Colon Tumor Cells Research Articles

Noninvasive Evaluation of EGFR Expression of Digestive Tumors Using 99mTc-MAG3-Cet-F(ab')2-Based SPECT/CT Imaging.

Purpose This study is aimed at investigating the feasibility of cetuximab (Cet) F(ab′)2 fragment- (Cet-F(ab′)2-) based single photon emission tomography/computed tomography (SPECT/CT) for assessing the epidermal growth factor receptor (EGFR) expression in digestive tumor mouse models. Methods Cet-F(ab′)2 was synthesized using immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS) protease and purified with protein A beads. The product and its in vitro stability in normal saline and 1% bovine serum albumin were analyzed with sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The EGFR expression in the human colon tumor cell line HT29 and the human stomach tumor cell line MGC803 were verified using western blotting and immunocytochemistry. Cet-F(ab′)2 was conjugated with 5(6)-carboxytetramethylrhodamine succinimidyl ester to demonstrate its binding ability to the MGC803 and HT29 cells. Cet-F(ab′)2 was conjugated with NHS-MAG3 for 99mTc radiolabeling. The best imaging time was determined using a biodistribution assay at 1, 4, 16, and 24 h after injection of the 99mTc-MAG3-Cet-F(ab′)2 tracer. Furthermore, 99mTc-MAG3-Cet-F(ab′)2 SPECT/CT was performed on MGC803 and HT29 tumor-bearing nude mice. Results HT29 cells had low EGFR expression while MGC803 cell exhibited the high EGFR expression. Cet-F(ab′)2 and intact cetuximab showed similar high binding ability to MGC803 cells but not to HT29 cells. Cet-F(ab′)2 and 99mTc-MAG3-Cet-F(ab′)2 showed excellent in vitro stability. The biodistribution assay showed that the target to nontarget ratio was the highest at 16 h (17.29 ± 5.72, n = 4) after tracer injection. The 99mTc-MAG3-Cet-F(ab′)2-based SPECT/CT imaging revealed rapid and sustained tracer uptake in MGC803 tumors rather than in HT29 tumors with high image contrast, which was consistent with the results in vitro. Conclusion SPECT/CT imaging using 99mTc-MAG3-Cet-F(ab′)2 enables the evaluation of the EGFR expression in murine EGFR-positive tumors, indicating the potential utility for noninvasive evaluation of the EGFR expression in tumors.

ANTITUMOR EFFECT IN VIVO OF THE NEW PYRIDINE COMPOUND

Abstract. The study was the antitumor activity of a new compound - a pyridine derivative (LHT-13-19). On the culture of human colon cancer HCT116, the suppressive concentration of LHT-13-19 was determined during incubation of tumor cells in the concentration range from 10-9 to 10-7 M. The survival of tumor cells was also determined in the MTT test. Antitumor activity in vivo was studied on thirty athymic humanized 12-week-old female mice of the BALB / c nu / nu line with xenograft adenocarcinoma of the large intestine, divided into 3 groups, 10 mice each, which received intraperitoneal injections of 0.9% sodium chloride solution in a volume of 0.5 ml, cyclophosphamide at a dose of 10 mg / kg, LHT-13-19 at a daily dose of 3.7 mg / kg, respectively, for 10 days. Daily incubation of human colon tumor cells with LHT-13-19 is accompanied by the formation of the cytotoxic effect of the compound. In a xenograft model of colon adenocarcinoma, 10-day intraperitoneal administration of compound LHT-13-19 at a daily dose of 3.7 mg / kg was accompanied by the formation of an antitumor effect in the form of an increase in the doubling time of the tumor size, the survival rate of animals - carriers of xenograft, and a decrease in the frequency of metastasis. Keywords. Pyridine derivatives, LHT-13-19, cell culture, anticancer activity, inhibitory concentration

ON ANTITUMOR ACTIVITY OF A NOVEL COMPOUND – A PYRIDINE DERIVATIVE

The work studied the antitumor activity of a new compound, a pyridine derivative LHT-13-19. To determine the effective inhibitory concentration, a culture of human colon cancer HCT116 was used. Incubation of tumor cells was carried out in the presence of LHT-13-19 in the concentration range from 10-9 to 10-7 M. The survival of tumor cells was determined in MTT test. In vivo experiments were performed on thirty athymic humanized 12-week-old female BALB/c nu/nu mice with xenograft colorectal adenocarcinoma. All animals were divided into 3 groups, 10 mice in each: animals of the control group were injected daily intraperitoneally with 0.5 ml of PBS, animals of the second group were treated with a cyclophosphamide at a dose of 10 mg/kg, mice of the third the groups received LHT-13-19 as experimental therapy at a daily dose of 3.7 mg/kg during 10 days. Animals’ survival and antitumor activity were assessed. Tumor-doubling time, the number of liver superficial metastases and survival time were estimated. Daily incubation of human colon tumor cells with LHT-13-19 is accompanied by the formation of the cytotoxic effect of the compound. On a xenograft model of colon adenocarcinoma in humanized BALB/c nu/nu mice, a 10-day course of 3.7 mg/kg LHT-13-19 leads to the formation of an antitumor effect due to increase in the time of tumor doubling, animal survival and a decrease in the frequency of metastasis

Dnmt3a Mutations in the Hematopoietic System Promote Colitis-Associated Colon Cancer: A Model of Clonal Hematopoiesis in Solid Tumors

Clonal hematopoiesis (CH) is a condition wherein hematopoietic stem cells (HSCs) acquire mutations, often in presumed leukemia driver genes such as in DNMT3A, leading to clonal dominance and disproportionate contribution to peripheral blood without overt hematologic abnormalities (Jaiswal et al. 2014). In large cohorts of patients with solid tumors including colorectal cancer, presence of CH is associated with inferior overall survival due to progression of primary malignancy (Coombs et al. 2017). While potential involvement of CH in the pathogenesis of solid tumors driven by unrelated genetic alterations has far-reaching translational implications, the mechanistic understanding of this relationship is lacking.Colorectal cancer is a leading cause of cancer deaths worldwide and can arise from preceding inflammatory bowel disease. More than 20% of patients with colon cancer have detectable CH, which is notably more prevalent than in age-matched general population. We hypothesized that bone marrow-derived cells harboring alterations in DNMT3A, the most commonly disrupted gene in CH (up to 40% of cases), contribute to the pathogenesis of colorectal cancer. To address this, we combined a bone marrow transplantation-based model of CH driven by heterozygous loss of Dnmt3a with a well-established induction of colitis-associated colon cancer (CAC) by azoxymethane (AOM, a carcinogen) and dextran sodium sulfate (DSS, a colitogen). High resolution colonoscopy demonstrated increased colon wall opacity, visible bleeding, numerous fibrin patches and earlier onset of tumor formation with higher penetrance in animals with partial ablation of Dnmt3a in the hematopoietic compartment. Heightened colon pathology was reflected by modified murine endoscopic index of CAC severity (MEICS) (Fung and Putoczki 2018) (MEICS scores 7.5±2.0 in WT vs 10.0±3.2 in Dnmt3a +/-, p=0.05, n=14,13). We observed elevated incidence of tumors >2mm in diameter (40.2±22.2% in WT vs 74.2±15.7% in Dnmt3a +/-, p=0.03, in 5 independent trials) and a higher tumor burden in Dnmt3a +/- chimerae (7.4±7.8 mm 3 in WT vs 14.5±13.9 mm 3 in Dnmt3a +/-, p=0.0017, n=53,55) at end point. Histopathological analysis revealed marked immune infiltration, extensive ulceration and dysplasia of colonic epithelium, and more frequent adenocarcinoma formation with occasional submucosal invasion (histology score 20.2±4.4 in WT vs 24.6±6.9 in Dnmt3a +/-, p=0.03, n=10,9). To uncover signaling pathways deregulated by experimental Dnmt3a-driven CH, we surveyed the auto- and paracrine stimulatory loops by multiplex cytokine/chemokine assays and profiled transcriptomes of colon tumors by bulk RNA-seq. These studies identified gene signatures commonly associated with colon carcinogenesis including accentuated epithelial-to-mesenchymal transition (gene set enrichment analysis (GSEA) NES=2.42, padj=0.0096), Wnt/b-catenin (NES=2.07, padj=0.0096), and Vegf/angiogenesis (NES=1.83, padj=0.0096). Elevated proliferation (Ki67) and increased vascularization (CD31) were validated by immunohistochemistry. These aspects of tumor pathophysiology along with the composition and functional characteristics of the tumor-infiltrating leukocytes derived from animals with and without hematopoietic-specific Dnmt3a partial loss are being further investigated. Preliminary data showed bone marrow-derived macrophages (BMDMs) from Dnmt3a +/- chimera exhibit impaired ability to phagocytose syngeneic MC38 colon tumor cells, compared to WT.Our study provides new insights into the non-tumor-cell-autonomous mechanisms of carcinogenesis effected by alterations in the hematopoietic system and into the interplay between the immune system and the aggressive phenotype of colorectal cancer. Our findings may aid the development of novel therapeutic options to improve outcomes in solid tumor patients with CH. [Display omitted] DisclosuresBowman: Mission Bio: Honoraria, Speakers Bureau. Levine: Ajax: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria; Prelude: Membership on an entity's Board of Directors or advisory committees; Auron: Membership on an entity's Board of Directors or advisory committees; Mission Bio: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Zentalis: Membership on an entity's Board of Directors or advisory committees; Imago: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Lilly: Honoraria; QIAGEN: Membership on an entity's Board of Directors or advisory committees; Morphosys: Consultancy; Astellas: Consultancy; Janssen: Consultancy; Incyte: Consultancy; Roche: Honoraria, Research Funding.

Synthesis, crystal structure, DFT and anticancer study of dihydroartermisinin-paracetamol hybrids

A pair of C-10 epimers of dihydroartermisinin-paracetamol hybrids (A and B) were prepared in this work, and both of which were characterized by MS, 1H NMR, 13C NMR, IR and ECD spectra as well as by a single crystal X-ray structural analysis. Subsequently, the structures of the two compounds were analyzed with DFT-based approaches. The analytical results on conformations indicated that the DFT-optimized molecular structures were consistent with the crystal structures confirmed by X-ray diffraction. The packing of the molecules in the solid state was stabilized by hydrogen bonds. The signs of the experimental IR and ECD spectra was also efficiently simulated by the DFT methods. A primary evaluation on anticancer effects of A and B was conducted towards human breast tumor cell line MCF-7 and human colon tumor cell line HCT116, the results demonstrated that 10-α-O-(4-acetylamino) phenyl dihydroartemisinin (A) was slightly less potent than its 10-β counterpart (B) in both tested cell lines.

Antiproliferative Activity of Stokesia laevis Ethanolic Extract in Combination with Several Food-Related Bioactive Compounds; In Vitro (Caco-2) and In Silico Docking (TNKS1 and TNKS2) Studies

This study evaluates in vitro cytotoxic and antiproliferative activity on human colon tumor cell line Caco-2 (ATCC-HTB-37) of a standardized (5 mg GAE/mL) ethanolic extract from Stokesia laevis (Slae26), of five polyphenols compounds (reference substances, ref.), namely luteolin-7-O-glucoside, luteolin-8-C-glucoside, caffeic acid, gentisic acid, and p-aminobenzoic acid (PABA), as well as of Slae26 combinations with the five reference substances, 1:1 mass rate (GAE, ref.). Cell viability studies (MTS test) have revealed IC50 values of 36 μg GAE/mL in the case of Slae26 ethanolic extract, while Slae26 combinations with the five phenolics indicated IC50 values around 5 μg GAE/mL. In silico docking studies on the molecular targets human tankyrase 1 (TNKS1) and human tankyrase 2 (TNKS2) in complex with their native ligands, Co-crystallized 3J5A and Co-crystallized FLN, indicated score values of −104.15 and −76.97, respectively; in the series of the reference compounds studied, luteolin-7-O-glucoside was revealed with the best score values on both molecular targets (−80.49 and −85.17), together signifying real antiproliferative potential against human colon cancer of Slae26, of luteolin-7-O-glucoside, and of Slae26 combinations with all food-related bioactive compounds tested.

Phytochemical Screening of Himatanthus sucuuba (Spruce) Woodson (Apocynaceae) Latex, In Vitro Cytotoxicity and Incision Wound Repair in Mice.

Himatanthus sucuuba, also known as “Bellaco caspi”, is a medicinal plant whose latex, stem bark, and leaves possess phenolic acids, lupeol, β-dihydro-plumbericinic acid, plumericin, and plumeride, among other components. Some of these have been linked to such biological activities as antiulcer, anti-inflammatory, and wound healing. The aim of this study was to determine the phytochemical compounds of H. sucuuba latex, as well as its in vitro cytotoxicity and wound healing effect in mice. Latex was collected in the province of Iquitos, Peru. Phytochemical analysis was carried out with UPLC-ESI-MS/MS. The cytotoxicity was evaluated on two colon tumor cell lines (SW480 and SW620) and non-malignant cells (human keratinocytes, HaCaT, and Chinese hamster ovary, CHO-K1). The mice were distributed into two groups, as follows: Group I—control (n = 10; without treatment); II—(n = 10) H. sucuuba latex; wounds were induced with a scalpel in the dorsal–cervical area and treatments were applied topically twice a day on the incision for 10 days. Molecular docking was carried out on the glycogen synthase kinase 3β protein. Twenty-four chemical compounds were determined, mainly flavonoid-type compounds. Latex did not have a cytotoxic effect on tumor cells with IC50 values of more than 500 µg/mL. The latex had a regenerative effect on wounds in mice. Acacetin-7-O-neohesperidoside had the best docking score of −9.9 kcal/mol. In conclusion, H. sucuuba latex had a wound healing effect in mice, as confirmed by histological study. However, a non-cytotoxic effect was observed on colon tumor cells SW480 and SW620.

Inflammatory Mediators and Gut Microbial Toxins Drive Colon Tumorigenesis by IL-23 Dependent Mechanism.

Simple SummaryWestern-style diet, rich in high fat, is the major cause of obesity and enhanced risk of colon cancer in the USA and worldwide. The inflammatory molecules are a well-established link between obesity and the modulation of colon tumorigenesis. In particular, IL-23 plays an important role in the impact of a western-style diet on obesity, the gut microbiome, and colon tumorigenesis. However, the underlying mechanism of IL-23 production for colon tumor progression and whether IL-23 can be a potential target is not clear. Our findings signify the role of pro-tumorigenic innate immune cells, including dendritic cells and macrophages in IL-23 production by bacterial toxins and eicosanoids. IL-23 knockdown in the tumorigenic dendritic cells and macrophages inhibited the colon tumor cell and organoids growth. Taken together, targeting IL-23 may be a promising option for the prevention and treatment of high-fat/obesity-associated colon cancer in clinical trials.Obesity-associated chronic inflammation predisposes colon cancer risk development. Interleukin-23 (IL-23) is a potential inflammatory mediator linking obesity to chronic colonic inflammation, altered gut microbiome, and colon carcinogenesis. We aimed to elucidate the role of pro-inflammatory eicosanoids and gut bacterial toxins in priming dendritic cells and macrophages for IL-23 secretion to promote colon tumor progression. To investigate the association of IL-23 with obesity and colon tumorigenesis, we utilized TCGA data set and colonic tumors from humans and preclinical models. To understand IL-23 production by inflammatory mediators and gut microbial toxins, we performed several in vitro mechanistic studies to mimic the tumor microenvironment. Colonic tumors were utilized to perform the ex vivo experiments. Our findings showed that IL-23 is elevated in obese individuals, colonic tumors and correlated with reduced disease-free survival. In vitro studies showed that IL-23 treatment increased the colon tumor cell self-renewal, migration, and invasion while disrupting epithelial barrier permeability. Co-culture experiments of educated dendritic cells/macrophages with colon cancer cells significantly increased the tumor aggression by increasing the secretory levels of IL-23, and these observations are further supported by ex vivo rat colonic tumor organotypic experiments. Our results demonstrate gut microbe toxins and eicosanoids facilitate IL-23 production, which plays an important role in obesity-associated colonic tumor progression. This newly identified nexus represents a potential target for the prevention and treatment of obesity-associated colon cancer.

Hyperprogression of a mismatch repair-deficient colon cancer in a humanized mouse model following administration of immune checkpoint inhibitor pembrolizumab.

Immunotherapy is an established treatment modality in oncology. However, in addition to primary or acquired therapy resistance with immune checkpoint blockade (ICB), hyperprogressive disease (HPD) or hyperprogression (HP) with acceleration of tumor growth occurs in a subset of patients receiving ICB therapy. A validated and predictive animal model would help investigate HPD/HP to develop new approaches for this challenging clinical entity. Using human cytotoxic T-cell line TALL-104 injected intraperitoneally into immunodeficient NCRU-nude athymic mice bearing mismatch repair-deficient (MMR-d) human colon carcinoma HCT116 p53-null (but not wild-type p53) tumor xenograft, we observed accelerated tumor growth after PD-1 blockade with pembrolizumab administration. There was increased colon tumor cell proliferation as determined by immunohistochemical Ki67 staining of tumor sections. There was no increase in MDM2 or MDM4/MDMX in the p53-null HCT116 cells versus the wild-type p53-expressing isogenic tumor cells, suggesting the effects in this model may be MDM2 or MDM4/MDMX-independent. Human cytokine profiling revealed changes in IFN-γ, TRAIL-R2/TNFRSF10B, TRANCE/TNFSF11/RANK L, CCL2/JE/MCP-1, Chitinase 3-like 1, IL-4 and TNF-α. This represents a novel humanized HPD mouse model with a link to deficiency of the p53 pathway of tumor suppression in the setting of MMR-d. Our novel humanized preclinical TALL-104/p53-null HCT116 mouse model implicates p53-deficiency in an MMR-d tumor as a possible contributor to HPD/HP and may help with evaluating therapeutic strategies in cancer immunotherapy to extend clinical benefits of ICB’s in a broader patient population.

Extracellular Polymeric Substances (EPS) produced by Streptomyces sp. biofilms: Chemical composition and anticancer properties

The extracellular polymeric substances (EPS) have shown free radical scavenging and antitumor activity against both breast and colon cell lines. In this regard, actinobacteria have become an increasingly popular sources of EPS. Therefore, in this study four Streptomyces strains isolated from contaminated soil (M7, A5, A14 and MC1) were evaluated for determining its biofilm-forming capacity including under pesticide stress. In addition, chemical composition of EPS and its cytotoxic effects over 4T1 breast cancer cell and Caco-2 human tumor colon cells were evaluated. The results demonstrated that Streptomyces sp. A5 had the highest capability to develop biofilm more than other strains tested, even under pesticide stress. Moreover, this strain produced EPS with a total protein/total polysaccharide rate of 1.59 ± 0.05. On the other hand, cytotoxicity assays of EPS showed that Streptomyces sp. A5 display a higher toxic effect against 4T1 Breast cancer cells (96.2 ± 13.5 %), Caco-2 (73.9 ± 6.4 %) and low toxicity (29.9 % ± 9.1 %) against non-transformed intestinal cells (IEC-18). Data do not show cytotoxic effect relationship with biofilm-forming capabilities of strains, nor the chemical composition of EPS matrix. The gene that codes for polysaccharide deacetylase, parB-like and transRDD proteins, were identified. These results contribute to the knowledge about the variability of chemical composition and potential cytotoxic properties of EPS produced by Streptomyces biofilms. It proposes interesting future challenges for linking Streptomyces-based pesticide remediation technology with the development of new antitumor drugs.

Effect of non-competitive inhibitors of aminopeptidase N on viability of human and murine tumor cells

Abstract Cancer is the second leading cause of death worldwide. Peptidases participate in tumor development and growth. Mammalian neutral aminopeptidase (APN, EC 3.4.11.2, M1 family) catalyzes the cleavage of neutral and basic amino acids from the N-terminus of substrates. APN expression is dysregulated in several types of cancer, being a target for the development of new anticancer agents. Recently, we identified three new non-competitive inhibitors of soluble porcine APN (pAPN) by virtual screening (BTB11079, JFD00064, BTB07018, from Maybridge). In the present contribution we assayed their effect on the activity of APN in a microsomal preparation of porcine kidney cortex, a model of the physicochemical environment of the enzyme. These classical inhibitors had an IC50 value of 3–5 µM. Additionally, using a kinetic approach and a specific substrate, we quantified APN activity on the cell surface of human and murine lung, colon, prostate, and skin tumor cells. APN inhibitors reduced tumor cells viability, more efficiently in the higher APN activity tumor cell lines, but not in non-tumoral cells. BTB11079, JFD00064, BTB07018 effects on cell viability were stronger than that of bestatin, a positive control. Thus, these non-competitive APN inhibitors may be useful tools for cancer treatment.

Anticancer Activity of Some Ruthenium(III) Complexes with Quinolone Antibiotics: In Vitro Cytotoxicity, Cell Cycle Modulation, and Apoptosis-Inducing Properties in LoVo Colon Cancer Cell Line

Cisplatin is one of the most effective anticancer agents used to treat colon cancer, which is the third malignancy between the most common human cancers in the world, but the resistance developed represents an obstacle against the full success of chemotherapy. An emerging interest appeared in finding other metallic compounds, such as ruthenium(III) complexes, for chemotherapeutic application in cancer. Our study focused on the anticancer activity of several ruthenium (Ru) complexes with quinolone antibiotics in colon tumor cell cultures. Real-time cell analysis and drug-mediated cytotoxicity tests monitored the inhibitory effects in the drug-treated LoVo colon cancer cells. Flow cytometry assays were performed to evaluate cell cycle phases distribution and apoptotic events. The obtained results showed dose-dependent increased levels of cell lysis and induction of apoptosis in LoVo cancer cells treated with the Ru(III) complexes. In addition, data showed a major decrease in cell proliferation, since the percentages of cells distributed in the S cell cycle phase diminished, and a G0/G1 cell arrest was observed. Therefore, our results strongly suggest that the newly synthesized Ru(III) complexes might play an important role in future chemotherapeutic approaches, since their activity is based on diminishing cell proliferation, induction of apoptosis, and modulation of cell cycle phases.

Determination of therapeutic agents efficiencies of microsatellite instability high colon cancer cells in post‐metastatic liver biochip modeling

Two distinct genetic mutational pathways characterized by either chromosomal instability or high-frequency microsatellite instability (MSI-H) are recognized in the pathogenesis of colorectal cancer (CRC). Recently, it has been shown that patients with primary CRC that displays MSI-H have a significant, stage-independent, multivariate survival advantage. Biological properties of CMS1 (MSI-H type) can affect therapeutic efficiencies of agents used in the treatment of CRC, and therefore become a new predictive factor of the treatment. But, the predictive impact of MSI-H status for adjuvant chemotherapy remains controversial. This study will assess whether there is any unnecessary or inappropriate use of treatment agents recommended for adjuvant therapy of stage 2 and 3 of disease and for palliative or curative treatment of liver metastatic disease in microsatellite instability high group, a molecular subtype of colon cancer. Within this scope, the efficiencies of fluorouracil- and oxaliplatin-based chemotherapeutic agents will be shown on stage 3 microsatellite instability high colon tumor cell lines first, and then a microfluidic model will be created, imitating the metastasis of colon cancer to the liver. In the microfluidic chip model, we will create in liver tissue, where the metastasis of microsatellite instability high colon cancer will be simulated; the effectiveness of chemotherapeutic agents, immunotherapy agents, and targeted agents on tumor cells as well as drug response will be assessed according to cell viability through released biomarkers from the cells. The proposed hypothesis study includes the modeling and treatment of patient-derived post-metastatic liver cancer in microfluidics which has priority at the global and our region and consequently develop personal medication.

Abstract 2901: Pdcd4 inhibits slug translation to suppress E-cadherin expression and invasion in colon tumor cells

Abstract Programmed cell death 4 (Pdcd4) has been demonstrated to function as a tumor suppressor and protein translation inhibitor. We previously reported that knockdown of Pdcd4 down-regulated E-cadherin expression. E-cadherin is a key regulator for tumor cell invasion and metastasis, whose expression is controlled by transcription repressors Snail, Slug, ZEB1/2, and Twist. Both Slug and Snail have been shown to repress E-cadherin expression and contribute to tumor progression in various cancers including colon cancer. Here, we reported that knockdown of Pdcd4 increased Slug protein level but not SLUG mRNA level in colon tumor cells. Conversely, over-expression of Pdcd4 decreased Slug expression. Using sucrose gradient fractionation assays, Pdcd4 knockdown was found to increase the population of SLUG mRNA in the polysomal fractions. These results indicate that Pdcd4 translationally regulates Slug expression. To understand the mechanism by which Pdcd4 regulates Slug translation, the SLUG 5' untranslated region (5'UTR) was cloned and fused with luciferase reporter and named as 5'UTR-Slug-Luc. Knockdown of Pdcd4 significantly enhanced the translation of 5'UTR-Slug-Luc while ectopic expression of Pdcd4 repressed the translation of 5'UTR-Slug-Luc, suggesting that SLUG 5'UTR is an essential element for Pdcd4 to inhibit Slug translation. Moreover, cells treated with silvestrol, an inhibitor for translation initiation factor 4A (eIF4A), significantly suppressed Slug translation. In addition, ectopic expression of Pdcd4 inhibited Slug translation and thereby decreased E-cadherin expression. By contrast, Pdcd4(D418A), a mutant that does not bind with eIF4A, failed to suppress the expression of Slug and E-cadherin. These findings suggest that Pdcd4-inhibited Slug translation is through suppressing eIF4A. Furthermore, knockdown of Slug in Pdcd4 knockdown cells restored the E-cadherin expression and reversed the invasive capacity induced by Pdcd4 knockdown, indicating that up-regulation of Slug contributes to Pdcd4-knockdown enhanced invasion. Taken together, this study demonstrates that Pdcd4 inhibits translation of Slug to regulate E-cadherin expression and invasion in colon tumor cells. Citation Format: Qing Wang, Jiang Zhu, Hsin-Sheng Yang. Pdcd4 inhibits slug translation to suppress E-cadherin expression and invasion in colon tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2901.

Antitumor Activity against Human Colorectal Adenocarcinoma of Silver Nanoparticles: Influence of [Ag]/[PVP] Ratio.

Silver nanoparticles (AgNPs) not only have shown remarkable results as antimicrobial and antiviral agents but also as antitumor agents. This work reports the complete characterization of five polyvinylpyrrolidone-coated AgNP (PVP-AgNP) formulations, their cytotoxic activity against human colon tumor cells (HCT-15), their cytotoxic effect on primary mouse cultures, and their lethal dose on BALB/c mice. The evaluated AgNP formulations have a composition within the ranges Ag: 1.14–1.32% w/w, PVP: 19.6–24.5% and H2O: 74.2–79.2% with predominant spherical shape within an average size range of 16–30 nm according to transmission electron microscopy (TEM). All formulations assessed increase mitochondrial ROS concentration and induce apoptosis as the leading death pathway on HCT-15 cells. Except for AgNP1, the growth inhibition potency of AgNP formulations of human colon tumor cancer cells (HCT-15) is 34.5 times higher than carboplatin, one of the first-line chemotherapy agents. Nevertheless, 5–10% of necrotic events, even at the lower concentration evaluated, were observed. The cytotoxic selectivity was confirmed by evaluating the cytotoxic effect on aorta, spleen, heart, liver, and kidney primary cultures from BALB/c mice. Despite the cytotoxic effects observed in vitro, the lethal dose and histopathological analysis showed the low toxicity of these formulations (all of them on Category 4 of the Globally Harmonized System of Classification and Labelling of Chemicals) and minor damage observed on analyzed organs. The results provide an additional example of the rational design of safety nanomaterials with antitumor potency and urge further experiments to complete the preclinical studies for these AgNP formulations.

Abstract 2868: A cell based phenotypic assay platform for cancer metastasis drug discovery and diagnostics

Abstract Metastatic cancers are difficult to treat and are the cause of about 90% of cancer related deaths. Furthermore, predictive diagnostics are not readily available, and lack of good translational assays has impaired drug discovery. Mestastop has developed a platform of cell based functional assays that aims to address these drug discovery and diagnostic gaps. The platform is comprised of a set of 14 cell based phenotypic assays that capture multiple aspects of the complex process of metastasis, including characterization of the plasticity ratios (EMT, MET) and stemness of cells, invasion, intravasation, circulation, extravasation, and survival in secondary tissues (e.g metabolic shift, exosome secretion). Wild type tumor cells (initially a set of colon tumor cell lines) are utilized in the platform. Non-metastatic wild type cells (e.g. HT-29) that have been engineered into metastatic cells are also utilized to further elucidate metastatic processes, thereby creating two baseline data sets for each assay; one for the normal growing tumor and the other for the select metastatic population. Patient derived tumors are now being analyzed in the platform to help build a translational model. Preclinical animal models are being jointly developed with Immunobiome Inc. (South Korea). As the data from all these assays present a multivariate problem, we are developing a machine learning algorithm, METSCANTM, to analyze the results. The platform will be applied to the screening of novel compounds for drug discovery and of approved drugs for repurposing. Analysis of patient derived tumors may allow for the early detection of patients with high metastatic potential. Four colon cancer cell lines and several standard chemotherapy drugs have been used to validate the platform. A group of triple negative breast cancer cell lines is now being tested. Preliminary data for a set of approved, marketed drugs has further validated the potential of the platform for drug discovery and repurposing. The Mestastop proprietary assay platform will empower biopharma in the discovery of novel drug candidates and repurposing of approved drugs for the treatment of metastasis, and the early diagnosis of cancers with high metastatic potential, thereby addressing the unmet need in the area of cancer metastasis. Citation Format: Arnab Roy Chowdhury, Debabani Roy Chowdhury, Manoj Pandre, Samrat Roy, Sundarajan Kannan, John W. Ellingboe. A cell based phenotypic assay platform for cancer metastasis drug discovery and diagnostics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2868.

SARS-CoV-2 Host Receptor ACE2 Protein Expression Atlas in Human Gastrointestinal Tract.

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects host cells through interactions with its receptor, Angiotensin-converting enzyme 2 (ACE2), causing severe acute respiratory syndrome and death in a considerable proportion of people. Patients infected with SARS-CoV-2 experience digestive symptoms. However, the precise protein expression atlas of ACE2 in the gastrointestinal tract remains unclear. In this study, we aimed to explore the ACE2 protein expression pattern and the underlying function of ACE2 in the gastrointestinal tract, including the colon, stomach, liver, and pancreas.MethodsWe measured the protein expression of ACE2 in the gastrointestinal tract using immunohistochemical (IHC) staining with an ACE2-specific antibody of paraffin-embedded colon, stomach, liver, and pancreatic tissues. The correlation between the protein expression of ACE2 and the prognosis of patients with gastrointestinal cancers was analyzed by the log-rank (Mantel–Cox) test. The influence of ACE2 on colon, stomach, liver, and pancreatic tumor cell line proliferation was tested using a Cell Counting Kit 8 (CCK-8) assay.ResultsACE2 presented heterogeneous expression patterns in the gastrointestinal tract, and it showed a punctate distribution in hepatic cells. Compared to that in parallel adjacent non-tumor tissues, the protein expression of ACE2 was significantly increased in colon cancer, stomach cancer, and pancreatic cancer tissues but dramatically decreased in liver cancer tissues. However, the expression level of the ACE2 protein was not correlated with the survival of patients with gastrointestinal cancers. Consistently, ACE2 did not affect the proliferation of gastrointestinal cancer cells in vitro.ConclusionThe ACE2 protein is widely expressed in the gastrointestinal tract, and its expression is significantly altered in gastrointestinal tumor tissues. ACE2 is not an independent prognostic marker of gastrointestinal cancers.

Cadmium-Associated Molecular Signatures in Cancer Cell Models

Simple SummaryThe exposure of cancer cells to cadmium compounds may be associated with the acceleration of tumor progression. It is known that cadmium is a transcriptional regulator, and the study of differentially expressed genes has enabled the identification and classification of cadmium-associated molecular signatures as useful biomarkers that are potentially transferable to clinical research. This review recapitulates the studies that report the detection of such signatures in breast, gastric, colon, liver, lung, and nasopharyngeal tumor cell models, as specifically demonstrated by individual gene or whole genome expression profiling.The exposure of cancer cells to cadmium and its compounds is often associated with the development of more malignant phenotypes, thereby contributing to the acceleration of tumor progression. It is known that cadmium is a transcriptional regulator that induces molecular reprogramming, and therefore the study of differentially expressed genes has enabled the identification and classification of molecular signatures inherent in human neoplastic cells upon cadmium exposure as useful biomarkers that are potentially transferable to clinical research. This review recapitulates selected studies that report the detection of cadmium-associated signatures in breast, gastric, colon, liver, lung, and nasopharyngeal tumor cell models, as specifically demonstrated by individual gene or whole genome expression profiling. Where available, the molecular, biochemical, and/or physiological aspects associated with the targeted gene activation or silencing in the discussed cell models are also outlined.

Enhancement of innate and adaptive anti-tumor immunity by serum obtained from vascular photodynamic therapy-cured BALB/c mouse.

Photodynamic therapy (PDT) is a clinically approved treatment for various types of cancer. Besides killing the tumor cells directly, PDT has also been reported to trigger anti-tumor immunity. In our previous study, BAM-SiPc-based PDT was shown to induce immunogenic cell death on CT26 murine colon tumor cells in vitro. Using the BALB/c mouse animal model and a vascular-PDT (VPDT) approach, it could also eradicate tumor in ∼ 70% of tumor-bearing mice and elicit an anti-tumor immune response. In the present study, the serum obtained from the VPDT-cured mice was studied and found to possess various immunomodulatory properties. In in vitro studies, it stimulated cytokine secretions of IL-6 and C-X-C motif chemokine ligands 1-3 in CT26 cells through the NF-κB and MAPK pathways. The complement protein C5a boosted in the serum was shown to be involved in the process. The serum also induced calreticulin exposure on CT26 cells and activated dendritic cells. It contained CT26-targeting antibodies which, through the Fc region, induced macrophage engulfment of the tumor cells. In in vivo studies, inoculation of the serum-treated CT26 cells to mice demonstrated a retarded tumor growth with leukocytes, particularly T cells, attracted to the tumor site. In addition, the VPDT-cured mice showed different degrees of resistance against challenge of other types of murine tumor cells, for example, the breast tumor 4T1 and EMT6 cells.

Mitophagy protein PINK1 suppresses colon tumor growth by metabolic reprogramming via p53 activation and reducing acetyl-CoA production.

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in the US. Understanding the mechanisms of CRC progression is essential to improve treatment. Mitochondria is the powerhouse for healthy cells. However, in tumor cells, less energy is produced by the mitochondria and metabolic reprogramming is an early hallmark of cancer. The metabolic differences between normal and cancer cells are being interrogated to uncover new therapeutic approaches. Mitochondria targeting PTEN-induced kinase 1 (PINK1) is a key regulator of mitophagy, the selective elimination of damaged mitochondria by autophagy. Defective mitophagy is increasingly associated with various diseases including CRC. However, a significant gap exists in our understanding of how PINK1-dependent mitophagy participates in the metabolic regulation of CRC. By mining Oncomine, we found that PINK1 expression was downregulated in human CRC tissues compared to normal colons. Moreover, disruption of PINK1 increased colon tumorigenesis in two colitis-associated CRC mouse models, suggesting that PINK1 functions as a tumor suppressor in CRC. PINK1 overexpression in murine colon tumor cells promoted mitophagy, decreased glycolysis and increased mitochondrial respiration potentially via activation of p53 signaling pathways. In contrast, PINK1 deletion decreased apoptosis, increased glycolysis, and reduced mitochondrial respiration and p53 signaling. Interestingly, PINK1 overexpression in vivo increased apoptotic cell death and suppressed colon tumor xenograft growth. Metabolomic analysis revealed that acetyl-CoA was significantly reduced in tumors with PINK1 overexpression, which was partly due to activation of the HIF-1α-pyruvate dehydrogenase (PDH) kinase 1 (PDHK1)-PDHE1α axis. Strikingly, treating mice with acetate increased acetyl-CoA levels and rescued PINK1-suppressed tumor growth. Importantly, PINK1 disruption simultaneously increased xenografted tumor growth and acetyl-CoA production. In conclusion, mitophagy protein PINK1 suppresses colon tumor growth by metabolic reprogramming and reducing acetyl-CoA production.