Depletion of ABCC10 in Human Colorectal Caco-2 Cancer Cells Using the CRISPR-Cas9 System
type: CreativeWork
description: Colorectal cancer (CRC) is one of the most prominent cancers detected worldwide. Cancer treatment with standard chemotherapy has led to improved CRC patients' overall survival rate. However, in more than 40% of patients, the standard chemotherapy achieves only a brief or no tumour response or unexpected toxicity, leading to therapeutic failure. Accumulating evidence identified a family of cell membrane transporter proteins (ABC transporters) that transport various chemotherapeutics across cell membranes and, in this way, may control the cellular accumulation, antitumour activity, and toxicity of front-line therapeutics. Multi-drug resistant proteins (MRPs) belong to the ABCC subfamily, and overexpression of ABCC transporters in tumour cells results in multi-drug resistance, which is when tumour cells are able to resist the antitumour cytotoxicity of a range of structurally diverse anticancer drugs (e.g.: Oxaliplatin, 5-Fluorouracil). MRP7 (encoded by ABCC10 gene) has recently been reported to confer resistance to the antitumor drug docetaxel and oxaliplatin. However, previous ABCC10 work was undertaken by using siRNA gene knockdown and pharmacological inhibitors, but those experiments were confounded by the off-target effects. Therefore, we hypothesized that CRISPR-cas9 gene editing technology could be used to achieve precise and permanent disruption of the functional expression of ABCC10 transporter protein and reverse the drug resistance in human colorectal cancer Caco-2 cells. To test our hypothesis, Caco-2 cells were transfected with ABCC10 guideRNA-Cas9 protein ribonucleoprotein complexes through liposome-mediated delivery. The efficiency of the ABCC10 gene disruption was analysed using the T7 Endoneuclease1 cleavage efficiency assay. The single clones of ABCC10 knockouts were obtained from limiting dilution. The variation of the ABCC10 (MRP7) protein expression in the wild Caco-2 cells and the transfected clones was assessed following western blot analysis. Th…
abstract: Colorectal cancer (CRC) is one of the most prominent cancers detected worldwide. Cancer treatment with standard chemotherapy has led to improved CRC patients' overall survival rate. However, in more than 40% of patients, the standard chemotherapy achieves only a brief or no tumour response or unexpected toxicity, leading to therapeutic failure. Accumulating evidence identified a family of cell membrane transporter proteins (ABC transporters) that transport various chemotherapeutics across cell membranes and, in this way, may control the cellular accumulation, antitumour activity, and toxicity of front-line therapeutics. Multi-drug resistant proteins (MRPs) belong to the ABCC subfamily, and overexpression of ABCC transporters in tumour cells results in multi-drug resistance, which is when tumour cells are able to resist the antitumour cytotoxicity of a range of structurally diverse anticancer drugs (e.g.: Oxaliplatin, 5-Fluorouracil). MRP7 (encoded by ABCC10 gene) has recently been reported to confer resistance to the antitumor drug docetaxel and oxaliplatin. However, previous ABCC10 work was undertaken by using siRNA gene knockdown and pharmacological inhibitors, but those experiments were confounded by the off-target effects. Therefore, we hypothesized that CRISPR-cas9 gene editing technology could be used to achieve precise and permanent disruption of the functional expression of ABCC10 transporter protein and reverse the drug resistance in human colorectal cancer Caco-2 cells. To test our hypothesis, Caco-2 cells were transfected with ABCC10 guideRNA-Cas9 protein ribonucleoprotein complexes through liposome-mediated delivery. The efficiency of the ABCC10 gene disruption was analysed using the T7 Endoneuclease1 cleavage efficiency assay. The single clones of ABCC10 knockouts were obtained from limiting dilution. The variation of the ABCC10 (MRP7) protein expression in the wild Caco-2 cells and the transfected clones was assessed following western blot analysis. Th…
author: Narasinghe, Sajeevani
date published: 2024
date modified: 2026-07-27T18:46:03Z
provider: AUT University
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: AUT University