Colorectal Cancer Cell Lines Research Articles

ADAMTSL2 is a potential prognostic biomarker and immunotherapeutic target for colorectal cancer: Bioinformatic analysis and experimental verification.

The ADAMTS Like 2 (ADAMTSL2) mutation has been identified to be associated with different human genetic diseases. The role of ADAMTSL2 is unclear in colorectal cancer (CRC). The study investigated the expression of ADAMTSL2 in both pan cancer and CRC, using data from The Cancer Genome Atlas (TCGA) database to assess its diagnostic value. The study examined the correlation between ADAMTSL2 expression levels and clinical characteristics, as well as prognosis in CRC. The study explored potential regulatory networks involving ADAMTSL2, including its association with immune infiltration, immune checkpoint genes, tumor mutational burden (TMB) / microsatellite instability (MSI), tumor stemness index (mRNAsi), and drug sensitivity in CRC. ADAMTSL2 expression was validated using GSE71187 and quantitative real-time PCR (qRT-PCR). ADAMTSL2 was aberrantly expressed in pan cancer and CRC. An increased level of ADAMTSL2 expression in patients with CRC was significantly associated with the pathologic N stage (p < 0.001), pathologic stage (p < 0.001), age (p < 0.001), histological type (p < 0.001), and neoplasm type (p = 0.001). The high expression of ADAMTSL2 in patients with CRC was found to be significantly associated with a poorer overall survival (OS) (HR: 1.67; 95% CI: 1.18-2.38; p = 0.004), progression-free survival (PFS) (HR: 1.55; 95% CI: 1.14-2.11; p = 0.005) and disease-specific survival (DSS) (HR: 1.83; 95% CI: 1.16-2.89; p = 0.010). The expression of ADAMTSL2 in patients with CRC (p = 0.009) was identified as an independent prognostic determinant. ADAMTSL2 was associated with extracellular matrix receptor (ECM-receptor) interaction, transforming growth factor β (TGF-β) signaling pathway, and more. ADAMTSL2 expression was correlated with immune infiltration, immune checkpoint genes, TMB / MSI and mRNAsi in CRC. ADAMTSL2 expression was significantly and negatively correlated with 1-BET-762, Trametinib, and WZ3105 in CRC. ADAMTSL2 was significantly upregulated in CRC cell lines. The high expression of ADAMTSL2 is significantly correlated with lower OS and immune infiltration of CRC. ADAMTSL2 may be a potential prognostic biomarker and immunotherapeutic target for CRC patients.

&lt;i&gt;In-vitro&lt;/i&gt; Anticancer and &lt;i&gt;In silico&lt;/i&gt; Assessment of &lt;i&gt;Oxalis corniculata&lt;/i&gt; Against Colorectal Cancer (HCT 116) Cell line

&lt;i&gt;In-vitro&lt;/i&gt; Anticancer and &lt;i&gt;In silico&lt;/i&gt; Assessment of &lt;i&gt;Oxalis corniculata&lt;/i&gt; Against Colorectal Cancer (HCT 116) Cell line

Cytotoxicity assay of Turkish rare endemic Helianthemum germanicopolitanum Bornm. plant extract on HT-29 cell line

Objective: The aim of this study was to investigate the cytotoxic effect of Helianthem germanicopolitanum Bornm., a rare endemic plant in Turkey, against colon cancer. Materials and Methods: After the extraction of Helianthemum germanicopolitanum Bornm. plant, the phytochemical profile of the plant was analyzed by High Performance Liquid Chromatography (HPLC). Cytotoxicity analysis on HT-29 human colon cancer cell line was performed by WST 1 assay. The results were compared with other studies on similar plant species in the literature. Results: H. germanicopolitanum plant contains various flavonoids and these flavonoids have cytotoxic effects on colorectal cancer cells. These cytotoxic effects provide anticarcinogenic effects by activating cell death pathways at certain concentrations. These findings provide an important insight into the therapeutic potential of the plant. Conclusion: Cytotoxicity studies of flavonoids in extracts obtained from extraction procedures on colorectal cancer cell lines show that flavonoids offer anticarcinogenic effects by activating cell death pathways at certain concentrations.

Identification and Validation of SLC9A2 as A Potential Tumor Suppressor in Colorectal Cancer: Integrating Bioinformatics Analysis with Experimental Confirmation.

To uncover the mechanisms underlying the development of colorectal cancer (CRC), we applied bioinformatic analyses to identify key genes and experimentally validated their possible roles in CRC onset and progression. We performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis on differentially expressed genes (DEGs), constructed a protein-protein interaction (PPI) network to find the top 10 hub genes, and analyzed their expression in colon adenocarcinoma (COAD) and rectum adenocarcinoma (READ). We also studied the correlation between these genes and immune cell infiltration and prognosis and validated the expression of SLC9A2 in CRC tissues and cell lines using qRT-PCR and Western blotting. Functional experiments were conducted in vitro to investigate the effects of SLC9A2 on tumor growth and metastasis. We found 130 DEGs, with 45 up-regulated and 85 down-regulated in CRC. GO analysis indicated that these DEGs were primarily enriched in functions related to the regulation of cellular pH, zymogen granules, and transmembrane transporter activity. KEGG pathway analysis revealed that the DEGs played pivotal roles in pancreatic secretion, rheumatoid arthritis, and the IL-17 signaling pathway. We identified 10 hub genes: CXCL1, SLC26A3, CXCL2, MMP7, MMP1, SLC9A2, SLC4A4, CLCA1, CLCA4, and ZG16. GO enrichment analysis showed that these hub genes were predominantly involved in the positive regulation of transcription. Gene expression analysis revealed that CXCL1, CXCL2, MMP1, and MMP7 were highly expressed in CRC, whereas CLCA1, CLCA4, SLC4A4, SLC9A2, SLC26A3, and ZG16 were expressed at lower levels. Survival analysis revealed that 5 key genes were significantly associated with the prognosis of CRC. Both mRNA and protein expression levels of SLC9A2 were markedly reduced in CRC tissues and cell lines. Importantly, SLC9A2 overexpression in SW480 cells led to a notable inhibition of cell proliferation, migration, and invasion. Western blotting analysis revealed that the expression levels of phosphorylated ERK (p-ERK) and phosphorylated JNK (p-JNK) proteins were significantly increased, whereas there were no significant changes in the expression levels of ERK and JNK following SLC9A2 overexpression. Correlation analysis indicated a potential link between SLC9A2 expression and the MAPK signaling pathway. Our study suggests that SLC9A2 acts as a tumor suppressor through the MAPK pathway and could be a potential target for CRC diagnosis and therapy.

SGLT2 inhibitor promotes mitochondrial dysfunction and ER-phagy in colorectal cancer cells

BackgroundSodium-glucose transporter 2 (SGLT2) inhibitors (iSGLT2) are approved medications for type 2 diabetes. Recent studies indicate that iSGLT2 inhibit the growth of some cancer cells. However, the mechanism(s) remains to be fully elucidated.MethodsThe SGLT2 levels were determined in normal colon CCD 841 CoN and, HCT 116, HT-29, SW480 and LoVo colorectal cancer (CRC) cell lines by quantitative real-time PCR and western blot. The effect of iSGLT2 canagliflozin on cell proliferation was examined using CCK-8, as its role on CRC cells metabolism and tumorigenesis has been evaluated by XF HS Seahorse Bioanalyzer and flow cytometric analyses. Transient gene silencing experiments and analysis of protein–protein interaction network were conducted to evaluate the SGLT2 molecular targets in CRC cells.ResultsData showed that the treatment with iSGLT2 (50 µM) for 72 h induced cell cycle arrest (p < 0.001), impaired glucose and energetic metabolism (p < 0.001), promoted apoptotic cell death and ER stress flowing into autophagy (p < 0.001) in HCT 116 and HT-29 cells. These cellular events were accompanied by sirtuin 3 (SIRT3) upregulation (p < 0.01), as also supported by SIRT3 transient silencing experiments resulting in the attenuation of the effects of iSGLT2 on the cellular metabolic/energetic alterations and the induction of programmed cell death. The identification and validation of dipeptidyl peptidase 4 (DPP4) as potential common target of SGLT2 and SIRT3 were also assessed.ConclusionsThese results deepened knowledge on the iSGLT2 contribution in limiting CRC tumorigenesis unveiling the SGLT2/SIRT3 axis in the cytotoxic mechanisms.Graphical

Synthesis and in vitro cytotoxicity of benzoxazole-based PPARα/γ antagonists in colorectal cancer cell lines.

A series of benzoxazole-based amides and sulfonamides were synthesized and evaluated for their human peroxisome proliferator-activated receptor (PPAR)α and PPARγ activity. All tested compounds showed a dual antagonist profile on both PPAR subtypes; based on transactivation results, seven compounds were selected to test their in vitro antiproliferative activity in a panel of eight cancer cell lines with different expression rates of PPARα and PPARγ. 3f was identified as the most cytotoxic compound, with higher potency in the colorectal cancer cell lines HT-29 and HCT116. Compound 3f induced a concentration-dependent activation of caspases and cell-cycle arrest in both colorectal cancer models. Docking experiments were also performed to shed light on the putative binding mode of this novel class of dual PPARα/γ antagonists.

Extracellular vesicles containing MFGE8 from colorectal cancer facilitate macrophage efferocytosis

BackgroundColorectal cancer (CRC) commonly exhibits tolerance to cisplatin treatment, but the underlying mechanisms remain unclear. Within the tumor microenvironment, macrophages play a role in resisting the cytotoxic effects of chemotherapy by engaging in efferocytosis to clear apoptotic cells induced by chemotherapeutic agents. The involvement of extracellular vesicles (EVs), an intercellular communicator within the tumor microenvironment, in regulating the efferocytosis for the promotion of drug resistance has not been thoroughly investigated.MethodsWe constructed GFP fluorescent-expressing CRC cell lines (including GFP-CT26 and GFP-MC38) to detect macrophage efferocytosis through flow cytometric analysis. We isolated and purified CRC-secreted EVs using a multi-step ultracentrifugation method and identified them through electron microscopy and nanoflow cytometry. Proteomic analysis was conducted to identify the protein molecules carried by CRC-EVs. MFGE8 knockout CRC cell lines were constructed using CRISPR-Cas9, and their effects were validated through in vitro and in vivo experiments using Western blotting, immunofluorescence, and flow cytometric analysis, confirming that these EVs activate the macrophage αvβ3-Src-FAK-STAT3 signaling pathway, thereby promoting efferocytosis.ResultsIn this study, we found that CRC-derived EVs (CRC-EVs) enhanced macrophage efferocytosis of cisplatin-induced apoptotic CRC cells. Analysis of The Cancer Genome Atlas (TCGA) database revealed a high expression of the efferocytosis-associated gene MFGE8 in CRC patients, suggesting a poorer prognosis. Additionally, mass spectrometry-based proteomic analysis identified a high abundance of MFGE8 protein in CRC-EVs. Utilizing CRISPR-Cas9 gene edition system, we generated MFGE8-knockout CRC cells, demonstrating that their EVs fail to upregulate macrophage efferocytosis in vitro and in vivo. Furthermore, we demonstrated that MFGE8 in CRC-EVs stimulated macrophage efferocytosis by increasing the expression of αvβ3 on the cell surface, thereby activating the intracellular Src-FAK-STAT3 signaling pathway.ConclusionsTherefore, this study highlighted a mechanism in CRC-EVs carrying MFGE8 activated the macrophage efferocytosis. This activation promoted the clearance of cisplatin-induced apoptotic CRC cells, contributing to CRC resistance against cisplatin. These findings provide novel insights into the potential synergistic application of chemotherapy drugs, EVs inhibitors, and efferocytosis antagonists for CRC treatment.

Ubiquitin-specific peptidase 15 regulates the TFAP4/PCGF1 axis facilitating liver metastasis of colorectal cancer and cell stemness

The tumor recurrence and metastasis of colorectal cancer (CRC) are responsible for most of CRC-linked mortalities. It is an urgent need to deeply investigate the pathogenesis of CRC metastasis and look for novel targets for its treatment. The current study aimed to investigate the effects of ubiquitin-specific peptidase 15 (USP-15) on the CRC progression. In vivo, a mouse model of liver metastasis of CRC tumor was established to investigate the role of USP-15. In vitro, the migrated and invasive abilities of CRC cells were assessed by transwell assay. Cell stemness was evaluated by using sphere formation assay. The underlying mechanism was further explored by employing the co-immunoprecipitation, dual luciferase reporter assay, oligonucleotide pull-down assay, and chromatin immunoprecipitation assay. The results showed that USP-15 was upregulated in CRC patients with liver metastasis and high metastatic potential cell lines of CRC. Loss of USP-15 repressed the epithelial-to-mesenchymal transition (EMT), migration, invasion, and stemness properties of CRC cells in vitro. Downregulation of USP-15 reduced the liver metastasis of mice in vivo. USP-15 upregulation obtained the contrary effects. Subsequently, USP-15 deubiquitinated transcription factor AP-4 (TFAP4) and enhanced its protein stability. TFAP4 could transcriptionally activated polycomb group ring finger 1 (PCGF1). The pro-cancer effects of USP-15 were rescue by the knockdown of TFAP4 or PCGF1. In conclusions: USP-15 facilitated the liver metastasis by the enhancement of cell stemness and EMT in CRC, which was at least partly mediated by the deubiquitination of TFAP4 upon the upregulation of PCGF1.

Casearia sylvestris var. lingua (Càmbess.) Eichler leaves aqueous extract improves colon inflammation through mucogenic, antioxidant and anti-inflammatory actions in TNBS- induced IBD rats

Ethnopharmacological relevanceCasearia sylvestris var. lingua (Cambess.) Eichler, a member of the Salicaceae family, holds a prominent place in traditional medicine across various cultures due to its versatile therapeutic properties. Historically, indigenous communities have utilized different parts of the plant, including leaves, bark, and roots, to address a wide array of health conditions. Traditional uses of C. sylvestris var. lingua encompasses the treatment of gastrointestinal disorders, respiratory infections, wound healing, inflammation, and stomach ulcers. Pharmacological studies have demonstrated the plant's antimicrobial, anti-inflammatory, antioxidant, analgesic, gastroprotective, and immunomodulatory effects. This signifies the first scientific validation report for C. sylvestris var. lingua regarding its effectiveness against ulcerative colitis. The report aims to affirm the traditional use of this plant through pre-clinical experiments. Aim of the researchThis work uses an aqueous extract from C. sylvestris var. lingua leaves (AECs) to evaluate the acute anti-ulcerative colitis efficacy in rat and HT-29 (human colorectal cancer cell line) models. MethodsTo determine the secondary metabolites of AECs, liquid chromatography with a diode array detector (LC-DAD) study was carried out. 2,4,6-trinitrobenzenesulfonic acid (TNBS, 30 mg/0.25 mL EtOH 30% v/v) was used as an enema to cause acute colitis. Three days were spent giving the C. sylvestris var. lingua extract orally by gavage at dosages of 3, 30, and 300 mg/kg. The same route was used to deliver distilled water to the vehicle and naïve groups. After the animals were sacrificed on the fourth day, intestinal tissues were taken for histological examination and evaluation of biochemical tests such as those measuring superoxide dismutase (SOD), reduced glutathione (GSH), catalase (CAT), malondialdehyde (MDA), nitrite/nitrate, myeloperoxidase (MPO) activity. Additionally, interleukin 1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), and interleukin 10 (IL-10), were conducted on the intestinal tissues. Additionally, an MTT assay was used to evaluate the effect of AECs on the viability of HT-29 cells. Additionally, a molecular docking study was carried out to compare some potential target proteins with identified chemicals found in AECs. ResultsLC-DAD analysis identified five compounds (caffeic acid, ellagic acid, ferulic acid, gallic acid, and quercetin) in AECs. Pre-administration of AECs (3; 30; 300 mg/kg) and mesalazine (500 mg/kg) reduced macroscopic scores (55%, 47%, 45%, and 52%, p < 0.001) and ulcerated areas (70.3%, 70.5%, 57%, and 56%, p < 0.001), respectively. It also increased SOD, GSH, and CAT activities (p < 0.01), while decreasing MDA (p < 0.001), nitrite/nitrate (p < 0.05), and MPO (p < 0.001) activities compared to the colitis group. Concerning inflammatory markers, significant modulations were observed: AECs (3, 30, and 300 mg/kg) lowered levels of IL-1β and TNF-α (p < 0.001) and increased IL-10 levels (p < 0.001) compared to the colitis groups. The viability of HT-29 cells was suppressed by AECs with an IC50 of 195.90 ± 0.01 μg/mL (48 h). During the molecular docking analysis, quercetin, gallic acid, ferulic acid, caffeic acid, and ellagic acid demonstrated consistent binding affinities, forming stable interactions with the 3w3l (TLR8) and the 3ds6 (MAPK14) complexes. ConclusionThese results imply that the intestinal mucogenic, anti-inflammatory, and antioxidant properties of the C. sylvestris var. lingua leaf extract may be involved in its therapeutic actions for ulcerative colitis. The results of the in silico study point to the possibility of quercetin and ellagic acid interacting with P38 and TLR8, respectively, in a beneficial way.

Evaluation of Anti-Cancer Potential of Homeopathic Medicines by In-Vitro Study on Human Breast, Lung and Colorectal Cancer Cells

The purpose of this study is to assess the cytotoxic effects of homoeopathic drugs on cancer cell lines by In-vitro experiment. Background: Homoeopathy is a renowned therapeutic approach in many nations across the world. However, the approach has sparked much scientific discussion concerning its efficacy and methods of action. This is particularly true when it comes to the application of homeopathy in cancer treatment. Objective: The purpose of this study was to evaluate the cytotoxic effect of homoeopathic remedies on cancer cells and to emphasise the therapeutic effects of 50 Millesimal potencies. Materials &amp; Methods: Carcinosinum, Conium maculatum, and Asterias rubens were the drugs used in 0/1 potency against the MCF 7 breast cancer cell line. Carcinosinum, Nitric acid, and Ruta were the drugs in 0/1 potency used against the HCT 116 colorectal cancer cell line and the NCI-H460 lung cancer cell line. 90% alcohol medicated on poppy-sized globules, dried fully and used as a control for all cell lines. The cytotoxicity has been assessed using the standard MTT test. Results: The LC50 values of the medicines tested on breast and lung cancer cells were not obtained up to 40000 ppm concentrations, as a result, cytotoxicity evaluation was not possible. The LC50 values of the medicines tested on Colo-rectal cancer cells were obtained around 20,000 ppm concentration and showed a gradual increase in Cell Death with increasing Concentration of exposure of the drugs. As a result, these drugs revealed cytotoxic qualities as well as anti-cancer actions. Conclusion: Despite the objective facts, we cannot reach a definite conclusion about the anti-cancer effectiveness of these drugs in vitro because several aspects of homeopathy's action are unlikely to be replicated in In-vitro study. Determination of reasons for the same or understanding the molecular mechanism of resistance of the cell lines to the cytotoxic effects of the drugs were not within the purview of the study and these can be addressed in future studies. Keywords: Homeopathy, Cancer, MCF-7, HCT 116, NCI-H 460, In-Vitro, Carcinosinum, Conium maculatum, Asteria rubens, Ruta graveolens, Nitric acid, 50 Millesimal Potency, MTT Assay.

FERMENTATION, PURIFICATION AND IN-VITRO EVALUATION OF ANTIMICROBIAL, ANTITUMOR AND ANTIOXIDANT COMPOUNDS FROM A HIMALAYAN ACTINOBACTERIA STRAIN STREPTOMYCES SP. PU-AK14

This study reports the laboratory scale fermentation, compounds purification and bioactivity screening of the strain Streptomyces sp. PU-AK14 isolated from Himalayan mountains range, Pakistan, for its potential to produce antimicrobial, antioxidant and antitumor compounds. For this purpose, laboratory fermentation (20 L working volume) was performed followed by extraction of the compounds by XAD adsorbent gel using the solvents methanol and ethyl acetate. Further purification of the compounds was done by silica gel column chromatography, sephadex column and preparative TLC. A total of 18 partially purified compounds were retrieved from crude extract and were screened for biological activity. The crude extract was more active against gram negative bacteria with MIC of 0.1 mg/ml against Klebsiella pneumoniae and Escherichia coli. Eight of the partially purified compounds exhibited activity against MRSA whereas five fractions were active against gram negative bacteria. Seven partially purified fractions exhibited promising antioxidant activity with radical scavenging activity ranging from 78.2 to 42.34 percent. Cytotoxicity of partially purified compounds ranged from 75% to 100% larval mortality at 100mg/ml against Artemia salina and 50% to 52% cell mortality against HCT 116 colorectal cancer cell line at 100mg/ml. The best anti-tumor activity was exhibited by the fraction AK141111L. The study suggested that Streptomyces sp. PU-AK14 is a potential source of chemotherapeutically useful compounds.

Limited impact of cancer-derived gangliosides on anti-tumor immunity in colorectal cancer.

Aberrant glycosylation is a key mechanism employed by cancer cells to evade immune surveillance, induce angiogenesis and metastasis, among other hallmarks of cancer. Sialic acids, distinctive terminal glycan structures located on glycoproteins or glycolipids, are prominently upregulated across various tumor types, including colorectal cancer (CRC). Sialylated glycans modulate anti-tumor immune responses through their interactions with Siglecs, a family of glycan-binding receptors with specificity for sialic acid-containing glycoconjugates, often resulting in immunosuppression. In this paper, we investigated the immunomodulatory function of ST3Gal5, a sialyltransferase that catalyzes the addition of α2-3 sialic acids to glycosphingolipids, since lower expression of ST3Gal5 is associated with better survival of CRC patients. We employed CRISPR/Cas9 to knock out the ST3Gal5 gene in two murine CRC cell lines MC38 and CT26. Glycomics analysis confirmed the removal of sialic acids on glycolipids, with no discernible impact on glycoprotein sialylation. Although knocking out ST3Gal5 in both cell lines did not affect in vivo tumor growth, we observed enhanced levels of regulatory T cells in CT26 tumors lacking ST3Gal5. Moreover, we demonstrate that the absence of ST3Gal5 affected size and blood vessel density only in MC38 tumors. In summary, we ascertain that sialylation of glycosphingolipids has a limited influence on the anti-tumor immune response in CRC, despite detecting alterations in the tumor microenvironment, possibly due to a shift in ganglioside abundance.

#883 P-cresyl sulphate and Indoxyl sulphate: two uremic toxins stimulating colon cancer progression in patients with chronic renal failure

Abstract Background and Aims Chronic kidney disease (CKD) leads to the accumulation and production of uremic toxins, such as Indoxyl Sulphate (IS) and P-cresyl sulphate (p-CS). These toxins have been found to activate various processes that promote tumor growth. The literature has reported a two-fold increase in the incidence of colon cancer (CRC) and a worse prognosis in CKD patients, although the specific mechanisms underlying this association have not been fully elucidated. Given the pro-oncogenic activities of p-CS and IS at both the systemic and intestinal levels, it is plausible that these toxins contribute to the heightened aggressiveness of CRC in CKD patients. Method In order to assess the impact of toxins detected in uremic patients, we conducted experiments using three different colorectal cancer (CRC) cell lines (HT29, Hct116, LoVo). We tested various concentrations of IS and p-CS and measured their effects on tumor cell proliferation through a colorimetric assay called WST-8. For further analysis of cell survival, we performed a clonogenic assay. To investigate the migratory abilities of tumor cells after toxins treatment, we conducted a wound healing assay. Additionally, we assessed the invasive capacities of the three CRC cell lines post treatment with IS and p-CS using an invasion assay. In order to identify the specific pathways affected by toxins exposure, we conducted Real Time PCR and Western blot analysis to evaluate the inflammatory pathway and the epithelial-mesenchymal transition (EMT), respectively. Results The results of this study are remarkable, as we observed a notable increase in cell growth after exposure of cultured cells to various concentrations of toxins, as compared to the control group. Furthermore, the concentrations of IS and p-CS induced a substantial enhancement in cell migration and invasion. In the case of HCT116 cells treated with uremic toxins, there was an up-regulation of Vimentin protein expression, while E-cadherin expression was down-regulated compared to the control group. These proteins are crucial in the process of Epithelial-Mesenchymal Transition (EMT). Additionally, we evaluated the expression of genes associated with the inflammatory process (TNF-alpha, INOS, and IL-6) in the HT-29 and LoVo cell lines. Interestingly, we observed an up-regulation of these genes. Of particular importance, in the LoVo cell line, IL-6 exhibited the most significant increase in expression. Conclusion Based on these preliminary results, it appears that IS and p-CS play a key role in the progression of CRC in patients with CKD.

Mechanism of the Radioresistant Colorectal Cancer Cell Line SW480RR Established after Fractionated X Irradiation.

Radioresistant cancer cells are risk factors for recurrence and are occasionally detected in recurrent tumors after radiotherapy. Intratumor heterogeneity is believed to be a potential cause of treatment resistance. Heterogeneity in DNA content has also been reported in human colorectal cancer; however, little is known about how such heterogeneity changes with radiotherapy or how it affects cancer radioresistance. In the present study, we established radioresistant clone SW480RR cells after fractionated X-ray irradiation of human colorectal cancer-derived SW480.hu cells, which are composed of two cell populations with different chromosome numbers, and examined how cellular radioresistance changed with fractionated radiotherapy. Compared with the parental cell population, which mostly comprised cells with higher ploidy, the radioresistant clones showed lower ploidy and less initial DNA damage. The lower ploidy cells in the parental cell population were identified as having radioresistance prior to irradiation; thus, SW480RR cells were considered intrinsically radioresistant cells selected from the parental population through fractionated irradiation. This study presents a practical example of the emergence of radioresistant cells from a cell population with ploidy heterogeneity after irradiation. The most likely mechanism is the selection of an intrinsically radioresistant population after fractionated X-ray irradiation, with a background in which lower ploidy cells exhibit lower initial DNA damage.

Mutational signatures of colorectal cancers according to distinct computational workflows.

Tumor mutational signatures have gained prominence in cancer research, yet the lack of standardized methods hinders reproducibility and robustness. Leveraging colorectal cancer (CRC) as a model, we explored the influence of computational parameters on mutational signature analyses across 230 CRC cell lines and 152 CRC patients. Results were validated in three independent datasets: 483 endometrial cancer patients stratified by mismatch repair (MMR) status, 35 lung cancer patients by smoking status and 12 patient-derived organoids (PDOs) annotated for colibactin exposure. Assessing various bioinformatic tools, reference datasets and input data sizes including whole genome sequencing, whole exome sequencing and a pan-cancer gene panel, we demonstrated significant variability in the results. We report that the use of distinct algorithms and references led to statistically different results, highlighting how arbitrary choices may induce variability in the mutational signature contributions. Furthermore, we found a differential contribution of mutational signatures between coding and intergenic regions and defined the minimum number of somatic variants required for reliable mutational signature assignment. To facilitate the identification of the most suitable workflows, we developed Comparative Mutational Signature analysis on Coding and Extragenic Regions (CoMSCER), a bioinformatic tool which allows researchers to easily perform comparative mutational signature analysis by coupling the results from several tools and public reference datasets and to assess mutational signature contributions in coding and non-coding genomic regions. In conclusion, our study provides a comparative framework to elucidate the impact of distinct computational workflows on mutational signatures.

Erk Inhibition as a Promising Therapeutic Strategy for High IL-8-Secreting and Low SPTAN1-Expressing Colorectal Cancer.

Tumor recurrence and drug resistance are responsible for poor prognosis in colorectal cancer (CRC). DNA mismatch repair (MMR) deficiency or elevated interleukin-8 (IL-8) levels are characteristics of CRCs, which have been independently correlated with treatment resistance to common therapies. We recently demonstrated significantly impaired therapeutical response and increased IL-8 release of CRC cell lines with reduced expression of MMR protein MLH1 as well as cytoskeletal non-erythrocytic spectrin alpha II (SPTAN1). In the present study, decreased intratumoral MLH1 and SPTAN1 expression in CRCs could be significantly correlated with enhanced serum IL-8. Furthermore, using stably reduced SPTAN1-expressing SW480, SW620 or HT-29 cell lines, the RAS-mediated RAF/MEK/ERK pathway was analyzed. Here, a close connection between low SPTAN1 expression, increased IL-8 secretion, enhanced extracellular-signal-regulated kinase (ERK) phosphorylation and a mesenchymal phenotype were detected. The inhibition of ERK by U0126 led to a significant reduction in IL-8 secretion, and the combination therapy of U0126 with FOLFOX optimizes the response of corresponding cancer cell lines. Therefore, we hypothesize that the combination therapy of FOLFOX and U0126 may have great potential to improve drug efficacy on this subgroup of CRCs, showing decreased MLH1 and SPTAN1 accompanied with high serum IL-8 in affected patients.

Monastrol suppresses invasion and metastasis in human colorectal cancer cells by targeting fascin independent of kinesin-Eg5 pathway

Rearrangement of the actin cytoskeleton is a prerequisite for carcinoma cells to develop cellular protrusions, which are required for migration, invasion, and metastasis. Fascin is a key protein involved in actin bundling and is expressed in aggressive and invasive carcinomas. Additionally, fascin appears to be involved in tubulin-binding and microtubule rearrangement. Pharmacophoric-based in silico screening was performed to identify compounds with better fascin inhibitory properties than migrastatin, a gold-standard fascin inhibitor. We hypothesized that monastrol displays anti-migratory and anti-invasive properties via fascin blocking in colorectal cancer cell lines. Biophysical (thermofluor and ligand titration followed by fluorescence spectroscopy), biochemical (NMR), and cellular assays (MTT, invasion of human tissue), as well as animal model studies (zebrafish invasion) were performed to characterize the inhibitory effect of monastrol on fascin activity. In silico analysis revealed that monastrol is a potential fascin-binding compound. Biophysical and biochemical assays demonstrated that monastrol binds to fascin and interferes with its actin-bundling activity. Cell culture studies, including a 3D human myoma disc model, showed that monastrol inhibited fascin-driven cytoplasmic protrusions as well as invasion. In silico, confocal microscopy, and immunoprecipitation assays demonstrated that monastrol disrupted fascin-tubulin interactions. These anti-invasive effects were confirmed in vivo. In silico confocal microscopy and immunoprecipitation assays were carried out to test whether monastrol disrupted the fascin-tubulin interaction. This study reports, for the first time, the in vitro and in vivo anti-invasive properties of monastrol in colorectal tumor cells. The number and types of interactions suggest potential binding of monastrol across actin and tubulin sites on fascin, which could be valuable for the development of antitumor therapies.

Combination of FOLFOXIRI Drugs with Oncolytic Coxsackie B3 Virus PD-H Synergistically Induces Oncolysis in the Refractory Colorectal Cancer Cell Line Colo320.

FOLFOXIRI chemotherapy is a first-line therapy for advanced or metastatic colorectal cancer (CRC), yet its therapeutic efficacy remains limited. Immunostimulatory therapies like oncolytic viruses can complement chemotherapies by fostering the infiltration of the tumor by immune cells and enhancing drug cytotoxicity. In this study, we explored the effect of combining the FOLFOXIRI chemotherapeutic agents with the oncolytic coxsackievirus B3 (CVB3) PD-H in the CRC cell line Colo320. Additionally, we examined the impact of the drugs on the expression of microRNAs (miRs), which could be used to increase the safety of oncolytic CVB3 containing corresponding miR target sites (miR-TS). The measurement of cytotoxic activity using the Chou-Talalay combination index approach revealed that PD-H synergistically enhanced the cytotoxic activity of oxaliplatin (OX), 5-fluorouracil (5-FU) and SN-38. PD-H replication was not affected by OX and SN-38 but inhibited by high concentrations of 5-FU. MiR expression levels were not or only slightly elevated by the drugs or with drug/PD-H combinations on Colo320 cells. Moreover, the drug treatment did not increase the mutation rate of the miR-TS inserted into the PD-H genome. The results demonstrate that the combination of FOLFOXIRI drugs and PD-H may be a promising approach to enhance the therapeutic effect of FOLFOXIRI therapy in CRC.

Connection between MiR-490 and CCND1 and GSK3β genes play an effective role in Wnt signaling pathway in colorectal cancer.

The Wnt signaling pathway is identified as one of the main disrupted pathways in Colorectal cancer (CRC). Results from studies focusing on this route will aid greatly in the detection and treatment of CRC. MicroRNAs (MiRs), particularly MiR-490, has emerged as key regulator of gene expression in biological pathways, making it an attractive research target. This is notably true for the Wnt signaling pathway, which is usually disordered in CRC tissues. This study aimed to evaluate the expression level of MiR-490 isomiRs and determine some of its key target genes involved in Wnt signaling pathway in CRC tissues and cell lines, based on experimental and bioinformatics analysis. Elevated expression of GSK3β and CCND1 indicate that the progression of CRC tumor is associated with the inhibitory effect of MiR-490 isomiRs on the Wnt/β-catenin signaling pathway. This finding was supported by the observation of a positive connection between the expression pattern of miR-490-3p and 5p, and CCND1 and GSK3β in CRC. The valuable results of this study provide a means of identifying biomarkers with the potential to either inhibit or activate CRC cellular pathways.

High expression of fragile X mental retardation protein inhibits ferroptosis of colorectal tumor cells by activating the RAS/MAPK signaling pathway

To investigate the mechanism by which fragile X mental retardation protein (FMRP) regulates ferroptosis evasion in colorectal cancer (CRC) cells. We examined FMRP expression levels in CRC cell lines using RT-qPCR and Western blotting and analyzed the biological functions and signaling pathways involved in FMRP-mediated regulation of CRC progression using the TCGA database. A lentiviral FMRP overexpression vector (Lv-FMRP) and 3 knockdown vectors (siFMRP-1, siFMRP-2, and siFMRP-3) were constructed, and their effects on proliferation of HCT116 cells were examined using CCK8 assay and plate clone formation assay; the changes in cell ferroptosis level was determined using MDA/ROS/GSH/Fe2+ kits, mitochondrial membrane potential changes were detected using JC-1 fluorescence staining, and the expressions of proteins associated with ferroptosis and the RAS/MAPK signaling pathway were detected using Western blotting. The subcutaneous tumorigenic potential of the transfected cells was evaluated in nude mice. Compared with normal colonic mucosal epithelial NCM460 cells, the CRC cell lines had significantly higher FMRP expression level. Bioinformatics analysis suggested the involvement of FMRP in regulation of reactive oxygen, oxidative stress-induced cell death, mitochondrial respiration, and glutathione metabolism pathways. In the cell experiments, FMRP knockdown significantly inhibited proliferation of HCT116 cells, lowered cellular GSH content, increased MDA and ROS levels, Fe2+ fluorescence intensity, and mitochondrial membrane potential, and decreased SLC7A11/GPX4 protein expressions and the phosphorylation levels of ERK, MEK, MAPK, and RAS proteins; FMRP overexpression resulted in the opposite changes in the cells. In the tumor-bearing nude mice, HCT116 cells with FMRP knockdown showed attenuated tumorigenic potential with lowered xenograft growth rate and reduced SLC7A11 expression in the xenograft. The high expression of FMRP inhibits ferroptosis in CRC cells and promotes progression of CRC by activating the RAS/MAPK signaling pathway.