Part 1 - Expression analysis of LASP1 in human cancer cell lines Cancer takes place by malignant transformation of normal cells; it remains one of the most common causes of death worldwide. LIM and SH3 protein (LASP1), is an actin-binding protein. It is believed that this protein plays a crucial role in cancer dissemination, progression, metastasis, and angiogenesis. I have studied the expression levels of LASP1 by using the Western blot analysis in different breast cancer lines and oral carcinoma line. Two cell lines of breast cancer with different malignancy levels were chosen: MDA-MB-231 (triple-negative for oestrogen receptor, progesterone receptor and human epidermal GF receptor 2) and MCF-7 (poorly aggressive and non-invasive cell line). And the expression levels in oral carcinoma cell lines (HSC-2 (non-invasive, non-metastatic squamous carcinoma) and CAL-27 (poorly differentiated tongue carcinoma)) were analysed: The preliminary hypothesis can be done that the overexpression of LASP1 is correlated with the malignancy of cancer cell line which was confirmed both in breast cancer cell lines and oral carcinoma cell lines. Part 2 - Role of Src signaling pathways in endothelial cell permeability SFKs are the group of nonreceptor tyrosine kinases involved in the regulation of various signaling pathways involved in proliferation, survival, migration, angiogenesis, and metastasis. Src associates with adherens junctions by interacting with VE-cadherin. Upon association in protein complexes, Src can phosphorylate VE-cadherin. Upon VEGF-A binding to its cognate receptors (VEGFR1 and VEGFR2 in vascular ECs), the intrinsic tyrosine kinase activity of these receptors is activated, leading to trans-phosphorylation and direct interaction with many SH-2-containing signalling molecules, including Src. Once bound to VEGFR1 or VEGFR2, Src undergoes a conformational change leading to its activation and the subsequent phosphorylation of VE-cadherin. To understand the role of SFKs member YES in the vascular permeability, the Western blot analyses was performed. The levels of pVEGFR2, total VEGFR2, pSTAT1, STAT1 were studied in HUVECs after VEGF stimulation and in the siYES and siSRC silenced cells. It was noticeable, that siRNA-mediated YES knock-down HUVECs have the lowest expression levels of pSTAT1. Both siYES HUVECs and siSRC HUVECs showed lower pSTAT1 expression in comparison with control. This data suggests that SFKs are involved in the activation of STAT1. Additionally, after Immunostaining analysis of pPAX, it was evident the higher concentratin of pPAX within siYES HUVECs than in control. It was also plausible, that in siYES cells pPAX is more distributed in the focal adhesion sites. The highest level of PAX phosphorylation is observed in siYES cells stimulated for 5 min with VEGFA. The increased adhesion site size phosphorylation of paxillin might provoke increased cell motility. After the studying of cell motility of siYES HUVECs by performing the scratch assay, it was noticeable that the movements of HUVEC siYES were less orchestrated, and the cells were more tended to lose and break adherens junctions. In the contrast, the movements of HUVEC siCTRL were more organised, more structured, and the adherens junctions were more stable in comparison to HUVEC siYES. Better understanding of the mechanisms of SFK influence on cell-cell connection remodelling has an enormous interest in the therapeutic potential of SFK moderation treatment within inflammationrelated illnesses and neoplastic diseases. Part 3 - Natural compounds in chemoprevention Another area of my research focus were natural compounds in chemoprevention/chemotherapy. To structure and highlight all studied information I did literature overview of two natural compounds: piperine and ginger extracts. The chemopreventive molecular mechanisms of these natural compounds include cell cycle arrest, induction of cancer cell death, misbalancing of redox homeostasis, inhibition of cell proliferation, angiogenesis, migration, and dissemination of cancer cells in different cancer types.

Expression analysis of LASP1 in human cancer cell lines, role of Src signaling pathways in endothelial cell permeability and natural compounds in chemoprevention

ZADOROZHNA, MARIIA
2022-01-01

Abstract

Part 1 - Expression analysis of LASP1 in human cancer cell lines Cancer takes place by malignant transformation of normal cells; it remains one of the most common causes of death worldwide. LIM and SH3 protein (LASP1), is an actin-binding protein. It is believed that this protein plays a crucial role in cancer dissemination, progression, metastasis, and angiogenesis. I have studied the expression levels of LASP1 by using the Western blot analysis in different breast cancer lines and oral carcinoma line. Two cell lines of breast cancer with different malignancy levels were chosen: MDA-MB-231 (triple-negative for oestrogen receptor, progesterone receptor and human epidermal GF receptor 2) and MCF-7 (poorly aggressive and non-invasive cell line). And the expression levels in oral carcinoma cell lines (HSC-2 (non-invasive, non-metastatic squamous carcinoma) and CAL-27 (poorly differentiated tongue carcinoma)) were analysed: The preliminary hypothesis can be done that the overexpression of LASP1 is correlated with the malignancy of cancer cell line which was confirmed both in breast cancer cell lines and oral carcinoma cell lines. Part 2 - Role of Src signaling pathways in endothelial cell permeability SFKs are the group of nonreceptor tyrosine kinases involved in the regulation of various signaling pathways involved in proliferation, survival, migration, angiogenesis, and metastasis. Src associates with adherens junctions by interacting with VE-cadherin. Upon association in protein complexes, Src can phosphorylate VE-cadherin. Upon VEGF-A binding to its cognate receptors (VEGFR1 and VEGFR2 in vascular ECs), the intrinsic tyrosine kinase activity of these receptors is activated, leading to trans-phosphorylation and direct interaction with many SH-2-containing signalling molecules, including Src. Once bound to VEGFR1 or VEGFR2, Src undergoes a conformational change leading to its activation and the subsequent phosphorylation of VE-cadherin. To understand the role of SFKs member YES in the vascular permeability, the Western blot analyses was performed. The levels of pVEGFR2, total VEGFR2, pSTAT1, STAT1 were studied in HUVECs after VEGF stimulation and in the siYES and siSRC silenced cells. It was noticeable, that siRNA-mediated YES knock-down HUVECs have the lowest expression levels of pSTAT1. Both siYES HUVECs and siSRC HUVECs showed lower pSTAT1 expression in comparison with control. This data suggests that SFKs are involved in the activation of STAT1. Additionally, after Immunostaining analysis of pPAX, it was evident the higher concentratin of pPAX within siYES HUVECs than in control. It was also plausible, that in siYES cells pPAX is more distributed in the focal adhesion sites. The highest level of PAX phosphorylation is observed in siYES cells stimulated for 5 min with VEGFA. The increased adhesion site size phosphorylation of paxillin might provoke increased cell motility. After the studying of cell motility of siYES HUVECs by performing the scratch assay, it was noticeable that the movements of HUVEC siYES were less orchestrated, and the cells were more tended to lose and break adherens junctions. In the contrast, the movements of HUVEC siCTRL were more organised, more structured, and the adherens junctions were more stable in comparison to HUVEC siYES. Better understanding of the mechanisms of SFK influence on cell-cell connection remodelling has an enormous interest in the therapeutic potential of SFK moderation treatment within inflammationrelated illnesses and neoplastic diseases. Part 3 - Natural compounds in chemoprevention Another area of my research focus were natural compounds in chemoprevention/chemotherapy. To structure and highlight all studied information I did literature overview of two natural compounds: piperine and ginger extracts. The chemopreventive molecular mechanisms of these natural compounds include cell cycle arrest, induction of cancer cell death, misbalancing of redox homeostasis, inhibition of cell proliferation, angiogenesis, migration, and dissemination of cancer cells in different cancer types.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11369/425849
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