Examining the effects of small molecule tyrosine kinase inhibitors on glucose metabolism in skeletal muscle and liver cell lines in vitro
- Authors: Mugiya, Takudzwa Cleophas
- Date: 2025-04-02
- Subjects: Diabetes , Blood glucose , Cancer , Insulin , Protein kinase B , Glucose transporters
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/478829 , vital:78227
- Description: Diabetes mellitus is rising due to aging, sedentary lifestyles, obesity, and unhealthy diets, posing a global health threat. Due to increase in prevalence together with shortfalls associated with current treatment options, there is still a necessity for a continuous search of new pharmacotherapies. Small molecule tyrosine kinase inhibitors are drugs, used in cancer chemotherapy and have been shown to affect glycaemic control and metabolism variably. Studies have shown that tyrosine kinase inhibitors can alter glycaemic control and glucose metabolism, with some demonstrating hypoglycaemic activities whilst others showing hyperglycaemic properties. The mechanism by which tyrosine kinase inhibitors cause glycaemic dysregulation is not well understood, therefore, the clinical significance of these chemotherapeutic agents on glucose handling is also poorly documented. Aims and objectives: This study aims to elucidate how small molecule tyrosine kinase inhibitors affect glucose metabolism in C2C12 and HepG2 cells in vitro, including their impact on glucose uptake, AKT, GLUT-4, and IL-6 expression, GLUT-4 translocation, and alpha-amylase and alpha-glucosidase activity. Methods: In this study, C2C12 and HepG2 cells were seeded in well plates and the initial media glucose concentration was recorded. Cells were then treated with small molecule tyrosine kinase inhibitors; imatinib, dasatinib, axitinib, and erlotinib for 24 hours. Thereafter, the effect of the test drugs was assessed on cell viability, glucose uptake, expression of AKT GLUT-4 and IL-6, and translocation of GLUT-4. Furthermore, effects of the drugs were assessed on the activities of alpha amylase and glucosidase using calometric assays. Results and Discussion: Cells treated with small molecule tyrosine kinase inhibitors were viable after 24 hours. A concentration-dependent increase in glucose uptake in C2C12 cells treated with imatinib was observed as the concentration of imatinib increased. Axitinib, dasatinib, and erlotinib demonstrated glucose uptake levels comparable to the control across all concentrations. Small molecule tyrosine kinase inhibitors demonstrated an increase in GLUT4 translocation in the absence of insulin. GLUT4 expression was comparable in cells treated with small molecule tyrosine kinase inhibitors and the control. Small molecule tyrosine kinase inhibitors showed an increase in AKT expression. C2C12 cells treated with small molecule tyrosine kinase inhibitors were observed to have elevated IL-6 expression compared to the control. The HepG2 cells treated with erlotinib and imatinib demonstrated elevated glucose uptake while cells treated with axitinib and dasatinib were observed to have a lower glucose uptake. Treatment with dasatinib led to a decrease in Akt expression as concentration increased. Small molecule tyrosine kinase inhibitors demonstrated inhibition of alpha-amylase, while only dasatinib and axitinib showed inhibition of alpha-glucosidase. Conclusion: The results show that small molecule tyrosine kinase inhibitors impact glucose metabolism in C2C12 and HepG2 cells via their effect on GLUT-4 translocation and expression and AKT expression. Dasatinib showed promising potential with regard to antidiabetic capabilities. Further research is needed to better understand these mechanisms' effects on metabolic homeostasis and inform future therapeutic strategies. , Thesis (MSc (Pharm)) -- Faculty of Pharmacy, Pharmacy, 2025
- Full Text:
- Date Issued: 2025-04-02
- Authors: Mugiya, Takudzwa Cleophas
- Date: 2025-04-02
- Subjects: Diabetes , Blood glucose , Cancer , Insulin , Protein kinase B , Glucose transporters
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/478829 , vital:78227
- Description: Diabetes mellitus is rising due to aging, sedentary lifestyles, obesity, and unhealthy diets, posing a global health threat. Due to increase in prevalence together with shortfalls associated with current treatment options, there is still a necessity for a continuous search of new pharmacotherapies. Small molecule tyrosine kinase inhibitors are drugs, used in cancer chemotherapy and have been shown to affect glycaemic control and metabolism variably. Studies have shown that tyrosine kinase inhibitors can alter glycaemic control and glucose metabolism, with some demonstrating hypoglycaemic activities whilst others showing hyperglycaemic properties. The mechanism by which tyrosine kinase inhibitors cause glycaemic dysregulation is not well understood, therefore, the clinical significance of these chemotherapeutic agents on glucose handling is also poorly documented. Aims and objectives: This study aims to elucidate how small molecule tyrosine kinase inhibitors affect glucose metabolism in C2C12 and HepG2 cells in vitro, including their impact on glucose uptake, AKT, GLUT-4, and IL-6 expression, GLUT-4 translocation, and alpha-amylase and alpha-glucosidase activity. Methods: In this study, C2C12 and HepG2 cells were seeded in well plates and the initial media glucose concentration was recorded. Cells were then treated with small molecule tyrosine kinase inhibitors; imatinib, dasatinib, axitinib, and erlotinib for 24 hours. Thereafter, the effect of the test drugs was assessed on cell viability, glucose uptake, expression of AKT GLUT-4 and IL-6, and translocation of GLUT-4. Furthermore, effects of the drugs were assessed on the activities of alpha amylase and glucosidase using calometric assays. Results and Discussion: Cells treated with small molecule tyrosine kinase inhibitors were viable after 24 hours. A concentration-dependent increase in glucose uptake in C2C12 cells treated with imatinib was observed as the concentration of imatinib increased. Axitinib, dasatinib, and erlotinib demonstrated glucose uptake levels comparable to the control across all concentrations. Small molecule tyrosine kinase inhibitors demonstrated an increase in GLUT4 translocation in the absence of insulin. GLUT4 expression was comparable in cells treated with small molecule tyrosine kinase inhibitors and the control. Small molecule tyrosine kinase inhibitors showed an increase in AKT expression. C2C12 cells treated with small molecule tyrosine kinase inhibitors were observed to have elevated IL-6 expression compared to the control. The HepG2 cells treated with erlotinib and imatinib demonstrated elevated glucose uptake while cells treated with axitinib and dasatinib were observed to have a lower glucose uptake. Treatment with dasatinib led to a decrease in Akt expression as concentration increased. Small molecule tyrosine kinase inhibitors demonstrated inhibition of alpha-amylase, while only dasatinib and axitinib showed inhibition of alpha-glucosidase. Conclusion: The results show that small molecule tyrosine kinase inhibitors impact glucose metabolism in C2C12 and HepG2 cells via their effect on GLUT-4 translocation and expression and AKT expression. Dasatinib showed promising potential with regard to antidiabetic capabilities. Further research is needed to better understand these mechanisms' effects on metabolic homeostasis and inform future therapeutic strategies. , Thesis (MSc (Pharm)) -- Faculty of Pharmacy, Pharmacy, 2025
- Full Text:
- Date Issued: 2025-04-02
Investigating the role of Hsp90 and LRP1 in FN matrix dynamics
- Authors: Boël, Natasha Marie-Eraine
- Date: 2016
- Subjects: Extracellular matrix , Molecular chaperones , Heat shock proteins , Cancer , Fibronectins
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/2713 , vital:20319
- Description: Fibronectin (FN), a matrix protein responsible for regulating processes including migration and differentiation, is secreted as a soluble dimer which is assembled into an insoluble extracellular matrix. The dynamics of FN matrix assembly and degradation play a large role in cell migration and invasion contributing to the metastatic potential of cancer cells. Previous studies from our group have shown the direct binding of Hsp90 and FN in vitro and that inhibition of Hsp90 with novobiocin (NOV) caused internalisation of the FN matrix. However, the receptor mediating this internalisation is currently unknown. Low density lipoprotein 1 (LRP1) is a likely candidate as it is a ubiquitous receptor responsible for regulating internalisation of diverse ligands and is known to bind both Hsp90 and FN. We used wild type and knockout LRP1 cell lines to study the endocytosis of FN via this receptor. Here, we demonstrate that LRP1-deficient cells accumulated greatly increased levels of FN and were found to be less sensitive to pharmacological inhibition of Hsp90 by NOV. LRP1-expressing MEF-1 and Hs578T breast cancer cells experienced an increase in total FN in response to NOV, at concentrations below the EC50 value, followed by a dose-dependent loss of FN. We attributed greater FN levels to a loss of extracellular FN matrix coupled with increased internalisation of FN. Cell-surface biotinylation and DOC assays showed that loss of extracellular FN was specific to LRP1-expressing MEF-1 cells. Furthermore, we demonstrate that the loss of extracellular FN is not affected by changes in FN mRNA levels as determined by qRT-PCR, and that treatment with NOV resulted in the accelerated degradation of FN in the presence of cycloheximide. Immunoprecipitation studies reveal a putative complex exists between FN, Hsp90 and LRP1 in both cancer and non-cancer cells which is not perturbed by NOV. Western analyses revealed increased proteolytic processing of LRP1 in response to NOV which we proposed, based on literature, to modulate signalling pathways as a potential mechanism for regulating FN turnover. Moreover, using wound healing assays we identified increased migration to be one of the consequences associated with loss of extracellular FN by Hsp90 inhibition but only in cells containing LRP1. In summary, this study provides new insights into the Hsp90-LRP1 mediated loss of FN matrix and also reveals for the first time the functional consequence related to FN turnover by NOV was an increase in migration in LRP1-expressing cells.
- Full Text:
- Date Issued: 2016
- Authors: Boël, Natasha Marie-Eraine
- Date: 2016
- Subjects: Extracellular matrix , Molecular chaperones , Heat shock proteins , Cancer , Fibronectins
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/2713 , vital:20319
- Description: Fibronectin (FN), a matrix protein responsible for regulating processes including migration and differentiation, is secreted as a soluble dimer which is assembled into an insoluble extracellular matrix. The dynamics of FN matrix assembly and degradation play a large role in cell migration and invasion contributing to the metastatic potential of cancer cells. Previous studies from our group have shown the direct binding of Hsp90 and FN in vitro and that inhibition of Hsp90 with novobiocin (NOV) caused internalisation of the FN matrix. However, the receptor mediating this internalisation is currently unknown. Low density lipoprotein 1 (LRP1) is a likely candidate as it is a ubiquitous receptor responsible for regulating internalisation of diverse ligands and is known to bind both Hsp90 and FN. We used wild type and knockout LRP1 cell lines to study the endocytosis of FN via this receptor. Here, we demonstrate that LRP1-deficient cells accumulated greatly increased levels of FN and were found to be less sensitive to pharmacological inhibition of Hsp90 by NOV. LRP1-expressing MEF-1 and Hs578T breast cancer cells experienced an increase in total FN in response to NOV, at concentrations below the EC50 value, followed by a dose-dependent loss of FN. We attributed greater FN levels to a loss of extracellular FN matrix coupled with increased internalisation of FN. Cell-surface biotinylation and DOC assays showed that loss of extracellular FN was specific to LRP1-expressing MEF-1 cells. Furthermore, we demonstrate that the loss of extracellular FN is not affected by changes in FN mRNA levels as determined by qRT-PCR, and that treatment with NOV resulted in the accelerated degradation of FN in the presence of cycloheximide. Immunoprecipitation studies reveal a putative complex exists between FN, Hsp90 and LRP1 in both cancer and non-cancer cells which is not perturbed by NOV. Western analyses revealed increased proteolytic processing of LRP1 in response to NOV which we proposed, based on literature, to modulate signalling pathways as a potential mechanism for regulating FN turnover. Moreover, using wound healing assays we identified increased migration to be one of the consequences associated with loss of extracellular FN by Hsp90 inhibition but only in cells containing LRP1. In summary, this study provides new insights into the Hsp90-LRP1 mediated loss of FN matrix and also reveals for the first time the functional consequence related to FN turnover by NOV was an increase in migration in LRP1-expressing cells.
- Full Text:
- Date Issued: 2016
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