Colo205 Cells Research Articles

Antiproliferative effects of combinational therapy of Lycopodium clavatum and quercetin in colon cancer cells.

Background Colorectal cancer (CRC) is the third most prevalent form of cancer and fourth leading cause of morbidity worldwide. Surgical resection remains the only curative approach for CRC, but recurrence following surgery is the main problem and ultimate cause of death. Lycopodium clavatum and quercetin have been found to exert its anticancer properties. The aim of the present study is to investigate whether quercetin or L. clavatum extract and combination of both have any profound role in reducing major inflammatory cytokines in Colo-320 cells. Methods L. clavatum and Quercetin alone or in combination was administered to colon cancer cells and various toxicity markers, gene expression analyses of apototic genes and gelatin zymmography were performed. Results Quercetin (50 μm) in combination with L. clavatum extract (10 μL) distinctly reduced cell growth and highlighted their potential effects in extirpation of colon cancer cells. Treatment with increased dose of L. clavatum extract in combination with quercetin reduced the colony size and proliferation potential when compared to the sole treatment of plant extracts. In the antimicrobial assays, it was observed that Lycopodium alone exhibited antimicrobial activity against Escherichia coli and Pseudomonas aeruginosa. Characterization of L. clavatum extract and quercetin was performed and confirmed the presence of flavonoids and alkaloids. Treatment with Lycopodium and quercetin combination induced significant down-regulation in activities of MMP2 and MMP9 tested by gelatin zymography. The combined treatment greatly affected the mRNA expression of p53, Bcl2, Bax, Caspase 3, Wnt 1, Cyclin D1, and Catalase genes in colon cancer cells. Conclusion The synergistic effect between Lycopodium and quercetin might bring forward the enhanced antitumorigenic properties of combinational therapy with natural products to successfully combat the cancer progression with minimal side effects and resistance to drugs.

Long Non-Coding RNA LEF1-AS1 Promotes Migration, Invasion and Metastasis of Colon Cancer Cells Through miR-30-5p/SOX9 Axis.

IntroductionAberrant expression of long non-coding RNAs (lncRNAs) has been implicated in the tumorigenesis and progression of colon cancer. Lymphoid enhancer-binding factor 1 antisense RNA 1 (LEF1-AS1), a highly conserved and newly discovered long non-coding RNA, has been reported to be upregulated and correlated with poor prognosis in colon cancer, but the exact role of it remains uncertain.Materials and MethodsIn our study, the biological functions of LEF1-AS1 in colon cancer were analyzed by cell viability assay, colony formation assay, scratch wound healing assay, transwell cell invasion assay, soft agar assay, luciferase reporter assay, pull down assay, tumor xenograft model and Western blot.ResultsWe found that LEF1-AS1 was upregulated in colon cancer patients and correlated with poor overall survival and recurrent-free survival. Besides, enforced expression of LEF1-AS1 in HT29 and T84 cells promoted migration, invasion, anchorage-independent growth, tumor xenograft formation and lung metastasis, while knockdown of LEF1-AS1 in COLO320 cells suppressed cell migration, invasion, anchorage-independent growth and tumor xenograft formation. In addition, LEF1-AS1 was directly interacted and inversely correlated with miR-30-5p in colon cancer, and SOX9 was a downstream target for miR-30-5p. LEF1-AS1 overexpression increased the expression level of SOX9, and restoration of SOX9 attenuated the effects caused by LEF1-AS1 knockdown in cell migration, invasion, anchorage-independent growth and tumor xenograft formation.ConclusionOur results indicated that LEF1-AS1 promoted migration, invasion and metastasis of colon cancer cells partially through miR-30-5p/SOX9 axis. The oncogenic LEF1-AS1 could be a potential prognostic biomarker for colon cancer.

20(S)-ginsenoside Rh2 as agent for the treatment of LMN-CRC via regulating epithelial-mesenchymal transition.

The lymph node metastasis of colorectal cancer (LMN-CRC) seriously threatens the prognosis of patients. Chemotherapy, as the most common treatment, results in severe bone marrow suppression. 20(S)-ginsenoside Rh2 (SGRh2), a major effective constituent of ginseng, has demonstrated therapeutic effects on a variety of diseases, including some tumours. SGRh2 treatment had no effect on other organs. Therefore, ginsenosides are considered a safe and effective antineoplastic drug. However, the effects of SGRh2 on LMN-CRC remain unknown. The present study investigated the potential effect of SGRh2 on LMN-CRC in vitro and in vivo. SW480 and CoLo205 cell lines were treated with SGRh2. SGRh2 dose-dependently decreased CRC cell proliferation by CCK-8, colony formation and Edu assays. The Transwell and scratch assays revealed that SGRh2 inhibits the migratory and invasive abilities of CRC cells in a dose-dependent manner. Furthermore, the results of Western blotting revealed that SGRh2 decreased the expression of matrix metalloproteinase (MMP)-2 and MMP9. In terms of the underlying mechanisms, SGRh2 regulates CRC metastasis by affecting epithelial–mesenchymal transition (EMT), which significantly up-regulated epithelial biomarkers (E-cadherin) and down-regulated mesenchymal biomarkers (N-cadherin and vimentin) and EMT transcriptional factors (Smad-3, Snail-1, and Twist-1). In vivo, SGRh2 significantly inhibited LMN-CRC without affecting other normal organs. Immunohistochemical results showed that SGRh2 treats LMN-CRC by regulating EMT. These results demonstrate that SGRh2 has therapeutic potential for LMN-CRC.

MiR-524-5p inhibits angiogenesis through targeting WNK1 in colon cancer cells.

There is increasing evidence that microRNA (miRNA) abnormity is involved in the occurrence and the development of various malignancies, including colon cancer. MiRNA-524-5p has been reported to possess anticancer activity in various tumors, which function is seldom investigated in colon cancer cells. The aim of this study was to explore the effect of the miRNA-524-5p/with-no-lysine kinase 1 (WNK1) system on angiogenesis in a colon cancer cell line (HT-29 and COLO205 cells) and further investigate the potential mechanisms. We found miRNA-524-5p expression was relatively high in COLO205 cells and relatively low in HT-29 cells. Elevating miRNA-524-5p expression inhibited proliferation, induced cycle arrest, diminished vascular endothelial growth factor production, and thereby suppressed angiogenesis in HT-29 cells. WNK1 silencing exerted the ability of antiangiogenesis in HT-29 cells. Besides, miRNA-524-5p deficiency-induced angiogenesis was impeded by WNK1 silence in COLO205 cells. In a murine tumor model, miRNA-524-5p agomir treatment significantly suppressed colon cancer tumorigenicity with the downregulation of WNK1 expression. In summary, our results indicated that miRNA-524-5p inhibited angiogenesis in colon cancer cells via targeting WNK1.NEW & NOTEWORTHY MiRNA-524-5p inhibited angiogenesis in colon cancer cells via targeting with-no-lysine kinase 1.

Synthesis, Cytotoxic, Antibacterial and Free Radical Scavenging Activities of New 1,2,4-Triazole Schiff Bases

Twelve new 1,2,4-triazole Schiff bases bearing a fluorinated indole ring were successfully synthesized. The 1,2,4-triazole Schiff bases were synthesized from the condensation reaction of 4-amino-5-mercapto-3-[(5-fluoro-2-methyl-1H-indol-3-yl)methyl]-1,2,4-triazole with a series of benzaldehyde derivatives in the presence of (+)-tartaric acid as the catalyst. The structures of Schiff bases were elucidated by FTIR, NMR and mass spectral data. All newly synthesized Schiff bases were screened for their cytotoxic, antibacterial and free radical scavenging activities. Schiff bases 6b, 6c, 6i and 6j with hydroxyl group at ortho or meta position of the phenyl ring demonstrated higher cytotoxic activity against COLO-205 cell lines with IC50 94.0-144.3 µmol/mL. Schiff base bearing 2-OH and 5-Cl groups showed moderate antibacterial activity against Bacillus cereus at MIC 151 µmol/mL. On the other hand, compounds 6b (IC50 150.4 µmol/mL), 6e (IC50 146.4 µmol/mL), 6f (IC50 120.9 µmol/mL) and 6g (IC50 146.2 µmol/mL) displayed a better free radical scavenging activity than the standard BHT.

Atractylenolide I inhibits colorectal cancer cell proliferation by affecting metabolism and stemness via AKT/mTOR signaling

BackgroundAtractylenolide I (ATL-1) is a natural herbal compound used in traditional Chinese medicine that has exhibited anti-cancer properties. The anti-tumorigenic activity of ATL-1 against colorectal cancer (CRC) and the underlying signaling pathways involved in its mechanisms are examined here. HypothesisATL-1 exerts therapeutic effect against CRC by disrupting glucose metabolism and cancer stem cell maintenance via AKT/mTOR pathway regulation. Study designIn vitro studies were performed in COLO205 and HCT116 CRC cell lines and in vivo studies were conducted in a mouse xenograft model of CRC tumor. MethodsCRC cells were treated with ATL-1 at various concentrations, with or without inhibitors of AKT or mTOR. Cell proliferation, apoptosis, invasion, stemness maintenance, glucose metabolism, and AKT/mTOR signaling were evaluated. CRC tumor-xenografted mice were treated with an AKT inhibitor and/or ATL-1, and glucose metabolism and stemness maintenance were examined in tumor tissues. ResultsATL-1 significantly inhibited the invasion of CRC cells by inducing their apoptosis, possibly via the excessive production of reactive oxygen species. Glucose metabolism (Warburg effect) was also altered and stem-like traits were suppressed by ATL-1. In addition, ATL-1 effectively acted as an inhibitor or AKT/mTOR by downregulating the phosphorylation of proteins related to the AKT/mTOR pathway. In vivo studies showed that tumor weight and volume were reduced by ATL-1 and that aerobic glycolysis, stemness maintenance, and AKT/mTOR activation were impaired by ATL-1 in colorectal tumors. ConclusionsATL-1 acts as an effective agent to suppress colorectal tumor progression, mainly by inhibiting CRC cell proliferation through altering apoptosis, glucose metabolism, and stem-like behavior. These processes were mediated by the AKT/mTOR signaling pathway both in vitro and in vivo. ATL-1 may be a potential agent to be used in molecular-targeted strategies for cancer treatment.

Oral delivery of pH-responsive alginate microbeads incorporating folic acid-grafted solid lipid nanoparticles exhibits enhanced targeting effect against colorectal cancer: A dual-targeted approach

To achieve an enhanced anticancer effect of drug to treat colorectal cancer, a dual-targeted (i.e., ligand-tailored and pH-triggered) multiparticulate system has been designed to deliver drug directly into the colon domain. In this regard, folic acid–grafted solid lipid nanoparticles (SLNs) bearing irinotecan were encapsulated in microbeads of alginates. Afterward, these microbeads were coated with enteric polymer (i.e., Eudragit S100) to make them pH-responsive. COLO-205 cells were used to determine in vitro cytotoxicity potential of various formulations. Findings for IHT loaded FA-coupled SLNs suggested significantly (p < 0.05) higher cytotoxic effect against COLO-205 cells (in vitro) as compared to drug solution and uncoupled SLNs. Further, the microbeads incorporating SLNs were evaluated for drug release in various simulated gastrointestinal fluids (i.e., pH, 1.2, 4.5, 7.4, and 6.8). Findings confirmed the release of the drug in the intestinal region only (i.e., pH > 7.0). In therapeutic experiments (in vivo), the optimized radiolabeled microbeads (99mTc-EuBIRSLN3 and 99mTc-EuBIRSLNF3) were administered via oral route to Balb/c mice. The results suggested that FA-coupled microbeads (99mTc-EuBIRSLNF3) distributed higher (19.62 ± 0.78%) amount of drug (i.e., 99mTc-IHT/g of tissue) as compared to uncoupled microbeads (99mTc-EuBIRSLN3, 7.63 ± 0.49%) in the colon tumor after 48 h, which confirmed its targeting ability to the tumor in the colon region. Further, 99mTc-EuBIRSLNF3 showed significantly (p < 0.01) higher antitumor effect against HT-29 cells bearing Balb/c mice over uncoupled microbeads and advocated their potential for enhanced antitumor efficacy for the treatment of colorectal cancer.

Antiproliferative activity of protein hydrolysates derived from fish by-products on human colon and breast cancer cells.

AbstractThe consumption of fish is encouraged in dietary guidelines because of its association with health benefits; however, fish processing generates by-products (gills, bones, skin, head), which can rise up to 50–60% of fish weight after filleting. The investigation of bioactivity of fish by-products and subsequent utilization for the development of high added-value food ingredients and/or supplements will reduce waste and create new products of high nutritional value. We studied the antiproliferative activity of fish by-product protein hydrolysates (FPH) on human colon and breast cancer cells. A total of 6 Meagres “Argirosomus Regius” (1256.45 ± 232.32g) and 16 Gilthead Sea Breams “Sparus Aurata” (403.47 ± 72.92g) were obtained by HCMR Institute in Crete, Greece. The fins, skin, bones, head, and gills were isolated, lyophilized, homogenized and stored separately at -80oC. The protein hydrolysates from each byproduct were prepared using a pH extraction method. Human colorectal adenocarcinoma cells (COLO320 cells) and human breast adenocarcinoma cells (MCF7A cells) were treated with different FPH for 48 hours, and cellular proliferation was measured using the MTT assay. Results are given as mean percentage of triplicate independent analyses. Statistical significance of growth inhibition was estimated following Wilcoxon Signed-Ranks test with α = 0.05. In COLO320 cells, FPH induced a the significant antiproliferative activity at 0.5g/L. Specifically, FPH from bones, gills and skin of Meagre showed a growth inhibition of 23.7%, 9.9% and 26.4%, respectively, after 48 h of treatment. The inhibition by the same by-products from the Gilthead Sea bream was 24.6%, 21.9% and 26.4%, respectively after 48 h. In MCF7A cells, FPH exerted a significant growth inhibition at 1g/L. The skin and fins of Meagre induced a growth inhibition of 25.5% and 39.0% respectively; the gills, skin and fins of Gilthead Sea Bream showed a growth inhibition of 28.5%, 47.1% and 39.6%, respectively, after 48 h. These values were in the same range with etoposide (reference anticancer molecule) in the concentration of 3.1μM for COLO320 cells and 50μM for MCF7A. These preliminary data suggest that certain fish by-products could represent a source of anticancer peptides and may cause growth inhibition of human cancer cells. The isolation of responsible bioactive peptides from fish byproducts and the integration of them into food supplements can have beneficial effects on human health.

Novel NBQ-48 as marker of hypoxic cells in 2D and 3D colon cancer cells.

This study presents the applicability of a novel nitro-substituted heterocyclic compound NBQ48, member of the family of compounds identified as 3 nitrobenzazolo[3, 2-a] quinolinium chloride salts (NBQS) as a screening tool to identify hypoxic tumor cells. The applicability was tested on COLO 205 colon cancer cells 2D and 3D cultures treated with NBQ48 to assess the formation of a bio-reduction fluorescent metabolite under hypoxic conditions in contrast, to those under aerobic environment. Hypoxic environment was created applying a controlled hypoxic gas chamber. Prior to testing the applicability of NBQ48 as a hypoxic fluorescent marker, cytotoxic studies where performed to identify a low-toxicity dose at 24 hours under aerobic and hypoxic environments that would allow the bio-reduction process with little toxicity. The differences in fluorescence emission after treatment was measured by fluorometer and fluorescence microscopy. Results indicated that cell treatments up to 24 hours with NBQ48 under aerobic environment did not reach an IC50 dose in COLO205 cells, however under hypoxic environment the IC50 reached at 100μM. The significant fluorescence increment after 24 and 48 hrs in 2D and 3D cultures treated with NBQ48 (75uM) at hypoxic in contrast to aerobic environments clearly demonstrated the need of a low oxygen content for the bio-reduction of the parent NBQ48. This study confirms the applicability of NBQ48 as markers for hypoxia in metabolically active 2D and 3D cultures. This hydrophilic hypoxic marker could additionally aid researchers in testing hypoxia activated pro-drugs for therapeutic applications in cancer as well as on other diseases where cellular hypoxia is a significant risk factor.

Activation of PERK Contributes to Apoptosis and G2/M Arrest by Microtubule Disruptors in Human Colorectal Carcinoma Cells

Microtubule-targeting agents (MTAs) are widely used in cancer chemotherapy, but the therapeutic responses significantly vary among different tumor types. Protein kinase RNA-like endoplasmic reticular (ER) kinase (PERK) is an ER stress kinase, and the role of PERK in the anticancer effects of MTAs is still undefined. In the present study, taxol (TAX) and nocodazole (NOC) significantly induced apoptosis with increased expression of phosphorylated PERK (pPERK; Tyr980) in four human colon cancer cell lines, including HCT-15, COLO205, HT-20, and LOVO cells. Induction of G2/M arrest by TAX and NOC with increases in phosphorylated Cdc25C and cyclin B1 protein were observed in human colon cancer cells. Application of the c-Jun N-terminal kinase (JNK) inhibitors SP600125 (SP) and JNK inhibitor V (JNKI) significantly reduced TAX- and NOC-induced apoptosis and G2/M arrest of human colon cancer cells. Interestingly, TAX- and NOC-induced pPERK (Tyr980) protein expression was inhibited by adding the JNK inhibitors, SP and JNKI, and application of the PERK inhibitor GSK2606414 (GSK) significantly reduced apoptosis and G2/M arrest by TAX and NOC, with decreased pPERK (Tyr980) and pJNK, phosphorylated Cdc25C, and Cyc B1 protein expressions in human colon cancer cells. Decreased viability by TAX and NOC was inhibited by knockdown of PERK using PERK siRNA in COLO205 and HCT-15 cells. Disruption of the mitochondrial membrane potential and an increase in B-cell lymphoma-2 (Bcl-2) protein phosphorylation (pBcl-2; Ser70) by TAX and NOC were prevented by adding the PERK inhibitor GSK and JNK inhibitor SP and JNKI. A cross-activation of JNK and PERK by TAX and NOC leading to anti-CRC actions including apoptosis and G2/M arrest was first demonstrated herein.

ANTIPROLIFERATIVE ACTIVITY OF ELEPHANTOPUS SCABER MEDIATED SILVER NANOPARTICLES AGAINST MCF-7, A-549, SCC-40, AND COLO-205 HUMAN CANCER CELL LINES

Objective: To unearth the applications of nanotechnology in medicine has become imperative with all the advancements in the technique. In the current study, we have attempted to exploit the anticancer ability of the green synthesized silver nanoparticles (AgNPs).&#x0D; Methods: The AgNPs were synthesized using 60% methanol (H-MeOH) Elephantopus scaber leaf extract, characterized, and discussed priorly. The effect of AgNPs was studied on the human breast (MCF-7), lung (A-549), oral (SCC-40), and colon (COLO-205) cancer cell lines through sulforhodamine B assay. We also carried out the synergistic activity with standard drug adriamycin (ADR).&#x0D; Results: According to the results obtained, AgNPs showed good antiproliferative activity with GI50 &lt;10 μg/ml on MCF-7, A-549, and SCC-40 cell lines when compared with the standard drug ADR. However, for COLO-205 cell line, the impact was 17.4 μg/ml and thus the treatment was less effective.&#x0D; Conclusion: The synergistic effect of AgNPs+ADR was even better for all the four cell lines than that of the AgNPs alone.

Uracil-coumarin based hybrid molecules as potent anti-cancer and anti-bacterial agents

In the current report, we have rationally designed a series of uracil-coumarin based bifunctional molecular hybrids roped by 1,2,3-triazole moiety. The designed compounds were synthesized and tested against a panel of six human cancer cell lines namely Colo-205, MCF-7, A-549, PA-1, PC-3 and Hela cells by Sulforhodamine B assay. The results indicated that the hybrid molecules can specifically inhibit the MCF-7 cancer cell proliferation amongst which A-2 was found to be most potent hybrid (GI50 = 1.55 µM) with fluorine atom as R with two carbon chain length between triazole and coumarin moieties. Cell cycle analysis revealed that A-2 significantly arrest the G2/M phase to inhibit proliferation of MCF-7 cells. Due to its mitotic arrest, A-2 was further analyzed to predict its various binding interactions within the active site of tubulin, which revealed its best binding pattern within the vinblastine binding site. In addition to this, antibacterial potential of all the synthetics was also evaluated which resulted in two hit lead molecules A-2 (MIC = 11.7 μg/mL) and A-3 (MIC = 7.23 μg/mL) that can significantly inhibit the bacterial strain Staphylococcus aureus comparable to that of standard drug levofloxacin (MIC = 3.12 μg/mL). Binding interactions within the active site of dihydrofolate reductase (DHFR) were also streamlined by using molecular docking studies. Overall studies revealed some interesting features of synthetics to be active which stated that, the compounds with electronegative atom on R and compounds with two carbon chain length between triazole and coumarin showed best results.

Abstract A130: Cannabidiol overcomes oxaliplatin resistance by enhancing autophagy through reduction NOS3 and SOD2

Abstract Although oxaliplatin is an effective chemotherapeutic drug for colorectal cancer (CRC) treatment, patients often develop resistance to it. Therefore, a new strategy for CRC treatment is needed. The purpose of this study was to determine the effect of cannabidiol (CBD), one of the components of the cannabis plant, in overcoming oxaliplatin resistance in CRC cells. We established oxaliplatin-resistant cell lines, DLD-1 R and colo205 R, in CRC DLD-1 and colo205 cells. Autophagic cell death was induced when oxaliplatin-resistant cells were treated with both oxaliplatin and CBD. Additionally, phosphorylation of nitric oxide synthase 3 (NOS3) was increased in oxaliplatin-resistant cells compared to that in parent cells. Combined treatment with oxaliplatin and CBD reduced phospho-NOS3 levels and NO production and resulted in the production of reactive oxygen species (ROS) by reducing the levels of superoxide dismutase 2, an antioxidant present in the mitochondria, causing mitochondrial dysfunction. Taken together, these results suggest that elevated phosphorylation of NOS3 is essential for oxaliplatin resistance. The combination of oxaliplatin and CBD decreased NOS3 phosphorylation, which resulted in autophagy, by inducing the overproduction of ROS through mitochondrial dysfunction, thus overcoming oxaliplatin resistance. Citation Format: Bu Gyeom Kim, Soyeon Jeong, Dae Yeong Kim, Bo Ram Kim, Jung Lim Kim, Seong Hye Park, Yoo Jin Na, Min Jee Jo, Hye Kyeong Yun, Yoon A Jeong, Sun Il Lee, Yoon A Jeong, Hong Jun Kim, Han Do Kim, Dae Hyun Kim, Dae-Hee Lee, Sang Cheul Oh. Cannabidiol overcomes oxaliplatin resistance by enhancing autophagy through reduction NOS3 and SOD2 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A130. doi:10.1158/1535-7163.TARG-19-A130

Abstract A093: Potential combination partners for a novel CDC7-selective Inhibitor, TAK-931

Abstract Background: Cell division cycle 7 (CDC7) is a serine/threonine kinase, which plays important roles in initiation of DNA replication. We developed a CDC7-selective inhibitor, TAK-931, designed to inhibit CDC7 activity in a time-dependent and ATP-competitive manner. In this study, we aimed to identify potential combination partners for TAK-931 to guide clinical combination strategies. Methods: In vitro combination studies of various agents with TAK-931 were carried out in COLO205, A549, SW620, SW48, H460, and HCT116 cancer cells using a fully automated system for assay execution and data analysis (Double Agent studies). Agents tested in combination with TAK-931 included: DNA damaging agents, tubulin binders, mitosis inhibitors, cell signaling modulators, proteasome inhibitors, and other targeted agents with significant single-agent activity against these cell lines. Treated stable isotope labeling using amino acids in cell culture (SILAC)-labeled H460 cells were subjected to quantitative mass spectrometry. In vivo combination studies of TAK-931 using DNA damaging agents or IR were conducted in multiple human xenograft models including patient-derived xenograft models (PDXs). Results: The Double Agent studies revealed that DNA damaging agents, such as topoisomerase inhibitors and platinum compounds, had the highest occurrence of synergistic antiproliferative effects in combination with TAK-931. In the phosphoproteomics analysis, gene ontology (GO) enrichment analysis revealed that DNA repair (p&amp;lt;0.01) and double-strand break (DSB) repair (p&amp;lt;0.01) were significantly enriched in the combination-treated cells with irradiation and TAK-931 (IR+TAK-931) compared with the untreated cells after 24 hours, whereas the enrichment of these pathways was not observed in the IR-treated cells. The functional analysis demonstrated that TAK-931 prolonged IR-induced 53BP1 foci formation (a DSB index), suppressed homologous recombination repair (HRR) activities, and hyperactivated DNA-damage response (DDR) pathways. These results suggest that TAK-931 could suppress DNA repair activity to enhance the biological activity of the DNA damaging agents for antiproliferation in cancer cells. Consistent with our hypothesis from in vitro studies, in vivo combination treatments of TAK-931 with DNA damaging agents, such as platinum compounds, topoisomerase inhibitors, and IR, also enhanced antitumor activities in multiple human xenograft models, including PDXs. Conclusions: Our findings suggest that chemotherapeutic drugs of DNA damaging agents could be potential candidates for combination with TAK-931 to expand its activity. Citation Format: Kenichi Iwai, Kurt Eng, Tadahiro Nambu, Jie Yu, Masamitsu Gotou, Sakiyo Tsukamoto, Saomi Murai, Shun-Ichiro Kageyama, Yukie Kashima, Susumu Kobayashi, Akihiro Ohashi. Potential combination partners for a novel CDC7-selective Inhibitor, TAK-931 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A093. doi:10.1158/1535-7163.TARG-19-A093

Inhibition of DNA Double-Strand Break Repair by Niclosamide in Human Colorectal Cancer Cells

The radiosensitizing effect of niclosamide against human rectal adenocarcinoma cells SW837 and human sigmoid colon carcinoma cells COLO 320 HSR and the underlying molecular mechanisms were investigated in this study. The survival rate of tumor cells under treatment with niclosamide, γ-radiation, and their combination was evaluated. The level of MDR1, Bcl-2, β-catenin, and γH2AX histone, a marker of DNA double-strand breaks (DSBs), was determined by flow cytometry using immunocytochemical methods. It was shown that the combined treatment with niclosamide and γ-radiation led to more intense tumor cell death than when niclosamide and γ-radiation were used separately. A decrease in the level of β-catenin was detected in the cells of the COLO 320 HSR line under treatment with niclosamide, which was more intense under combined treatment with niclosamide and irradiation. At the same time, the content of β-catenin in the cells of the SW837 line remained unchanged under treatment with niclosamide and γ-radiation and under their combined use. Niclosamide did not lead to the formation of DNA DSBs in unirradiated cells and did not increase their level 1 h after radiation exposure. The treatment of the cells with niclosamide before irradiation led to the fact that 24 h after irradiation the level of γH2AX histone, reflecting the number of DNA DSBs, was 1.6 times higher than that in intact cells and the cells exposed only to radiation. This evidences the inhibition of DNA DSB repair induced by γ-radiation. Thus, niclosamide possesses a radiosensitizing effect on human colorectal cancer cells, which is based on inhibition of DNA DSB repair. The inhibition of the Wnt/β-catenin signaling pathway under treatment with niclosamide is not detected in all types of tumor cells.

Overactivation of Akt Contributes to MEK Inhibitor Primary and Acquired Resistance in Colorectal Cancer Cells.

RAS and BRAF-mutated colorectal cancers are associated with resistance to chemotherapy and poor prognosis, highlighting the need for new therapeutic strategies. Although these cancers sometimes respond to mitogen activated protein kinase kinase (MEK) inhibitor treatment, they often acquire resistance via mechanisms, which are poorly understood. Here, we investigated the mechanism of MEK inhibitor resistance in primary- and acquired-resistant cells. Cell viability was examined using the trypan blue dye exclusion assay. Protein expression was analyzed by western blotting. Somatic mutations in colorectal cancer cells were investigated using the polymerase chain reaction array. PD0325901 and trametinib induced cell death in LoVo and Colo-205 cells but not in DLD-1 and HT-29 cells, which have a PIK3CA mutation constitutively activating Akt and NF-κB. Treatment with PD0325901 and trametinib suppressed ERK1/2 activation in all four cell lines but only induced Akt and NF-κB activation in DLD-1 and HT-29 cells. Inhibition of Akt but not NF-κB, overcame MEK inhibitor resistance in DLD-1 and HT-29 cells. Acquired-resistant LoVo/PR, Colo-205/PR and LoVo/TR cells have constitutively active Akt due to a M1043V mutation in the kinase activation loop of PIK3CA and Akt inhibitor resensitized these cells to MEK inhibitor. These results demonstrate that the overactivation of Akt plays a critical role in MEK inhibitor primary and acquired resistance and implicate combined Akt/MEK inhibition as a potentially useful treatment for RAS/BRAF-mutated colorectal cancer.

Identification of Tn antigen O-GalNAc-expressing glycoproteins in human carcinomas using novel anti-Tn recombinant antibodies.

The Tn antigen is a neoantigen abnormally expressed in many human carcinomas and expression correlates with metastasis and poor survival. To explore its biomarker potential, new antibodies are needed that specifically recognize this antigen in tumors. Here we generated two recombinant antibodies to the Tn antigen, Remab6 as a chimeric human IgG1 antibody and ReBaGs6 as a murine IgM antibody and characterized their specificities using multiple biochemical and biological approaches. Both Remab6 and ReBaGs6 recognize clustered Tn structures, but most importantly do not recognize glycoforms of human IgA1 that contain potential cross-reactive Tn antigen structures. In flow cytometry and immunofluorescence analyses, Remab6 recognizes human cancer cell lines expressing the Tn antigen, but not their Tn-negative counterparts. In immunohistochemistry (IHC), Remab6 stains many human cancers in tissue array format but rarely stains normal tissues and then mostly intracellularly. We used these antibodies to identify several unique Tn-containing glycoproteins in Tn-positive Colo205 cells, indicating their utility for glycoproteomics in future biomarker studies. Thus, recombinant Remab6 and ReBaGs6 are useful for biochemical characterization of cancer cells and IHC of tumors and represent promising tools for Tn biomarker discovery independently of recognition of IgA1.

Chemical characterization of main bioactive constituents in Paeonia ostii seed meal and GC-MS analysis of seed oil.

The seeds of tree peony (Paeonia ostii) are promulgated as emerging edible oil crops. However, biological properties of principal constituents of peony seeds were not well studied. Fifteen main constituents including suffruticosols A and B, trans-ε-viniferin, ampelopsin E, resveratrol, trans-resveratrol-4'-O-β-d-glucopyranoside, paeoniflorin, luteolin, luteolin-4'-O-β-d-glucopyranoside, apigenin, kaempferol, oleanic acid, betulinic acid, hederagenin, and caffeic acid were isolated and identified. Their cytotoxicity against human tumor cell lines (COLO205, HT-29, HepG2, AGS, and HL-60) were evaluated. Among them, trans-ε-viniferin showed the most potent cytotoxicity against HL-60 cells (IC50 5.6μM); ampelopsin E exhibited the most obvious antiproliferative properties on COLO205 (IC50 78.1μM) and HT-29 (IC50 4.2μM) cells, and betulinic acid showed the strongest growth inhibitory effects on HepG2 (IC50 6.6μM) and AGS (IC50 5.4μM) cells. Three enzymes (tyronsinase, α-glucosidase, and acetylcholinesterase) inhibitory activities of 12 compounds were also screened. Stilbene compounds, especially suffruticosols A and B, showed a significant inhibitory activity on all three enzymes. PRACTICAL APPLICATIONS: The cytotoxicity of 15 main constituents from peony seeds against COLO205, HT-29, HepG2, AGS, and HL-60 cells were evaluated. Among them, trans-ε-viniferin showed the most potent cytotoxicity against HL-60 cells (IC50 5.6μM); ampelopsin E exhibited the most obvious antiproliferative properties on COLO205 (IC50 78.1μM) and HT-29 (IC50 4.2μM) cells, and betulinic acid showed the strongest growth inhibitory effects on HepG2 (IC50 6.6μM) and AGS (IC50 5.4μM) cells. Collectively, these results suggested that Paeonia ostii seed (POS) extracts are potential candidates for anticancer agents.

BZW2 promotes the malignant progression of colorectal cancer via activating the ERK/MAPK pathway.

Colorectal cancer (CRC) is one of the most prevalent malignant solid cancers worldwide involving the dysregulation of multiple signaling molecules. However, the role and corresponding mechanism of basic leucine zipper and W2 domains 2 (BZW2) in CRC development, to our knowledge, has not been reported. We found BZW2 was overexpressed in human CRC tissues compared with that in paired adjacent colorectal samples. BZW2 overexpression was closely associated with tumor T stage (p = .030), metastatic lymph nodes (p = .037), TNM stage (p = .018) and the worse prognosis of CRC patients (p = .009). Moreover, BZW2 was an independent disadvantage prognostic factor (p = .031). BZW2 also showed an increased expression in different invasive CRC cell lines. Its silencing and overexpression diminished and increased cell proliferation, invasion, and migration in Colo205 and HCT116 cells via specifically activating of extracellular-signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) signaling. Moreover, ERK/MAPK inhibitor PD98059 reverse the enhancement of cell proliferation, invasion, and migration in BZW2 overexpressing HCT116 cells. BZW2 silencing also inhibited subcutaneous tumors growth and p-ERK expression in vivo. BZW2 promotes the malignant progression of CRC via activating ERK/MAPK signaling, which provided a promising gene target therapy for CRC.

The membrane effects of melittin on gastric and colorectal cancer.

The cytotoxic effects of melittin, a bee-venom peptide, have been widely studied towards cancer cells. Typically, these studies have examined the effect of melittin over extended-time courses (6–24 hours), meaning that immediate cellular interactions have been overlooked. In this work, we demonstrate the rapid effects of melittin on both gastric and colorectal cancer, specifically AGS, COLO205 and HCT-15 cell lines, over a period of 15 minutes. Melittin exhibited a dose dependent effect at 4 hours of treatment, with complete cellular death occurring at the highest dose of 20 μg/mL. Interestingly, when observed at shorter time points, melittin induced cellular changes within seconds; membrane damage was observed as swelling, breakage or blebbing. High-resolution imaging revealed treated cells to be compromised, showing clear change in cellular morphology. After 1 minute of melittin treatment, membrane changes were observed, and intracellular material could be seen expelled from the cells. Overall, these results enhance our understanding of the fast acting anti-cancer effects of melittin.