Characterization of the molecular mechanism(s) of cannabinoid-induced paraptosis in breast cancer cells
- Authors: de la Harpe, Amy
- Date: 2024-12
- Subjects: Cannabinoids , Cannabinoids -- Therapeutic use , Cancer cells
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10948/68818 , vital:77120
- Description: Natural products have been extensively studied for their anticancer potential, and several widely used anticancer drugs have natural origins. Many studies have demonstrated that natural compounds can induce paraptosis in various tumour cell lines. Paraptosis is a caspase-independent cell death mechanism characterised by cytoplasmic vacuolation arising from the endoplasmic reticulum (ER) and the mitochondria. The molecular mechanism of paraptosis is unclear; however, the literature indicates that dysregulation of calcium signalling plays an important role in paraptosis induction, particularly calcium-mediated signalling between the ER and mitochondria. This study aimed to identify and characterise the mechanism of cell death induced by a phytocannabinoid ratio which induced significant cytoplasmic vacuolation in the MCF7 breast cancer cell line. Several techniques were employed to address the aim of the study. The crystal violet assay was used to detect changes in viability. Several pathway inhibitors, as well as fluorescent staining and analysis using high-content screening (HCS), were used to measure the induction of various cell death mechanisms. Morphological changes were investigated using light and transmission electron microscopy. The phytocannabinoid ratio induced significant cell death and cytoplasmic vacuolation in MCF7 cells; however, the same trend was not observed in the MCF10A non-tumourigenic breast cell line. No cell cycle arrest, apoptosis, necrosis, autophagy, or ferroptosis induction was detected in MCF7 cells, suggesting that an alternative mechanism of cell death was induced. Vacuolation and cell death induced by the phytocannabinoid ratio were inhibited by cycloheximide, suggesting a dependence on protein synthesis, which is characteristic of paraptosis induction. The mechanism of paraptosis induction by the phytocannabinoid ratio was investigated, and it was found that treatment 1) induced ER dilation and mitochondrial swelling; 2) induced significant ER stress, mitochondrial calcium overload, and mitochondrial dysfunction which appeared to be mediated by the voltage-dependent anion channel (VDAC); and 3) significantly impaired all mitochondrial metabolic pathways. Overall, the data demonstrated that paraptosis induced by the cannabinoid ratio is mediated by calcium flux from the ER to the mitochondria. This highlighted a novel mechanism of phytocannabinoid-induced cell death and emphasised the anti-cancer potential of the use of phytocannabinoid ratios as opposed to individual phytocannabinoids, expanding their potential for use as anticancer agents. , Thesis (PhD) -- Faculty of Science, School of Biomolecular & Chemical Sciences, 2024
- Full Text:
- Date Issued: 2024-12
- Authors: de la Harpe, Amy
- Date: 2024-12
- Subjects: Cannabinoids , Cannabinoids -- Therapeutic use , Cancer cells
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10948/68818 , vital:77120
- Description: Natural products have been extensively studied for their anticancer potential, and several widely used anticancer drugs have natural origins. Many studies have demonstrated that natural compounds can induce paraptosis in various tumour cell lines. Paraptosis is a caspase-independent cell death mechanism characterised by cytoplasmic vacuolation arising from the endoplasmic reticulum (ER) and the mitochondria. The molecular mechanism of paraptosis is unclear; however, the literature indicates that dysregulation of calcium signalling plays an important role in paraptosis induction, particularly calcium-mediated signalling between the ER and mitochondria. This study aimed to identify and characterise the mechanism of cell death induced by a phytocannabinoid ratio which induced significant cytoplasmic vacuolation in the MCF7 breast cancer cell line. Several techniques were employed to address the aim of the study. The crystal violet assay was used to detect changes in viability. Several pathway inhibitors, as well as fluorescent staining and analysis using high-content screening (HCS), were used to measure the induction of various cell death mechanisms. Morphological changes were investigated using light and transmission electron microscopy. The phytocannabinoid ratio induced significant cell death and cytoplasmic vacuolation in MCF7 cells; however, the same trend was not observed in the MCF10A non-tumourigenic breast cell line. No cell cycle arrest, apoptosis, necrosis, autophagy, or ferroptosis induction was detected in MCF7 cells, suggesting that an alternative mechanism of cell death was induced. Vacuolation and cell death induced by the phytocannabinoid ratio were inhibited by cycloheximide, suggesting a dependence on protein synthesis, which is characteristic of paraptosis induction. The mechanism of paraptosis induction by the phytocannabinoid ratio was investigated, and it was found that treatment 1) induced ER dilation and mitochondrial swelling; 2) induced significant ER stress, mitochondrial calcium overload, and mitochondrial dysfunction which appeared to be mediated by the voltage-dependent anion channel (VDAC); and 3) significantly impaired all mitochondrial metabolic pathways. Overall, the data demonstrated that paraptosis induced by the cannabinoid ratio is mediated by calcium flux from the ER to the mitochondria. This highlighted a novel mechanism of phytocannabinoid-induced cell death and emphasised the anti-cancer potential of the use of phytocannabinoid ratios as opposed to individual phytocannabinoids, expanding their potential for use as anticancer agents. , Thesis (PhD) -- Faculty of Science, School of Biomolecular & Chemical Sciences, 2024
- Full Text:
- Date Issued: 2024-12
The inhibitory effects of cannabinoids from cannabis sativa on the enzymes dipeptidyl peptidase-IV, sucrase and maltase as a new therapeutic treatment for type 2 diabetes
- Authors: Viljoen, Zenobia
- Date: 2024-12
- Subjects: Diabetes -- Treatment , Cannabinoids -- Therapeutic use , Medical Marijuana -- therapeutic use
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10948/69516 , vital:77264
- Description: Type 2 diabetes is one of the most prevalent diseases worldwide. The treatments used to manage diabetes often have severe side effects and patients develop resistance to traditional treatment. The project aimed to test if phytocannabinoids from Cannabis sativa inhibited key enzymes involved in glycaemic homeostatic regulation, namely dipeptidyl peptidase 4 (DPP-4), sucrase, and maltase. This study investigated the inhibitory effects of 3 M-128 M cannabidiol (CBD), cannabinol (CBN), cannabigerol (CBG), and Δ9- tetrahydrocannabinol (THC). CD spectroscopy was used to investigate the changes in the secondary structure of DPP-4 with interacting inhibitors. The effect of 1.25, 2.5, and 5 mg/kg rat THC cannabis extract on the activity of DPP-4 in blood plasma and rat pancreatic tissue of the diabetic rat model and obese rat model. The effect of 1.25, 2.5, and 5 mg/kg rat THC cannabis extract on glucagon concentration in the blood plasma of the diabetic rat model and obese rat model was investigated. The carbohydrate digestive enzymes namely -amylase, -glucosidase and maltase are not inhibited by any of the cannabinoids. CBN had inhibitory effects on sucrase. CBN, CBG, and CBD are mixed inhibitors of DPP-4, thus they can inhibit DPP-4 competitively and uncompetitively depending on the concentration of the cannabinoid. THC was shown in kinetic and rat model studies to be a very weak inhibitor of DPP-4. CD spectroscopy showed that sitagliptin (FDA-approved drug and competitive inhibitor) and CBG mimic the denatured structure of DPP-4. CBD, CBN and THC mimic the free (active) form of DPP-4. A reduction in pancreatic DPP-4 activity was observed with 2.5 and 5 mg/kg rat THC (diabetic model). This study showed that diet plays a role in glycaemic dysregulation (obese rat model) and that insulin-resistant rats had four times higher glucagon levels compared to the lean control (diabetic model). 1.25 mg/kg rat THC reduced blood plasma DPP-4 activity and blood plasma glucagon. Cannabis sativa can be a feasible treatment to help manage type 2 diabetes by inhibiting DPP-4, especially medical strains of Cannabis sativa with high concentrations of CBD and CBG. , Thesis (MSc) -- Faculty of Science, School of Biomolecular & Chemical Sciences, 2024
- Full Text:
- Date Issued: 2024-12
- Authors: Viljoen, Zenobia
- Date: 2024-12
- Subjects: Diabetes -- Treatment , Cannabinoids -- Therapeutic use , Medical Marijuana -- therapeutic use
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10948/69516 , vital:77264
- Description: Type 2 diabetes is one of the most prevalent diseases worldwide. The treatments used to manage diabetes often have severe side effects and patients develop resistance to traditional treatment. The project aimed to test if phytocannabinoids from Cannabis sativa inhibited key enzymes involved in glycaemic homeostatic regulation, namely dipeptidyl peptidase 4 (DPP-4), sucrase, and maltase. This study investigated the inhibitory effects of 3 M-128 M cannabidiol (CBD), cannabinol (CBN), cannabigerol (CBG), and Δ9- tetrahydrocannabinol (THC). CD spectroscopy was used to investigate the changes in the secondary structure of DPP-4 with interacting inhibitors. The effect of 1.25, 2.5, and 5 mg/kg rat THC cannabis extract on the activity of DPP-4 in blood plasma and rat pancreatic tissue of the diabetic rat model and obese rat model. The effect of 1.25, 2.5, and 5 mg/kg rat THC cannabis extract on glucagon concentration in the blood plasma of the diabetic rat model and obese rat model was investigated. The carbohydrate digestive enzymes namely -amylase, -glucosidase and maltase are not inhibited by any of the cannabinoids. CBN had inhibitory effects on sucrase. CBN, CBG, and CBD are mixed inhibitors of DPP-4, thus they can inhibit DPP-4 competitively and uncompetitively depending on the concentration of the cannabinoid. THC was shown in kinetic and rat model studies to be a very weak inhibitor of DPP-4. CD spectroscopy showed that sitagliptin (FDA-approved drug and competitive inhibitor) and CBG mimic the denatured structure of DPP-4. CBD, CBN and THC mimic the free (active) form of DPP-4. A reduction in pancreatic DPP-4 activity was observed with 2.5 and 5 mg/kg rat THC (diabetic model). This study showed that diet plays a role in glycaemic dysregulation (obese rat model) and that insulin-resistant rats had four times higher glucagon levels compared to the lean control (diabetic model). 1.25 mg/kg rat THC reduced blood plasma DPP-4 activity and blood plasma glucagon. Cannabis sativa can be a feasible treatment to help manage type 2 diabetes by inhibiting DPP-4, especially medical strains of Cannabis sativa with high concentrations of CBD and CBG. , Thesis (MSc) -- Faculty of Science, School of Biomolecular & Chemical Sciences, 2024
- Full Text:
- Date Issued: 2024-12
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