Growth Of Colon Carcinoma Cells Research Articles

Novel read through agent: ZKN-0013 demonstrates efficacy in APCmin model of familial adenomatous polyposis.

Familial adenomatous polyposis (FAP) is a precancerous, colorectal disease characterized by hundreds to thousands of adenomatous polyps caused by mutations in the tumor suppressor gene adenomatous polyposis coli (APC). Approximately 30% of these mutations are premature termination codons (PTC), resulting in the production of a truncated, dysfunctional APC protein. Consequently, the β-catenin degradation complex fails to form in the cytoplasm, leading to elevated nuclear levels of β-catenin and unregulated β-catenin/wnt-pathway signaling. We present in vitro and in vivo data demonstrating that the novel macrolide, ZKN-0013, promotes read through of premature stop codons, leading to functional restoration of full-length APC protein. Human colorectal carcinoma SW403 and SW1417 cells harboring PTC mutations in the APC gene showed reduced levels of nuclear β-catenin and c-myc upon treatment with ZKN-0013, indicating that the macrolide-mediated read through of premature stop codons produced bioactive APC protein and inhibited the β-catenin/wnt-pathway. In a mouse model of adenomatous polyposis coli, treatment of APCmin mice with ZKN-0013 caused a significant decrease in intestinal polyps, adenomas, and associated anemia, resulting in increased survival. Immunohistochemistry revealed decreased nuclear β-catenin staining in the epithelial cells of the polyps in ZKN-0013-treated APCmin mice, confirming the impact on the β-catenin/wnt-pathway. These results indicate that ZKN-0013 may have therapeutic potential for the treatment of FAP caused by nonsense mutations in the APC gene. KEY MESSAGES: • ZKN-0013 inhibited the growth of human colon carcinoma cells with APC nonsense mutations. • ZKN-0013 promoted read through of premature stop codons in the APC gene. • In APCmin mice, ZKN-0013 treatment reduced intestinal polyps and their progression to adenomas. • ZKN-0013 treatment in APCmin mice resulted in reduced anemia and increased survival.

Combination of PAKs inhibitors IPA-3 and PF-3758309 effectively suppresses colon carcinoma cell growth by perturbing DNA damage response

Purpose PAKs proteins are speculated as new promising targets for cancer therapy due to their central role in many oncogenic pathways. Because PAKs proteins are very significant during carcinogenesis, we aimed to investigate the hypothesis that inhibition of PAKs with IPA-3 and PF-3758309 treatment could synergistically reduce colon carcinoma cell growth. Materials and methods The cytotoxic effects of both drugs were determined by a cell viability assay. Cell cycle and apoptosis were analyzed by flow cytometry. The effects of inhibitor drugs on marker genes of apoptosis, autophagy, cell cycle, and DNA damage were tested via immunoblotting. Results and conclusions We found out the synergistic effect of these drugs in pair on five colon cancer cell lines. Combined treatment with IPA-3+PF-3758309 in SW620 and Colo 205 cells markedly suppressed colon formation and induced apoptosis, cell cycle arrest, and autophagy compared with treatment with each drug alone. Additionally, this combination sensitized colon cancer cells to ionizing radiation that resulted in inhibition of cell growth. Significance Collectively, our findings show for the first time that cotreatment of IPA-3 with PF-3758309 exhibits superior inhibitory effects on colon carcinoma cell growth via inducing DNA damage-related cell death and also enforces a cell cycle arrest.

Combination of PAKs Inhibitors IPA-3 and PF-3758309 Effectively Suppresses Colon Carcinoma Cell Growth by Perturbing DNA Damage Response

Combination of PAKs Inhibitors IPA-3 and PF-3758309 Effectively Suppresses Colon Carcinoma Cell Growth by Perturbing DNA Damage Response

Heptamethine Cyanine Dye MHI-148-Mediated Drug Delivery System to Enhance the Anticancer Efficiency of Paclitaxel.

IntroductionPaclitaxel (PTX) is a conventional chemotherapeutic drug that effectively treats various cancers. The cellular uptake and therapeutic potential of PTX are limited by its slow penetration and low solubility in water. The development of cancer chemotherapy methods is currently facing considerable challenges with respect to the delivery of the drugs, particularly in targeting the tumor site without exerting detrimental effects on the healthy surrounding cells. One possibility for improving the therapeutic potential is through the development of tumor-targeted delivery methods.MethodsWe successfully synthesized paclitaxel-MHI-148 conjugates (PTX-MHI) by coupling PTX with the tumor-targeting heptamethine cyanine dye MHI-148. Synthesis and purification were characterized using the absorbance spectrum and the results of time-of-flight mass spectrometry. Cellular uptake and cytotoxicity studies were conducted in vitro and in vivo.ResultsPTX-MHI accumulates in tumor cells but not in normal cells, as observed by in vitro near-infrared fluorescent (NIRF) imaging along with in vivo NIRF imaging and organ biodistribution studies. We observed that MHI-148-conjugated PTX shows greater efficiency in cancer cells than PTX alone, even in the absence of light treatment. PTX-MHI could also be used for specific drug delivery to intracellular compartments, such as the mitochondria and lysosomes of cancer cells, to improve the outcomes of tumor-targeting therapy.ConclusionThe results indicated that PTX-MHI-mediated cancer therapy exerts an excellent inhibitory effect on colon carcinoma (HT-29) cell growth with low toxicity in normal fibroblasts (NIH3T3).

Camel whey protein hydrolysates induced G2/M cellcycle arrest in human colorectal carcinoma

Camel milk has been gaining immmense importance due to high nutritious value and medicinal properties. Peptides from milk proteins is gaining popularity in various therapeutics including human cancer. The study was aimed to investigate the anti-cancerous and anti-inflammatory properties of camel whey protein hydrolysates (CWPHs). CWPHs were generated at three temperatures (30 ℃, 37 ℃, and 45 ℃), two hydrolysis timepoints (120 and 360 min) and with three different enzyme concentrations (0.5, 1 and 2 %). CWPHs demonstrated an increase in anti-inflammatory effect between 732.50 (P-6.1) and 3779.16 (P-2.1) µg Dicolfenac Sodium Equivalent (DSE)/mg protein. CWPHs (P-4.3 & 5.2) inhibited growth of human colon carcinoma cells (HCT116) with an IC50 value of 231 and 221 μg/ml, respectively. P-4.3 induced G2/M cell cycle arrest and modulated the expression of Cdk1, p-Cdk1, Cyclin B1, p-histone H3, p21 and p53. Docking of two peptides (AHLEQVLLR and ALPNIDPPTVER) from CWPHs (P-4.3) identified Polo like kinase 1 as a potential target, which strongly supports our in vitro data and provides an encouraging insight into developing a novel peptide-based anticancer formulation. These results suggest that the active component, CWPHs (P-4.3), can be further studied and modeled to form a small molecule anti-cancerous therapy.

Regio-specific biotransformation of alizarin to alizarin methoxide with enhanced cytotoxicity against proliferative cells.

Alizarin has been reported to have an antigenotoxic activity along with an inhibitory effect on the tumor cell growth of human colon carcinoma cells. Alizarin was biotransformed into an O-methoxide derivative using O-methyltransferase from Streptomyces avermitilis MA4680 (SaOMT2) to enhance its bioefficacy. The biotransformed product was extracted, purified, and characterized using various chromatographic and spectroscopic analyses, and confirmed to be an alizarin 2-O-methoxide. The antiproliferative activity of the compound against gastric cancer cells (AGS), uterine cervical cancer (Hela), liver cancer (HepG2), and normal cell lines was investigated. Alizarin 2-O-methoxide showed an inhibitory effect on all three cancer-cell lines at very low concentrations, from 0.078µM, with no cytotoxicity against 267B1 (human prostate epithelial) and MRC-5 (normal human fetal lung fibroblast).

Cell Wall Membrane Fraction of Chlorella sorokiniana Enhances Host Antitumor Immunity and Inhibits Colon Carcinoma Growth in Mice.

A colon cancer growth inhibitor partially purified from the isolated cell wall membrane fraction of Chlorella sorokiniana, here referred to as Chlorella membrane factor (CMF), was evaluated for its antitumor and immunomodulatory effects in cell culture and in a colon carcinoma mouse model. The CMF treatment dose- and time-dependently inhibited colon carcinoma cell growth in 2-dimensional cultures. Treatment with CMF also significantly inhibited the growth of colon carcinoma spheroids in 3-dimensional cell culture in coculture with T lymphocytes. In a mouse CT26 colon carcinoma peritoneal dissemination model, intraperitoneal injection of CMF (10 or 30 mg dry weight/kg body weight, every other day) dose-dependently and significantly attenuated the growth of tumor nodules via induction of tumor cell apoptosis. Evaluation of immune cell populations in ascites showed that CMF treatment tended to increase T lymphocytes but lower granulocyte populations. The present study suggests that the cell wall membrane fraction of Chlorella sorokiniana contains a bioactive material that inhibits colon carcinoma growth via direct cell growth inhibition and stimulation of host antitumor immunity. Hence, it is suggested that the Chlorella cell wall membrane extract or a bioactive substance in the extract is an attractive complementary medicine for cancer therapy.

New sesquiterpenes from Asteriscus graveolens

Asteriscus graveolens (Forsk) Less. is a Saharan medicinal plant of Asteraceae family. A new acyclic sesquiterpene [7,12-dihydroxy-6,7-dihydro-5,(6) E-dehydronerolidol (3)] and sesquiterpene germacranolide lactone derivatives [9β-hydroxy-11β,13-dihydroparthenolide-9-O-β-D-glucopyranoside (7) and 9α-hydroxy-11β,13-dihydroparthenolide-9-O-β-D-glucopyranoside (8)] along with eight known compounds were isolated from polar extracts of aerial parts. Their structures were established by the analysis of 1 D- 2 D-NMR and high-resolution mass spectrometry data. A. graveolens extracts and compounds showed a significant (P < 0.05) and concentration dependent inhibitory effect on the growth of Human Colon Carcinoma (HCT116) and Human Colorectal Adenocarcinoma (DLD1) cells with IC50 in a concentration range from 89.4 to 296.0 µg/mL for extracts and from 32.6 to 728.1 µg/mL for compounds. No cytotoxic effects was evidenced in normal Primary Human Dermal Fibroblast (HDFa) up to 0.050 mg/mL for extracts and 1.0 mg/mL for pure compounds.

Molecular characterization, recombinant expression and bioactivity profile of an antimicrobial peptide, Ss-arasin from the Indian mud crab, Scylla serrata

Antimicrobial peptides (AMP) are potential alternatives to conventional antibiotics with the prospect to treat infections caused by multidrug resistant bacteria. This is the report of the first arasin sequence from the mud crab, Scylla serrata, designated as Ss-arasin. The complete cDNA sequences of the open reading frame (ORF) is comprised of 198 bp encoding 65 amino acid with a predicted molecular weight of 7 kDa and a predicted isoelectric point of 10.68. The sequence of the N-terminal 24 amino acid residues is indicative of a signal sequence directing the newly synthesize protein toward the secretory pathway. The 41-residue mature peptide is composed of two domains, an N-terminal Gly/Arg-rich domain and a C-terminal cysteine-rich domain. Challenging the mud crab with lipopolysaccharide (LPS) increased expression of Ss-arasin mRNA in haemocytes, reaching the highest level at 6 h, before dropping to basal levels at 24 h. Recombinant rSs-arasin showed antimicrobial activity against three bacterial species Staphylococcus aureus (40 mM), Pseudomonas aeruginosa (40 mM) and Escherichia coli (40 mM) implying significant anti-bacterial action. In addition, recombinant rSs-arasin inhibited human cervical carcinoma (HeLa) and colon carcinoma (HT-29) cell growth. These initial findings are encouraging to further study the structure-activity relationships to optimize these biological functions for future drug development.

Epigenetic inactivation of HOXD10 is associated with human colon cancer via inhibiting the RHOC/AKT/MAPK signaling pathway

BackgroundTo examine the influence of HOXD10 on the metabolism and growth of colon carcinoma cells by suppressing the RHOC/AKT/MAPK pathway.MethodsThirty-seven paired colon cancer and its adjacent samples from The Cancer Genome Atlas (TCGA) were analyzed. Chip Analysis Methylation Pipeline (ChAMP) analysis was employed for differential methylated points (DMPs) and the differential methylation regions (DMRs) screening. The HOXD10 mRNA expression and DNA methylation levels were detected by RT-PCR. The Cell proliferation, migration, invasion and apoptosis were respectively measured by MTT assay, transwell assay, wound healing assay and flow cytometry assay in carcinoma cell lines after treated with 5-aza-2′-deoxycytidine (5-Aza-dC) or transfected with HOXD10-expressing plasmid. The expression of HOXD10 and RHOC was revealed by immunohistochemistry in disparate differentiation colon carcinoma tissues, and the dephosphorylation of AKT and MAPK pathways were detected by RT-PCR and western blot.ResultsThe bioinformatics analysis demonstrated that HOXD10 was hypermethylated and low-expressed in colorectal cancer tissues. The detection of RT-PCR indicated the similar results in colorectal cancer cell lines and tissues. The induction of demethylation was recovered by treatment with 5-Aza-dC and the HOXD10 in colorectal cancer cell lines was re-expressed by transfection with a HOXD10 expression vector. The demethylation or overexpression of HOXD10 suppressed proliferation, migration, invasion and promoted apoptosis in colorectal cancer cells. HXOD10 suppressed the tumor growth and detected an opposite trend of protein RHOC. AKT and MAPK pathways were notably inactivated after the dephosphorylation due to the overexpression of HOXD10.ConclusionsHOXD10 was suppressed in colon adenocarcinoma cells, which down-regulated RHOC/AKT/MAPK pathway to enhance colon cancer cells apoptosis and constrain the proliferation, migration and invasion.

RETRACTED: Triptolide inhibits the growth and migration of colon carcinoma cells by down-regulation of miR-191

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Editor-in-Chief. Concerns were raised about the background pattern of the Western Blots from Figure 2A. Given the comments of Dr Elisabeth Bik regarding this article “This paper belongs to a set of over 400 papers (as per February 2020) that share very similar Western blots with tadpole-like shaped bands, the same background pattern, and striking similarities in title structures, paper layout, bar graph design, and - in a subset - flow cytometry panels”, the journal requested the authors to provide the raw data. However, the authors were not able to fulfil this request and therefore the Editor-in-Chief decided to retract the article.

Cyanidin-3-O-glucoside chloride acts synergistically with luteolin to inhibit the growth of colon and breast carcinoma cells.

In this study, we investigated whether the combination of cyanidin-3-O-glucoside chloride and luteolin could inhibit the growth of HCT-8 colon and MCF-7 breast carcinoma cells, and whether the effect of the combination was greater than the effect of either drug on its own. Growth inhibition was assessed using the CCK-8 assay, level of apoptosis and cell cycle distribution were determined using flow cytometry, and the mechanism of apoptosis induction was explored using a colorimetric assay of caspases-3 and -9. Experiments indicated that combination treatment inhibited proliferation and increased apoptosis in both cell lines to a greater extent than either drug on its own. These results suggest that luteolin and cyanidin-3-O-glucoside chloride synergistically inhibitthe growth of colon cancer and breast cancer cells. This work justifies further effort to develop this potential combination therapy.

Forced Expression of CXCL10 Prevents Liver Metastasis of Colon Carcinoma Cells by the Recruitment of Natural Killer Cells

CXC chemokine ligand 10 (CXCL10) is a CXC chemokine family protein that transmits signals by binding to its specific receptor, CXCR3. CXCL10 is also known as an interferon-γ-inducible chemokine involved in various biological phenomena, including chemotaxis of natural killer (NK) cells and cytotoxic T lymphocytes, that suppress tumor growth and inhibition of angiogenesis. In this study, we examined the effects of forced expression of CXCL10 in a murine colon carcinoma cell line (CT26) on growth and metastasis in syngeneic mice. We first established CT26 cells that were stably expressing murine CXCL10 (CT26/CXCL10) and compared their growth with their parental CT26 cells in vitro and in vivo. The in vitro growth of the CT26/CXCL10 and CT26 cells was comparable, whereas the in vivo growth of the CT26/CXCL10 cells in the skin was strongly suppressed. Liver metastasis of the CT26/CXCL10 cells was also significantly suppressed after intra-splenic implantation. Removal of NK cells by the administration of anti-asialo GM1 antibody canceled the suppression of subcutaneous growth and liver metastasis of CT26/CXCL10 cells. Immunofluorescence clearly showed that abundant NKp46-positive NK cells were recruited into the liver metastatic lesions of the CT26/CXCL10 cells, consistent with specific NK cell migration towards the culture supernatant from the CT26/CXCL10 cells in the chemotaxis assay using transwells. These findings indicate that CXCL10 prevents in vivo growth and metastasis of colon carcinoma cells by recruiting NK cells, suggesting that forced expression of CXCL10 in the colon tumors by gene delivery should lead to a favorable clinical outcome.

Divergent impact of actin isoforms on cell cycle regulation

ABSTRACTWe have shown that cytoplasmic actin isoforms play different roles in neoplastic cell transformation. β-Cytoplasmic actin acts as a tumor suppressor, affecting epithelial differentiation, cell growth, cell invasion and tumor growth of colon and lung carcinoma cells. In contrast, γ-cytoplasmic actin enhances malignant features of tumor cells whose actin network regulation is carried out via the γ-actin isoform. The goal of this study was to describe the role of cytoplasmic actins in cell cycle regulation of breast cancer cell lines MCF-7 and MDA-MB-231. The distinct roles of each cytoplasmic actin in the cell cycle driving were observed. β-Actin as well as γ-actin down-regulation inhibited proliferation of breast cancer cells, but only down-regulation of β-actin induced a significant decrease in diploid cell population and accumulation of tetraploid cells. Down-regulation of β-actin stimulated cyclin A2, B1 and D3 expression, whereas down-regulation of γ-actin reduced expression of these cyclins in both cell lines. Moreover, cyclin B1 and γ-actin were co-localized in mitotic control and β-actin-deficient cells. In mitotic MCF-7 cells down-regulation of β-actin caused an enrichment of prophase/metaphase population compared with control. γ-Actin down-regulation induced telophase enrichment.ERK1/2 and γ-actin co-localization and possible selective binding were revealed in MCF7 cells. β-Actin down-regulation induced ERK1/2 activation, while γ-actin down-regulation led to reduction of p-ERK1/2. A direct interaction of ERK1/2 with γ-actin and cyclin A2 in the same protein complex was also discovered. We suggest that γ-actin down-regulation leads to decrease of cyclin A2 level, inhibits ERK1/2 signaling and deceleration of breast cancer cells proliferation.

Upregulation of human glycolipid transfer protein (GLTP) induces necroptosis in colon carcinoma cells

Human GLTP on chromosome 12 (locus 12q24.11) encodes a 24 kD amphitropic lipid transfer protein (GLTP) that mediates glycosphingolipid (GSL) intermembrane trafficking and regulates GSL homeostatic levels within cells. Herein, we provide evidence that GLTP overexpression inhibits the growth of human colon carcinoma cells (HT-29; HCT-116), but spares normal colonic cells (CCD-18Co). Mechanistic studies reveal that GLTP overexpression arrested the cell cycle at the G1/S checkpoint via upregulation of cyclin-dependent kinase inhibitor-1B (Kip1/p27) and cyclin-dependent kinase inhibitor 1A (Cip1/p21) at the protein and mRNA levels, and downregulation of cyclin-dependent kinase-2 (CDK2), cyclin-dependent kinase-4 (CDK4), cyclin E and cyclin D1 protein levels. Assessment of the biological fate of HCT-116 cells overexpressing GLTP indicated no increase in cell death suggesting induction of quiescence. However, HT-29 cells overexpressing GLTP underwent cell death by necroptosis as revealed by phosphorylation of human mixed lineage kinase domain-like protein (pMLKL) via receptor-interacting protein kinase-3 (RIPK-3), elevated cytosolic calcium, and plasma membrane permeabilization by pMLKL oligomerization. Overexpression of W96A-GLTP, an ablated GSL binding site mutant, failed to arrest the cell cycle or induce necroptosis. Sphingolipid assessment (ceramide, monohexosylceramide, sphingomyelin, ceramide-1-phosphate, sphingosine, and sphingosine-1-phosphate) of HT-29 cells overexpressing GLTP revealed large decreases (>5-fold) in sphingosine-1-phosphate with minimal change in 16:0-ceramide, tipping the ‘sphingolipid rheostat’ (S1P/16:0-Cer ratio) towards cell death. Depletion of RIPK-3 or MLKL abrogated necroptosis induced by GLTP overexpression. Our findings establish GLTP upregulation as a previously unknown suppressor of human colon carcinoma HT-29 cells via interference with cell cycle progression and induction of necroptosis.

Terpenes from essential oils and hydrolate ofTeucrium alopecurustriggered apoptotic events dependent on caspases activation and PARP cleavage in human colon cancer cells through decreased protein expressions

This study focused on characterizing the Hydrophobic and Hydrophilic fractions of Teucrium alopecurus in the context of cancer prevention and therapy. The goal was also to elucidate the molecular mechanisms involved and to determine its efficacy against cancer by triggering apoptosis and suppressing tumorigenesis in human colon cancer. The data here clearly demonstrated that oily fractions of Teucrium alopecurus act as free radical scavengers, antibacterial agent and inhibited the proliferation of HCT-116, U266, SCC4, Panc28, KBM5, and MCF-7 cells in a time- and concentration-dependent manner. The results of live/dead and colony formation assays further revealed that Teucrium essential oil has the efficacy to suppress the growth of colon carcinoma cells. In addition, essential oil of Teucrium alopecurus induced apoptosis, as indicated by cleavage of caspases-3, -8, and -9 and poly-adenosine diphosphate ribose polymerase. Moreover, Teucrium alopecurus essential oil suppressed gene expression involved in survival, proliferation, invasion, angiogenesis, and metastasis in human colon cancer cells. No sign of toxicity was detected in vivo after treatment with increasing concentrations of essential oil. Oral administration of T.alopecurus inhibited LPS-induced colon inflammation. This anticancer property of this specie Teucrium alopecurus fractions could be due to their phenolic and/or sesquiterpene content (d-limonene, α-Bisabolol, Humulene, Thymol, and (+)-epi-Bicyclosesquiphellandrene). Hence our study reveals the anticancer activity of Teucrium alopecurus oil mediated through the suppression of cell growth, cell proliferation, and the induction of apoptosis of cancer cells. Thus, it has potential to be developed as an anticancer agent; however more in vitro and in vivo studies are warranted.

Abstract 4966: The SUV39H1-H3K9me3 axis mediates colon carcinoma cell intrinsic apoptosis and immune evasion

Abstract Human colon cancer, except for the small subset of microsatellite instable (MSI) colon cancer, does not respond to anti-PD-L1/PD-1 immune checkpoint inhibitor (ICI) immunotherapy. A well-established notion is that MSI colon cancer harbors various mutations that serve as neoantigens to generate tumor-specific cytotoxic T lymphocytes (CTLs). Because CTLs suppress tumor through inducing tumor cell apoptosis, we hypothesized that colon cancer cell intrinsic apoptosis resistance is potentially another mechanism underlying microsatellite stable (MSS) colon cancer nonresponse to ICI immunotherapy. If tumor cells are not sensitive to apoptosis induction, then tumor cells cannot be killed by CTLs regardless of how potent the CTLs are. To test this hypothesis, we analyzed MSI and MSS human colon carcinoma specimens and developed a small molecule that targets H3K9me3-mediated cell death pathways as a sensitizer for colon cancer ICI immunotherapy. As expected, we observed high level of CTL infiltration in all eight MSI colon carcinoma specimens examined. However, five of the nine MSS colon carcinoma specimens also exhibited high level of CTL infiltration, suggesting that CTL level is not the sole mechanism underlying MSS colon cancer non-response to ICI immunotherapy. Analysis of the TCGA database revealed that SUV39H1, a histone methyltransferase that catalyzes H3K9me3, is significantly elevated in human colon carcinoma specimens compared to normal human colon tissues. A SUV39H1 structure-based virtual chemical library screening in combination with functional assays identified a small-molecule SUV39H1 inhibitor. Further analysis of the top forty-three hits identified a potent SUV39H1 inhibitor. Structure modifications were then performed and a chemical synthesis procedure was developed to synthesize a novel SUV39H1 inhibitor, termed F5446. F5446 has an EC50 of 0.496 µM against SUV39H1 enzymatic activity and suppresses human colon carcinoma cell growth in a dose-dependent manner in vitro. F5446 suppresses colon carcinoma growth through upregulating key cell cycle regulators to induce cell cycle arrest at the S phase. Furthermore, F5446 also decreased H3K9me3 at the FAS promoter to upregulate Fas expression. Consequently, a sublethal dose of F5446 effectively overcame human colon carcinoma cell resistance to apoptosis induced by FasL, a ligand on activated CTLs in vitro. Treatment of colon carcinoma-bearing mice with F5446 and anti-PD-1 both significantly suppress tumor growth in vivo. Our data determined that F5446 is a novel SUV39H1 inhibitor that has the potential to be further developed as a sensitizer to overcome colon carcinoma resistance to ICI immunotherapy through activating the Fas-FasL pathway. Citation Format: Chunwan Lu, Dafeng Yang, John D. Klement, Aaron H. Colby, Mark W. Grinstaff, Cedric Pearce, Nicholas H. Oberlies, Thomas Albers, Iryna Lebedyeva, Kebin Liu. The SUV39H1-H3K9me3 axis mediates colon carcinoma cell intrinsic apoptosis and immune evasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4966.

Study of isobutyrylshikonin inhibiting proliferation of colon carcinoma cells through PI3K/Akt/m-TOR pathway

To investigate the inhibitory effect of isobutyrylshikonin on the growth of human colon carcinoma cells in vitro and its effect on the PI3K/Akt/m-TOR pathway. MTT assay was used to detect the inhibitory effect of different concentrations (0, 6.25, 12.5, 25, 50, 100 mg·L⁻¹) of isobutyrylshikonin on the proliferation of human colon carcinoma cell HT29 at 24, 48 h. CCK-8 method was used to detect the inhibitory effect of isobutyrylshikonin on HT29, HCT116, DLD-1 and Caco-2 at 48 h. AnnexinV/propidium iodide staining was applied in detecting the apoptoticrate of HT29 cells treated with different concentrations of isobutyrylshikonin at 24 h and 48 h. Cycletest plus DNA was employed to analyze HT29 apoptosis and cell cycle after 48 h treatment with isobutyrylshikonin at different concentrations. Western blot and RT-PCR assay were used to examine the protein and mRNA expressions of PI3K, p-PI3K, Akt, p-Akt and m-TOR. The results showed that isobutyrylshikonin inhibited the proliferation of different human colon carcinoma cells, and the inhibition rate was in a dose-dependent manner. Isobutyrylshikonin induced apoptosis mainly in the early stage and blocked cells in the G₀/G₁ or G₂/M phase. Isobutyrylshikonin reduced the protein expressions of PI3K, p-PI3K, Akt, p-Akt, m-TOR and the mRNA expressions of PI3K, Akt, m-TOR in a dose-dependent manner. Isobutyrylshikonin can significantly inhibit the proliferation, induce the early apoptosis and change the cycle distribution in colon carcinoma cells.This biological effect may be correlated with the inhibition of PI3K/AKT/m-TOR pathway.

Abstract B17: A dual in vivo and in silico system to model ectopic lymph node structure formation and antitumor immune response in the murine tumor microenvironment

Abstract The tumor microenvironment is a dynamic and complex system consisting of the tumor bed, surrounding stroma, vasculature, and immune infiltrate that self-organizes in response to contextual queues and polarizing factors. The nature and extent of this organization can determine if an immune response is beneficial, suppressed, or tumor promoting. Previous efforts have identified a gene expression signature (GES) that is composed of twelve chemokines and that is associated with the presence of ectopic lymph node structures (ELNS) within the tumor microenvironment. These ELNS are predictive of increased survival across a spectrum of solid tumors, and are thought to be foci of active antigen presentation, lymphocyte recruitment, and immune activation. Understanding how and why these structures form is paramount for improving existing immunotherapies and identifying novel treatment modalities. Most in vivo models utilize subcutaneous orthotopic inoculations that, while producing a tumorous mass, lack the surrounding context of a physiologically relevant microenvironment and completely lack ELNS. Alternatively, naturally occurring or chemically induced models offer a more physiologically relevant setting, but are often highly variable systems in which controlling experimental parameters becomes problematic. Here, we build upon insights gained from the GES using an implantable three-dimensional bioscaffold system to study the interaction of a developing tumor with endogenous immune infiltrates and implanted stroma. This allows for the introduction of carefully controlled microenvironmental elements in a more consistent mouse model. In this system, we can selectively interrogate individual chemokines using slow-release microparticles, and visualize immune infiltrates in real time using intravital microscopy. Concurrently, we use this system to parameterize an integrated mathematical model of the tumor microenvironment, which can then reinform our three-dimensional bioscaffold model. This dual-model system has identified an important role for three components of the GES--CCL19, CCL21, and CXCL13--in the induction of ELNS, and suggests that the type and extent of stromal activation can greatly enhance or prevent effective antitumor immunity. In particular, stromal activation using the lymphotoxin beta receptor ligand, LTα1β2, can induce organized aggregates of endogenous lymphocytes; significantly increase infiltration of T cells, B cells, and dendritic cells (all p&amp;lt;0.05); and prevent the growth of MC-38 colon carcinoma cells (p&amp;lt;0.001 by implant weight). In silico model runs parameterized by ex vivo microtaxis assays and in vivo ELNS formation assays predict that polarized activation of stromal cells is sufficient to compartmentalize lymphoid aggregates into discrete B cell and T cell zones and promote antitumor activity. Conversely, stromal activation in the presence of certain type-II promoting factors, such as all-trans retinoic acid, can result in the infiltration of suppressive populations that promote tumorigenesis. Taken together, this novel dual-model system of the tumor microenvironment suggests that the context of stromal activation in the tumor microenvironment can either promote effective antitumor immunity by inducing ELNS, or suppress antitumor immune response by recruiting suppressive populations. Citation Format: Adam W. Mailloux, Falahat Rana, Yohsuke Yagawa, Mark Robertson-Tessi, Zhou L. Susan, Alexander R. A. Anderson, James J. Mulé. A dual in vivo and in silico system to model ectopic lymph node structure formation and antitumor immune response in the murine tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr B17.

Formononetin inhibits colon carcinoma cell growth and invasion by microRNA‑149‑mediated EphB3 downregulation and inhibition of PI3K/AKT and STAT3 signaling pathways.

Formononetin (Form), a phytoestrogen extracted from the roots of Astragalus membranaceus, is one of the fundamental herbs used in traditional Chinese medicine because of its protective effects against certain malignant tumors. However, its role in colon carcinoma cells and the underlying molecular mechanisms have not been completely elucidated. The present study aimed to demonstrate that Form significantly inhibited the proliferation and invasion of the colon carcinoma cell lines SW1116 and HCT116. Mechanistic studies have suggested that Form suppresses colon carcinoma cell growth by downregulating cell cycle-associated protein (cyclin D1) expression and arresting the cell cycle at the G0-G1 checkpoint. Further studies revealed that treatment with Form inhibits matrix metalloproteinase (MMP)2 and MMP9 expression. Aditionally, the results demonstrated that Form significantly increased microRNA (miR)-149 expression. Following miR-149 overexpression in SW1116 and HCT116 cells using an miR-149 mimic, cell viability and Ephrin type-B receptor 3 (EphB3) levels decreased. Furthermore, the inhibitory effects of Form were associated with phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and signal transducer and activator of transcription 3 (STAT3) signaling pathways. These results indicated the suppressive effect of Form on colon carcinoma cell proliferation and invasion, possibly via miR-149-induced EphB3 downregulation and the inhibition of the PI3K/AKT and STAT3 signaling pathways. Overall, Form may be used as a novel candidate for the clinical treatment of colorectal cancer in the future.