activity. Catalase activity assay was performed to measure the oxidative stress and antioxidant defense mechanisms in SW480 and SW620 cell lines. Subsequently, up or downregulation of cancer-specific gene expression such as BAX, Caspase-3, BCL-2, CDK-6, and PCNA genes were assessed after the treatment of small peptide ANP in SW480 and SW620 colon cancer cell lines. The ANP treatment decreased the colon cancer cell proliferation, upregulated the apoptosis-related gene expression (Caspase-3, BAX), downregulated the anti-apoptotic gene (BCL-2) expression, and proliferation-related genes (CDK-6, PCNA) in SW480 and SW620 colon cancer cell lines, and the differences were found to be statistically significantly. Further, increased levels of catalase in the colon cancer cell lines after ANP-treatment suggested the therapeutic role of ANP. Subsequently, the LDH analysis showed the potential of ANP in inducing colon cancer cell damage. Collectively, the current study clearly shows that ANP is a potential molecule in reducing uncontrolled cancer cell growth. However, additional research using animal models and additional colon cancer cell lines is needed to validate its potential usage in clinical studies.

Targeting FGFR1 by β,β-dimethylacrylalkannin suppresses the proliferation of colorectal cancer in cellular and xenograft models

BackgroundColorectal cancer (CRC) continues to be a major global health challenge, ranking as a top cause of cancer-related mortality. Alarmingly, the five-year survival rate for CRC patients hovers around a mere 10–30 %. The disruption of fibroblast growth factor receptor (FGFRs) signaling pathways is significantly implicated in the onset and advancement of CRC, presenting a promising target for therapeutic intervention in CRC management. Further investigation is essential to comprehensively elucidate FGFR1′s function in CRC and to create potent therapies that specifically target FGFR1. PurposeThis study aims to demonstrate the oncogenic role of FGFR1 in colorectal cancer and to explore the potential of β,β-dimethylacrylalkannin (β,β-DMAA) as a therapeutic option to inhibit FGFR1. MethodsIn this research, we employed a comprehensive suite of techniques including tissue array, kinase profiling, computational docking, knockdown assay to predict and explore the inhibitor of FGFR1. Furthermore, we utilized kinase assay, pull-down, cell proliferation tests, and Patient derived xenograft (PDX) mouse models to further investigate a novel FGFR1 inhibitor and its impact on the growth of CRC. ResultsIn our research, we discovered that FGFR1 protein is markedly upregulated in colorectal cancer tissues, suggesting a significant role in regulating cellular proliferation, particularly in patients with colorectal cancer. Furthermore, we conducted a computational docking, kinase profiling analysis, simulation and identified that β,β-DMAA could directly bind with FGFR1 within ATP binding pocket domain. Cell-based assays confirmed that β,β-DMAA effectively inhibited the proliferation of colon cancer cells and also triggered cell cycle arrest, apoptosis, and altered FGFR1-mediated signaling pathways. Moreover, β,β-DMAA effectively attenuated the development of PDX tumors in mice that were FGFR1-positive, with no notable toxicity observed. In summary, our study highlights the pivotal role of FGFR1 in colorectal cancer, suggesting that inhibiting FGFR1 activity could be a promising strategy for therapeutic intervention. We present strong evidence that targeting FGFR1 with β,β-DMAA is a viable approach for the management of colorectal cancer. Given its low toxicity and high efficacy, β,β-DMAA, as an FGFR1 inhibitor, warrants further investigation in clinical settings for the treatment of FGFR1-positive tumors.

Transcriptomic analysis and experiments revealed that remimazolam promotes proliferation and G1/S transition in HCT8 cells.

Remimazolam is a new ultrashort-acting benzodiazepine for sedation and anesthesia. The effects of remimazolam and the mechanism by which it functions in cancer cells have not been determined. This research aimed to explore the mechanism of remimazolam action in colon cancer treatment, using bioinformatics analysis and in vitro experiments. Cell cycle progression, colony formation, self-renewal capacity, and apoptosis detection were performed in HCT8 cells treated with or without remimazolam. Transcriptome sequencing, Gene Ontology, Kyoto Encyclopedia of Genes and Genome, Protein-Protein Interaction, Gene Set Enrichment Analysis, Western blotting, and qPCR were performed to investigate the mechanism of action of remimazolam in HCT8 colon cancer cells. Remimazolam promoted proliferation and cell-cycle progression of HCT8 cells. After remimazolam treatment, a total of 1,096 differentially expressed genes (DEGs) were identified: 673 genes were downregulated, and 423 genes were upregulated. The DEGs were enriched mainly in "DNA replication", "cell cycle", and "G1/S transition" related pathways. There were 15 DEGs verified by qPCR, and representative biomarkers were detected by Western Bloting. The remimazolam-mediated promotion of cell proliferation and cell cycle was reversed by G1T28, a CDK4/6 inhibitor. Remimazolam promoted cell-cycle progression and proliferation in HCT8 colon cancer cells, indicating that the long-term use of remimazolam has potential adverse effects in the anesthesia of patients with colon cancer.

Promising anticancer activity of cromolyn in colon cancer: in vitro and in vivo analysis

PurposeColon cancer is a prevalent cancer globally, representing approximately 10% of all cancer cases and accounting for 10% of all cancer-related deaths. Therefore, finding new therapeutic methods with high efficiency will be very valuable. Cromolyn (C), a common anti-allergic and mast cell membrane stabilizing drug, has recently shown valuable anti-cancer effects in several studies. This study was designed to investigate the anti-cancer activity of cromolyn on colon cancer in vitro and in vivo and to determine values such as selectivity index and survival effect.MethodsHT-29 (colon cancer) and MCF-10 (normal epithelial) cell lines were treated with C and Doxorubicin (DOX; Positive control). IC50 values and the effects of C and DOX on apoptosis were explored using methyl thiazole diphenyl-tetrazolium bromide (MTT) assay and Annexin V/PI Apoptosis Assay Kit. To investigate in an animal study, colon cancer was subcutaneously induced by CT26 cells (mouse colon cancer) in bulb/c mice. Mice were treated with 0.05 LD50 intraperitoneal every other day for 35 days. After the death of mice, tumor volume, tumor weight, and survival rate were evaluated.ResultsC selectively and significantly suppressed the proliferation of cancer cells in a dose-dependent manner. The IC50 values for the MCF-10 and HT29 cell lines were 7.33 ± 0.78 μM and 2.33 ± 0.6 μM, respectively. Notably, the selective index (SI) highlighted that C displayed greater selectivity in inhibiting cancer cell growth compared to DOX, with SI values of 3.15 and 2.60, respectively. C exhibited higher effectiveness and selectivity in inducing apoptosis in cancer cells compared to DOX, with a significant p-value (61% vs. 52%, P-value ≤ 0.0001). Also, in mice bearing colon cancer, C reduced the tumor volume (6317 ± 1685mm3) and tumor weight (9.8 ± 1.6 g) compared to the negative control group (weight 12.45 ± 0.9 g; volume 7346 ± 1077) but these values were not statistically significant (P ≤ 0.05).ConclusionOur study showed that cromolyn is a selective and strong drug in inhibiting the proliferation of colon cancer cells. Based on our results, the efficacy of C in vitro analysis (MTT assays and apoptosis), as well as animal studies is competitive with the FDA-approved drug doxorubicin. C is very promising as a low-complication and good-efficacy drug for cancer drug repositioning. This requires clinical research study designs to comprehensively evaluate its anti-cancer effects.

NXPH4 can be used as a biomarker for pan-cancer and promotes colon cancer progression.

NXPH4 promotes cancer proliferation and invasion. However, its specific role and mechanism in cancer remain unclear. Transcriptome and clinical data for pan-cancer were derived from the TCGA database. K-M survival curve and univariate Cox were used for prognostic analysis. CIBERSORT and TIMER algorithms were employed to calculate immune cell infiltration. Gene set enrichment analysis (GSEA) was employed for investigating the function of NXPH4. Western blot verified differential expression of NXPH4 in colon cancer. Functional assays (CCK-8, plate clonogenicity assay, wound healing assay, and Transwell assay) confirmed the impact of NXPH4 on proliferation, invasion, and migration of colon cancer cells. Dysregulation of NXPH4 in pan-cancer suggests its potential as a diagnostic and prognostic marker for certain cancers, including colon and liver cancer. High expression of NXPH4 in pan-cancer might be associated with the increase in copy number and hypomethylation. NXPH4 expression in pan-cancer is substantially linked to immune cell infiltration in the immune microenvironment. NXPH4 expression is associated with the susceptibility to immunotherapy and chemotherapy. Western blot further confirmed the higher expression of NXPH4 in colon cancer. Knockdown of NXPH4 significantly suppresses proliferation, invasion, and migration of colon cancer cell lines HT-29 and HCT116, as validated by functional assays.

LncRNA EBLN3P Accelerates Cell Proliferation and Invasion of Colon Cancer through Modulating the miR-519d-3p/ZFP91 Axis.

Purpose: The study aims to explore the roles and underlying mechanisms of long noncoding RNAs endogenous bornavirus-like nucleoprotein (lncRNA EBLN3P) in colon cancer, emphasizing the potential impact of these insights on advancing colon cancer treatment strategies. By shedding light on lncRNA EBLN3P's involvement, this research could contribute to the development of novel therapeutic approaches, enhancing the efficacy of interventions for colon cancer patients. Methods: We employed quantitative reverse transcription polymerase chain reaction to assess the levels of lncRNA EBLN3P, zinc finger protein (ZFP91), and miR-519d-3p, alongside CCK-8 and EdU assays for cell proliferation, flow cytometry for apoptosis, and Transwell and wound healing assays for migration and invasion. The in vivo function of lncRNA EBLN3P was investigated through a xenograft model, and protein levels were evaluated via Western blot analysis. Results: LncRNA EBLN3P was found to be upregulated in colon cancer tissues and cells, promoting cell proliferation and metastasis while inhibiting apoptosis. Downregulation of lncRNA EBLN3P reduced tumor size, volume, and weight in a mouse model. MiR-519d-3p, which negatively interacts with lncRNA EBLN3P, was found to be downregulated in colon cancer tissues and cell lines. Its upregulation hindered cancer cell proliferation and metastasis while enhancing apoptosis. ZFP91, a binding partner of miR-519d-3p, was upregulated in colon cancer and inversely related to miR-519d-3p levels. Rescue experiments indicated that the effects of lncRNA EBLN3P silencing could be reversed by miR-519d-3p suppression, but were mitigated by ZFP91 downregulation. Conclusion: LncRNA EBLN3P facilitates colon cancer progression via the miR-519d-3p/ZFP91 axis, presenting a novel understanding of lncRNA EBLN3P's role and offering potential therapeutic insights for colon cancer treatment. This study fills a critical gap by linking lncRNA EBLN3P with the miR-519d-3p/ZFP91 axis in the context of colon cancer, thereby broadening our understanding of the molecular mechanisms underlying colon cancer progression.

Hsa_circ_0101050 accelerates the progression of Colon cancer by targeting the miR-140–3 p/MELK axis

BackgroundCircular RNAs (circRNAs) are involved in the progression of colon cancer (CC). This study aimed to examine the role of a new circRNA circ_0101050 in CC. MethodsDual-luciferase reporter and RNA immunoprecipitation analyses were performed to validate the target relationships among maternal embryonic leucine zipper kinase (MELK), microRNA (miR)-140–3 p, and circ_0101050. Expression levels were calculated using western blotting and/or quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Western blotting was performed to evaluate the relative expression of Bcl-2 and Bax proteins to determine cell death. Cell Counting Kit-8 (CCK-8) and colony formation assays were performed to determine the proliferative potential of CC cells. The migration rate of CC cells was evaluated using wound healing assays. Tumor formation tests were performed to determine the effect of circ_0101050 on tumor development in vivo. ResultsElevated levels of circ_0101050 and MELK were observed in CC. By inhibiting circ 0,101,050 or MELK, CC cell proliferation and migration were inhibited, but CC cell apoptosis was promoted. Silencing circ_0101050 also inhibited CC growth in vivo. We also found that miR-140–3 p was downregulated, which alleviated the repressive effects of circ_0101050 knockdown on proliferating and migrating CC cells, as well as the stimulating effect on apoptosis. In addition, the absence of MELK alleviated the effects of miR-140–3 p downregulation, which enhanced CC cell malignancy. ConclusionsCirc_0101050 exacerbates malignant phenotypes in CC by targeting the miR-140–3 p/MELK axis. These findings suggested that the circ_0101050/miR-140–3 p/MELK network may be a prospective target for CC treatment.

The RNA-binding protein RBMS3 inhibits the progression of colon cancer by regulating the stability of LIMS1 mRNA.

The RNA-binding motif single-stranded interacting protein 3 (RBMS3) is a constituent of the RNA-binding motif (RBM) protein family, which assumes a pivotal role in governing cellular biogenesis processes such as the cell cycle and apoptosis. Despite an abundance of studies elucidating RBMS3's divergent roles in the genesis and advancement of various tumors, its involvement in colon cancer remains enigmatic. The present investigation employed data analysis from TCGA and GTEx to unveil that RBMS3 expression demonstrated a diminished presence in colon cancer tissues when juxtaposed with normal colon tissues. The effect of RBMS3 and LIM zinc finger domain 1 (LIMS1) on colon cancer was substantiated via animal models and cellular experiments. The connection between RBMS3 and LIM zinc finger domain 1 (LIMS1) was verified by molecular biology methods. The study conclusively ascertained that augmenting RBMS3 expression quells the proliferation, migration, and invasion of colon cancer cells. Furthermore, the inquiry unveiled a plausible mechanism through which RBMS3 impacts the expression of LIMS1 by modulating its mRNA stability. The investigation ascertained that RBMS3 inhibits the progression of colon cancer by regulating LIMS1. The inhibitory function of LIMS1 and RBMS3 is closely intertwined in colon cancer, with knocking down LIMS1 being able to rescue the inhibitory effect of RBMS3 overexpression on the functionality of colon cancer cell CONCLUSIONS: The discernments delineate RBMS3 as a novel suppressor of cancer via LIMS1, thereby bestowing fresh therapeutic possibilities and illuminating the intricacies of colon cancer.

F1 fraction isolated from Mesobuthus eupeus scorpion venom induces macrophage polarization toward M1 phenotype and exerts anti-tumoral effects on the CT26 tumor cell line

Scorpion venoms identified as agents with anti-tumor and anti-angiogenic features. Tumor microenvironment (TME) plays a pivotal role in the process of tumorigenesis, tumor development, and polarization of M2 phenotype tumor associated macrophages (TAMs). M2 polarized cells are associated with tumor growth, invasion, and metastasis. The fractionation process was performed by gel filtration chromatography on a Sephadex G50 column. To elucidate whether scorpion venom can alter macrophage polarization, we treated interleukin (IL)-4-polarized M2 cells with isolated fractions from Mesobuthus eupeus. Next, we evaluated the cytokine production and specific markers expression for M2 and M1 phenotype using enzyme linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (PCR), respectively. The phagocytic capacity of macrophages was also assessed. In addition, the migration assay and MTT analysis were performed to investigate the effects of reprogrammed macrophages on the CT-26 colon cancer cells. The results indicated that F1 fraction of venom significantly upregulated the levels and expression of M1-associated cytokines and markers, including tumor necrosis factor-alpha (TNF-α) (p < 0.001), IL-1 (p < 0.01), interferon regulatory factor 5 (IRF5) (p < 0.0001), induced nitric oxide synthase (iNOS) (p < 0.0001), and CD86 (p < 0.0001), and downregulated M2-related markers, including transforming growth factor-beta (TGF-β) (p < 0.05), IL-10 (p < 0.05), Fizz1 (p < 0.0001), arginase-1 (Arg-1) (p < 0.0001), and CD206 (p < 0.001). The macrophage phagocytic capacity was enhanced after treatment with F1 fraction (p < 0.01). Moreover, incubation of CT-26 cell line with conditioned media of F1-treated macrophages suppressed migration (p < 0.0001) and proliferation (p < 0.01) of tumor cells. In conclusion, our findings demonstrated the potential of Mesobuthus eupeus venom in M2-to-M1 macrophage polarization as a promising therapeutic approach against proliferation and metastasis of colon cancer cells.

Abstract 6967: Role of CXCL7 in colon cancer cell proliferation

Abstract Chemokine (C-X-C motif) ligand 7 (CXCL7) - a protein product of the pro platelet basic protein (PPBP) gene - is a potential biomarker for colorectal cancer (CRC) diagnosis. However, its role in CRC progression is not known. Hence, we examined the role of CXCL7 in colon cancer cell proliferation through enhanced aerobic glycolysis. We first examined the effect of CXCL7 on cellular proliferation and glycolytic flux using human colon cancer (HT-29) cells transfected with CXCL7 or an empty vector as a control. Stable CXCL7- and vector-expressing cells were selected using hygromycin B. In addition, we treated HT-29 cells with CXCL7 obtained from the conditioned media (CM) from CXCL7-transfected HEK-293T cells or CM from vector-transfected (control) cells. CXCL7 expression was confirmed by analyzing PPBP expression using RT-qPCR, and CXCL7 level in the conditioned media was quantified by ELISA. The specificity of CXCL7 actions were examined using a CXCL7 neutralizing antibody (R&amp;D Systems) and an antagonist (SB225002) to C-X-C motif chemokine receptor 2 (CXCR2). Cancer cell proliferation was assessed by measuring DNA synthesis (EdU incorporation). Glycolytic flux was monitored by measuring glucose uptake using a fluorescent derivative of 2-deoxyglucose (2-NBDG), Seahorse glycolysis stress test analysis, lactate accumulation in the cultured cell’s media, and Western blotting for glycolytic enzyme protein expression. To further investigate CXCL7-mediated proliferation, we examined EdU incorporation and colony size in 3D engineered tissues and tumor xenograft growth in nude mice injected with CXCL7- and vector-expressing HT29 cells. The proliferation rate in CXCL7-expressing HT-29 cells and HT-29 cells treated with the CXCL7-CM were significantly higher (1.2-fold and 1.8-fold, respectively) compared to controls (p&amp;lt;0.01). Consistently, the proliferation rate in CXCL7-expressing HT-29 3D engineered tissues was significantly enhanced compared to vector-expressing tissues (p&amp;lt;0.05). Importantly, blocking the CXCL7/CXCR2 axis using a CXCL7 neutralizing antibody and a CXCR2 inhibitor abrogated CXCL7-stimulated proliferation. Lactate levels were two-fold higher in the culture media from both CXCL7-expressing HT-29 cells and HT-29 cells treated with CXCL7-CM compared to the controls (p&amp;lt;0.05). Glucose uptake was also greater in CXCL7-overexpressing cells and CXCL7-CM treated cells compared to vector controls. Data from Seahorse assay and Western blots confirmed that CXCL7-expressing HT-29 cells and HT-29 cells treated with CXCL7-CM have higher glycolytic capacity and increased glycolytic enzymes’ protein expression compared to the controls, respectively. In vivo, CXCL7-expressing HT-29 xenograft tumor weight and PCNA protein levels were 100% and 60% greater, respectively, than vector-expressing tumors (p&amp;lt;0.05). Our study for the first time showed that CXCL7 stimulates colon cancer cell proliferation and enhances aerobic glycolysis. Citation Format: Hadeel Aldhowayan, Jannatul F. Nipa, Lauren Jun, Ifeoluwa Odeniyi, Parker L. Jones, Ramesh Jeganathan, Elizabeth A. Lipke, Michael W. Greene. Role of CXCL7 in colon cancer cell proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6967.

Abstract 1826: New inhibitors of oncology targets discovered using the affinity-mediated selection of DNA-encoded chemical libraries followed by machine-learning

Abstract X-Chem’s DNA-Encoded Chemical Library platform generates a large amount of binding data for each target it is screened against. Here, we report the application of machine learning to these large datasets with case studies that include the identification of novel inhibitors of two oncology targets. One, ERα, is an estrogen-activated receptor that controls transcription and stimulates breast cell proliferation including in breast cancer. The second, DCAF1, is the substrate-recognition component of an E3 ligase complex that is associated with the proliferation of colon cancer cells. Over 100 billion DNA-Encoded compounds were synthesized and screened for their affinity to each target. The selection output data was then featurized and used to train multiple machine-learned models. Models with demonstrated predictive power were then used to score virtual catalogue compounds which were subsequently acquired and tested. A range of successful data-science strategies will be described including those that lead to double-digit nM inhibitors of each of DCAF1 and ERα. Citation Format: Anthony D. Keefe, Mohamed AbouZleikha, AJ Baghaie, Matthew A. Clark, Marie-Aude Guie, John P. Guilinger, Ryan Walsh, Ying Zhang. New inhibitors of oncology targets discovered using the affinity-mediated selection of DNA-encoded chemical libraries followed by machine-learning [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1826.

Abstract 4377: AT rich interaction domain 5a promotes gastrointestinal tumorigenesis by regulating YAP and STAT3 pathways

Abstract Background: Chronic inflammation and an inflammatory cytokine, interleukin(IL)-6, play important roles in gastrointestinal tumorigenesis and metastasis. Recently, autocrine IL-6 has been reported to promote cancer development and progression in gastrointestinal cancer. However, it is still unknown how IL-6 level is regulated in gastrointestinal cancer cells. RNA-binding proteins (RBPs) are known to be one of the regulatory mechanisms of cytokine production, including IL-6, especially in immune cells. AT rich interaction domain 5a (Arid5a), one of the RBPs, has been reported to bind to IL6 3’UTR and regulates its stability and expression. Therefore, we aimed to elucidate the role of Arid5a in gastrointestinal cancer, especially gastric and colon cancer. Methods: Human gastric and colon cancer cell lines were used in this study. Arid5a knocked-down gastric and colon cancer cells were generated with lentiviral transduction of shRNAs targeting Arid5a and their phenotypes were investigated by several assays including MTT, apoptosis and sphere formation assays. Bulk RNA-seq analysis was performed using Arid5a knocked-down gastric and colon cancer cells. Results: In both human gastric and colon cancer cell lines, Arid5a knockdown suppressed cancer cell proliferation and spheroid formation, and increased apoptosis. Unexpectedly, IL6 mRNA expression was not correlated with Arid5a expression level. We found that Arid5a knockdown suppresses the activation of YAP and STAT3 by western blot analysis. We identified several new Arid5a target genes by bulk RNA-seq analysis. Conclusion: Arid5a plays an important role in the regulation of cancer cell proliferation, apoptosis and stemness in human gastric and colon cancer cells, perhaps independently of IL-6. Arid5a regulates YAP and STAT3 pathways by unknown mechanisms. We are now investigating how Arid5a regulates YAP and STAT3 pathways by focusing new Arid5a target genes. Citation Format: Koji Taniguchi, Kazuya Hamada, Shugo Tanaka, Hiroyuki Kurosu, Issei Kawakita, Kentaro Kumagai, Kentaro Nakazono, Sari Iwasaki, Satoshi Tanaka. AT rich interaction domain 5a promotes gastrointestinal tumorigenesis by regulating YAP and STAT3 pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4377.

Effects of Viable and Heat-Inactivated Bifidobacterium longum D42 on Proliferation and Apoptosis of HT-29 Human Colon Cancer Cells.

Bifidobacterium longum is a common probiotic; both viable and heat-inactivated Bifidobacterium longum have many probiotic effects, such as anticancer effects. But some mechanisms of anticancer effects are still unclear, especially for heat-inactivated probiotics. In this study, we analyzed the effects of viable and heat-inactivated Bifidobacterium longum D42 on human colon cancer cells (HT-29). Cell proliferation, membrane permeability and apoptosis were detected by using the CCK-8 method, LDH method and Annexin V-FITC/PI kits. The ROS level and mitochondrial membrane potential were examined using the fluorescent probes DCFH-DA and JC-1. Real-time fluorescence quantitative PCR (RT-qPCR) and Western blot were used to detect the expression of mitochondrial apoptosis pathway genes and proteins. The results showed that viable and heat-inactivated Bifidobacterium longum D42 at concentrations of 1 × 106 CFU/mL significantly inhibited the proliferation of and increased the level of LDH release of HT-29 colon cancer cells. We found that they could increase the apoptosis rate of HT-29 cells. Moreover, they could also induce apoptosis by inducing cells to produce ROS and destroying the mitochondrial membrane potential of cells. Further studies found that they could increase the mRNA transcription and protein expression levels of the Caspase-3, Caspase-9 and Bax genes in cells, and reduce the mRNA transcription and protein expression levels of the Bcl-2 gene. In summary, our findings revealed that viable and heat-inactivated Bifidobacterium longum D42 have inhibitory effects on proliferation and promote the apoptosis of human colon cancer cells, and also have certain adjuvant drug therapeutic effects and have potential application value in the adjuvant treatment of colon cancer.

Cafestol inhibits colon cancer cell proliferation and tumor growth in xenograft mice by activating LKB1/AMPK/ULK1-dependent autophagy

Chemotherapy failure in colorectal cancer patients is the major cause of recurrence and poor prognosis. As a result, there is an urgent need to develop drugs that have a good chemotherapy effect while also being extremely safe. In this study, we found cafestol inhibited colon cancer growth and HCT116 proliferation in vivo and in vitro, and improved the composition of intestinal flora. Further metabolomic data showed that autophagy and AMPK pathways were involved in the process of cafestol's anti-colon cancer effects. The functional validation studies revealed that cafestol increased autophagy vesicles and LC3B-II levels. The autophagic flux induced by cafestol was prevented by using BafA1. The autophagy inhibitor 3-MA blocked the cafestol-induced increase in LC3B-II and cell proliferation inhibition. Then we found that cafestol induced the increased expressions of LKB1, AMPK, ULK1, p-LKB1, p-AMPK, and p-ULK1 proteins in vivo and in vitro. Using the siRNA targeted to the Lkb1 gene, the levels of AMPK, ULK1, and LC3B-II were suppressed under cafestol treatment. These results indicated that the effect of cafestol is through regulating LKB1/AMPK/ULK1 pathway-mediated autophagic death. Finally, a correlation matrix of the microbiome and autophagy-related proteins was conducted. We found that cafestol-induced autophagic protein expression was positively correlated with the beneficial intestinal bacteria (Muribaculaceae, Bacteroides, Prevotellacece, and Alloprevotella) and negatively correlated with the hazardous bacteria. Conclusions: This study found that cafestol inhibited colon cancer in vitro and in vivo by the mechanism that may be related to LKB1/AMPK/ULK1 pathway-mediated autophagic cell death and improved intestinal microenvironment.

The Impact of Atmospheric Cadmium Exposure on Colon Cancer and the Invasiveness of Intestinal Stents in the Cancerous Colon.

Inhalation exposure to carcinogenic metals such as cadmium (Cd) is a significant global health concern linked to various cancers. However, the precise carcinogenic mechanism underlying inhalation exposure remains elusive. In this study, CT26 mouse colon cancer (CC) cells were implanted into BALB/c mice to establish CC mouse models. Some of the CC mice were implanted with intestinal stents. The mice were exposed to atomized oxygen and nitrogen (O2/N2) gas containing Cd. Atmospheric Cd intensified inflammation in CC cells and heightened Nicotinamide Adenine Dinucleotide Phosphate (NADPH) Oxidase 1 (NOX1) activity, which is an indirect measurement of increased reactive oxygen species (ROS) production. This escalated ROS production triggered abnormal Wnt protein secretion, activated the Wnt/β-catenin signaling pathway, and stimulated CC cell proliferation. No discernible body weight effect was seen in the CC mice, possibly due to the later-stage tumor weight gain, which masked the changes in body weight. Cd facilitated colon tumor restructuring and cell migration at the later stage. The implantation of intestinal stents inhibited the expression of Superoxide Dismutase 1 (SOD1) in the colon tumors of the CC mice, with no evident effects on the expression levels of NOX1, SOD2, and Catalase (CAT) enzymes. Elevated ROS levels, indirectly reflected by enzyme activity, did not substantially impact the Wnt/β-catenin signaling pathway and even contributed to slowing its imbalance. Stent implantation eased the inflammation occurring in colon tumors by reducing CC cell proliferation but it induced discomfort in the mice, leading to a reduction in food intake and weight. Cd partially fosters CC tumorigenesis via the ROS-mediated Wnt/β-catenin signaling pathway. The effect of Cd on the invasive effect of intestinal stents in the cancerous colon is not significant.

KLK10 derived from tumor endothelial cells accelerates colon cancer cell proliferation and hematogenous liver metastasis formation.

Tumor endothelial cells (TECs), which are thought to be structurally and functionally different from normal endothelial cells (NECs), are increasingly attracting attention as a therapeutic target in hypervascular malignancies. Although colorectal liver metastasis (CRLM) tumors are hypovascular, inhibitors of angiogenesis are a key drug in multidisciplinary therapy, and TECs might be involved in the development and progression of cancer. Here, we analyzed the function of TEC in the CRLM tumor microenvironment. We used a murine colon cancer cell line (CT26) and isolated TECs from CRLM tumors. TECs showed higher proliferation and migration than NECs. Coinjection of CT26 and TECs yielded rapid tumor formation invivo. Immunofluorescence analysis showed that coinjection of CT26 and TECs increased vessel formation and Ki-67+ cells. Transcriptome analysis identified kallikrein-related peptide 10 (KLK10) as a candidate target. Coinjection of CT26 and TECs after KLK10 downregulation with siRNA suppressed tumor formation invivo. TEC secretion of KLK10 decreased after KLK10 downregulation, and conditioned medium after KLK10 knockdown in TECs suppressed CT26 proliferative activity. Double immunofluorescence staining of KLK10 and CD31 in CRLM tissues revealed a significant correlation between poor prognosis and positive KLK10 expression in TECs and tumor cells. On multivariate analysis, KLK10 expression was an independent prognostic factor in disease-free survival. In conclusion, KLK10 derived from TECs accelerates colon cancer cell proliferation and hematogenous liver metastasis formation. KLK10 in TECs might offer a promising therapeutic target in CRLM.