scholarly journals Targeting REV7 effectively reverses 5-FU and oxaliplatin resistance in colorectal cancer

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Xianjun Sun ◽  
Wenhou Hou ◽  
Xin Liu ◽  
Jie Chai ◽  
Hongliang Guo ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Viability ◽  
Research Effort ◽  
Translesion Synthesis ◽  
Xenograft Model ◽  
Mice Model ◽  
Cell Viability Assay ◽  
Viability Assay ◽  
Xenograft Mice Model

Abstract Background Despite an enormous research effort, patients diagnosed with advanced colorectal cancer (CRC) still have low prognosis after surgical resection and chemotherapy. The major obstacle for CRC treatment is chemoresistance to front line anti-cancer drugs, such as 5-fluorouracil (5-FU) and oxaliplatin. However, the mechanism of chemoresistance to these drugs remains unclear. Methods Cell viability to 5-FU and oxaliplatin was measured by the CellTiter-Glo® 2.0 Cell Viability Assay. The endogenous REV7 protein in CRC cells was detected by western blotting. The translesion synthesis (TLS) events were measured by plasmid-based TLS efficiency assay. Cell apoptosis was evaluated by caspase3/7 activity assay. The in vivo tumor progression was analyzed by HT29 xenograft mice model. Results In this study, we found that expression of REV7, which is a key component of translesion synthesis (TLS) polymerase ζ (POL ζ), is significantly increased in both 5-FU and oxaliplatin resistant CRC cells. TLS efficiency analysis revealed that upregulated REV7 protein level results in enhanced TLS in response to 5-FU and oxaliplatin. Importantly, inhibition of REV7 by CRISPR/Cas9 knockout exhibited significant synergy with 5-FU and oxaliplatin in cell culture and murine xenograft model. Conclusion These results suggest that combination of REV7 deficiency and 5-FU or oxaliplatin has strong inhibitory effects on CRC cells and identified REV7 as a promising target for chemoresistant CRC treatment.

scholarly journals β-Adrenergic Receptor Inhibitor and Oncolytic Herpesvirus Combination Therapy Shows Enhanced Antitumoral and Antiangiogenic Effects on Colorectal Cancer

2021 ◽  
Vol 12 ◽  
Author(s):  
Jiali Hu ◽  
Cuiyu Chen ◽  
Ruitao Lu ◽  
Yu Zhang ◽  
Yang Wang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Oncolytic Virus ◽  
Caspase 3 ◽  
Therapeutic Potential ◽  
Tumor Regression ◽  
Oncolytic Viruses ◽  
Cell Viability Assay ◽  
Viability Assay

Oncolytic viruses (OVs) are considered a promising therapeutic alternative for cancer. However, despite the development of novel OVs with improved efficacy and tumor selectivity, their limited efficacy as monotherapeutic agents remains a significant challenge. This study extended our previously observed combination effects of propranolol, a nonselective β-blocker, and the T1012G oncolytic virus into colorectal cancer models. A cell viability assay showed that cotreatment could induce synergistic killing effects on human and murine colorectal cell lines. Moreover, cotreatment caused sustained tumor regression compared with T1012G monotherapy or propranolol monotherapy in human HCT116 and murine MC38 tumor models. The propranolol activity was not via a direct effect on viral replication in vitro or in vivo. Western blotting showed that cotreatment significantly enhanced the expression of cleaved caspase-3 in HCT116 and MC38 cells compared with the propranolol or T1012G alone. In addition, propranolol or T1012G treatment induced a 35.06% ± 0.53% or 35.49% ± 2.68% reduction in VEGF secretion in HUVECs (p < 0.01/p < 0.01). Cotreatment further inhibited VEGF secretion compared with the monotherapies (compared with propranolol treatment: 75.06% ± 1.50% decrease, compared with T1012G treatment: 74.91% ± 0.68%; p＜0.001, p < 0.001). Consistent with the in vitro results, in vivo data showed that cotreatment could reduce Ki67 and enhance cleaved caspase 3 and CD31 expression in human HCT116 and murine MC38 xenografts. In summary, β-blockers could improve the therapeutic potential of OVs by enhancing oncolytic virus-mediated killing of colorectal cancer cells and colorectal tumors.

scholarly journals Polyphenolic Fraction Obtained From Thalassia testudinum Marine Plant and Thalassiolin B Exert Cytotoxic Effects in Colorectal Cancer Cells and Arrest Tumor Progression in a Xenograft Mouse Model

2020 ◽  
Vol 11 ◽  
Author(s):  
Livan Delgado-Roche ◽  
Kethia González ◽  
Fernando Mesta ◽  
Beatriz Couder ◽  
Zaira Tavarez ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Antitumor Activity ◽  
Thalassia Testudinum ◽  
Ros Generation ◽  
Mice Model ◽  
Marine Plant ◽  
Colorectal Cancer Cells ◽  
Xenograft Mice Model ◽  
Xenograft Mice

Marine plants are important sources of pharmacologically active metabolites. The aim of the present work was to evaluate the cytotoxic and antitumor activity of a polyphenolic fraction obtained from Thalassia testudinum marine plant and thalassiolin B in human colorectal cancer cells. Human cancer cell lines, including HCT15, HCT116, SW260, and HT29 were treated with tested products for cytotoxicity evaluation by crystal violet assay. The potential proapoptotic effect of these natural products was assessed by flow cytometry in HCT15 cells at 48 h using Annexin V-FITC/propidium iodide. In addition, reactive oxygen species (ROS) generation was measured by fluorescence using DCFH-DA staining, and sulfhydryl concentration by spectrophotometry. The in vivo antitumor activity of the polyphenolic fraction (25 mg/kg) was evaluated in a xenograft model in nu/nu mice. In vivo proapoptotic effect was also evaluated by immunohistochemistry using anti-caspase 3 and anti-Bcl-2 antibodies. The results showed that tested products exert colorectal cancer cell cytotoxicity. Besides, the tested products induced a significant increase (p < 0.05) of intracellular ROS generation, and a depletion of sulfhydryl concentration in HCT15 cells. The polyphenolic fraction arrested tumor growth and induced apoptosis in the xenograft mice model. These results demonstrate the cytotoxic activity of T. testudinum metabolites associated, at least, with ROS overproduction and pro-apoptotic effects. Here we demonstrated for the first time the antitumor activity of a T. testudinum polar extract in a xenograft mice model. These results suggest the potential use of T. testudinum marine plant metabolites as adjuvant treatment in cancer therapy.

Do We have a Satisfactory Cell Viability Assay? Review of the Currently Commercially-Available Assays

2020 ◽  
Vol 17 (1) ◽  
pp. 2-22 ◽  
Author(s):  
Abdel-Baset Halim
Keyword(s):  
Cell Viability ◽  
Data Interpretation ◽  
Positive Control ◽  
Live Cells ◽  
Proof Of Concept ◽  
Cell Viability Assay ◽  
Viability Assay ◽  
Current Cell ◽  
Dying Cells ◽  
Differential Permeability

:Cell-based assays are an important part of the drug discovery process and clinical research. One of the main hurdles is to design sufficiently robust assays with adequate signal to noise parameters while maintaining the inherent physiology of the cells and not interfering with the pharmacology of target being investigated.:A plethora of assays that assess cell viability (or cell heath in general) are commercially available and can be classified under different categories according to their concepts and principle of reactions. The assays are valuable tools, however, suffer from a large number of limitations. Some of these limitations can be procedural or operational, but others can be critical as those related to a poor concept or the lack of proof of concept of an assay, e.g. those relying on differential permeability of dyes in-and-out of viable versus compromised cell membranes. While the assays can differentiate between dead and live cells, most, if not all, of them can just assess the relative performance of cells rather than providing a clear distinction between healthy and dying cells. The possible impact of relatively high molecular weight dyes, used in most of the assay, on cell viability has not been addressed. More innovative assays are needed, and until better alternatives are developed, setup of current cell-based studies and data interpretation should be made with the limitations in mind. Negative and positive control should be considered whenever feasible. Also, researchers should use more than one orthogonal method for better assessment of cell health.

scholarly journals Oncogenic lncRNA ZNF561-AS1 is essential for colorectal cancer proliferation and survival through regulation of miR-26a-3p/miR-128-5p-SRSF6 axis

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Zizhen Si ◽  
Lei Yu ◽  
Haoyu Jing ◽  
Lun Wu ◽  
Xidi Wang
Keyword(s):  
Colorectal Cancer ◽  
Rna Binding ◽  
Xenograft Model ◽  
Luciferase Reporter ◽  
Cancer Proliferation ◽  
Mouse Xenograft ◽  
Mouse Xenograft Model

Abstract Background Long non-coding RNAs (lncRNA) are reported to influence colorectal cancer (CRC) progression. Currently, the functions of the lncRNA ZNF561 antisense RNA 1 (ZNF561-AS1) in CRC are unknown. Methods ZNF561-AS1 and SRSF6 expression in CRC patient samples and CRC cell lines was evaluated through TCGA database analysis, western blot along with real-time PCR. SRSF6 expression in CRC cells was also examined upon ZNF561-AS1 depletion or overexpression. Interaction between miR-26a-3p, miR-128-5p, ZNF561-AS1, and SRSF6 was examined by dual luciferase reporter assay, as well as RNA binding protein immunoprecipitation (RIP) assay. Small interfering RNA (siRNA) mediated knockdown experiments were performed to assess the role of ZNF561-AS1 and SRSF6 in the proliferative actives and apoptosis rate of CRC cells. A mouse xenograft model was employed to assess tumor growth upon ZNF561-AS1 knockdown and SRSF6 rescue. Results We find that ZNF561-AS1 and SRSF6 were upregulated in CRC patient tissues. ZNF561-AS1 expression was reduced in tissues from treated CRC patients but upregulated in CRC tissues from relapsed patients. SRSF6 expression was suppressed and enhanced by ZNF561-AS1 depletion and overexpression, respectively. Mechanistically, ZNF561-AS1 regulated SRSF6 expression by sponging miR-26a-3p and miR-128-5p. ZNF561-AS1-miR-26a-3p/miR-128-5p-SRSF6 axis was required for CRC proliferation and survival. ZNF561-AS1 knockdown suppressed CRC cell proliferation and triggered apoptosis. ZNF561-AS1 depletion suppressed the growth of tumors in a model of a nude mouse xenograft. Similar observations were made upon SRSF6 depletion. SRSF6 overexpression reversed the inhibitory activities of ZNF561-AS1 in vivo, as well as in vitro. Conclusion In summary, we find that ZNF561-AS1 promotes CRC progression via the miR-26a-3p/miR-128-5p-SRSF6 axis. This study reveals new perspectives into the role of ZNF561-AS1 in CRC.

scholarly journals EXTH-46. ARTIFICIAL INTELLIGENCE-BASED IDENTIFICATION OF COMBINED VANDETANIB AND EVEROLIMUS IN THE TREATMENT OF ACVR1-MUTANT DIFFUSE INTRINSIC PONTINE GLIOMA

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii97-ii97
Author(s):  
Diana Carvalho ◽  
Peter Richardson ◽  
Nagore Gene Olaciregui ◽  
Reda Stankunaite ◽  
Cinzia Emilia Lavarino ◽  
...  
Keyword(s):  
Artificial Intelligence ◽  
Cell Viability ◽  
Xenograft Model ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Pontine Glioma ◽  
Serine Threonine Kinase ◽  
Orthotopic Xenografts ◽  
Extended Survival

Abstract Somatic mutations in ACVR1, encoding the serine/threonine kinase ALK2 receptor, are found in a quarter of children with the currently incurable brain tumour diffuse intrinsic pontine glioma (DIPG). Treatment of ACVR1-mutant DIPG patient-derived models with multiple inhibitor chemotypes leads to a reduction in cell viability in vitro and extended survival in orthotopic xenografts in vivo, though there are currently no specific ACVR1 inhibitors licensed for DIPG. Using an Artificial Intelligence-based platform to search for approved compounds which could be used to treat ACVR1-mutant DIPG, the combination of vandetanib and everolimus was identified as a possible therapeutic approach. Vandetanib, an approved inhibitor of VEGFR/RET/EGFR, was found to target ACVR1 (Kd=150nM) and reduce DIPG cell viability in vitro, but has been trialed in DIPG patients with limited success, in part due to an inability to cross the blood-brain-barrier. In addition to mTOR, everolimus inhibits both ABCG2 (BCRP) and ABCB1 (P-gp) transporter, and was synergistic in DIPG cells when combined with vandetanib in vitro. This combination is well-tolerated in vivo, and significantly extended survival and reduced tumour burden in an orthotopic ACVR1-mutant patient-derived DIPG xenograft model. Based on these preclinical data, three patients with ACVR1-mutant DIPG were treated with vandetanib and everolimus. These cases may inform on the dosing and the toxicity profile of this combination for future clinical studies. This bench-to-bedside approach represents a rapidly translatable therapeutic strategy in children with ACVR1 mutant DIPG.

scholarly journals PRMT1 enhances oncogenic arginine methylation of NONO in colorectal cancer

Oncogene ◽  
2021 ◽  
Author(s):  
Xin-Ke Yin ◽  
Yun-Long Wang ◽  
Fei Wang ◽  
Wei-Xing Feng ◽  
Shao-Mei Bai ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Locally Advanced ◽  
Xenograft Model ◽  
Arginine Methylation ◽  
Mass Spectrometry Analysis ◽  
Migration And Invasion ◽  
Poor Overall Survival ◽  
Potential Marker

AbstractArginine methylation is an important posttranslational modification catalyzed by protein arginine methyltransferases (PRMTs). However, the role of PRMTs in colorectal cancer (CRC) progression is not well understood. Here we report that non-POU domain-containing octamer-binding protein (NONO) is overexpressed in CRC tissue and is a potential marker for poor prognosis in CRC patients. NONO silencing resulted in decreased proliferation, migration, and invasion of CRC cells, whereas overexpression had the opposite effect. In a xenograft model, tumors derived from NONO-deficient CRC cells were smaller than those derived from wild-type (WT) cells, and PRMT1 inhibition blocked CRC xenograft progression. A mass spectrometry analysis indicated that NONO is a substrate of PRMT1. R251 of NONO was asymmetrically dimethylated by PRMT1 in vitro and in vivo. Compared to NONO WT cells, NONO R251K mutant-expressing CRC cells showed reduced proliferation, migration, and invasion, and PRMT1 knockdown or pharmacological inhibition abrogated the malignant phenotype associated with NONO asymmetric dimethylation in both KRAS WT and mutant CRC cells. Compared to adjacent normal tissue, PRMT1 was highly expressed in the CRC zone in clinical specimens, which was correlated with poor overall survival in patients with locally advanced CRC. These results demonstrate that PRMT1-mediated methylation of NONO at R251 promotes CRC growth and metastasis, and suggest that PRMT1 inhibition may be an effective therapeutic strategy for CRC treatment regardless of KRAS mutation status.

scholarly journals Human Recombinant Fab Fragment Neutralizes Shiga Toxin Type 2 Cytotoxic Effects in vitro and in vivo

Toxins ◽  
2018 ◽  
Vol 10 (12) ◽  
pp. 508 ◽  
Author(s):  
Daniela Luz ◽  
Maria Amaral ◽  
Flavia Sacerdoti ◽  
Alan Bernal ◽  
Wagner Quintilio ◽  
...  
Keyword(s):  
Shiga Toxin ◽  
Lethal Dose ◽  
Dependent Manner ◽  
Fab Fragment ◽  
Cell Viability Assay ◽  
Cytotoxic Effects ◽  
Morphological Alterations ◽  
Viability Assay

Shiga toxin (Stx) producing Escherichia coli (STEC) is responsible for causing hemolytic uremic syndrome (HUS), a life-threatening thrombotic microangiopathy characterized by thrombocytopenia, hemolytic anemia, and acute renal failure after bacterially induced hemorrhagic diarrhea. Until now, there has been neither an effective treatment nor method of prevention for the deleterious effects caused by Stx intoxication. Antibodies are well recognized as affinity components of therapeutic drugs; thus, a previously obtained recombinant human FabC11:Stx2 fragment was used to neutralize Stx2 in vitro in a Vero cell viability assay. Herein, we demonstrated that this fragment neutralized, in a dose-dependent manner, the cytotoxic effects of Stx2 on human glomerular endothelial cells, on human proximal tubular epithelial cells, and prevented the morphological alterations induced by Stx2. FabC11:Stx2 protected mice from a lethal dose of Stx2 by toxin-antibody pre-incubation. Altogether, our results show the ability of a new encouraging molecule to prevent Stx-intoxication symptoms during STEC infection.

scholarly journals Promising biocompatible hydrogels of crosslinked polyelectrolytes for biomedical applications

2017 ◽  
Vol 3 (2) ◽  
pp. 695-698
Author(s):  
Andreas Brietzke ◽  
Christian von der Ehe ◽  
Sabine Illner ◽  
Claudia Matschegewski ◽  
Niels Grabow ◽  
...  
Keyword(s):  
Aqueous Solution ◽  
Ionic Liquids ◽  
Cell Viability ◽  
Biomedical Applications ◽  
High Potential ◽  
Cell Viability Assay ◽  
Mouse Fibroblasts ◽  
Viability Assay ◽  
Intelligent Implant ◽  
L929 Mouse

AbstractFor the development of intelligent implant systems hydrogels (HG) from crosslinked ionic liquids feature a high potential to be utilised as a drug depot. Biocompatibility of the HGs is one key prerequisite for biomedical applications. HGs were polymerised from a variety of different ionic monomers based on methacrylate, methacrylamide, styrene or vinyl imidazolium derivatives in aqueous solution. N,N'-methylenebisacrylamide was used as crosslinker. CellQuanti-Blue™ Cell Viability Assay Kit was implemented to proof viability of L929 mouse fibroblasts. The predominant part of the HG eluates generated only a marginal reduction of less than 15% cell viability at 100% eluate concentration. This underlines the excellent suitability of these HGs for biomedical applications and revealed some promising candidates for the development of drug depots for implants.

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