SW480 Colorectal Cancer Cells Research Articles

RUNX1-induced upregulation of PTGS2 enhances cell growth, migration and invasion in colorectal cancer cells

Colorectal cancer (CRC) arises via the progressive accumulation of dysregulation in key genes including oncogenes and tumor-suppressor genes. Prostaglandin-endoperoxide synthase 2 (PTGS2, also called COX2) acts as an oncogenic driver in CRC. Here, we explored the upstream transcription factors (TFs) responsible for elevating PTGS2 expression in CRC cells. The results showed that PTGS2 silencing repressed cell growth, migration and invasion in HCT116 and SW480 CRC cells. The two fragments (499–981 bp) and (1053–1434 bp) were confirmed as the core TF binding profiles of the PTGS2 promoter. PTGS2 expression positively correlated with RUNX1 level in colon adenocarcinoma (COAD) samples using the TCGA-COAD dataset. Furthermore, RUNX1 acted as a positive regulator of PTGS2 expression by promoting transcriptional activation of the PTGS2 promoter via the 1086–1096 bp binding motif. In conclusion, our study demonstrates that PTGS2 upregulation induced by the TF RUNX1 promotes CRC cell growth, migration and invasion, providing an increased rationale for the use of PTGS2 inhibitors in CRC prevention and treatment.

Machine Learning-Based Integrated Multiomics Characterization of Colorectal Cancer Reveals Distinctive Metabolic Signatures.

The metabolic signature identification of colorectal cancer is critical for its early diagnosis and therapeutic approaches that will significantly block cancer progression and improve patient survival. Here, we combined an untargeted metabolic analysis strategy based on internal extractive electrospray ionization mass spectrometry and the machine learning approach to analyze metabolites in 173 pairs of cancer samples and matched normal tissue samples to build robust metabolic signature models for diagnostic purposes. Screening and independent validation of metabolic signatures from colorectal cancers via machine learning methods (Logistic Regression_L1 for feature selection and eXtreme Gradient Boosting for classification) was performed to generate a panel of seven signatures with good diagnostic performance (the accuracy of 87.74%, sensitivity of 85.82%, and specificity of 89.66%). Moreover, seven signatures were evaluated according to their ability to distinguish between cancer and normal tissues, with the metabolic molecule PC (30:0) showing good diagnostic performance. In addition, genes associated with PC (30:0) were identified by multiomics analysis (combining metabolic data with transcriptomic data analysis) and our results showed that PC (30:0) could promote the proliferation of colorectal cancer cell SW480, revealing the correlation between genetic changes and metabolic dysregulation in cancer. Overall, our results reveal potential determinants affecting metabolite dysregulation, paving the way for a mechanistic understanding of altered tissue metabolites in colorectal cancer and design interventions for manipulating the levels of circulating metabolites.

LINC01021 Attenuates Expression and Affects Alternative Splicing of a Subset of p53-Regulated Genes.

Loss of the p53-inducible LINC01021 in p53-proficient CRC cell lines results in increased sensitivity to DNA-damaging chemotherapeutics. Here, we comprehensively analyze how LINC01021 affects the p53-induced transcriptional program. Using a CRISPR/Cas9-approach, we deleted the p53 binding site in the LINC01021 promoter of SW480 colorectal cancer cells and subjected them to RNA-Seq analysis after the activation of ectopic p53. RNA affinity purification followed by mass spectrometry was used to identify proteins associated with LINC01021. Loss of the p53-inducibility of LINC01021 resulted in an ~1.8-fold increase in the number of significantly regulated mRNAs compared to LINC01021 wild-type cells after ectopic activation of p53. A subset of direct p53 target genes, such as NOXA and FAS, displayed significantly stronger induction when the p53-inducibility of LINC01021 was abrogated. Loss of the p53-inducibility of LINC01021 resulted in alternative splicing of a small number of mRNAs, such as ARHGAP12, HSF2, and LYN. Several RNA binding proteins involved in pre-mRNA splicing were identified as interaction partners of LINC01021 by mass spectrometry. Our results suggest that LINC01021 may restrict the extent and strength of p53-mediated transcriptional changes via context-dependent regulation of the expression and splicing of a subset of p53-regulated genes.

Wighteone, a prenylated flavonoid from licorice, inhibits growth of SW480 colorectal cancer cells by allosteric inhibition of Akt

Ethnopharmacological relevanceLicorice is a frequently used herbal medicine worldwide, and is used to treat cough, hepatitis, cancer and influenza in clinical practice of traditional Chinese medicine. Modern pharmacological studies indicate that prenylated flavonoids play an important role in the anti-tumor activity of licorice, especially the tumors in stomach, lung, colon and liver. Wighteone is one of the main prenylated flavonoids in licorice, and its possible effect and target against colorectal cancer have not been investigated. Aim of the studyThis study aimed to investigate the anti-colorectal cancer effect and underlying mechanism of wighteone. Materials and methodsSW480 human colorectal cancer cells were used to evaluate the in vitro anti-colorectal cancer activity and Akt regulation effect of wighteone by flow cytometry, phosphoproteomic and Western blot analysis. Surface plasmon resonance (SPR) assay, molecular docking and dynamics simulation, and kinase activity assay were used to investigate the direct interaction between wighteone and Akt. A nude mouse xenograft model with SW480 cells was used to verify the in vivo anti-colorectal cancer activity of wighteone. ResultsWighteone inhibited phosphorylation of Akt and its downstream kinases in SW480 cells, which led to a reduction in cell viability. Wighteone had direct interaction with both PH and kinase domains of Akt, which locked Akt in a “closed” conformation with allosteric inhibition, and Gln79, Tyr272, Arg273 and Lys297 played the most critical role due to their hydrogen bond and hydrophobic interactions with wighteone. Based on Akt overexpression or activation in SW480 cells, further mechanistic studies suggested that wighteone-induced Akt inhibition led to cycle arrest, apoptosis and autophagic death of SW480 cells. Moreover, wighteone exerted in vivo anti-colorectal cancer effect and Akt inhibition activity in the nude mouse xenograft model. ConclusionWighteone could inhibit growth of SW480 cells through allosteric inhibition of Akt, which led to cell cycle arrest, apoptosis and autophagic death. The results contributed to understanding of the anti-tumor mechanism of licorice, and also provided a rationale to design novel Akt allosteric inhibitors for the treatment of colorectal cancer.

Cancer-associated fibroblasts drive colorectal cancer cell progression through exosomal miR-20a-5p-mediated targeting of PTEN and stimulating interleukin-6 production.

This study evaluated the clinical relevance of a set of five serum-derived circulating microRNAs (miRNAs) in colorectal cancer (CRC). Additionally, we investigated the role of miR-20a-5p released by exosomes derived from cancer-associated fibroblasts (CAFs) in the context of CRC. The expression levels of five circulating serum-derived miRNAs (miR-20a-5p, miR-122-5p, miR-139-3p, miR-143-5p, and miR-193a-5p) were quantified by real-time quantitative PCR (RT-qPCR), and their associations with clinicopathological characteristics in CRC patients were assessed. The diagnostic accuracy of these miRNAs was determined through Receiver Operating Characteristic (ROC) curve analysis. CAFs and normal fibroblasts (NFs) were isolated from tissue samples, and subsequently, exosomes derived from these cells were isolated and meticulously characterized using electron microscopy and Western blotting. The cellular internalization of fluorescent-labeled exosomes was visualized by confocal microscopy. Gain- and loss-of-function experiments were conducted to elucidate the oncogenic role of miR-20a-5p transferred by exosomes derived from CAFs in CRC progression. The underlying mechanisms were uncovered through luciferase reporter assay, Western blotting, enzyme-linked immunosorbent assays, as well as proliferation and migration assays. The expression levels of serum-derived circulating miR-20a-5p and miR-122-5p were significantly higher in CRC and were positively correlated with advanced stages of tumorigenesis and lymph node metastasis (LNM). In contrast, circulating miR-139-3p, miR-143-5p, and miR-193a-5p were down-regulated in CRC and associated with early tumorigenesis. Except for miR-139-3p, they showed a negative correlation with LNM status. Among the candidate miRNAs, significantly elevated levels of miR-20a-5p were observed in both cellular and exosomal fractions of CAFs. Our findings indicated that miR-20a-5p induces the expression of EMT markers, partly by targeting PTEN. Exosomal miR-20a secreted by CAFs emerged as a key factor enhancing the proliferation and migration of CRC cells. The inhibition of miR-20a impaired the proliferative and migratory potential of CAF-derived exosomes in SW480 CRC cells, suggesting that the oncogenic effects of CAF-derived exosomes are mediated through the exosomal transfer of miR-20a. Furthermore, exosomes originating from CAFs induced increased nuclear translocation of the NF-kB p65 transcription factor in SW480 CRC cells, leading to increased interleukin-6 (IL-6) production. We established a set of five circulating miRNAs as a non-invasive biomarker for CRC diagnosis. Additionally, our findings shed light on the intricate mechanisms underpinning the oncogenic impacts of CAF-derived exosomes and underscore the pivotal role of miR-20a-5p in CRC progression.

23-Hydroxybetulinic acid attenuates 5-fluorouracil resistance of colorectal cancer by modulating M2 macrophage polarization via STAT6 signaling

Macrophage polarization is closely associated with the inflammatory processes involved in the development and chemoresistance of colorectal cancer (CRC). M2 macrophages, the predominant subtype of tumor-associated macrophages (TAMs) in a wide variety of malignancies, have been demonstrated to promote the resistance of CRC to multiple chemotherapeutic drugs, such as 5-fluorouracil (5-FU). In our study, we investigated the potential of 23-Hydroxybetulinic Acid (23-HBA), a significant active component of Pulsatilla chinensis (P. chinensis), to inhibit the polarization of M2 macrophages induced by IL-4. Our results showed that 23-HBA reduced the expression of M2 specific marker CD206, while downregulating the mRNA levels of M2 related genes (CD206, Arg1, IL-10, and CCL2). Additionally, 23-HBA effectively attenuated the inhibitory effects of the conditioned medium from M2 macrophages on apoptosis in colorectal cancer SW480 cells. Mechanistically, 23-HBA prevented the phosphorylation and nuclear translocation of the STAT6 protein, resulting in the inhibition of IL-10 release in M2 macrophages. Moreover, it interfered with the activation of the IL-10/STAT3/Bcl-2 signaling pathway in SW480 cells, ultimately reducing M2 macrophage-induced resistance to 5-FU. Importantly, depleting STAT6 expression in macrophages abolished the suppressive effect of 23-HBA on M2 macrophage polarization, while also eliminating its ability to decrease M2 macrophage-induced 5-FU resistance in cancer cells. Furthermore, 23-HBA significantly diminished the proportion of M2 macrophages in the tumor tissues of colorectal cancer mice, simultaneously enhancing the anti-cancer efficacy of 5-FU. The findings presented in this study highlight the capacity of 23-HBA to inhibit M2 macrophage polarization, a process that contributes to reduced 5-FU resistance in colorectal cancer.

Abstract 4095: Targeting alternative classes of tumor antigens to improve immunotherapy for microsatellite stable, mismatch repair-proficient colorectal cancer

Abstract Background: The clinical outcomes of immunotherapy for microsatellite stable (MSS) colorectal cancer (CRC) remains unsatisfactory. The development of more effective immunotherapies is hampered by limited knowledge of the appropriate target antigens, also considering the low burden of mutations generating neoantigens of these tumors. Alternative classes of MSS CRC antigens may provide actionable epitopes. Methods: We have characterized the immunopeptidome of 2 MSS CRC cell lines derived from the primary tumor (SW480) and a metastatic lymph node (SW620) of the same patient. Given the low mutational burden of MSS, we focused on peptides belonging to alternative sources of HLA-A*02 epitopes, such as proteasomal cis-spliced peptides derived from cancer testis antigens and linear epitopes derived from Human Endogenous Retroviruses (HERVs), which are frequently reactivated in CRC. Peptide immunogenicity was functionally validated based on their ability to expand HLA-A*02+ donor T cells (>3) specifically killing targets cells presenting the relevant peptides or HLA-matched CRC cell lines. In vivo epitope immunogenicity was verified by analyzing polyfunctional responses of human CD8+ T cells obtained from NSG-A2 mice humanized with A*02+ donor PBMCs and immunized with epitope-loaded nano-emulsions selectively targeting human Clec9A+ dendritic cells. Therapeutic exploitation of the novel epitopes was investigated by adoptive transfer of epitope-specific T cells in NSG-A2 mice injected with SW620 cells. Epitope-specific T cell responses were investigated in PBMCs from 10 MSS CRC patients and 7 donors. Results: Among >50,000 HLA-bound peptides identified by in depth immunopeptidomics analysis, 11 cis-spliced peptides and 5 HERV peptides were functionally validated as immunogenic. Epitope-specific T cells showed specific cytotoxic activity against SW480 and SW620 CRC cells. Globally, cis-spliced peptides were more immunogenic in vitro than HERV-derived peptides. The immunogenicity of both groups of peptides was confirmed in vivo in humanized mice. Adoptively transferred HERV or cis-spliced epitope specific T cells significantly inhibited the growth of SW620 cells in vivo, with cis-spliced epitope-specific T cells being more effective. Therapeutic efficacy of Clec9A-targeting vaccines exploiting these epitopes is currently under investigation. T cell responses specific for cis-spliced and HERV epitopes were detected in PBMCs from 3/4 MSS CRC HLA-A*02+ patients but in none of 4 HLA-A*02- MSS CRC patients or 7 healthy donors (4 HLA-A*02+ and 3 HLA-A*02-). Conclusions: We have identified and validated a new class of immunogenic HLA-A*02 epitopes derived from alternative classes of MSS CRC antigens. We also provide the proof of principle supporting their therapeutic exploitation to improve the management of these poorly immunogenic tumors. Citation Format: Riccardo Dolcetti, Bijun Zeng, Jingran Ye, Tima Shamekhi, Madeleine Harpur, Davide Moi, Sara Roth, Rob Ramsay, Divya Duscharla, Antony W. Purcell, Pouya Faridi, Roberta Mazzieri. Targeting alternative classes of tumor antigens to improve immunotherapy for microsatellite stable, mismatch repair-proficient colorectal cancer [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 4095.

Abstract 5452: AXIN2 differentially modulates stem cell subpopulations providing a potential therapeutic opportunity for targeting colorectal cancer stem cells

Abstract Constitutively activated WNT-signaling promotes stem cell (SC) overpopulation which drives most colorectal cancer (CRC) development. It is not known, however, how WNT-signaling activity regulates different SC sub-populations. We previously reported that SC population size decreases as AXIN2 is knocked down, and increases when AXIN2 is overexpressed. Changes in cell cycle and apoptotic rate were also observed. Here, we present our recent results on changes between different SC subpopulations (ALDH+ vs. LGR5+) that occur due to AXIN2 modulation. We hypothesize that modulation of AXIN2 promotes differential expansion between different CRC SC subpopulations (ALDH+ and LGR5+). We observed that AXIN2 not only increases LGR5+ SC population size, but also expands the ALDH+ SC population to a greater extent as shown by an increased ALDH+-to-LGR5+ SCs ratio. This indicates that AXIN2, via WNT signaling, regulates SC population size, particularly ALDH+ SCs. Since the ALDH SC marker is also a key component in the retinoic acid (RA) signaling pathway, we surmised that increased AXIN2 would not only lead to elevated ALDH levels, but also upregulate the RA-signaling pathway components in CRC cells. To this end, we conducted NanoString mRNA profiling to study the effects of AXIN2 knockdown and overexpression in HT29 and SW480 CRC cells. The results showed that increased AXIN2 significantly up-regulated several key components of the RA-signaling pathway. Taken together, our results indicate that AXIN2 drives a shift in SC subpopulation sizes favoring the ALDH+ SCs. It also promotes the RA-signaling component expression - a pathway known to regulate SC differentiation. As a potential treatment strategy for CRC, therapeutically inducing high levels of AXIN2 could shift the balance between SC populations towards ALDH+ SCs, which may provide a way to target cancer SCs with retinoids to induce their differentiation. Citation Format: Chi Zhang, Caroline O. Facey, Lynn M. Opdenaker, Bruce M. Boman. AXIN2 differentially modulates stem cell subpopulations providing a potential therapeutic opportunity for targeting colorectal cancer stem cells [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 5452.

Abstract 6015: Effect of antineoplastic drugs on cell-to-cell communication in the progression of colorectal cancer

Abstract Background: In the past few years, there has been notable progress in colorectal cancer research, resulting in novel development and delivery of drugs specifically designed for individual patient requirements. However, one of the major roadblocks in designing and treating colorectal cancer is the drug resistance found in advanced stages. Thus, there is a profound need to establish new methods to overcome this barrier. Previous work illustrated that cell-to-cell communications are dysfunctional for multitude of reasons in colorectal cancer cells. Restoration of cell-to-cell communication is a mean to deliver antineoplastic drugs via bystanding effect. Prior research has demonstrated that restoring gap junctions of cell-to-cell communication in breast cancer cells enhances the efficacy of cisplatin, a drug previously deemed resistant. Hence, this study seeks to assess the potency and efficacy of antineoplastic agents known to show resistance in colorectal cancer, with the intention to investigate how gap junctions can increase the efficacy of antineoplastic agents via restoration of cell-to-cell communication. Method: We assessed cell viability utilizing the RealTime Glo MT Cell Viability assay (Promega, Inc.). Four specific colorectal cancer cell lines, Caco2, HT-29, SW620, and SW480, were used. Four antineoplastic agents, doxorubicin, cediranib, nilotinib, and erlotinib, were selected for the study and subsequently in the combination study of gap junction enhancer. The lethal dose at 50% (LD50) was calculated for each antineoplastic agent in each cell line. Results: The results show significant variation of LD50. HT-29 cells dosed with doxorubicin showed LD50 of 8.9 µM, 1.6 µM, and 0.5 µM at 24 hours, 36 hours, and 48 hours of exposure, respectively. HT-29 cells dosed with erlotinib showed LD50 of 15.8 µM and 8.9 µM at 48 hours and 60 hours of exposure, respectively. SW620 dosed with doxorubicin showed LD50 is 0.5 µM at 36 hours of exposure. SW620 dosed with erlotinib showed LD50 of 15.8 µM and 0.5 µM at 48 hours and 60 hours of exposure, respectively. Caco2 cells dosed with cediranib showed LD50 of 2.8 µM and 1.6 µM at 36 hours and 48 hours of exposure, respectively. Conclusion: The findings from this study revealed varying LD50 values for these cell lines when treated with the four distinct drugs. This enabled us to determine a foundational effect of these drugs on these cell lines, which had not been previously identified. Discovering this foundational effect will allow us to investigate the synergy effect when combined with gap junction enhancer by setting a baseline effect using these results. Citation Format: Tomas Lugo, Annelise Nguyen. Effect of antineoplastic drugs on cell-to-cell communication in the progression of colorectal cancer [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 6015.

Curcumin activates the JNK signaling pathway to promote ferroptosis in colon cancer cells.

Recent evidence has proved that curcumin as a natural polyphenol have a great anticancer and anti-proliferative effects in cancer cells. Ferroptosis, a new form of regulated cell death, plays a vital role in the pathogenesis and therapy of cancers. In this study, we aimed to examine the effects of curcumin in ferroptosis of human colorectal cancer cells and its underlying molecular mechanisms. SW-480 colorectal cancer cells were treated by curcumin with different concentrations. Cell viability was determined by using MTT assay. The concentrations of reactive oxygen species (ROS) and intracellular iron were measured using specific related kits. Real-time PCR and Western blot analysis were used to determine the expression of ferroptosis-related proteins including ACSL4, GPx4 and FTH1 and activation of JNK protein. Curcumin suppressed SW-480 cancer cells viability in dose-dependent manner. Cell treatment with curcumin led to accumulation of ROS and iron within cells and increase in the intracellular levels of lipid peroxidation. In addition, curcumin modulated the mRNA and protein expression levels of ferroptosis-related proteins including ACSL4, GPx4 and FTH1 and suppression of JNK signaling. Curcumin may exhibit its anticancer effect on colorectal cancer by downregulating JNK signaling to induce ferroptosis in SW-480 cells.

Peptide mimetic NC114 induces growth arrest by preventing PKCδ activation and FOXM1 nuclear translocation in colorectal cancer cells.

The peptide mimetic, NC114, is a promising anticancer compound that specifically kills colorectal cancer cells without affecting normal colon epithelial cells. In our previous study, we observed that NC114 inhibited the Wnt/β-catenin pathway, with significant downregulation of both Ser 675-phosphorylated β-catenin and its target genes, cyclin D1 and survivin. However, the molecular mechanism responsible for its cytotoxic effect has not yet been fully characterized. In the present study, we demonstrated that NC114 prevented cell cycle progression from S to G2/M phase by downregulating cell cycle-related gene expression, and also induced growth arrest in SW480 and HCT-116 colorectal cancer cells. A novel covariation network analysis combined with transcriptome analysis revealed a series of signaling cascades affected by NC114 treatment, and identified protein kinase C-δ (PKCδ) and forkhead box protein M1 (FOXM1) as important regulatory factors for NC114-induced growth arrest. NC114 treatment inhibits the activation of PKCδ and its kinase activity, which suppresses MEK/ERK signaling. Attenuated MEK/ERK signaling then results in a reduction in FOXM1 phosphorylation and subsequent nuclear translocation of FOXM1 and β-catenin. Consequently, formation of a T-cell factor-4 (TCF4)/β-catenin transcription complex in the nucleus is inhibited and transcription of its target genes, such as cell cycle-related genes, is downregulated. The efficacy of NC114 on tumor growth was confirmed in a xenograft model. Collectively, elucidation of the mechanism by which NC114 induces growth arrest in colorectal cancer cells should provide a novel therapeutic strategy for colorectal cancer treatment.

Effect of some aspirin analogues on SW480 Colorectal cancer cell line: Tip of the iceberg

Abstract Colorectal cancer (CRC) is the third most common cancer and second leading cause of death with approximately 1.9 million cases and almost a million deaths worldwide in 2020. It is predicted that the global incidences of CRC will be over 3 million in 2040. Aspirin has been shown to have cytotoxic and immunomodulatory effects on CRC cell lines. However, due to side effects such as gastrointestinal bleeding in older patients, there has always a quest for safer and more effective analogues, leading to the synthesis of aspirin analogues. One of the primary regulators of the mitochondria-mediated pathway to apoptosis is the family known as BCL-2 proteins, which are broadly grouped into pro-apoptotic proteins such as BAX, BAK, BIK, BAD and anti-apoptotic proteins such as BCL-2 and BCL-XL. Phase contrast images of the SW480 CRC cells treated with aspirin, meta-aspirin, para-aspirin, ortho-thioaspirin, meta-thioaspirin and para-thioaspirin at 0.5 mM for 24 h, 48 h and 72 h were taken at 100X magnification. Western blot was used to detect BAX and BCL-2 proteins by treating CRC cells with aspirin and its analogues, and analysed by SDS-PAGE and immunoblotting. The blots were then probed with primary antibodies BCL-2 and BAX, after which the HRP bound protein-labelled antibody was visualised using ECL and exposed to CL-XPosure™ Film. It was observed that the isomers of aspirin (O-ASP), meta-aspirin (M-ASP) and para-aspirin (P-ASP) significantly increased the density of BAX Ab. O-ASP, M-ASP, P-ASP and ortho-thioaspirin (O-TASP) significantly reduced the density of BCL-2 Ab, thus proposing that the anti-apoptotic effect of this cell line is reversed/reduced by these aspirin analogues. The results of this study suggest that M-ASP and P-ASP have its antiproliferative effect on the SW480 CRC cell line via driving the pro-apoptotic effect of BAX protein and decreasing the expression of anti-apoptotic of BCL-2 protein, thus, encouraging apoptosis in this CRC cell line.

Sample Preparation of Adherent Cell Lines for Flow Cytometry: Protocol Optimization—Our Experience with SW-480 colorectal cancer cell line

Sample Preparation of Adherent Cell Lines for Flow Cytometry: Protocol Optimization—Our Experience with SW-480 colorectal cancer cell line

Curcumin affects apoptosis of colorectal cancer cells through ATF6-mediated endoplasmic reticulum stress.

Colorectal cancer (CRC) is the main cause of cancer-associated death. Herein, we treated SW620 and HT-29 CRC cells with different curcumin concentrations, followed by treatment with the half maximal inhibitory concentration (IC50) curcumin/endoplasmic reticulum stress (ERS) inhibitor 4-phenyl butyric acid (4-PBA)/activating transcription factor 6 (ATF6) interference plasmid (si-ATF6). We detected cell proliferation/apoptosis, ATF6 cellular localization/nuclear translocation, ion concentration, ATF6 protein/apoptotic protein (Bax/Bcl-2/Cleaved Caspase-3) levels, and ERS-related proteins (glucose-regulated protein 78 [Grp78]/C/EBP homologous protein [CHOP]). We discovered inhibited cell proliferation/growth, enhanced cell apoptosis/(Bax/Bcl-2) ratio/Cleaved Caspase-3 levels/Ca2+ concentration in the cytoplasm/ERS-related protein (Grp78/CHOP) levels, and activated ERS following treatment with IC50 curcumin. 4-PBA partially reversed the inhibitory effect of curcumin on SW620 cells by restraining ERS. Curcumin stimulated ATF6 expression and its nuclear translocation to activate ERS. ATF6 silencing partly annulled the inhibitory effect of curcumin on SW620 cells. Our study explored the molecular mechanism of curcumin affecting CRC cell apoptosis through ATF6.

HPLC-DAD Analysis and Investigation of Biological Properties of the Leaves of Globularia alypum (L.), Infusion Extract.

Globularia alypum L. (GA) belonging to the Globulariaceae family is a Mediterranean plant which is widely used in traditional Tunisian medicine. The aim of this study was to investigate the phytochemical composition, antioxidant, anti-arthritic, antiproliferative, antibacterial and antibiofilm potential of aqueous GA leaf extracts (AGAL). Quantitative analyses of the different constituents of extracts were evaluated by high-performance liquid chromatography with photodiode-array detection (HPLC-DAD). Spectrophotometric methods and chemical tests were used for antioxidant and anti-arthritic activities. The antiproliferative study was evaluated using colorectal cancer SW620 cells, while the antibacterial assessment and analysis of the antibiofilm effects were determined by the microdilution method and the crystal violet assay, respectively. AGAL extracts presented several components, mainly Nepetin-7-Glucoside and trans-ferrulic acid. The results showed that they had an important antioxidant (IC50 = 0.34; 0.38 and 1.20 mg/mL) and anti-arthritic (IC50 = 2.94 mg/mL) properties, and these effects are displayed in a dose-dependent manner. In addition, this extract demonstrated significant antiproliferative (IC50 = 50 µg/mL), antibacterial (MIC = 6.25 mg/mL and MBC = 6.25 mg/mL), and antibiofilm (59.70% at 25 mg/mL) properties especially against S. aureus. The results achieved confirm the important role of this plant as a source of therapeutic activities.

The power of mumps virus: Matrix protein activates apoptotic pathways in human colorectal cell lines

New therapeutic approaches can significantly impact the control of colorectal cancer (CRC), which is increasing worldwide. In this study, we investigated the potential of targeting viral proteins to combat cancer cells. Specifically, we examined the anticancer potential of the matrix (M) protein of the mumps virus Hoshino strain in SW480 CRC cell lines. To begin, we individually transfected SW480 cells with pcDNA3 plasmids containing the mumps virus M gene. We then investigated the percentage of cell death, caspase activity, and the expression levels of genes involved in apoptosis pathways. Following this, we performed bioinformatics analysis on the M protein to identify any similarities with Bcl-2 family members and their viral homologs. Our diagnostic methods showed that treatment with the mumps M protein induced apoptosis and upregulated the expression and activity of pro-apoptotic proteins in SW480 CRC cells compared to the control and vector groups. Based on our bioinformatics studies, we proposed that the BH3 motif in the M protein may trigger apoptosis in CRC cells by interacting with cellular Bax. Overall, our study showed for the first time that the mumps virus M protein could be considered as a targeted treatment for CRC by inducing apoptotic pathways.

New gold(III) complexes TGS 121, 404, and 702 show anti-tumor activity in colitis-induced colorectal cancer: an in vitro and in vivo study

BackgroundChronic inflammation in the course of inflammatory bowel disease may result in colon cancer, or colitis-associated colorectal cancer (CACRC). It is well established that CACRC is associated with oxidative stress and secretion of multiple pro-inflammatory cytokines, e.g. tumor necrosis factor-α. Recently, we proved that the administration of gold(III) complexes resulted in the alleviation of acute colitis in mice. The aim of the current study was to assess the antitumor effect of a novel series of gold(III) complexes: TGS 121, 404, 512, 701, 702, and 703.MaterialsAnalyzed gold(III) complexes were screened in the in vitro studies using colorectal cancer and normal colon epithelium cell lines, SW480, HT-29, and CCD 841 CoN, and in vivo, in the CACRC mouse model.ResultsOf all tested complexes, TGS 121, 404, and 702 exhibited the strongest anti-tumor effect in in vitro viability assay of colon cancer cell lines and in in vivo CACRC model, in which these complexes decreased the total number of colonic tumors and macroscopic score. We also evidenced that the mechanism of action was linked to the enzymatic antioxidant system and inflammatory cytokines.ConclusionsTGS 121, 404, and 702 present anti-tumor potential and are an attractive therapeutic option for colorectal cancer.

CYP1B1 promotes colorectal cancer liver metastasis by enhancing the growth of metastatic cancer cells via a fatty acids-dependent manner.

Liver metastasis (LM) accounts for most colorectal cancer (CRC)-related deaths. However, how metastatic CRC cells gain the ability to survive and grow in liver remains largely unknown. First, we screened differentially expressed genes (DEGs) between LM and paired primary tumors (PTs) in Gene Expression Omnibus (GEO) database, and identified cytochrome P450 1B1 (CYP1B1) as the only common differential gene. Then, we verified messenger RNA (mRNA) and protein expression level in clinical specimens. After constructing stable up-regulated CYP1B1 versions of HCT116 and RKO CRC cells and stable down-regulated CYP1B1 versions of SW480 and HT29 CRC cells, cell proliferation assays, subcutaneous tumor formation, and mouse LM models were used to comprehend its function. Next, we used RNA-seq to uncover specific mechanisms of growth; cell cycle, polymerase chain reaction (PCR), western blot (WB) and GEO series (GSE) datasets were used to verify its mechanism. Last, gas chromatography tandem mass spectrometry (GC-MS/MS) was adopted to examine which fatty acids were changed. A significantly higher level of CYP1B1 was found in LM than in PT in paired clinical CRC LM samples (P<0.05). After CYP1B1 overexpression in HCT116 and RKO cells, cell proliferation abilities in vitro and in vivo were enhanced; LM of NOD.Cg-PrkdcscidIl2rgem1Smoc (NSG) mice were enhanced. And knockdown of CYP1B1 in SW480 and HT29 cells, cell proliferation abilities in vitro and in vivo were reduced; LM of NSG mice were declined (P<0.05). RNA-seq showed 59 common genes from upregulated genes of RKO overexpression group and downregulated genes of SW480 knockdown group were enriched in cell cycle and DNA replication. Further investigation revealed CYP1B1 regulated alternation of MCM5, PCNA, and FEN1 genes, and G1/S transition in CRC cells. GC-MS/MS revealed long chain fatty acids (LCFAs) made a difference in SW480 knockdown group (P<0.05). Through adding LCFAs into SW480 and HT29 knockdown groups, cell proliferation abilities in vitro and in vivo were enhanced, and expressions of MCM5, PCNA, FEN1 were upregulated (P<0.05). CYP1B1 exerts a significant influence on LM of CRC by modulating tumor cell proliferation via "CYP1B1-LCFAs-G1/S transition". This finding suggests CYP1B1 could be a promising target for CRC LM.

Cellular Responses to Extracellular Vesicles as Potential Markers of Colorectal Cancer Progression

The development of novel screening tests aims to support early asymptomatic diagnosis and subtyping patients according to similar traits in the heterogeneous cancer cohort. Extracellular vesicles (EVs) are promising candidates for the detection of disease markers from bodily fluids, but limitations in the standardisation of isolation methods and the intrinsic EV heterogeneity obtained from liquid biopsies are currently obstacles to clinical adoption. Here, cellular responses to cancer EVs were initially explored as potential complementary biomarkers for stage separation using colorectal cancer (CRC) SW480 and SW620 cell line models. A pilot study on a small cohort of CRC patients and controls was then developed by performing a multivariate analysis of cellular responses to plasma-derived EVs. Several cell activities and markers involved in tumour microenvironment pathways were influenced by the treatment of cell line EVs in a stage-dependent manner. The multivariate analysis combining plasma EV markers and cellular responses to plasma EVs was able to separate patients according to disease stage. This preliminary study offers the potential of considering cellular responses to EVs in combination with EV biomarkers in the development of screening methods.

Mitochondrial Functionality Is Regulated by Alkylphospholipids in Human Colon Cancer Cells.

Alkylphospholipids (APLs) have been studied as anticancer drugs that interfere with biological membranes without targeting DNA. Although their mechanism of action is not fully elucidated yet, it is known that they disrupt the intracellular trafficking of cholesterol and its metabolism. Here, we analyzed whether APLs could also interfere with mitochondrial function. For this purpose, we used HT29 colorectal cancer cells, derived from a primary tumor, and SW620 colorectal cancer cells, derived from a metastasis site. After treatment with the APLs miltefosine and perifosine, we analyzed various mitochondrial parameters, including mitochondrial mass, cardiolipin content, mitochondrial membrane potential, H2O2 production, the levels of oxidative phosphorylation (OXPHOS) complexes, metabolic enzymes activity, the oxygen consumption rate, and the levels of apoptosis and autophagy markers. APLs, especially perifosine, increased mitochondrial mass while OXPHOS complexes levels were decreased without affecting the total oxygen consumption rate. Additionally, we observed an increase in pyruvate dehydrogenase (PDH) and isocitrate dehydrogenase (IDH) levels and a decrease in lactate dehydrogenase (LDH) activity, suggesting a metabolic rewiring induced by perifosine. These alterations led to higher mitochondrial membrane potential, which was potentiated by decreased uncoupling protein 2 (UCP2) levels and increased reactive oxygen species (ROS) production. Consequently, perifosine induced an imbalance in mitochondrial function, resulting in higher ROS production that ultimately impacted cellular viability.