An improved 3-(4,5-dmethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl )-2H-tetrazolium proliferation assay to overcome the interference of hydralazine
- Wang, Yutang, Nguyen, Dinh, Yang, Guang, Anesi, Jack, Chai, Zhonglin, Charchar, Fadi, Golledge, Jonathan
- Authors: Wang, Yutang , Nguyen, Dinh , Yang, Guang , Anesi, Jack , Chai, Zhonglin , Charchar, Fadi , Golledge, Jonathan
- Date: 2020
- Type: Text , Journal article
- Relation: Assay and Drug Development Technologies Vol. 18, no. 8 (Dec 2020), p. 379-384
- Relation: https://purl.org/au-research/grants/nhmrc/1062671
- Full Text:
- Reviewed:
- Description: The MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay is one of the most commonly used assays to assess cell proliferation and cytotoxicity, but is subject to interference by testing compounds. Hydralazine, an antihypertensive drug, is commonly investigated in multiple fields such as heart failure, cancer, and blood pressure research. This study reported interference of the MTS assay by hydralazine and a simple modification overcoming this interference. Vascular smooth muscle cells were cultured in the presence or absence of hydralazine (0, 10, 50,100, and 500 mu M) for 2 or 24 h. Cell numbers were analyzed using MTS, trypan blue exclusion, or microscopic assays. A modified version of the standard MTS assay was established, in which an additional step was added replacing the test medium, containing hydralazine, with fresh culture medium immediately before the addition of the MTS reagent. Culture with hydralazine at concentrations of 50, 100, and 500 mu M for 2 h increased absorbance (p< 0.05) in the standard MTS assay, whereas microscopy suggested no change in cell numbers. Culture with 500 mu m hydralazine for 24 h increased absorbance (p< 0.05) in the standard MTS assay, however, trypan blue exclusion and microscopy suggested a decrease in cell numbers. In a cell-free system, hydralazine (>= 10 mu M) increased absorbance in a concentration-dependent manner. The modified MTS assay produced results consistent with trypan blue exclusion and microscopy. In conclusion, a simple modification of the standard MTS assay overcame the interference of hydralazine and may be useful to avoid interference from other tested compounds.
- Authors: Wang, Yutang , Nguyen, Dinh , Yang, Guang , Anesi, Jack , Chai, Zhonglin , Charchar, Fadi , Golledge, Jonathan
- Date: 2020
- Type: Text , Journal article
- Relation: Assay and Drug Development Technologies Vol. 18, no. 8 (Dec 2020), p. 379-384
- Relation: https://purl.org/au-research/grants/nhmrc/1062671
- Full Text:
- Reviewed:
- Description: The MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay is one of the most commonly used assays to assess cell proliferation and cytotoxicity, but is subject to interference by testing compounds. Hydralazine, an antihypertensive drug, is commonly investigated in multiple fields such as heart failure, cancer, and blood pressure research. This study reported interference of the MTS assay by hydralazine and a simple modification overcoming this interference. Vascular smooth muscle cells were cultured in the presence or absence of hydralazine (0, 10, 50,100, and 500 mu M) for 2 or 24 h. Cell numbers were analyzed using MTS, trypan blue exclusion, or microscopic assays. A modified version of the standard MTS assay was established, in which an additional step was added replacing the test medium, containing hydralazine, with fresh culture medium immediately before the addition of the MTS reagent. Culture with hydralazine at concentrations of 50, 100, and 500 mu M for 2 h increased absorbance (p< 0.05) in the standard MTS assay, whereas microscopy suggested no change in cell numbers. Culture with 500 mu m hydralazine for 24 h increased absorbance (p< 0.05) in the standard MTS assay, however, trypan blue exclusion and microscopy suggested a decrease in cell numbers. In a cell-free system, hydralazine (>= 10 mu M) increased absorbance in a concentration-dependent manner. The modified MTS assay produced results consistent with trypan blue exclusion and microscopy. In conclusion, a simple modification of the standard MTS assay overcame the interference of hydralazine and may be useful to avoid interference from other tested compounds.
Knock down of TIMP-2 by siRNA and CRISPR/Cas9 mediates diverse cellular reprogramming of metastasis and chemosensitivity in ovarian cancer
- Escalona, Ruth, Chu, Simon, Kadife, Elif, Kelly, Jason, Kannourakis, George, Findlay, Jock, Ahmed, Nuzhat
- Authors: Escalona, Ruth , Chu, Simon , Kadife, Elif , Kelly, Jason , Kannourakis, George , Findlay, Jock , Ahmed, Nuzhat
- Date: 2022
- Type: Text , Journal article
- Relation: Cancer Cell International Vol. 22, no. 1 (2022), p.
- Full Text:
- Reviewed:
- Description: Background: The endogenous tissue inhibitor of metalloproteinase-2 (TIMP-2), through its homeostatic action on certain metalloproteinases, plays a vital role in remodelling extracellular matrix (ECM) to facilitate cancer progression. This study investigated the role of TIMP-2 in an ovarian cancer cell line in which the expression of TIMP-2 was reduced by either siRNA or CRISPR/Cas9. Methods: OVCAR5 cells were transiently and stably transfected with either single or pooled TIMP-2 siRNAs (T2-KD cells) or by CRISPR/Cas9 under the influence of two distinct guide RNAs (gRNA1 and gRNA2 cell lines). The expression of different genes was analysed at the mRNA level by quantitative real time PCR (qRT-PCR) and at the protein level by immunofluorescence (IF) and western blot. Proliferation of cells was investigated by 5-Ethynyl-2′-deoxyuridine (EdU) assay or staining with Ki67. Cell migration/invasion was determined by xCELLigence. Cell growth in vitro was determined by 3D spheroid cultures and in vivo by a mouse xenograft model. Results: Approximately 70–90% knock down of TIMP-2 expression were confirmed in T2-KD, gRNA1 and gRNA2 OVCAR5 ovarian cancer cells at the protein level. T2-KD, gRNA1 and gRNA2 cells exhibited a significant downregulation of MMP-2 expression, but concurrently a significant upregulation in the expression of membrane bound MMP-14 compared to control and parental cells. Enhanced proliferation and invasion were exhibited in all TIMP-2 knocked down cells but differences in sensitivity to paclitaxel (PTX) treatment were observed, with T2-KD cells and gRNA2 cell line being sensitive, while the gRNA1 cell line was resistant to PTX treatment. In addition, significant differences in the growth of gRNA1 and gRNA2 cell lines were observed in in vitro 3D cultures as well as in an in vivo mouse xenograft model. Conclusions: Our results suggest that the inhibition of TIMP-2 by siRNA and CRISPR/Cas-9 modulate the expression of MMP-2 and MMP-14 and reprogram ovarian cancer cells to facilitate proliferation and invasion. Distinct disparities in in vitro chemosensitivity and growth in 3D culture, and differences in tumour burden and invasion to proximal organs in a mouse model imply that selective suppression of TIMP-2 expression by siRNA or CRISPR/Cas-9 alters important aspects of metastasis and chemosensitivity in ovarian cancer. © 2022, The Author(s).
- Authors: Escalona, Ruth , Chu, Simon , Kadife, Elif , Kelly, Jason , Kannourakis, George , Findlay, Jock , Ahmed, Nuzhat
- Date: 2022
- Type: Text , Journal article
- Relation: Cancer Cell International Vol. 22, no. 1 (2022), p.
- Full Text:
- Reviewed:
- Description: Background: The endogenous tissue inhibitor of metalloproteinase-2 (TIMP-2), through its homeostatic action on certain metalloproteinases, plays a vital role in remodelling extracellular matrix (ECM) to facilitate cancer progression. This study investigated the role of TIMP-2 in an ovarian cancer cell line in which the expression of TIMP-2 was reduced by either siRNA or CRISPR/Cas9. Methods: OVCAR5 cells were transiently and stably transfected with either single or pooled TIMP-2 siRNAs (T2-KD cells) or by CRISPR/Cas9 under the influence of two distinct guide RNAs (gRNA1 and gRNA2 cell lines). The expression of different genes was analysed at the mRNA level by quantitative real time PCR (qRT-PCR) and at the protein level by immunofluorescence (IF) and western blot. Proliferation of cells was investigated by 5-Ethynyl-2′-deoxyuridine (EdU) assay or staining with Ki67. Cell migration/invasion was determined by xCELLigence. Cell growth in vitro was determined by 3D spheroid cultures and in vivo by a mouse xenograft model. Results: Approximately 70–90% knock down of TIMP-2 expression were confirmed in T2-KD, gRNA1 and gRNA2 OVCAR5 ovarian cancer cells at the protein level. T2-KD, gRNA1 and gRNA2 cells exhibited a significant downregulation of MMP-2 expression, but concurrently a significant upregulation in the expression of membrane bound MMP-14 compared to control and parental cells. Enhanced proliferation and invasion were exhibited in all TIMP-2 knocked down cells but differences in sensitivity to paclitaxel (PTX) treatment were observed, with T2-KD cells and gRNA2 cell line being sensitive, while the gRNA1 cell line was resistant to PTX treatment. In addition, significant differences in the growth of gRNA1 and gRNA2 cell lines were observed in in vitro 3D cultures as well as in an in vivo mouse xenograft model. Conclusions: Our results suggest that the inhibition of TIMP-2 by siRNA and CRISPR/Cas-9 modulate the expression of MMP-2 and MMP-14 and reprogram ovarian cancer cells to facilitate proliferation and invasion. Distinct disparities in in vitro chemosensitivity and growth in 3D culture, and differences in tumour burden and invasion to proximal organs in a mouse model imply that selective suppression of TIMP-2 expression by siRNA or CRISPR/Cas-9 alters important aspects of metastasis and chemosensitivity in ovarian cancer. © 2022, The Author(s).
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