HT-29 Colon Cancer Cells Research Articles

Anti-tumor effects of an antagonistic mAb against the ASCT2 amino acid transporter on KRAS-mutated human colorectal cancer cells.

KRAS mutations are detected in numerous human cancers, but there are few effective drugs for KRAS‐mutated cancers. Transporters for amino acids and glucose are highly expressed on cancer cells, possibly to maintain rapid cell growth and metabolism. Alanine‐serine‐cysteine transporter 2 (ASCT2) is a primary transporter for glutamine in cancer cells. In this study, we developed a novel monoclonal antibody (mAb) recognizing the extracellular domain of human ASCT2, and investigated whether ASCT2 can be a therapeutic target for KRAS‐mutated cancers. Rats were immunized with RH7777 rat hepatoma cells expressing human ASCT2 fused to green fluorescent protein (GFP). Splenocytes from the immunized rats were fused with P3X63Ag8.653 mouse myeloma cells, and selected and cloned hybridoma cells secreting Ab3‐8 mAb were established. This mAb reacted with RH7777 transfectants expressing ASCT2‐GFP proteins in a GFP intensity‐dependent manner. Ab3‐8 reacted with various human cancer cells, but not with non‐cancer breast epithelial cells or ASCT2‐knocked out HEK293 and SW1116 cells. In SW1116 and HCT116 human colon cancer cells with KRAS mutations, treatment with Ab3‐8 reduced intracellular glutamine transport, phosphorylation of AKT and ERK, and inhibited in vivo tumor growth of these cells in athymic mice. Inhibition of in vivo tumor growth by Ab3‐8 was not observed in HT29 colon and HeLa uterus cancer cells with wild‐type KRAS. These results suggest that ASCT2 is an excellent therapeutic target for KRAS‐mutated cancers.

AGS-30, an andrographolide derivative, suppresses tumor angiogenesis and growth in vitro and in vivo

Poor bioavailability and limited efficacy are challenges associated with using andrographolide as a therapeutic agent. We recently synthesized AGS-30, a new andrographolide derivative, in our laboratory. In this study we investigated the potential anti-tumor effect of AGS-30 and the underlying mechanisms, particularly those related to angiogenesis. Results from our in vitro experiments showed that AGS-30 exerted anti-angiogenic effects by inhibiting endothelial cell proliferation, migration, invasion, and tube formation. Phosphorylation and activation of angiogenesis-related signaling molecules (e.g., vascular endothelial growth factor [VEGF] receptor 2, mitogen-activated protein kinase kinase 1/2, extracellular signal-regulated kinase 1/2, mechanistic target of rapamycin [mTOR], protein kinase B [Akt], and p38) were markedly reduced by AGS-30. Meanwhile, AGS-30 potently inhibited cell proliferation and phosphorylation of cell survival-related proteins (e.g., Akt, mTOR, and ERK1/2) and decreased the expression of VEGF in HT-29 colon cancer cells. AGS-30 blocked microvessel sprouting in a rat aortic ring model and blood vessel formation in zebrafish embryos and a mouse Matrigel plug model. Additionally, AGS-30 suppressed tumor growth and angiogenesis in HT-29 colon cancer cell xenografts in nude mice. These effects were not observed when same concentration of andrographolide, the parent compound of AGS-30, was used. Thus, AGS-30 exerted a strong antitumor effect by inhibiting tumor cell growth and angiogenesis and is a candidate compound for the treatment of cancer.

Pristimerin suppresses colorectal cancer through inhibiting inflammatory responses and Wnt/β-catenin signaling

Pristimerin, a triterpenoid, has exhibited potential anti-inflammatory and anti-tumor activities. Nevertheless, the role and mechanism of pristimerin in intestinal inflammation and colon cancer require further investigation. Here, we found that pristimerin protected mice from dextran sulfate sodium (DSS)-induced colitis, restoring epithelial damage and reducing tissue inflammation and inflammatory cell infiltration. In addition, pristimerin dramatically reduced the number and size of the tumors in a azoxymethane (AOM)/DSS-induced colitis-associated colorectal cancer (CAC) model. Furthermore, we found that pristimerin suppressed Wnt/β-catenin signaling by RNA-Seq. Pristimerin inhibited Wnt/β-catenin signaling via activation of GSK3β, thereby suppressing Wnt target gene expression in colon cancer HCT116 and HT-29 cells. In HCT116 colon cancer xenografts and APCmin/+ mice, which undergo spontaneous intestinal tumorigenesis, administration of pristimerin reduced the tumor progression and decreased the expression of phosphorylated GSK3β Ser 9, β-catenin, cyclin D1 and c-Myc. These results suggest that pristimerin is a potent agent for preventing colon inflammation and carcinogenesis.

Indocyanine green loaded APTMS coated SPIONs for dual phototherapy of cancer

Superparamagnetic iron oxide nanoparticles (SPIONs) have been recently recognized as highly efficient photothermal therapy (PTT) agents. Here, we demonstrate, for the first time to our knowledge, dose and laser intensity dependent PTT potential of small, spherical, 3-aminopropyltrimethoxysilane coated cationic superparamagnetic iron oxide nanoparticles (APTMS@SPIONs) in aqueous solutions upon irradiation at 795 nm. Indocyanine green (ICG) which has been recently used for photodynamic therapy (PDT), was loaded to APTMS@SPIONs to improve the stability of ICG and to achieve an effective mild PTT and PDT (dual therapy) combination for synergistic therapeutic effect on cancer cells via a single laser treatment in the near infrared (NIR). Neither APTMS@SPIONs nor ICG-APTMS@SPIONs showed dark toxicity on MCF7 breast and HT29 colon cancer cell lines. A safe laser procedure was determined as 10 min irradiation at 795 nm with 1.8 W/cm2 of laser intensity, at which APTMS@SPION did not cause a significant cell death. However, free ICG reduced cell viability at and above 10 μg/ml under these conditions along with generation of reactive oxygen species (ROS), more effectively in MCF7. ICG-APTMS@SPION treated cells showed 2-fold increase in ROS generation and near complete cell death at and below 5 μg/ml ICG dose, even in less sensitive HT29 cells after a single laser treatment at NIR, which would be safe for the healthy tissue and provide a longer penetration depth. Besides, both components can be utilized for diagnosis and the overall composition may be used for optical-image guided phototherapy in the NIR region.

Adjuvant therapy with γ-tocopherol-induce apoptosis in HT-29 colon cancer via cyclin-dependent cell cycle arrest mechanism.

Resistance to chemotherapy with 5-fluorouracil (5-FU) in patients with colorectal cancer (CRC) is the major obstacle to reach the maximum efficiency of CRC treatment. Combination therapy has emerged as a novel anticancer strategy. The present study evaluates the cotreatment of γ-tocopherol and 5-FU in enhancing the efficacy of chemotherapy against HT-29 colon cancer cells. Cytotoxic effect of this combination was examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and a synergistic effect was evaluated by a combination index technique. Nuclear morphology was studied via 4',6-diamidino-2-phenylindole staining and flow cytometric assays were conducted to identify molecular mechanisms of apoptosis and cell cycle progression. We investigated the expression of Cyclin D1, Cyclin E, Bax, and Bcl-2 by a quantitative real-time polymerase chain reaction. The IC50 values for 5-FU and γ-tocopherol were 21.8 ± 2.5 and 14.4 ± 2.6 μM, respectively, and also this combination therapeutic increased the percentage of apoptotic cells from 35% ± 2% to 40% ± 4% (P < .05). Furthermore, incubation HT-29 colon cells with combined concentrations of two drugs caused significant accumulation of cells in the subGsubG1 phase. Our results presented the combination therapy with 5-FU and γ-tocopherol as a novel therapeutic approach, which can enhance the efficacy of chemotherapy.

Anti-proliferative bioactivity against HT-29 colon cancer cells of a withanolides-rich extract from golden berry (Physalis peruviana L.) calyx investigated by Foodomics

Abstract A comprehensive Foodomics study was carried out in this work to investigate the changes induced at gene and metabolite expression levels on HT29 colon cancer cell lines upon treatment with a bioactives-enriched extract from goldenberry calyx. As a result of the proposed multi-omics approach, the tested extract induced transcriptional activation of pro-apoptotic genes, and altered the expression of several genes related to the oxidative stress response in treated cancer cells. Metabolomics data confirm altered cellular redox homeostasis, providing additional evidence to transcriptomic results. Foodomics data integration also revealed alteration on relevant metabolic processes, suggesting inactivation of aminoacyl tRNA charging pathway, dysfunction on carnitine shuttle and beta-oxidation of fatty acids, and pyrimidine ribonucleotide interconversion impairment. These observations are in line with functional analysis and anti-proliferative activity results, where the viability of HT-29 colon cancer cells was notably reduced after 48 h treatment without affecting the viability of normal human colon fibroblast cells.

Synthesis and cellular effects of novel 1,3,5-triazine derivatives in DLD and Ht-29 human colon cancer cell lines

SummaryThis study provides new information on the cellular effects of 1,3,5-triazine nitrogen mustards with different peptide groups in DLD and Ht-29 human colon cancer cell lines. A novel series of 2,4,6-trisubstituted 1,3,5-triazine derivatives bearing 2-chloroethyl and oligopeptide moieties was designed and synthesized. The most cytotoxic derivative was triazine with an Ala-Ala-OMe substituent on the ring (compound 7b). This compound induced time- and dose-dependent cytotoxicity in the DLD-1 and HT-29 colon cancer cell lines. The triazine derivative furthermore induced apoptosis through intracellular signaling pathway attenuation. Compound 7b may be a candidate for further evaluation as a chemotherapeutic agent against colorectal cancer.

Adenine Inhibits the Growth of Colon Cancer Cells via AMP-Activated Protein Kinase Mediated Autophagy.

Background Adenine is involved in a variety of cell biological processes and has been explored for pharmacological uses. Its therapeutic use for managing cancer is of great interest. In the present study, we investigated the anticancer effects of adenine and the underlying mechanism in colon cancer cells. Methods Cell viability was measured using the MTT assay. Levels of phosphorylation and protein expression were determined using western blotting. qPCR was carried out to determine the changes in mRNA expression of genes of interest. Results Adenine significantly inhibited the viability of colon cancer cells, HT29 and Caco-2 cells, in a dose-dependent manner. Adenine induced significant apoptosis in HT29 cells, whereas Caco-2 cells exhibited less apoptotic responses. The data showed that adenine activated AMP-activated protein kinase (AMPK) signaling contributing to autophagic cell death through mTOR in both colon cancer cell lines. Conclusions Our findings suggest that adenine inhibits the growth of colon cancer cells. Anticancer activity of adenine in colon cancer cells is attributable to the activation of apoptotic signaling and in turn the AMPK/mTOR pathway. Adenine represents a natural compound with anticancer potency.

Germline variability and tumor expression level of ribosomal protein gene RPL28 are associated with survival of metastatic colorectal cancer patients

This study investigated the potential of single nucleotide polymorphisms as predictors of survival in two cohorts comprising 417 metastatic colorectal cancer (mCRC) patients treated with the FOLFIRI (folinic acid, 5-fluorouracil and irinotecan) regimen. The rs4806668G > T of the ribosomal protein gene RPL28 was associated with shorter progression-free survival and overall survival by 5 and 9 months (P = 0.002), with hazard ratios of 3.36 (P < 0.001) and 3.07 (P = 0.002), respectively. The rs4806668T allele was associated with an increased RPL28 expression in transverse normal colon tissues (n = 246, P = 0.007). RPL28 expression was higher in colorectal tumors compared to paired normal tissues by up to 124% (P < 0.001) in three independent datasets. Metastatic cases with highest RPL28 tumor expression had a reduced survival in two datasets (n = 88, P = 0.009 and n = 56, P = 0.009). High RPL28 was further associated with changes in immunoglobulin and extracellular matrix pathways. Repression of RPL28 reduced proliferation by 1.4-fold to 5.6-fold (P < 0.05) in colon cancer HCT116 and HT-29 cells. Our findings suggest that the ribosomal RPL28 protein may influence mCRC outcome.

Radiosensitizing effects of trabectedin on human A549 lung cancer cells and HT-29 colon cancer cells

Background and Purpose Trabectedin is a unique alkylating agent with promising effects against a range of solid tumors. In this study, we aimed to examine the cytotoxic and radiosensitizing effects of trabectedin on two human epithelial tumor cell lines in vitro, and its effects on DNA repair capacity. Methods Cancer cells (A549: human lung cancer cells, HT-29: colon cancer cells) were treated with either trabectedin alone for the determination of their growth, or in combination with radiation for the determination of their metabolic activity, proliferation, and clonogenic survival. Besides, the γH2AX foci assay was performed for the assessment of ionizing radiation-induced DNA damage and to evaluate the influence of trabectedin on DNA damage repair. Results Treatment with trabectedin resulted in a growth-inhibiting effect on both cell lines, with the IC50 values remaining within a low nanomolar range. Analyses of metabolic activity confirmed a cytotoxic influence of trabectedin and a BrdU assay demonstrated an antiproliferative effect. When combined with radiation, incubation with trabectedin was found to enhance the radiosensitivity of the tumor cells. The γH2AX foci assay resulted in an increased number of DNA double-strand breaks (DSBs) in cells treated with trabectedin. Conclusion The results of this study underline the antitumor activity of trabectedin at low nanomolar concentrations. We demonstrated that trabectedin enhanced radiation response in human lung (A549) cancer cells and colon (HT-29) cancer cells. Further studies are necessary to examine trabectedin as a potential candidate for future applications in radiotherapy.

Inhibition of Proliferation of Colorectal Cancer Cells by Phenolic Extracts of Mandarin (&lt;i&gt;Citrus reticulate&lt;/i&gt;)&lt;i&gt; &lt;/i&gt;and Lime (&lt;i&gt;Citrus aurantifolia&lt;/i&gt;) Fruit Waste

Several epidemiological studies have suggested that phenolics have antineoplastic activities. The objective of this study was to determine the antiproliferative effect of rich phenolics extracts of mandarin (Citrus reticulate) and lime (Citrus aurantifolia) fruit waste in colorectal cancer cells, RKO (carcinom) and HT-29 (adenocarcinom). Antiproliferative effect was evaluated using the methylthiazolydiphenyl-tetrazolium bromide (MTT) assay and testing phenolics extracts from different mandarin and lime fruit waste drying temperatures (fresh, 60, 90, 120ºC), at different times (24, 48 and 72 h). Mandarin and lime phenolics extracts inhibit cell viability of colon cancer cell lines, HT-29 and RKO, at 24 h to 72 h. Results suggested that phenolics extracts had different action mechanisms on each type of cells and therefore, more studies were required to elucidate such mechanisms.

The microRNA-200 family acts as an oncogene in colorectal cancer by inhibiting the tumor suppressor RASSF2.

This study aimed to determine whether manipulation of the microRNA-200 (miR-200) family could influence colon adenocarcinoma cell behavior. The miR-200 family has a significant role in tumor suppression and functions as an oncogene. In vitro studies on gain and loss of function with small interfering RNA demonstrated that the miR-200 family could regulate RASSF2 expression. Knockdown of the miR-200 family in the HT-29 colon cancer cell line increased KRAS expression but decreased signaling in the MAPK/ERK signaling pathway through reduced ERK phosphorylation. Increased expression of the miR-200 family in the CCD-841 colon epithelium cell line increased KRAS expression and led to increased signaling in the MAPK/ERK signaling pathway but increased ERK phosphorylation. Functionally, knockdown of the miR-200 family led to decreased cell proliferation in the HT-29 cells; therefore, increased miR-200 family expression could increase cell proliferation in the CCD-841 cell line. The present study included a large paired miR array dataset (n=632), in which the miR-200 family was significantly found to be increased in colon cancer when compared with normal adjacent colon epithelium. In a miR-seq dataset (n=199), the study found that miR-200 family expression was increased in localized colon cancer compared with metastatic disease. Decreased expression was associated with poorer overall survival. The miR-200 family directly targeted RASSF2 and was inversely correlated with RASSF2 expression (n=199, all P<0.001). Despite the well-defined role of the miR-200 family in tumor suppression, the present findings demonstrated a novel function of the miR-200 family in tumor proliferation.

Differences in virulence gene expression between human blood and stool Campylobacter coli clade 1 ST828CC isolates

BackgroundCampylobacter colonise the gastrointestinal tract of warm-blooded animals and are major enteropathogens in humans. C. coli is less common than C. jejuni and accounts for about 10% of the total number of Campylobacter infections although the two species seem to share many virulence determinants. Campylobacter bacteraemia is rare, estimated to occur in less than 1% of the infections, and the exact mechanisms regulating the progression of the infection from the gastrointestinal tract to the blood stream are unclear. Here, we looked at the contribution of C. coli to Campylobacter infections and further compared various virulence traits in C. coli clade 1 blood and stool isolates.ResultsWe assessed the numbers of C. jejuni and C. coli among typed isolates in the PubMLST database and found that C. coli accounted for 25.9% of blood isolates, but only 8.9% of the stool isolates. Phylogenetic analysis of 128 C. coli clade 1 whole genome sequences deposited to NCBI revealed no specific clustering of the human blood, stool or animal isolates. Of the six C. coli isolates chosen for phenotypic analyses, stool isolates adhered significantly better to human HT-29 colon cancer cells than the blood isolates, while there was no difference in induced IL-8 levels between the isolates. Furthermore, the stool isolates had two- to fourfold higher RNA expression levels of the flpA, ciaB, iamA and cdt virulence genes than the blood isolates. Finally, we looked at the gene structure of the cdtA, B and C toxin genes and found numerous nucleotide additions and deletions disrupting the open reading frames. In contrast to 58% isolates of animal origin, only 38% and 32% of human blood and stool isolates, respectively, had all three cdt genes intact, a prerequisite to produce functional toxins.ConclusionsThis study reveals interesting differences between C. coli clade 1 isolates of human and animal origin on one hand, and also between human blood and stool isolates, on the other. The results suggest that C. coli might downregulate and/or inactivate various virulence determinants as the isolates pass from the animal host to the human gastrointestinal tract and enter the human blood stream.

Anticancer Activity of Copper Oxide Nanoparticles Synthesized from Brassia actinophylla Flower Extract

There are many methods to synthesize metal and metal oxide nanoparticles. In this paper, copper oxide nanoparticles have been synthesized by solution combustion method using Brassia actinophylla i.e. Schefflera actinophylla flower extract belongs to Araliaceae family. The importance of solution combustion is one of the easy and simplest methods for the synthesis of metal oxide nanoparticle. The CuO nanoparticles were synthesized at various temperatures and the characterization has been carried out by UV, FTIR, PXRD, SEM, TEM and EDAX analysis. At lower temperature, the peak was not observed but at 400 ºC, the UV peak was observed at 340 nm. The FTIR peaks observed at 1000-500 cm-1 confirms again the presence of CuO nanoparticles. The monoclinic phase and crystalline structure of nanoparticles were revealed by PXRD pattern, by Scherrer′s method the average crystalline sizes were found to be in the range of 15 to 24 nm. The size and the shape of nanoparticles were confirmed by SEM and TEM reports. The SEM images of nanoparticles show spherical in shape and free from agglomeration. TEM analysis reports the nanoparticle sizes ranging from 2 to 20 nm. The percentage of copper (52 %) and oxygen (26 %) elements were recorded in the EDAX analysis. The study of size and stability of nanoparticles were done by zeta potential values. The antibacterial activity of CuO nanoparticles were carried out against Staphylococcus aureus and Escherichia coli bacteria's by agar well diffusion method. The MTT assay was performed in order to check the anticancer activity of CuO nanoparticles against HT-29 colon cancer cells.

Pro-apoptotic effect of the novel benzylidene derivative MHY695 in human colon cancer cells.

The induction of apoptosis is a useful strategy in anti-cancer research. Various Moon Hyung Yang (MHY) compounds have been developed as novel anti-cancer drug candidates; in the present study, the pro-apoptotic effects of (Z)-5-(3-ethoxy-4- hydroxybenzylidene)-2-thioxothiazolidin-4-one (MHY695) on HCT116 human colon cancer cells were assessed. MTT assays were performed to investigate the dose-dependent cytotoxic effects of MHY695 on HCT116 cells. Immunofluorescence staining and flow cytometry analyses were performed to identify apoptotic cell death, and western blot analysis was used to investigate the apoptotic-signaling pathways. A mouse xenograft model was also used to determine the effects of MHY695 in vivo. MHY695 decreased the viability of HCT116 cells and induced apoptotic cytotoxicity. The apoptotic mechanisms induced by MHY695 involved the dephosphorylation of Bcl-2-associated agonist of cell death protein following protein kinase B inactivation, induced myeloid leukaemia cell differentiation protein and BH3-interacting domain death agonist truncation, caspase-3 and −9 activation and poly (ADP-ribose) polymerase cleavage. In addition, MHY695 significantly suppressed tumor growth in the mouse xenograft model, compared with the vehicle control. Notably, MHY695 exhibited potent anti-cancer effects in four different types of human colon cancer cell line, including Caco-2, DLD-1, HT-29 and HCT116. Additionally, MHY695 showed reduced cytotoxicity in NCM460, normal colonic epithelial cells. Furthermore, MHY-induced cytotoxicity in colon cancer cells was independent of the tumor suppressor protein p53. Collectively, these observations suggested that MHY695 may be a novel drug for the treatment of colon cancer.

Synthesis, biological evaluation, and docking studies of new raloxifene sulfonate or sulfamate derivatives as inhibitors of nucleotide pyrophosphatase/phosphodiesterase

A new series of raloxifene sulfonate/sulfamate derivatives were designed and synthesized. The target compounds were tested for inhibitory effect against nucleotide pyrophosphatase/phosphodiesterase-1 and -3 (NPP1 and NPP3) enzymes. Furthermore, all the ten target compounds were subjected to cytotoxic studies on various cancer cell lines, and the most potent derivatives were explored for their potency against these cancer cell lines as well as F180 fibroblasts to investigate the selectivity indexes. Compound 1f exerted the highest potency against HT-29 colon cancer cell line (IC50 = 1.4 μM) with 8.43-fold selectivity towards HT-29 than F180 fibroblasts. Compound 1f exerted sub-micromolar IC50 values against NPP1 and NPP3 (IC50 = 0.29 μM and 0.71 μM, respectively). The most potent inhibitors were docked in developed homology model of NPP1 and crystal structure of NPP3. All the docked analogues manifested remarkable interactions within the active pocket of NPP1 and NPP3.

Mesenchymal stem cell homing towards cancer cells is increased by enzyme activity of cathepsin D

Mesenchymal stem cells home towards inflammatory microenvironments, such as the tumour stroma, where they have been shown to have both pro- and anti-tumorigenic effects. Here, we demonstrate that the aspartic acid protease cathepsin D is part of the chemoattraction process. Using a Boyden chamber co-culture system, the migration of the mesenchymal stem cells and their invasion through Matrigel increased in the presence of breast cancer MDA-MB-231 cells, colon cancer HT29 cells or their conditioned media. Mesenchymal stem cell movement was reduced by protease inhibitors of matrix metalloproteinases and by pepstatin A, an inhibitor of cathepsin D. We confirmed a role for cathepsin D through addition of recombinant protein, upregulation of cathepsin D release using chloroquine and knockdown of cathepsin D expression. While all cell types expressed active cathepsin D, enzymatically inactive precursor procathepsin D was expressed only at low levels by mesenchymal stem cells. Expression in mesenchymal stem cells was increased following co-culture with cancer cells. The chemoattractive effect of cathepsin D required its enzymatic activity, but not changes in mesenchymal stem cell proliferation or adhesion rates. In conclusion, cathepsin D and its precursors enhance mesenchymal stem cell homing towards tumour sites, most likely by enzymatic mechanisms.

Isorhamnetin glycoside isolated from Opuntia ficus-indica (L.) MilI induces apoptosis in human colon cancer cells through mitochondrial damage

This work aimed to evaluate the mechanisms involved in the apoptosis induction of isorhamnetin-3-O-glucosyl-pentoside (IGP) in metastatic human colon cancer cells (HT-29). To achieve this, we assessed phosphatidylserine (PS) exposure, cell membrane disruption, chromatin condensation, cell cycle alterations, mitochondrial damage, ROS production, and caspase-dependence on cell death. Our results showed that IGP induced cell death on HT-29 cells through PS exposure (48%) and membrane permeabilization (30%) as well as nuclear condensation (54%) compared with control cells. Moreover, IGP treatment induced cell cycle arrest in G2/M phase. Bax/Bcl-2 ratio increased and the loss of mitochondrial membrane potential (63%) was observed in IGP-treated cells. Finally, as apoptosis is a caspase-dependent cell death mechanism, we used a pancaspase-inhibitor (Q-VD-OPh) to demonstrate that the cell death induced by IGP was caspase-dependent. Overall these results indicated that IGP induced apoptosis through caspase-dependent mitochondrial damage in HT-29 colon cancer cells.

Abstract 3126: A novel biomarker signature in predicting chemoresistance in colorectal cancer: Potential application in chemotherapy

Abstract Despite advances in the cytotoxic and targeted therapy, resistance to chemotherapy remains one of the greatest challenges in long-term management of metastatic colorectal cancer, which eventually contributes to patient death as tumors accumulate means of evading treatment. We have recently demonstrated that expression of the tight junction protein claudin-1 increases while claudin-7 expression decreases with human colon cancer (CRC) progression and metastasis. Epithelial-mesenchymal transition (EMT) and cancer stem cells (CSC) are critically implicated in cancer metastasis and chemoresistance. Taking this to account, we performed a computational assessment of 250 patient datasets from two different cancer centers (Vanderbilt and Moffitt) and identified the gene sets whose expression increased proportionally with claudin-1 expression and decreased with claudin-7, and vice versa, and correlated these gene sets it with chemoresistance, EMT and CSC markers. We identified a resulting 23-gene set biomarker signature, out of these gene clusters. Some of the important candidates based on literature and their function in colorectal cancer which we focused on were SLC6A6, PIK3CA, ASAP1, TMEM and E2F2. This signature was then validated using the TCGA database. To further evaluate functional relevance of this biomarker signature, we developed oxaliplatin resistant DLD-1 and HT29 colon cancer cells. The chemoresistance of these cells were confirmed by determining the IC50 values DLD (Parental) (4.5μM) and HT29 (Parental) (10.08 μM), after exposing them to increasing conc. of clinically relevant dose of oxaliplatin DLD (Oxaliplatin-R) (16.35 μM) and HT29 (Oxaliplatin-R) (20.05 μM). To our interest, we observed a significant upregulation of SLC6A6, PIK3CA, ASAP1, TMEM and downregulation of E2F2 in DLDOxaR and HT29OxaR cells compared to parental cells. Moreover, DLDOxaR and HT29OxaR cells possessed significantly increased expression of EMT markers such as α-SMA, vimentin, Snail and Slug along with enrichment of the stem cell markers like CD133, CD44 and Aldh1, colony and sphere forming ability. A concomitant decrease in E-cadherin characterized these cells. A similar increase in the expressions of SLC6A6, PIK3CA, ASAP1, TMEM and decrease in E2F2 was observed in colon cancer mouse models as well as chemoresistant patient samples. Taken together, we propose a new predictive biomarker signature which may offer insights into identifying new therapies required to overcome the acquired resistance of colon cancer towards Oxaliplatin and uncover potential molecular pathways involved in treatment failure to help guide therapeutic alternative. Citation Format: Saiprasad Gowrikumar, Kristina Pravoverov, Caroline Selisteda, Kiran D. Bastola, Steven Chen, Joshua J. Smith, Mary K. Washington, Amar B. Singh, Punita Dhawan. A novel biomarker signature in predicting chemoresistance in colorectal cancer: Potential application in chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3126.

Abstract 840: Identification of novel colorectal tumor suppressor genes through genome-wide promoter hypermethylation analysis

Abstract Cancer initiation and progression are driven by both genetic and epigenetic changes. Although recent genome/exome sequencing efforts have significantly contributed to the thorough characterization of the genetic changes associated with the oncogenic process, further investigation is required to systematically identify the driver genes regulated by promoter hypermethylation. Using genome-wide analysis of the levels of promoter methylation (HumanMethylation27, Illumina) and the levels of mRNA expression (microarray analysis) in a panel of 30 colorectal cancer cell lines and 223 primary colorectal tumors (TCGA), we found a subset of 553 (5.6%) genes whose levels of promoter methylation showed a significant negative association with their expression. Higher overall methylation levels were associated with microsatellite instability (MSI), a CpG methylator phenotype (CIMP), faster proliferation and absence of APC mutations. Next, because genes that are epigenetically silenced could represent important drivers of the oncogenic process we investigated the role of the zinc finger transcriptional regulator ZNF238/ZBTB18, a gene silenced by promoter methylation, on the growth of colon cancer cells. Reintroduction of ZNF238 in HCT116 and HT29 colon cancer cells with low endogenous levels and promoter methylation of ZNF238, resulted in a significant reduction of cell proliferation both in vitro and in a subcutaneous xenograft NOD/SCID mouse model. Moreover, using immunohistochemical analysis with a validated antibody we found that ZNF238 is lost or reduced in the majority of the 133 primary colorectal tumors included in a tissue microarray, and that lower ZNF238 expression is associated with lymph node metastasis and shorter survival of patients with locally advanced colorectal cancer. In summary, we identified a set of 553 genes putatively silenced by promoter methylation in colorectal tumors that could significantly contribute to the oncogenic process. Moreover, as a proof of concept, we demonstrate that the epigenetically silenced gene ZNF238 has tumor suppressor activity and is associated with the survival of colorectal cancer patients. Citation Format: Sarah Bazzocco, Jose Higinio Dopeso, Águeda Martínez-Barriocanal, Estefanía Anguita, Rocio Nieto, Alex Sanchez, John M. Mariadason, Diego Arango. Identification of novel colorectal tumor suppressor genes through genome-wide promoter hypermethylation analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 840.