Colon Cancer Cell Line DLD-1 Research Articles

Fucosylated Chondroitin Sulfate from Bohadschia ocellata: Structure Analysis and Bioactivities

Fucosylated chondroitin sulfate (FCS) was prepared from Bohadschia ocellata using protease hydrolysis. The structural characteristics of FCS were confirmed through chemical composition analysis using FTIR spectroscopy, 1H NMR, and 13C NMR. FCS from B. ocellata (FCS-Bo) exhibited an average molecular weight of approximately 122 kDa. The biological activities of FCS-Bo, including anticoagulant, anti-cancer, and Protein Tyrosine Phosphatase 1B (PTP1B) inhibition, were evaluated. FCS-Bo displayed potent anticoagulant properties, markedly extending activated partial thromboplastin time, prothrombin time, and thrombin time when compared to the heparin control. In anti-cancer bioactivity research, FCS-Bo efficiently inhibited colony formation in the colon cancer cell lines HCT-116, HT-29, and DLD-1, achieving inhibition rates of up to 65%. Additionally, FCS-Bo exhibited significant inhibition of PTP1B, with an IC50 as low as 0.0326 µg/mL, suggesting its potential for improving insulin sensitivity and managing conditions such as type 2 diabetes and obesity.

Rosmarinic Acid Potentiates Cytotoxicity of Cisplatin against Colorectal Cancer Cells by Enhancing Apoptotic and Ferroptosis.

Rosmarinic acid (RA) has demonstrated anticancer effects on several types of malignancies. However, whether RA promotes the anticancer effect of cisplatin on colorectal cancer cells remains sketchy. This study aimed to explore whether RA potentiates the cytotoxicity of cisplatin against colon cancer cells and the underlying mechanism. Cell viability, cell cycle progression, and apoptosis was evaluated using sulforhodamine B (SRB) assay, flow cytometric analysis, and propidium iodide/Annexin V staining, respectively. Western blotting was utilized to analyze signaling pathways. Our findings showed that RA significantly enhanced the inhibitory effect on cell viability and the induction of apoptosis on the colon cancer cell lines DLD-1 and LoVo. Signaling cascade analysis revealed that the combination of RA and cisplatin jointly induced Bax and caspase activation while downregulating Bcl-2, glutathione peroxidase 4 (GPX4), and SLC7A11 in DLD-1 cells. Moreover, caspase inhibitor and ferroptosis inhibitor significantly reversed the inhibition of cell viability in response to RA combined with cisplatin. Collectively, these findings demonstrate that RA enhances the cytotoxicity of cisplatin against colon cancer cells, attributing to the promotion of apoptosis and ferroptosis.

Oncofetal morphogenesis similar to embryonic gut formation by a subpopulation of DLD-1 human colon cancer cells

Cancer stem cells (CSC) are thought to be responsible for cancer phenotypes and cellular heterogeneity. Here we demonstrate that the human colon cancer cell line DLD1 contains two types of CSC-like cells that undergo distinct morphogenesis in the reconstituted basement membrane gel Matrigel. In our method with cancer cell spheroids, the parent cell line (DLD1-P) developed grape-like budding structures, whereas the other (DLD1-Wm) and its single-cell clones dynamically developed worm-like ones. Gene expression analysis suggested that the former mimicked intestinal crypt-villus morphogenesis, while the latter mimicked embryonic hindgut development. The organoids of DLD1-Wm cells rapidly extended in two opposite directions by expressing dipolar proteolytic activity. The invasive morphogenesis required the expression of MMP-2 and CD133 genes and ROCK activity. These cells also exhibited gastrula-like morphogenesis even in two-dimensional cultures without Matrigel. Moreover, the two DLD1 cell lines showed clear differences in cellular growth, tumor growth and susceptibility to paclitaxel. This study also provides a simple organoid culture method for human cancer cell lines. HT-29 and other cancer cell lines underwent characteristic morphogenesis in direct contact with normal fibroblasts. Such organoid cultures would be useful for investigating the nature of CSCs and for screening anti-cancer drugs. Our results lead to the hypothesis that CSC-like cells with both invasive activity and a fetal phenotype, i. e. oncofetal CSCs, are generated in some types of colon cancers.

Indoleamine 2,3-Dioxygenase Inhibitor Suppresses Colon Cancer Cell Migration, Invasion, and Epithelial-Mesenchymal Transition.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a key enzyme in tryptophan metabolism and plays an important role in immunosuppression. The effects of IDO1 on tumor invasion and metastasis have been studied in several types of malignancies. However, the role of IDO1 in these steps in colorectal cancer (CRC) has not been elucidated. Therefore, we aimed to investigate the effects of IDO1 on invasion, migration, and epithelial-mesenchymal transition (EMT) in CRC cells. All experiments were performed using the DLD-1 colon cancer cell line that expresses IDO1. We conducted a scratch wound healing assay and Boyden chamber assay to investigate the impact of IDO1 on DLD-1 cell migration and invasion, respectively, in the presence and absence of the IDO1 inhibitor L-1-methyl-tryptophan (L-1-MT). Additionally, western blotting was performed to analyze alterations in the expression of EMT-related markers caused by L-1-MT. High expression of IDO1 was confirmed in the cytoplasm of DLD-1 by immunofluorescence staining. In the scratch wound healing assay, the invasion ability of DLD-1 cells decreased to 62% after treatment with L-1-MT at 1,000 μM for 24 h. In the Boyden chamber assay, the migration of DLD-1 cells was suppressed by 85% after treatment with L-1-MT at 2,500 μM for 24 h. L-1-MT treatment increased the expression level of E-cadherin and decreased the expression levels of vimentin, Snail, and Slug. IDO1 inhibition reduced the invasion and migration ability of IDO1-expressing DLD-1 colon cancer cells, which was accompanied by altered expression of EMT-related proteins. IDO1 could be a potential target for the treatment of advanced CRC.

Phytochemical Profiling, in Vitro Biological Activities and in Silico Molecular Docking Studies of the Crude Extract of Crambe orientalis, an Endemic Plant in Turkey.

In this study, total phenolic and flavonoid analyses of flower, leaf, and stem aqueous extracts of C. orientalis were performed. Total phenolic contents of C. orientalis extracts ranged from 12.2±0.06 to 19.0±0.07 mg GAE/g extract. Total flavonoid values range between 2.0±0.11 and 6.6±0.19 mg CE/g extract. Urease, collagenase, tyrosinase, and α-glucosidase inhibition activities were determined in vitro and the relationship between them was examined. IC50 results for all enzymes were obtained between 0.18 and 3.53 μg/mL. The aqueous extract of the plant C. orientalis showed potent cytotoxic effects against the human colon cancer cell lines DLD-1. As the extract concentration increases, cell death increases. The main fatty acid composition by GC/MS analysis is erucic acid (36.5 %). The potential binding modes of the fatty acids in the plant extract to the enzymes and possible inhibition mechanisms were determined by molecular docking calculation.

The Expression of LINC00673 in Colon Cancer and the Melatonin's Effect on Its Expression

Objective: In this study, the cell lines SW620 and DLD-1 which belongs to colon cancer were employed with the aim of observing melatonin's effect on the growth of colon cancer cells and exploring the influence of melatonin about the autophagy of colon cancer. Besides. It uses qRT-PCR to identify LINCRNA00673 (LINC00673) expression in colon cancer and the influence of melatonin on LINC00673 expression.For the purpose of providing a fundamental theory for melatonin in anti-cancer therapy and also presenting as a biomarker for the early detection of colon cancer. Method: 1) Cultivating the colon cancer cell lines SW620 and DLD-1, and subdividing them in accordance with distinct concentrations of melatonin: 0 mM, 1mM, and 2mM, to detect the impact of melatonin for migration and growth of colon cancer cells by Cell Scratch Test and Colony Formation Assay.2) Detecting the expression level of autophagy-related molecules in colon cancer cells in response to melatonin employing Western Blot Assay. 3) Utilizing QT-PCR, extract RNA from two groups of cells and detect the expression of LINC00673 and LINC00673 following melatonin treatment. Result: 1) The Cell Scratch test and Colony Formation Assay indicated that compared with the normal group, the scratch healing of SW620 cells and DLD-1 cells decreased. Furthermore, following melatonin administration, the number of cell colonies decreased. And the results are statistically significant. 2) Western blotting results illustrated that after the interferon of melatonin, the expression level of the autophagy-related molecule Beclin-1 increased significantly, whereas the expression of P62 decreased. 3) QT-PCR results demonstrated that the expression of LINC00673 in colon cancer cells SW620 and DLD-1 was significantly increased. Moreover, melatonin had an inhibiting effect on it. Conclusion: These in vitro experiments managed to prove that melatonin possesses a powerful anti-colon cancer effect and can inhibit the growth and spread of colon cancer. Western blotting indicated that melatonin can inhibit colon cancer through autophagy. Besides which, it is discovered that melatonin may exert its anticancer effect by influencing LINC00673.

Stress Relaxation-Induced Colon Tumor Multicellular Spheroid Culture Based on Biomimetic Hydrogel for Nanoenzyme Ferroptosis Sensitization Evaluation.

Ferroptosis has recently become a research hotspot, and the induction of tumor cell ferroptosis has emerged as a powerful method for tumor therapy. However, the efficiency of tumor cell ferroptosis induction remains unmet for clinical use, which may be attributed to the large discrepancies between in vitro and in vivo models. To address this issue, in this study, a hydrogel platform with stress relaxation is utilized to develop a multicellular spheroid model of the DLD1 colon cancer cell line through cancer cell self-organization. The spheroids are highly similar to real tumor tissue, and ferroptosis resistance at the transcriptional, protein, and cellular levels. Collaboration of the ferroptosis induction reagent erastin and the nanoenzyme MnZnFe2 O4 @PEG-COOH to overcome the ferroptosis resistance of the spheroids is also demonstrated. Taken together, this study demonstrates the effectiveness of the model developed using this hydrogel platform for further mechanistic studies, and for the assessment of novel cancer treatment strategies based on ferroptosis.

Abstract 5987: Membrane spanning 4 domains A12 (MS4A12) c.C211T stop-gain single-nucleotide polymorphism (rs2298553) promotes metastasis in colorectal cancer via the Hippo pathway

Abstract Background: Colorectal cancer (CRC) is one of the most malignant tumors worldwide. The prognosis of patients with colorectal cancer remains poor, with a 5-year metastasis survival rate of 20%-30%. Membrane spanning 4 domains subfamily A (MS4A) is an evolving family of structurally related cell surface proteins. Previous studies demonstrated that MS4A12 expression in CRC was significantly higher than that in the normal tissues. And low expression of MS4A12 in CRC was associated with poor survival in patients. However, the impact and mechanism of MS4A12 c.C211T stop-gain single-nucleotide polymorphism (SNP) (rs2298553) on the metastatic phenotype of CRC have not previously been examined. Current opinion holds that SNP introducing premature termination codons could alter the stability and function of transcripts and proteins. The aim of this study was to investigate the correlation between MS4A12 stop-gain SNP and invasion and migration of CRC cells. Methods: Whole exome sequencing and target sequencing were used to screen SNP related to distant metastasis. Immunohistochemical staining of tissue microarray was used to detect proteins in CRC tumor cancer and normal tissues. Recombinant plasmids carrying wild-type (CC) and MS4A12 c.C211T stop-gain SNP (TT) were constructed and then transfected to colon cancer cell lines DLD1, RKO. Knockdown of MS4A12 used the short hairpin RNA (shRNA). Western blot was performed to detect the level of protein expression. Wound healing essay, migration and invasion assays were used to detect metastatic ability of CRC. RNA-seq analysis, co-immunoprecipitation and mass assays were performed to further mechanism. Results: MS4A12 c.C211T stop-gain SNP (rs2298553) was screened and identified to be associated with CRC metastasis. Tissue microarray revealed that the expression of MS4A12 was lower in the colorectal tumor tissues compared to normal tissues. The low expression of MS4A12 in CRC was associated with a poor prognosis in patients. The expression level of the TT group was close to that of the basal level of empty vector transfected groups, which suggested that the c.C211T stop-gain SNP (rs2298553) inhibited truncated MS4A12 protein expression. Compared to the CC group, the TT group showed significantly increased invasion and migration ability. Meanwhile, MS4A12 knockdown also promoted invasion and migration. GO and KEGG pathway analysis suggested that the Hippo signaling pathway might be associated with the inhibition of migration and invasion by MS4A12. Compared to the CC group, the TT group impedes YAP/TAZ phosphorylation and facilitates YAP/TAZ nuclear translocation. Conclusion: MS4A12 c.C211T stop-gain SNP (rs2298553) promotes the invasion and migration of CRC. These results provide an important theoretical basis for studying the invasion and migration of CRC. Citation Format: Shuang Liu, Ting Jiang, Yan Yuan, Haina Yu, Zhiwei Guo, Yuanhong Gao, Yan Li, Weiwei Xiao. Membrane spanning 4 domains A12 (MS4A12) c.C211T stop-gain single-nucleotide polymorphism (rs2298553) promotes metastasis in colorectal cancer via the Hippo pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5987.

GSTs, MRP and Apoptototic Markers in DLD-1 Human Colon Cancer Cell Line Before and After 5-FU Treatment

Multidrugresistance is an important factor limiting the effect of chemotherapy on cancer treatment. Disorders of drug transport and apoptosis, deterioration of redox homeostasis are among the main mechanisms that lead to multidrug resistance. The aim of this study was to determine the effect of 5-FU on GST isozymes, drug resistance proteins and apoptotic proteins before and after 5-Flourouracil application on DLD-1 colon cancer cell line. The cytotoxic effect of 5-FU was measured by WST-1test and, the efficiency of drug application was, also, proved by double staining via Hoechst 33342 with Propidium iodide. Next, the expression levels of GST isozymes, drug resistance proteins and apoptotic proteins were determined by immunocytochemistry. The cytotoxic effect of 5-FU at different doses on DLD-1 colon cancer cell line was determined by WST-1 method. MRP-2, 3, 6, 7 of drug resistance proteins; GSTA1, GSTM1, GSTT1, GSTZ1, GSTK1 and GSTO1 of GST proteins; bcl-2, caspase-3, p38, and p53, which are apoptotic proteins, have higher expression in the drug-treated DLD-1 cell line. GSTS1, MDR-1 and MRP-1expressions were not immunocytochemically different. It was determined that there is a direct correlation between the level of cytotoxicity and applied drug concentration. The cytotoxic effect of the drug increased with the increase in the dose of the drug. In this study, as first in the literature, the expression levels of some apoptotic markers, GST isozymes and drug resistance proteinswere evaluated togetherand except GSTS1, MDR-1 and MRP-1, they were all upregulated with respect to the control group after 5-FU administration.

BCL2A1 and CCL18 Are Predictive Biomarkers of Cisplatin Chemotherapy and Immunotherapy in Colon Cancer Patients.

Background: Cisplatin enhances the antitumor T cell response, and the combination of PD-L1 blockade produces a synergistic therapeutic effect. However, the clinical correlation between cisplatin and immunotherapy in colon cancer (CC) is unknown. Methods: Using the “pRRophetic” package, we calculated the IC50 of cisplatin. The correlation between cisplatin IC50, cisplatin resistance–related genes (CCL18 and BCL2A1), and immunotherapy were preliminarily verified in TCGA and further validated in independent cohorts (GSE39582 and GSE17538), cisplatin-resistant CC cell line DLD1, and our own clinical specimens. Classification performance was evaluated using the AUC value of the ROC curve. Scores of immune signatures, autophagy, ferroptosis, and stemness were quantified using the ssGSEA algorithm. Results: Based on respective medians of three CC cohorts, patients were divided into high- and low-IC50 groups. Compared with the high IC50 group, the low-IC50 group had significantly higher tumor microenvironment (TME) scores and lower tumor purity. Most co-signaling molecules were upregulated in low IC50 group. CC patients with good immunotherapy efficacy (MSI, dMMR, and more TMB) were more attributable to the low-IC50 group. Among seven shared differentially expressed cisplatin resistance–related genes, CCL18 and BCL2A1 had the best predictive efficacy of the above immunotherapy biomarkers. For wet experimental verification, compared with cisplatin-resistant DLD1, similar to PD-L1, CCL18 and BCL2A1 were significantly upregulated in wild-type DLD1. In our own CC tissues, the mRNA expression of CCL18, BCL2A1, and PD-L1 in dMMR were significantly increased. The high group of CCL18 or BCL2A1 had a higher proportion of MSI, dMMR, and more TMB. IC50, CCL18, BCL2A1, and PD-L1 were closely related to scores of immune-related pathways, immune signatures, autophagy, ferroptosis, and stemness. The microRNA shared by BCL2A1 and PD-L1, hsa-miR-137, were significantly associated with CCL18, BCL2A1, and PD-L1, and downregulated in low-IC50 group. The activity of the TOLL-like receptor signaling pathway affected the sensitivity of CC patients to cisplatin and immunotherapy. For subtype analysis, immune C2, immune C6, HM-indel, HM-SNV, C18, and C20 were equally sensitive to cisplatin chemotherapy and immunotherapy. Conclusions: CC patients sensitive to cisplatin chemotherapy were also sensitive to immunotherapy. CCL18 and BCL2A1 were novel biomarkers for cisplatin and immunotherapy.

Literature-based translation from synthetic lethality screening into therapeutics targets: CD82 is a novel target for KRAS mutation in colon cancer

Synthetic lethality (SL) is an emerging therapeutic paradigm in cancer. We introduced a different approach to prioritize SL gene pairs through literature mining and RAS-mutant high-throughput screening (HTS) data. We matched essential genes from text-mining and mutant genes from the COSMIC and CCLE HTS datasets to build a prediction model of SL gene pairs. CCLE gene expression data were used to enrich the essential-mutant SL gene pairs using Spearman’s correlation coefficient and literature mining. In total, 223 essential trigger terms were extracted and ranked. The threshold of the essential gene score (Sg) was set to 10. We identified 586 genes essential for the SL prediction model of colon cancer. Seven essential RAS-mutant SL gene pairs were identified in our model, including CD82-KRAS/NRAS, PEBP1-NRAS, MT-CO2-HRAS, IFI27-NRAS/KRAS, and SUMO1-HRAS gene pairs. Using RAS-mutant HTS data validation, we identified two potential SL gene pairs, including the CD82 (essential gene)–KRAS (mutant gene) pair and CD82–NRAS pair in the DLD-1 colon cancer cell line (Spearman’s correlation p-values = 0.004786 and 0.00249, respectively). Based on further annotations by PubChem, we observed that digitonin targeted the complex comprising CD82, especially in KRAS-mutated HCT116 cancer cells. Moreover, we experimentally demonstrated that CD82 exhibited selective vulnerability in KRAS-mutant colorectal cancer. We used literature mining and HTS data to identify candidates for SL targets for RAS-mutant colon cancer.

Synthesis and investigation of in vitro cytotoxic activities and thermal stability of novel pyridine derivative platinum (II) complexes vis a vis DFT studies

Bis(2-amino-5-nitropyridine)dichloroplatinum(II), [PtCl2L12] and bis(3,4-dimethylpyridine)dichloroplatinum(II), [PtCl2L22] complexes were synthesized and characterized via FT-IR, UV–Vis, 1H NMR, 13C NMR spectrometers. Single crystal X-ray diffraction measurements were conducted to determine the crystal structure of the [PtCl2L22] complex. The [PtCl2L22] was crystallized in the monoclinic crystal system with P21/c space group. The crystal parameters were Z = 2, a = 9.5881(5) Å, b = 12.5863(7) Å and c = 13.5363(7) Å. Experimental data are supported by the theoretical calculations. LanL2DZ based DFT/B3LYP method was used determine the molecular structures of the complexes for their probable most stable status. Potential energy distribution analysis was made for to confer the vibrational transitions for the complexes. Molecular electrostatic potential maps, frontier molecular orbitals and Mulliken charge distribution were calculated and the active regions of the molecules were determined. In this study, for to investigate the interaction mechanisms of [PtCl2L12] and [PtCl2L22] complexes with colon cancer protein molecular docking study was performed. The thermal analyses of the complexes were carried out via DTA/TGA/TG combined system. The activation energies of the complexes were calculated to be 134.31–175.40 kJ/mol via Flynn-Wall-Ozawa (FWO) and 132.42–179.41 kJ/mol via Kissenger-Akahira-Sunose (KAS) methods. The cytotoxic effects of the complexes against the colon cancer cell line (DLD-1) were investigated. The complexes were found to be cytotoxic against DLD-1 colon cancer cell lines, A549 human lung cancer cells and Beas-2Bhealthy human lung epithelial cells. The results indicate that the [PtCl2L12] complex, in which amino and nitro groups are bound as ligand on the pyridine ring, have higher cytotoxic effect.

Investigation of antioxidant activity and effect on cell viability of pyrrolidine compounds in MCF-7 (breast cancer) and DLD-1 (colon cancer) cell lines

The effect on cell viability of the pyrrolidine compounds was determined for 24 hours at different concentrations (25-100 µM) on cancer (MCF-7 and DLD-1) and normal (MCF-12A and CCD-18CO) cell lines by comparing MTT and RTCA assays. The antioxidant activities of the pyrrolidine compounds were determined at different concentrations (25-200 mM) with DPPH, metal chelating and reducing methods. In addition, the calculation of the structural properties of the new pyrrolidine scaffolding with the DFT method was presented. It has been determined that pyrrolidine compounds reduce the number of MCF-7 cancer cells according to a negative control by half of and inhibit the DLD-1 cancer cells with the MTT method. IC50 value for MCF-7 cell was determined as 100 µM. Metal chelation and DPPH activities have close results. The IC50 value of DPPH activity of the compounds ranged from 108 to 142 µg / mL.

Epibrassinolide activates AKT to trigger autophagy with polyamine metabolism in SW480 and DLD-1 colon cancer cell lines.

Epibrassinolide (EBR), a plant-derived polyhydroxylated derivative of 5α-cholestane, structurally shows similarities to animal steroid hormones. According to the present study, EBR treatment triggered a significant stress response via activating ER stress, autophagy, and apoptosis in cancer cells. EBR could also increase Akt phosphorylation in vitro. While the activation of Akt resulted in cellular metabolic activation in normal cells to proceed with cell survival, a rapid stress response was induced in cancer cells to reduce survival. Therefore, Akt as a mediator of cellular survival and death decision pathways is a crucial target in cancer cells. In this study, we determined that EBR induces stress responses through activating Akt, which reduced the mTOR complex I (mTORC1) activation in SW480 and DLD-1 colon cancer cells. As a consequence, EBR triggered macroautophagy and led to lipidation of LC3 most efficiently in SW480 cells. The cotreatment of spermidine (Spd) with EBR increased lipidation of LC3 synergistically in both cell lines. We also found that EBR promoted polyamine catabolism in SW480 cells. The retention of polyamine biosynthesis was remarkable following EBR treatment. We suggested that EBR-mediated Akt activation might determine the downstream cellular stress responses to induce autophagy related to polyamines.

A Detailed Chemical and Biological Investigation of Twelve Allium Species from Eastern Anatolia with Chemometric Studies.

Allium species are widely consumed as food all over the world. The phenolic profile of ethanol extracts of aerial parts and roots of 12 Allium species, collected from five different Eastern Anatolia regions, were studied using LC-MS/MS. In vitro antioxidant, anticholinesterase, cytotoxic and antimicrobial activities were also tested. The multivariate analyses were performed using principal component and hierarchical cluster analyses. Seventeen of 27 standard compounds were detected in all Allium species. The major components were mainly identified as quinic acid, malic acid, vanillin, and p-coumaric acid. The aerial parts possessed better antioxidant activity than roots. Aerial parts of A. atroviolaceum, A. chrysantherum, A. kharputense, and A. shirnakiense exhibited high cytotoxic activity against DLD-1 colon cancer cell lines (IC50 12.5 μg/mL). A. shatakiense and A. vineale demonstrated good antimicrobial activity against S. aureus and E. coli (MIC 75 μg/mL). According to chemometric analysis, differences were detected between aerial parts and the roots. The aerial parts of A. atroviolaceum, A. chrysantherum, A. kharputense, and A. shirnakiense could be potent in the pharmaceutical industry while A. shatakiense and A. vineale in the food industry after further investigations.

Small molecule 2,3-DCPE induces S phase arrest by activating the ATM/ATR-Chk1-Cdc25A signaling pathway in DLD-1 colon cancer cells.

In our previous study, it was reported that 2[[3-(2,3-dichlorophenoxy)propyl]amino]ethanol (2,3-DCPE) induces apoptosis and cell cycle arrest. The current study aimed to investigate the molecular mechanism involved in 2,3-DCPE-induced S phase arrest. The results demonstrated that 2,3-DCPE upregulated phosphorylated (p-)H2A histone family member X, a biomarker of DNA damage, in the DLD-1 colon cancer cell line. Western blotting revealed that 2,3-DCPE increased the checkpoint kinase (Chk)1 (Ser317 and Ser345) level and decreased the expression of M-phase inducer phosphatase 1 (Cdc25A) in a time-dependent manner. Subsequently, the results demonstrated that the ataxia-telangiectasia mutated (ATM) and ataxia-telangiectasia and Rad3-related (ATR) inhibitors wortmannin and caffeine had no effect on the cell cycle; however, the inhibitors partially abrogated 2,3-DCPE-induced S phase arrest. Flow cytometry assays revealed that caffeine (2 mM) reduced the proportion of S phase cells from 83 to 39.6% and that wortmannin (500 nM) reduced the proportion of S phase cells from 83 to 48.2%. Furthermore, wortmannin and caffeine inhibited the 2,3-DCPE-mediated phosphorylation of Chk1 and the degradation of Cdc25A. However, these ATM/ATR inhibitors had limited effect on 2,3-DCPE-induced apoptosis. Taken together, the data of the current study indicated that 2,3-DCPE caused DNA damage in colon cancer cells and that 2,3-DCPE-induced S phase arrest was associated with the activation of the ATM/ATR-Chk1-Cdc25A pathway.

Potential Role of PDGFRβ-Associated THBS4 in Colorectal Cancer Development.

Simple SummaryWe found increased levels of THBS4 and PDGFRb in tumor tissues compared to normal tissues of colon cancer patients. The relationship and the cause of the increase in these proteins had to be determined. Therefore, we performed several experiments and confirmed that excessive PDGFRb stimulation induces the THBS4 secretion through the intracellular Ca2+ signaling proteins. Our data show the possibility of post-translational modification of THBS4 by PDGFRb stimulation as there was no significant change in the THBS4 mRNA.Colorectal cancer is a significant cause of death since it frequently metastasizes to several organs such as the lung or liver. Tumor development is affected by various factors, including a tumor microenvironment, which may be an essential factor that leads to tumor growth, proliferation, invasion, and metastasis. In the tumor microenvironment, abnormal changes in various growth factors, enzymes, and cytokines can wield a strong influence on cancer. Thrombospondin-4 (THBS4), which is an extracellular matrix protein, also plays essential roles in the tumor microenvironment and mediates angiogenesis by transforming growth factor-β (TGFβ) signaling. Platelet-derived growth factor receptor β (PDGFRβ), which is a receptor tyrosine kinase and is also a downstream signal of TGFβ, is associated with invasion and metastasis in colorectal cancer. We identified that PDGFRβ and THBS4 are overexpressed in tumor tissues of colorectal cancer patients, and that PDGF-D expression increased after TGFβ treatment in the colon cancer cell line DLD-1. TGFβ and PDGF-D increased cellular THBS4 protein levels and secretion but did not increase THBS4 mRNA levels. This response was further confirmed by the inositol 1,4,5-triphosphate receptor (IP3R) and stromal interaction molecule 1 (STIM1) blockade as well as the PDGFRβ blockade. We propose that the PDGFRβ signal leads to a modification of the incomplete form of THBS4 to its complete form through IP3R, STIM1, and Ca2+-signal proteins, which further induces THBS4 secretion. Additionally, we identified that DLD-1 cell-conditioned medium stimulated with PDGF-D promotes adhesion, migration, and proliferation of colon myofibroblast CCD-18co cells, and this effect was intensified in the presence of thrombin. These findings suggest that excessive PDGFRβ signaling due to increased TGFβ and PDGF-D in colorectal tumors leads to over-secretion of THBS4 and proliferative tumor development.

In vitro co-delivery evaluation of PEGylated nano-liposome loaded by glycyrrhizic acid and cisplatin on cancer cell lines

Cisplatin, a platinum-based drug, is widely used in cancer treatment but is associated with significant side effects; new treatment delivery methods must be developed which enhance treatment efficiency while decreasing the sequelae of chemotherapy. The aim of this study was to develop novel liposomal nano-particles (NPs) loaded with two drugs including cisplatin and glycyrrhizic acid. This study hypothesized that introducing glycyrrhizic acid, as an adjuvant treatment, and cisplatin, as a chemotherapeutic agent, into a co-delivery system can enhance the therapeutic effect. In this study, the synthetic method and specifications of NPs were evaluated. After surface modification by polyethylene glycol (PEG), the size and the zeta potential of NPs were reported as 81.6 nm and − 30.7 mV, respectively. DLD-1 and LIM-2405 human colon cancer cell lines were then applied to evaluate the cytotoxicity of NPs using an MTT assay. The results revealed that IC50 for DLD-1, as a resistant cell line to cisplatin, decreased by 48% on co-delivery system treatment. Furthermore, to determine the proliferative potential of cell lines and DNA damage on cancerous cells after treatment by NPs and cisplatin in free form, the cell proliferation assay and immunocytochemistry method were performed. The results showed that a co-delivery system caused more than 43% DNA damage to cancerous cells compared with the free form of cisplatin. The finding of this study demonstrates that the co-delivery system has a significant anti-cancer efficiency compared with the free form of cisplatin. To conclude, the results of this study demonstrate a possible application of glycyrrhizic acid as an adjuvant treatment for cisplatin on co-delivery systems.

NMR-Based Chemical Profiling, Isolation and Evaluation of the Cytotoxic Potential of the Diterpenoid Siderol from Cultivated Sideritis euboea Heldr.

Diterpenes are characteristic compounds from the genus Sideritis L., possessing an array of biological activities. Siderol is the main constituent of the ent-kaurene diterpenes in Sideritis species. In order to isolate the specific compound and evaluate for the first time its cytotoxic activity, we explored the dichloromethane extract of cultivated Sideritis euboea Heldr. To track the specific natural bioactive agent, we applied NMR spectroscopy to the crude plant extract, since NMR can serve as a powerful and rapid tool both to navigate the targeted isolation process of bioactive constituents, and to also reveal the identity of bioactive components. Along these lines, from the rapid 1D 1H NMR spectrum of the total crude plant extract, we were able to determine the characteristic proton NMR signals of siderol. Furthermore, with the same NMR spectrum, we were able to categorize several secondary metabolites into chemical groups as a control of the isolation process. Therefore, this non-polar extract was explored, for the first time, revealing eleven compounds—one fatty acid ester; 2-(p-hydroxyphenyl)ethylstearate (1), three phytosterols; β-sitosterol (2), stigmasterol (3), and campesterol (4); one triterpenoid; ursolic acid (5), four diterpenoids; siderol (6), eubol (7), eubotriol (8), 7-epicandicandiol (9) and two flavonoids; xanthomicrol (10) and penduletin (11). The main isolated constituent was siderol. The antiproliferative potential of siderol was evaluated, using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay, on three human cancer cell lines DLD1, HeLa, and A549, where the IC50 values were estimated at 26.4 ± 3.7, 44.7 ± 7.2, and 46.0 ± 4.9 μΜ, respectively. The most potent activity was recorded in the human colon cancer cell line DLD1, where siderol exhibited the lowest IC50. Our study unveiled the beneficial potential of siderol as a remarkable cytotoxic agent and the significant contribution of NMR spectroscopy towards the isolation and identification of this potent anticancer agent.

The Effect of 4'-hydroxy-3,4,5-trimetoxystilbene, the Metabolite of Resveratrol Analogue DMU-212, on Growth, Cell Cycle and Apoptosis in DLD-1 and LOVO Colon Cancer Cell Lines.

Resveratrol is a phytoalexin that naturally occurs in grapes, blueberries, cranberries, peanuts and many other plants. Although resveratrol inhibits carcinogenesis in all three stages, its clinical application is restricted due to poor pharmacokinetics. The methylated analogues of resveratrol have been found to have higher bioavailability and cytotoxic activity than that of the prototupe compound. Among the various methoxy derivatives of resveratrol, 3,4,5,4′-tetrametoxystilbene (DMU-212) is suggested to be one of the strongest activators of cytotoxicity and apoptosis. DMU-212 has been shown to exert anti-tumor activity in DLD-1 and LOVO colon cancer cells. Since colorectal cancer is the third most common cause of cancer-related deaths worldwide, the development of new anticancer agents is nowadays of high significance. The aim of the present study was to assess the anticancer activity of 4′-hydroxy-3,4,5-trimetoxystilbene (DMU-281), the metabolite of DMU-212, in DLD-1 and LOVO cell lines. We showed for the first time the cytotoxic activity of DMU-281 triggered via cell cycle arrest at G2/M phase and apoptosis induction accompanied by the activation of caspases-9, -8, -3/7. Furthermore, DMU-281 has been found to change the expression pattern of genes and proteins related to intrinsic as well as extrinsic apoptosis. Since the activation of these pathways of apoptosis is still the most desired strategy in anticancer research, DMU-281 seems to provide a promising approach to the treatment of colon cancer.