TIMP-2 regulates proliferation, invasion and STAT3-mediated cancer stem cell-dependent chemoresistance in ovarian cancer cells
- Escalona, Ruth, Bilandzic, Maree, Western, Patrick, Kadife, Elif, Kannourakis, George, Findlay, Jock, Ahmed, Nuzhat
- Authors: Escalona, Ruth , Bilandzic, Maree , Western, Patrick , Kadife, Elif , Kannourakis, George , Findlay, Jock , Ahmed, Nuzhat
- Date: 2020
- Type: Text , Journal article
- Relation: BMC Cancer Vol. 20, no. 1 (2020), p.
- Full Text:
- Reviewed:
- Description: Background: The metzincin family of metalloproteinases and the tissue inhibitors of metalloproteinases (TIMPs) are essential proteins required for biological processes during cancer progression. This study aimed to determine the role of TIMP-2 in ovarian cancer progression and chemoresistance by reducing TIMP-2 expression in vitro in Fallopian tube secretory epithelial (FT282) and ovarian cancer (JHOS2 and OVCAR4) cell lines. Methods: FT282, JHOS2 and OVCAR4 cells were transiently transfected with either single or pooled TIMP-2 siRNAs. The expression of different genes after TIMP-2 knock down (T2-KD) or in response to chemotherapy was determined at the mRNA level by quantitative real time PCR (qRT-PCR) and at the protein level by immunofluorescence. Sensitivity of the cell lines in response to chemotherapy after TIMP-2 knock down was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 5-Ethynyl-2′-deoxyuridine (EdU) assays. Cell invasion in response to TIMP-2 knockdown was determined by xCELLigence. Results: Sixty to 90 % knock down of TIMP-2 expression was confirmed in FT282, OVCAR4 and JHOS2 cell lines at the mRNA and protein levels. TIMP-2 knock down did not change the mRNA expression of TIMP-1 or TIMP-3. However, a significant downregulation of MMP-2 in T2-KD cells occurred at both the protein and activation levels, compared to Control (Cont; scrambled siRNA) and Parental cells (P, transfection reagent only). In contrast, membrane bound MT1-MMP protein levels were significantly upregulated in T2-KD compared to Cont and P cells. T2-KD cells exhibited enhanced proliferation and increased sensitivity to cisplatin and paclitaxel treatments. Enhanced invasion was observed in the T2-KD-JOSH2 and OVCAR4 cells but not in T2-KD-FT282 cells. Treatment with cisplatin or paclitaxel significantly elevated the expression of TIMP-2 in Cont cells but not in T2-KD cells, consistent with significantly elevated expression of chemoresistance and CSC markers and activation of STAT3. Furthermore, a potent inhibitor of STAT3 activation, Momelotinib, suppressed chemotherapy-induced activation of P-STAT3 in OVCAR4 cells with concomitant reductions in the expression of chemoresistance genes and CSC markers. Conclusions: The above results suggest that TIMP-2 may have a novel role in ovarian cancer proliferation, invasion and chemoresistance. © 2020 The Author(s).
- Authors: Escalona, Ruth , Bilandzic, Maree , Western, Patrick , Kadife, Elif , Kannourakis, George , Findlay, Jock , Ahmed, Nuzhat
- Date: 2020
- Type: Text , Journal article
- Relation: BMC Cancer Vol. 20, no. 1 (2020), p.
- Full Text:
- Reviewed:
- Description: Background: The metzincin family of metalloproteinases and the tissue inhibitors of metalloproteinases (TIMPs) are essential proteins required for biological processes during cancer progression. This study aimed to determine the role of TIMP-2 in ovarian cancer progression and chemoresistance by reducing TIMP-2 expression in vitro in Fallopian tube secretory epithelial (FT282) and ovarian cancer (JHOS2 and OVCAR4) cell lines. Methods: FT282, JHOS2 and OVCAR4 cells were transiently transfected with either single or pooled TIMP-2 siRNAs. The expression of different genes after TIMP-2 knock down (T2-KD) or in response to chemotherapy was determined at the mRNA level by quantitative real time PCR (qRT-PCR) and at the protein level by immunofluorescence. Sensitivity of the cell lines in response to chemotherapy after TIMP-2 knock down was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 5-Ethynyl-2′-deoxyuridine (EdU) assays. Cell invasion in response to TIMP-2 knockdown was determined by xCELLigence. Results: Sixty to 90 % knock down of TIMP-2 expression was confirmed in FT282, OVCAR4 and JHOS2 cell lines at the mRNA and protein levels. TIMP-2 knock down did not change the mRNA expression of TIMP-1 or TIMP-3. However, a significant downregulation of MMP-2 in T2-KD cells occurred at both the protein and activation levels, compared to Control (Cont; scrambled siRNA) and Parental cells (P, transfection reagent only). In contrast, membrane bound MT1-MMP protein levels were significantly upregulated in T2-KD compared to Cont and P cells. T2-KD cells exhibited enhanced proliferation and increased sensitivity to cisplatin and paclitaxel treatments. Enhanced invasion was observed in the T2-KD-JOSH2 and OVCAR4 cells but not in T2-KD-FT282 cells. Treatment with cisplatin or paclitaxel significantly elevated the expression of TIMP-2 in Cont cells but not in T2-KD cells, consistent with significantly elevated expression of chemoresistance and CSC markers and activation of STAT3. Furthermore, a potent inhibitor of STAT3 activation, Momelotinib, suppressed chemotherapy-induced activation of P-STAT3 in OVCAR4 cells with concomitant reductions in the expression of chemoresistance genes and CSC markers. Conclusions: The above results suggest that TIMP-2 may have a novel role in ovarian cancer proliferation, invasion and chemoresistance. © 2020 The Author(s).
Introduced birds in urban remnant vegetation: Does remnant size really matter?
- Antos, Mark, Fitzsimons, John, Palmer, Grant, White, James
- Authors: Antos, Mark , Fitzsimons, John , Palmer, Grant , White, James
- Date: 2006
- Type: Text , Journal article
- Relation: Austral Ecology Vol. 31, no. 2 (2006), p. 254-261
- Full Text:
- Reviewed:
- Description: Introduced birds are a pervasive and dominant element of urban ecosystems. We examined the richness and relative abundance of introduced bird species in small (1-5 ha) medium (6-15 ha) and large (>15 ha) remnants of native vegetation within an urban matrix. Transects were surveyed during breeding and non-breeding seasons. There was a significant relationship between introduced species richness and remnant size with larger remnants supporting more introduced species. There was no significant difference in relative abundance of introduced species in remnants of different sizes. Introduced species, as a proportion of the relative abundance of the total avifauna (native and introduced species), did not vary significantly between remnants of differing sizes. There were significant differences in the composition of introduced bird species between the different remnant sizes, with large remnants supporting significantly different assemblages than medium and small remnants. Other variables also have substantial effects on the abundance of introduced bird species. The lack of significant differences in abundance between remnant sizes suggests they were all equally susceptible to invasion. No patches in the urban matrix are likely to be unaffected by introduced species. The effective long-term control of introduced bird species is difficult and resources may be better spent managing habitat in a way which renders it less suitable for introduced species (e.g. reducing areas of disturbed ground and weed dominated areas).
- Description: C1
- Description: 2003001638
- Authors: Antos, Mark , Fitzsimons, John , Palmer, Grant , White, James
- Date: 2006
- Type: Text , Journal article
- Relation: Austral Ecology Vol. 31, no. 2 (2006), p. 254-261
- Full Text:
- Reviewed:
- Description: Introduced birds are a pervasive and dominant element of urban ecosystems. We examined the richness and relative abundance of introduced bird species in small (1-5 ha) medium (6-15 ha) and large (>15 ha) remnants of native vegetation within an urban matrix. Transects were surveyed during breeding and non-breeding seasons. There was a significant relationship between introduced species richness and remnant size with larger remnants supporting more introduced species. There was no significant difference in relative abundance of introduced species in remnants of different sizes. Introduced species, as a proportion of the relative abundance of the total avifauna (native and introduced species), did not vary significantly between remnants of differing sizes. There were significant differences in the composition of introduced bird species between the different remnant sizes, with large remnants supporting significantly different assemblages than medium and small remnants. Other variables also have substantial effects on the abundance of introduced bird species. The lack of significant differences in abundance between remnant sizes suggests they were all equally susceptible to invasion. No patches in the urban matrix are likely to be unaffected by introduced species. The effective long-term control of introduced bird species is difficult and resources may be better spent managing habitat in a way which renders it less suitable for introduced species (e.g. reducing areas of disturbed ground and weed dominated areas).
- Description: C1
- Description: 2003001638
- Florentine, Singarayer, Westbrooke, Martin
- Authors: Florentine, Singarayer , Westbrooke, Martin
- Date: 2005
- Type: Text , Journal article
- Relation: Plant Protection Quarterly Vol. 20, no. 2 (2005), p. 42-45
- Full Text: false
- Reviewed:
- Description: The main objectives of this study were to investigate: (i) the distribution of exotic plant species in flooded and control (unflooded) areas in relation to a 1997 episodic flooding event and (ii) the influence of grazing in flooded and unflooded open and fenced plots on wed colonization.
- Description: 2003001069
The arid land invasive weed Nicotiana glauca R. Graham (Solanaceae) : Population and soil seed bank dynamics, seed germination patterns and seedling response to flood and drought
- Florentine, Singarayer, Westbrooke, Martin, Gosney, Kathleen, Ambrose, Graeme, O’Keefe, M.
- Authors: Florentine, Singarayer , Westbrooke, Martin , Gosney, Kathleen , Ambrose, Graeme , O’Keefe, M.
- Date: 2006
- Type: Text , Journal article
- Relation: Journal of Arid Environments Vol. 66, no. (2006), p. 218-230
- Full Text:
- Reviewed:
- Description: Disturbances in plant communities provide opportunities for weed germination, propagation, spread, and invasion. When the population density of a weed increases, fast-tracked and appropriate control management strategies are required. The objectives of this study were to: (i) examine the population and soil seed bank dynamics of Nicotiana glauca; (ii) compare the germination patterns of invasive N. glauca seeds collected from two states in Australia, and (iii) investigate the impact of a flood in September 1997 and subsequent drought on N. glauca seedlings. The density of N. glauca followed a steep positive increment during the sampling time (September 1999 to October 2004). The increment pattern was similar in flooded fenced and unfenced plots. Plant density increased over much of the observation period, but had declined to 80 and 432 stems ha−1, respectively, by October 2004. Stem density recorded in October 2004 along two transects radiating from the central point of the newly created lake demonstrated that a significant number of stems appeared to be dead. A soil seed bank study revealed that seed density varied significantly (p=0.0001) between flooded fenced (598.75±71) and flooded unfenced (327.5±66) plots. In contrast, no N. glauca seedlings were recruited from the soil collected from the control plots. Germination trials were undertaken on N. glauca seed collected from New South Wales. There was no significance difference detected between treatments light and temperature. Similarly, no interaction was found between light and temperature. A comparative study on seed germination patterns of N. glauca seeds collected from Ivanhoe, New South Wales, and the Flinders Ranges, South Australia, showed that temperature had a significant effect on N. glauca seed germination. The effect varied significantly with main variables (state, length and time) and also with different interactions, except state×light (p=0.3840). N. glauca seedlings exposed to flood were found to withstand partial flooding for at least 58 days. Under waterlogged conditions, the seedlings showed stem hypertrophy and produced adventitious roots. Only one seedling was found dead in the drought treatment. In conclusion, it is clear that N. glauca invaded the area after a rare flood event and began to function as a casual weed. Established seedlings in the field can withstand extreme ecological events such as flood and drought. Understanding the plants’ ecological characteristics through a study such as this at an early rather than late stage in the invasion will help us to take appropriate control measures for this species.
- Description: C1
- Description: 2003001616
- Authors: Florentine, Singarayer , Westbrooke, Martin , Gosney, Kathleen , Ambrose, Graeme , O’Keefe, M.
- Date: 2006
- Type: Text , Journal article
- Relation: Journal of Arid Environments Vol. 66, no. (2006), p. 218-230
- Full Text:
- Reviewed:
- Description: Disturbances in plant communities provide opportunities for weed germination, propagation, spread, and invasion. When the population density of a weed increases, fast-tracked and appropriate control management strategies are required. The objectives of this study were to: (i) examine the population and soil seed bank dynamics of Nicotiana glauca; (ii) compare the germination patterns of invasive N. glauca seeds collected from two states in Australia, and (iii) investigate the impact of a flood in September 1997 and subsequent drought on N. glauca seedlings. The density of N. glauca followed a steep positive increment during the sampling time (September 1999 to October 2004). The increment pattern was similar in flooded fenced and unfenced plots. Plant density increased over much of the observation period, but had declined to 80 and 432 stems ha−1, respectively, by October 2004. Stem density recorded in October 2004 along two transects radiating from the central point of the newly created lake demonstrated that a significant number of stems appeared to be dead. A soil seed bank study revealed that seed density varied significantly (p=0.0001) between flooded fenced (598.75±71) and flooded unfenced (327.5±66) plots. In contrast, no N. glauca seedlings were recruited from the soil collected from the control plots. Germination trials were undertaken on N. glauca seed collected from New South Wales. There was no significance difference detected between treatments light and temperature. Similarly, no interaction was found between light and temperature. A comparative study on seed germination patterns of N. glauca seeds collected from Ivanhoe, New South Wales, and the Flinders Ranges, South Australia, showed that temperature had a significant effect on N. glauca seed germination. The effect varied significantly with main variables (state, length and time) and also with different interactions, except state×light (p=0.3840). N. glauca seedlings exposed to flood were found to withstand partial flooding for at least 58 days. Under waterlogged conditions, the seedlings showed stem hypertrophy and produced adventitious roots. Only one seedling was found dead in the drought treatment. In conclusion, it is clear that N. glauca invaded the area after a rare flood event and began to function as a casual weed. Established seedlings in the field can withstand extreme ecological events such as flood and drought. Understanding the plants’ ecological characteristics through a study such as this at an early rather than late stage in the invasion will help us to take appropriate control measures for this species.
- Description: C1
- Description: 2003001616
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.
The Interleukin-11/IL-11 receptor promotes glioblastoma survival and invasion under glucose-starved conditions through enhanced glutaminolysis
- Stuart, Sarah, Bezawork-Geleta, Ayenachew, Areeb, Zammam, Gomez, Juliana, Tsui, Vanessa, Zulkifli, Ahmad, Paradiso, Lucia, Jones, Jordan, Nguyen, Hong, Putoczki, Tracy, Licciardi, Paul, Kannourakis, George, Morokoff, Andrew, Achuthan, Adrian, Luwor, Rodney
- Authors: Stuart, Sarah , Bezawork-Geleta, Ayenachew , Areeb, Zammam , Gomez, Juliana , Tsui, Vanessa , Zulkifli, Ahmad , Paradiso, Lucia , Jones, Jordan , Nguyen, Hong , Putoczki, Tracy , Licciardi, Paul , Kannourakis, George , Morokoff, Andrew , Achuthan, Adrian , Luwor, Rodney
- Date: 2023
- Type: Text , Journal article
- Relation: International Journal of Molecular Sciences Vol. 24, no. 4 (2023), p.
- Full Text:
- Reviewed:
- Description: Glioblastoma cells adapt to changes in glucose availability through metabolic plasticity allowing for cell survival and continued progression in low-glucose concentrations. However, the regulatory cytokine networks that govern the ability to survive in glucose-starved conditions are not fully defined. In the present study, we define a critical role for the IL-11/IL-11R
- Authors: Stuart, Sarah , Bezawork-Geleta, Ayenachew , Areeb, Zammam , Gomez, Juliana , Tsui, Vanessa , Zulkifli, Ahmad , Paradiso, Lucia , Jones, Jordan , Nguyen, Hong , Putoczki, Tracy , Licciardi, Paul , Kannourakis, George , Morokoff, Andrew , Achuthan, Adrian , Luwor, Rodney
- Date: 2023
- Type: Text , Journal article
- Relation: International Journal of Molecular Sciences Vol. 24, no. 4 (2023), p.
- Full Text:
- Reviewed:
- Description: Glioblastoma cells adapt to changes in glucose availability through metabolic plasticity allowing for cell survival and continued progression in low-glucose concentrations. However, the regulatory cytokine networks that govern the ability to survive in glucose-starved conditions are not fully defined. In the present study, we define a critical role for the IL-11/IL-11R
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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