Colorectal Tumor Cell Lines Research Articles

DNA demethylation and tri-methylation of H3K4 at the TACSTD2 promoter are complementary players for TROP2 regulation in colorectal cancer cells

TROP2 is a powerful cancer driver in colorectal cancer cells. Divergent epigenetic regulation mechanisms for the corresponding TACSTD2 gene exist such as miRNAs or DNA methylation. However, the role of TACSTD2 promoter hypermethylation in colorectal cancer has not been investigated yet. In this study, TROP2 expression strongly correlated with promoter methylation in different colorectal tumor cell lines. Treatment with 5-Azacytidine, a DNMT1 inhibitor, led to demethylation of the TACSTD2 promoter accompanied by an increase in TROP2 protein expression. TROP2 expression correlated with promoter methylation in vivo in human colon tumor tissue, thereby verifying promoter methylation as an important factor in the regulation of TROP2 expression in colorectal cancer. When performing a ChIP-Seq analysis in HCT116 and HT29 cells, we found that TACSTD2 promoter demethylation was accompanied by tri-methylation of H3K4. In silico analysis of GSE156613 data set confirmed that a higher binding of histone mark H3K4me3 around the TACSTD2 promoter was found in TACSTD2 high expressing tumors of colon cancer patients compared to the corresponding adjacent tumor tissue. Moreover, the link between TROP2 and the H3K4me3 code was even evident in tumors showing high intratumoral heterogeneity for TROP2 staining. Our data provide novel evidence for promoter demethylation and simultaneous gains of the active histone mark H3K4me3 across CpG-rich sequences, both being complementary mechanisms in the transcriptional regulation of TACSTD2 in colon cancer. The functional consequences of TROP2 loss in colorectal cancer needs to be further investigated.

Cellular Antioxidant, Anti-Inflammatory, and Antiproliferative Activities from the Flowers, Leaves and Fruits of Gallesia integrifolia Spreng Harms.

Gallesia integrifolia, a notable species in the Atlantic Forest, has been traditionally employed in folk medicine for treating rheumatism, asthma, and worms. This study investigated the cellular antioxidant, antiproliferative, and anti-inflammatory activities of the essential oils (EOs) and crude extracts (CEs) from G. integrifolia flowers, fruits, and leaves. The chemical identification of EOs was performed by GC-MS and CEs by UHPLC-MS. Cellular antioxidant and anti-inflammatory activities were assessed through mouse macrophage cell culture. In addition, the antiproliferative potential was evaluated in gastric, colorectal, breast, and lung tumor cell lines and non-tumor VERO cells. EOs predominantly contained organosulfur compounds in flowers (96.29%), fruits (94.94%), and leaves (90.72%). We found the main compound is 2,2'-Disulfanediyldiethanethiol in the EOs of flowers (47.00%), leaves (41.82%), and fruits (44.39%). Phenolic compounds were identified in CEs. The EOs and CEs demonstrated potential against the tumor cell lines tested (GI50 between 51 and 230 µg/mL). The selectivity index values were greater than 1.0 (1.01 to 3.37), suggesting a relative safety profile. Moreover, the anti-inflammatory activity IC50 ranged from 36.00 to 268 µg/mL, and the cellular oxidation inhibition ranged from 69% to 82%. The results suggest that oils and extracts derived from G. integrifolia have potential for use in various industrial sectors.

A Scalable Engineered Extracellular Matrix Platform to Expand Tumor Cells

The demand for high‐throughput and scalable cell expansion platforms that can accommodate diverse cell types remains a critical requirement across various biomedical fields. Fibronectin (Fn), an essential component of the extracellular matrix (ECM), has been used as a conformal surface coating for two‐dimensional (2D) cell culture systems. However, the soluble, globular Fn used for 2D coatings differs structurally from the native Fn, which possesses a three‐dimensional (3D) fibrillar structure. Herein, a large‐scale engineered ECM (EECM) cell expansion platform based on a 3D fibrillar Fn network spanning over centimeters is presented. Extended fibrillar networks are formed by shearing dilute Fn solutions over tessellated polymeric scaffolds, which are conveniently prepared by 3D printing. The structure and size of the Fn‐based 3D EECM scaffold are optimized by evaluating the proliferation of a colorectal tumor cell line, CT26, commonly used in the in vivo tumor immunotherapy models. The 3D EECM scaffolds support a fourfold more efficient tumor cell expansion than a conventional 2D culture system, demonstrating the potential efficacy in supporting the robust expansion of cancer cells ex vivo with an eye on cancer immunotherapy.

Proliferation Inhibitory Activity of Quinones from Blaps rynchopetera Defense Secretion on Colorectal Tumor Cells.

To explore the proliferation inhibitory effect of quinones from Blaps rynchopetera defense secretion on colorectal tumor cell lines. Human colorectal cancer cell HT-29, human colorectal adenocarcinoma cell Caco-2 and normal human colon epithelial cell CCD841 were chosen for the evaluation of inhibitory activity of the main quinones of B. rynchopetera defense secretion, including methyl p-benzoquinone (MBQ), ethyl p-benzoquinone (EBQ), and methyl hydroquinone (MHQ), through methyl thiazolyl tetrazolium assay. The tumor-related factors, cell cycles, related gene expressions and protein levels were detected by enzyme-linked immunosorbent assy, flow cytometry, RT-polymerase chain reaction and Western blot, respectively. MBQ, EBQ, and MHQ could significantly inhibit the proliferation of Caco-2, with half maximal inhibitory concentration (IC50) values of 7.04 ± 0.88, 10.92 ± 0.32, 9.35 ± 0.83, HT-29, with IC50 values of 14.90 ± 2.71, 20.50 ± 6.37, 13.90 ± 1.30, and CCD841, with IC50 values of 11.40 ± 0.68, 7.02 ± 0.44 and 7.83 ± 0.05 µg/mL, respectively. Tested quinones can reduce the expression of tumor-related factors tumor necrosis factor α, interleukin (IL)-10, and IL-6 in HT-29 cells, selectively promote apoptosis, and regulate the cell cycle which can reduce the proportion of cells in the G1 phase and increase the proportion of the S phase. Meanwhile, tested quinones could up-regulate mRNA and protein expression of GSK-3β and APC, while down-regulate that of β-catenin, Frizzled1, c-Myc, and CyclinD1 in the Wnt/β-catenin pathway of HT-29 cells. Quinones from B. rynchopetera defense secretion could inhibit the proliferation of colorectal tumor cells and reduce the expression of related factors, which would be functioned by regulating cell cycle, selectively promoting apoptosis, and affecting Wnt/β-catenin pathway-related mRNA and protein expressions.

MC38 colorectal tumor cell lines from two different sources display substantial differences in transcriptome, mutanome and neoantigen expression.

The cell line MC38 is a commonly used murine model for colorectal carcinoma. It has a high mutational burden, is sensitive to immune checkpoint immunotherapy and endogenous CD8+ T cell responses against neoantigens have been reported. Here, we re-sequenced exomes and transcriptomes of MC38 cells from two different sources, namely Kerafast (originating from NCI/NIH, MC38-K) and the Leiden University Medical Center cell line collection (MC38-L), comparing the cell lines on the genomic and transcriptomic level and analyzing their recognition by CD8+ T cells with known neo-epitope specificity. The data reveals a distinct structural composition of MC38-K and MC38-L cell line genomes and different ploidies. Further, the MC38-L cell line harbored about 1.3-fold more single nucleotide variations and small insertions and deletions than the MC38-K cell line. In addition, the observed mutational signatures differed; only 35.3% of the non-synonymous variants and 5.4% of the fusion gene events were shared. Transcript expression values of both cell lines correlated strongly (p = 0.919), but we found different pathways enriched in the genes that were differentially upregulated in the MC38-L or MC38-K cells, respectively. Our data show that previously described neoantigens in the MC38 model such as Rpl18mut and Adpgkmut were absent in the MC38-K cell line resulting that such neoantigen-specific CD8+ T cells recognizing and killing MC38-L cells did not recognize or kill MC38-K cells. This strongly indicates that at least two sub-cell lines of MC38 exist in the field and underlines the importance of meticulous tracking of investigated cell lines to obtain reproducible results, and for correct interpretation of the immunological data without artifacts. We present our analyses as a reference for researchers to select the appropriate sub-cell line for their own studies.

Sodium Butyrate Induces CRC Cell Ferroptosis via the CD44/SLC7A11 Pathway and Exhibits a Synergistic Therapeutic Effect with Erastin.

Human normal intestinal epithelial cell line FHT and colorectal tumor cell line HCT-116 were treated with NaB alone or in combination with different programmed cell death inhibitors. Cell activity was then assessed with MTT assays and PI staining; ferroptosis with Fe2+, glutathione (GSH), and lipid peroxidation assays; signaling pathway screening with PCR arrays; and CD44, SCL7A11, and GPX4 expression with Western blotting. A CD44-overexpressing HCT-116 cell line was constructed to determine the effect of the overexpression of CD44 on NaB-induced ferroptosis. The synergistic effect of co-treatment with NaB and Erastin was assessed by isobolographic analysis. NaB induced apoptosis and ferroptosis in HCT-116 cells but only induced low-level apoptosis in FHC cells. Moreover, NaB significantly increased intracellular Fe2+ and promoted GSH depletion and lipid peroxidation in HCT-116 cells. Ferroptosis-related qPCR array analysis identified CD44/SLC7A11 as a potential effector molecular of NaB-induced ferroptosis. NaB significantly inhibited the expression of CD44 and SLC7A11 in mouse CRC tissues. A CD44 overexpressed HCT-116 cell line was used to verify that CD44/SLC7A11 was a key signaling pathway that NaB-induced GSH depletion, lipid peroxidation accumulation, and ferroptosis in HCT-116 cells. Examination of whether NaB can increase the effect of ferroptosis agents showed that NaB, in combination with Erastin, a ferroptosis inducer, further promoted HCT-116 cell death and increased changes of ferroptosis markers. Our results suggest that NaB induces ferroptosis in CRC cells through the CD44/SLC7A11 signaling pathway and has synergistic effects with Erastin. These results may provide new insights into CRC prevention and the combined use of NaB and ferroptosis-inducing agents.

Dendritic cell-targeting chemokines inhibit colorectal cancer progression.

Recent progress in cancer immunotherapy has shown its promise and prompted researchers to develop novel therapeutic strategies. Dendritic cells (DCs) are professional antigen-presenting cells crucial for initiating adaptive anti-tumor immunity, therefore a promising target for cancer treatment. Here, anti-tumor activities of DC-targeting chemokines were explored in murine colorectal tumor models. The correlation of chemokine messenger RNA (mRNA) expression with DC markers was analyzed using The Cancer Genome Atlas (TCGA) dataset. Murine colorectal tumor cell lines (CT26 and MC38) stably overexpressing mouse C-C motif chemokine ligand 3 (CCL3), CCL19, CCL21, and X-C motif chemokine ligand 1 (XCL1) were established by lentiviral transduction. The effect of chemokines on tumor cell proliferation/survival was evaluated in vitro by cell counting kit-8 (CCK-8) assay and colony formation assay. Syngeneic subcutaneous tumor models were used to study the effects of these chemokines on tumor growth. Ki-67 expression in tumors was examined by immunohistochemistry. Immune cells in the tumor microenvironment (TME) and lymph nodes were analyzed by flow cytometry. Expression of the four chemokines was positively correlated with the two DC markers [integrin alpha X (ITGAX) and CLEC9A] in human colorectal tumor samples. Tumoral overexpression of DC-targeting chemokines had little or no effect on tumor cell proliferation/survival in vitro while significantly suppressing tumor growth in vivo. Fluorescence-activated cell sorting (FACS) analysis showed that CCL19, CCL21, and XCL1 boosted the ratios of DCs and T cells in CD45+ leukocytes while CCL3 increased the percentage of CD45+ leukocytes in total cells in MC38 tumor. XCL1 had an additional positive effect on antigen uptake by DCs in the TME and antigen transfer to tumor-draining lymph nodes. CCL3, CCL19, CCL21, and XCL1 exhibited potent anti-tumor activities in vivo, although they might differentially regulate immune cells in the TME and antigen transfer to lymph nodes.

Coumarins, furocoumarins and limonoids of Citrus trifoliata and their effects on human colon adenocarcinoma cell lines

Citrus trifoliata L. (Chinese or Japanese bitter orange) is a medicinal plant with furocoumarins and limonoids as characteristic secondary metabolites. The bitter taste of the fruit limits its use as food, however, it is applied in Asian traditional medicine for its antiphlogistic effect, to treat digestive ulcers and different gastrointestinal disorders and cancer. The phytochemical composition and pharmacological characteristics of this species have not been fully discovered, nevertheless its potential antiproliferative or cytotoxic effects might be related to furocoumarins or limonoids.Our aim was to isolate and identify secondary metabolites from C. trifoliata peel and seeds and to investigate their bioactivities that might be related to the supposed anticancer effect of the plant. By using different chromatographic methods, six pure compounds (phellopterin (2), scoparone (3), myrsellin (4), triphasiol (6), umbelliferone (7) and citropten (5,7-dimethoxycoumarin (8)) were isolated from the peel and four (imperatorin (1), auraptene (5), limonin (9) and deacetyl nomilin (10)) from the seeds of C. trifoliata fruits. These compounds are furocoumarin (1, 2), coumarin (3–8), and limonoid derivatives (9, 10). Scoparone (3) has been detected in this species for the first time.The furocoumarins (1–2) showed moderate activity on the human colorectal adenocarcinona tumor cell line COLO 320 in antiproliferative assays and 2 also had remarkable P-glycoprotein inhibitory activity and synergistic effect with doxorubicin. The coumarin 5 showed significant activity on the COLO 320 cell line in antiproliferative assays and P-glycoprotein inhibitory activity in the FACS (fluorescence activated cell sorting) assay.

Abstract 5505: ADAM10-targeting CAR-T cells inhibit colon cancer cell growth in vivo

Abstract Chimeric antigen receptor (CAR) T cell therapy has demonstrated a high degree of efficacy in patients with hematological malignancies such as acute lymphatic leukemia. However, its efficacy in solid tumors remains to be demonstrated. There are a number of issues currently limiting therapeutic treatment of solid tumors with CAR-T cells, none more important than target antigen selection. This is a major challenge in solid tumors due to their heterogenous characteristics and the potential for off-cancer effects. ADAM10, A Disintegrin And Metalloproteinase 10, plays critical roles in a wide range of pathophysiological processes during development and inflammation. These proteolytically active multifunctional proteins act as sheddases by cleaving transmembrane-bound proteins such as growth factors, adhesion molecules and chemokine receptors, which can then contribute to migration and invasion of cancer cells. An active form of ADAM10 that has undergone a conformational change has been identified in several cancer types, particularly colorectal cancer. In this study, a novel CAR was generated based on a previously characterized cancer-specific ADAM10 antibody. ADAM-10-targeting CAR-T cells were manufactured using T cells from the peripheral blood of the healthy donors. The CAR-T cells displayed the naïve and central memory phenotype that has been shown to differentiate and proliferate within tumor microenvironments, with little expression of exhaustion markers. These CAR-T cells were capable of killing several human colorectal tumor cell lines in vitro, assessed in a bioluminescence-based cytotoxicity assay. Furthermore, treatment of NSG mice bearing human LoVo colon cancer cell tumors with ADAM-10-targeting CAR-T cells led to significant inhibition of tumor growth compared with either untransduced control T cells or PBS treatment. Overall, these data suggest that targeting ADAM-10 via CAR-T cell therapy is a promising novel avenue of treatment of colorectal cancer. Citation Format: Elaheh Rohani Rad, Jade Foeng, Dylan McPeak, Timona Tyllis, Caitlin Abbott, Veronika bandara, Silvana Napoli, Batjargal gundsambuu, Simon Barry, Timothy Sadlon, shaun McColl. ADAM10-targeting CAR-T cells inhibit colon cancer cell growth in vivo [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 5505.

The nucleic acid sensor RIG-I as an inducer of anti-tumor response

Abstract The immune microenvironment plays a critical role in tumor progression. Recently, approaches to target Pattern Recognition Receptors (PRRs) have emerged as a promising strategy to enhance immune recognition of tumors. Among the PRRs, those which recognize DNA and RNA in the cells are unique in their ability to respond to both pathogenic and self-nucleic acids that are generated as a result of aberrant cell division/transcription. We previously showed that targeting the DNA sensor three prime exonuclease 1 (TREX1) enhanced immune responses in mouse tumor models. Here we show that the RNA sensor Retinoic acid Induced Gene 1 (RIG-I) acts as a robust immune activator across cancer types. According cBioportal data, expression of RIG-I seems to be a prognostic factor for progression free survival. To functional evaluate RIG-I response in tumor models, we used a hairpin RNA derived from H1N1 sequence. Interestingly, activation of RIG-I increased expression of TREX1 revealing a potential crosstalk between the two pathways. Both breast and colorectal tumor cell lines produce IFN-I after RIG-I activation. Consistently, RNAseq showed upregulation of IFN-I response genes and proinflammatory cytokines across different cell lines. Activation of RIG-I in the tumor cells decreased tumor burden with a concomitant broad cellular and molecular anti-tumor response. Thus, results showed an increase in NK and DC population, while the transcription profile was upregulation of innate immune response genes. Interestingly, tumor growth and NK results were consistent when tumor was implanted in immunocompromised mouse model. Taken together, our findings illustrate how tumor cell RIG-I is a potent inducer of cellular and molecular immune responses.

Antiproliferative Activity of Two Unusual Dimeric Flavonoids, Brachydin E and Brachydin F, Isolated from Fridericia platyphylla (Cham.) L.G.Lohmann: In Vitro and Molecular Docking Evaluation.

Despite the breakthrough in the development of anticancer therapies, plant-derived chemotherapeutics continue to be the basis of treatment for most types of cancers. Fridericia platyphylla is a shrub found in Brazilian cerrado biome which has cytotoxic, anti-inflammatory, and analgesic properties. The aim of this study was to investigate the antiproliferative potential of the crude hydroethanolic extract, subfraction (containing 59.3% of unusual dimeric flavonoids Brachydin E and 40.7% Brachydin F), as well as Brachydin E and Brachydin F isolated from F. platyphylla roots. The cytotoxic activity was evaluated in glioblastoma, lung, prostate, and colorectal human tumor cell lines. The crude hydroethanolic extract did not present cytotoxic activity, but its subfraction presented lower IC50 values for glioblastoma (U-251) and prostate adenocarcinoma (PC-3) cell lines. Brachydins E and F significantly reduced cell viability, proliferation, and clonogenic potential of PC-3, inducing them to the process of regulated cell death. In silico studies have indicated nuclear receptors as targets for Brachydins E and F, and molecular docking has pointed out their binding into glucocorticoid receptor (GR) ligand pocket. Targeting GR pathway has been described as a therapeutic strategy, especially for prostate cancer. These results suggest that Brachydin E and Brachydin F are promising compounds to be further explored for their antitumor effects.

AM22, a novel synthetic microRNA, inhibits the proliferation of colorectal cancer cells by targeting core binding factor subunit β (CBFB).

Our previous studies have revealed the important roles of the nonseed regions of microRNAs (miRNAs) in gene regulation, which provided novel insight into the development of miRNA analogs for cancer therapy. Here, we altered each nucleotide in the nonseed region of miR-34a and obtained novel synthetic miRNA analogs. Among them, AM22, with a base alteration from G to C at the 17th nucleotide of miR-34a, showed extensive antiproliferative activity against several colorectal tumor cell lines and achieved effective inhibition of core binding factor subunit β (CBFB) expression. Subsequent investigations demonstrated that AM22 directly targeted CBFB by binding to its 3'-untranslated region (3'-UTR). Inhibition of CBFB showed obvious antiproliferative activity on HCT-116 and SW620 cells. Furthermore, the antiproliferative effects of AM22 on these cells were also measured in xenograft mouse models. In conclusion, this study identified AM22 as a potential antitumor miRNA by targeting CBFB and provided a new design approach for miRNA-based cancer treatment by changing the nonseed region of miRNA.

Abstract P036: A high-throughput customized cytokinome screen of colon cancer cell responses to small-molecule oncology drugs

Abstract Inflammatory cytokines, immune stimulants, chemokines, and tumor growth/growth suppressing factors are molecular messengers that circulate and have the capability to modify the tumor microenvironment and impact response to therapeutics. The characterization of soluble mediators as biomarkers for diagnosis and prognosis is of interest in oncology. We utilize the cytokinome to characterize the response of colorectal tumor cell lines to selected small-molecules in oncology as a proof-of-concept dataset with immune synergy heat map rankings of analytes for drug and cell line combinations. We observed overall trends in drug-class effects with MEK-, BRAF-, PARP-inhibitors, and Imipridones in cytokine, chemokine, and growth factor responses that may help guide therapy selection. MEK-inhibitor treatment downregulated analytes VEGF, CXCL9/MIG, and IL-8/CXCL8 and upregulated CXCL14/BRAK, Prolactin, and CCL5/RANTES. BRAF-inhibitor treatment downregulated VEGF and IL-8/CXCL8, while increasing sTRAIL-R2. Treatment with PARP-inhibitors decreased CXCL9/MIG, IL-8/CXCL8, CCL3/MIP-1 alpha, VEGF, and CXCL14/BRAK, while treatment increased sTRAIL-R2 and prolactin. Treatment with Imipridones decreased CCL3/MIP-1 alpha, VEGF, CXCL14/BRAK, IL-8/CXCL8, and Prolactin and increased CXCL5/ENA-78. We also observed differential responses to therapeutics depending on the mutational profile of the cell line. In the future, a similar but larger data-set may be utilized in the clinic to aid in immune synergy prediction of a patient's response to therapy based on tumor genotype. Citation Format: Kelsey E. Huntington, Anna Louie, Lanlan Zhou, Wafik S. El-Deiry. A high-throughput customized cytokinome screen of colon cancer cell responses to small-molecule oncology drugs [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P036.

Novel 1,3,5-Triazinyl Aminobenzenesulfonamides Incorporating Aminoalcohol, Aminochalcone and Aminostilbene Structural Motifs as Potent Anti-VRE Agents, and Carbonic Anhydrases I, II, VII, IX, and XII Inhibitors.

A series of 1,3,5-triazinyl aminobenzenesulfonamides substituted by aminoalcohol, aminostilbene, and aminochalcone structural motifs was synthesized as potential human carbonic anhydrase (hCA) inhibitors. The compounds were evaluated on their inhibition of tumor-associated hCA IX and hCA XII, hCA VII isoenzyme present in the brain, and physiologically important hCA I and hCA II. While the test compounds had only a negligible effect on physiologically important isoenzymes, many of the studied compounds significantly affected the hCA IX isoenzyme. Several compounds showed activity against hCA XII; (E)-4-{2-[(4-[(2,3-dihydroxypropyl)amino]-6-[(4-styrylphenyl)amino]-1,3,5-triazin-2-yl)amino]ethyl}benzenesulfonamide (31) and (E)-4-{2-[(4-[(4-hydroxyphenyl)amino]-6-[(4-styrylphenyl)amino]-1,3,5-triazin-2-yl)amino]ethyl}benzenesulfonamide (32) were the most effective inhibitors with KIs = 4.4 and 5.9 nM, respectively. In addition, the compounds were tested against vancomycin-resistant Enterococcus faecalis (VRE) isolates. (E)-4-[2-({4-[(4-cinnamoylphenyl)amino]-6-[(4-hydroxyphenyl)amino]-1,3,5-triazin-2-yl}amino)ethyl]benzenesulfonamide (21) (MIC = 26.33 µM) and derivative 32 (MIC range 13.80–55.20 µM) demonstrated the highest activity against all tested strains. The most active compounds were evaluated for their cytotoxicity against the Human Colorectal Tumor Cell Line (HCT116 p53 +/+). Only 4,4’-[(6-chloro-1,3,5-triazin-2,4-diyl)bis(iminomethylene)]dibenzenesulfonamide (7) and compound 32 demonstrated an IC50 of ca. 6.5 μM; otherwise, the other selected derivatives did not show toxicity at concentrations up to 50 µM. The molecular modeling and docking of active compounds into various hCA isoenzymes, including bacterial carbonic anhydrase, specifically α-CA present in VRE, was performed to try to outline a possible mechanism of selective anti-VRE activity.

Di-2-pyridylketone 4, 4-dimethyl-3-thiosemicarbazone effectively induces human colorectal carcinoma cell apoptosis via mTOR pathway

Background: To investigate the anticancer mechanisms of di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) in human colon cancer cells. Human colorectal carcinoma (HCC) is one of the most commonly diagnosed cancers in both males and females. Current studies have found that iron chelators can be used as novel anticancer drugs; however, the anticancer activity of iron chelators and their target genes in HCC has been rarely reported. Methods: Dp44mT was used to treat two colorectal tumor cell lines, SW480 and HT-29. The proapoptotic effects of different concentrations of Dp44mt were measured using flow cytometry and Hoechst 33258 staining. Ferric ammonium citrate (FAC) was used as an additional iron donor to inhibit the effects of Dp44mT. Apoptosis and DNA damage-related proteins were examined by Western blot analysis. Results: In this study, we found that the iron chelators Dp44mT could induce the apoptosis in two colorectal tumor cell lines SW480 and HT-29, upregulate the expression level of p-histone H2A.X, and inhibit the phosphorylation level of mTOR in a dose-dependent way. Those effects could be reversed by the additional iron donor FAC. Conclusion: These data indicate that iron depletion and/or the presence of iron can modulate the HCC apoptosis progression in vitro, which may be a potential target for future HCC therapy.

A high-throughput customized cytokinome screen of colon cancer cell responses to small-molecule oncology drugs.

Inflammatory cytokines, chemokines, and growth factors are molecular messengers that circulate and have the capability to modify the tumor microenvironment and impact therapeutic response. The characterization of soluble mediators as biomarkers for diagnosis and prognosis is of interest in oncology. We utilize the cytokinome to characterize the response of colorectal tumor cell lines to selected small-molecules in oncology as a proof-of-concept dataset with immunomodulatory analyte heat map rankings for drug and cell line combinations. We observed overall trends in drug class effects with MEK-, BRAF-, PARP-inhibitors, and Imipridones in cytokine, chemokine, and growth factor responses that may help guide therapy selection. MEK-inhibitor treatment downregulated analytes VEGF, CXCL9/MIG, and IL-8/CXCL8 and upregulated CXCL14/BRAK, Prolactin, and CCL5/RANTES. BRAF-inhibitor treatment downregulated VEGF and IL-8/CXCL8, while increasing soluble TRAIL-R2. Treatment with PARP-inhibitors decreased CXCL9/MIG, IL-8/CXCL8, CCL3/MIP-1 alpha, VEGF, and CXCL14/BRAK, while treatment increased soluble TRAIL-R2 and prolactin. Treatment with Imipridones decreased CCL3/MIP-1 alpha, VEGF, CXCL14/BRAK, IL-8/CXCL8, and Prolactin and increased CXCL5/ENA-78. We also observed differential responses to therapeutics depending on the mutational profile of the cell line. In the future, a similar but larger dataset may be utilized in the clinic to aid in the prediction of patient response to immunomodulatory therapies based on tumor genotype.

Antitumor Activity of Fucus vesiculosus-Derived Phlorotannins through Activation of Apoptotic Signals in Gastric and Colorectal Tumor Cell Lines.

Seaweeds are one of the largest producers of biomass in the marine environment and a source of multiple bioactive metabolites with valuable health benefits. Among these, phlorotannins have been widely recognized for their promising bioactive properties. The potential antitumor capacity of Fucus vesiculosus-derived phlorotannins remains, however, poorly explored, especially in gastrointestinal tract-related tumors. Therefore, this work aimed to evaluate the cytotoxic properties and possible mechanisms by which F. vesiculosus crude extract (CRD), phlorotannin-rich extract (EtOAc), and further phlorotannin-purified fractions (F1–F9) trigger cell death on different tumor cell lines of the gastrointestinal tract, using flow cytometry. The results indicate that F. vesiculosus samples exert specific cytotoxicity against tumor cell lines without affecting the viability of normal cells. Moreover, it was found that, among the nine different phlorotannin fractions tested, F5 was the most active against both Caco-2 colorectal and MKN-28 gastric cancer cells, inducing death via activation of both apoptosis and necrosis. The UHPLC-MS analysis of this fraction revealed, among others, the presence of a compound tentatively identified as eckstolonol and another as fucofurodiphlorethol, which could be mainly responsible for the promising cytotoxic effects observed in this sample. Overall, the results herein reported contribute to a better understanding of the mechanisms behind the antitumor properties of F. vesiculosus phlorotannin-rich extracts.

Concomitant inhibition of B7-H3 and PD-L1 expression by a novel and synthetic microRNA delivers potent antitumor activities in colorectal tumor models.

The families of miR-34 and miR-449 share the same seed region. However, the members showed differential effects on the expression of B7-H3 and PD-L1 in HCT-116 cells. Using miR-34a as a template, the non-seed region was modified by nucleotide alteration, yielding four synthetic microRNA (miRNA) analogs. Among those, NS-MX3, with a base alteration from G to C at the 18th locus of miR-34a, showed the most potent inhibition on both B7-H3 and PD-L1 expression. Subsequent investigations demonstrated that NS-MX3 had a broad anti-proliferation activity against several colorectal tumor cell lines and its antitumor effect was consistently reflected by tumor growth inhibition (TGI) in the HCT-116 xenograft model. In addition, NS-MX3 displayed a synergistic effect on TGI when combined with bevacizumab or regorafenib. Further analysis revealed that the superior antitumor activity of NS-MX3 was correlated to concomitant suppression of both B7-H3 and PD-L1 expression in tumor tissues. Taken together, the present study indicates that the non-seed region of miRNAs plays an important role in the regulation of checkpoint genes, thus showcasing single nucleotide alteration of the non-seed region as a promising approach to discover and develop novel immunotherapies.

Antitumor, Immunomodulatory and Antiangiogenic Efficacy of Medicinal Mushroom Extract Mixtures in Advanced Colorectal Cancer Animal Model.

Due to frequent drug resistance and/or unwanted side-effects during conventional and targeted cancer treatments, development of multi-target therapies is an important research field. Medicinal mushrooms’ isolated specific compounds and mushroom extracts have been already proven as non-toxic multi-target inhibitors of specific oncogenic pathways, as well as potent immunomodulators. However, research on antitumor effects of multiple-species extract mixtures was limited so far. The aim of this study was therefore, a study of medicinal mushroom preparations AGARIKON.1 and AGARIKON PLUS on colorectal cell lines in vitro and colorectal mice model in vivo. We found a significant antiproliferative and pro-apoptotic effect of tested medicinal mushroom preparations on colorectal (HCT-116, SW620) tumor cell lines, while the effect on human fibroblast cell line (WI-38) was proliferative emphasizing a specificity towards tumor cell lines. We further investigated the effect of the medicinal mushroom preparations AGARIKON.1 and AGARIKON PLUS in various combinations with conventional cytostatic drug 5-fluorouracil in the advanced metastatic colorectal cancer mouse model CT26.WT. AGARIKON.1 and AGARIKON PLUS exhibited immunostimulatory and antiangiogenic properties in vivo which resulted in significantly increased survival and reduction in tumor volume. The antitumor effects of AGARIKON.1 and AGARIKON PLUS, with or without 5-fluorouracil, are based on M1 macrophage polarization enhancement, inhibition of M2 and tumor-associated macrophage (TAM) polarization, effects on T helper cell Th1/Th2/Th17 cytokine profiles, direct inhibition of CT26.WT tumor growth, inhibition of vascular endothelial growth factors (VEGF) and metalloproteinases 2 and 9 (MMP-2 and MMP-9) modulation. The administration of AGARIKON.1 and AGARIKON PLUS did not show genotoxic effect. This data provides good basis for an expanded translational study.

Piperine suppresses the Wnt/\u03b2-catenin pathway and has anti-cancer effects on colorectal cancer cells

More than 94% of colorectal cancer cases have mutations in one or more Wnt/β-catenin signaling pathway components. Inactivating mutations in APC or activating mutations in β-catenin (CTNNB1) lead to signaling overactivation and subsequent intestinal hyperplasia. Numerous classes of medicines derived from synthetic or natural small molecules, including alkaloids, have benefited the treatment of different diseases, including cancer, Piperine is a true alkaloid, derived from lysine, responsible for the spicy taste of black pepper (Piper nigrum) and long pepper (Piper longum). Studies have shown that piperine has a wide range of pharmacological properties; however, piperine molecular mechanisms of action are still not fully understood. By using Wnt/β-catenin pathway epistasis experiment we show that piperine inhibits the canonical Wnt pathway induced by overexpression of β-catenin, β-catenin S33A or dnTCF4 VP16, while also suppressing β-catenin nuclear localization in HCT116 cell line. Additionally, piperine impairs cell proliferation and migration in HCT116, SW480 and DLD-1 colorectal tumor cell lines, while not affecting the non-tumoral cell line IEC-6. In summary, piperine inhibits the canonical Wnt signaling pathway and displays anti-cancer effects on colorectal cancer cell lines.