Identification of novel Arf1 GTPase inhibitors for cancer target validation
- Authors: Mqwathi, Nomxolisi Vuyokasi
- Date: 2023-10-13
- Subjects: ARF1 , GTPase , Guanosine triphosphatase , Cancer Treatment
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/424666 , vital:72173
- Description: The key regulators of both anterograde and retrograde vesicular traffic, adenosine diphosphate-ribosylation factors (Arfs), also coordinate various signalling pathways and regulate cellular processes required for cell survival and function. In addition to its role in mediating secretory trafficking in the Golgi apparatus, the involvement of Arf1 in signalling pathways that contribute to the formation and progression of cancer has become apparent, and the overexpression and deregulation of Arf1 activity has been associated with cancer cell invasion, proliferation and metastasis. As with other small GTPases, Arf1 must cycle back and forth between an inactive (GDP-bound) and active (GTP-bound) conformation to carry out its function. However, the cycle of Arf1 inactivation and activation is controlled by Arf GTPase activating proteins (Arf-GAPs) that stimulate Arf1 to hydrolyse the bound GTP to GDP and Arf guanine nucleotide exchange factors (Arf-GEFs) that facilitate GDP for GTP exchange on Arf1, respectively. The identification of Arf1 inhibitors that indirectly disrupt Arf1 function by blocking its interaction with Arf-GAPs or Arf-GEFs has generated interest in their use as possible anti-cancer agents. The suppression of Arf1 activation (by targeting Arf-GEFs) has been investigated as a potential cancer therapeutic target and resulted in inhibitor compounds that have micromolar-range activity against cancer cells and targets and promising results in mouse models, but experience problems with bioavailability when used in vivo. This motivates the search for novel Arf1 inhibitors for validation purposes to question whether Arf1 is a viable target for cancer therapy. The purpose of the study was to employ a recently developed colourimetric screening assay to identify inhibitors of Arf1 activation (Arf-GEF inhibitors) and deactivation (Arf-GAP inhibitors), with a focus on evaluating the potential of Arf1 deactivation as an entirely novel anti-cancer target. The proteins required for the assay (Arf1, Arf-GEF and -GAP domains and a reporter protein, GST-GGA3) were expressed in E. coli. and purified using affinity chromatography. The assay could detect the activation of Arf1 by the catalytic Sec7 domain of the three Arf-GEFs chosen for this study, but reproducibility was compromised by the occasional spontaneous activation of Arf1 in the absence of the Arf-GEFs. By contrast, the assay could reproducibly detect Arf1 deactivation by an Arf-GAP domain (Arf-GAP1GAP) and was subsequently used to screen a library of α-helix mimetics. Thirteen hit compounds with IC50 values ranging from 0.53 to 20.95 μM were found to inhibit Arf-GAP1GAP-mediated stimulation of GTP hydrolysis by Arf1-GTP in this assay format, however, they did not effectively suppress the proliferation of three tested cell lines (HeLa, MCF-7 and MCF-12A). Interestingly, the results obtained from fluorescence microscopy studies suggested that the compounds disrupt Golgi structure and Arf1 localisation, presumably by keeping Arf1 in its active conformation by blocking Arf-GAP1 function. This suggests that the compounds affect Arf1 function in cells, and may be used to explore the feasibility of targeting Arf1 deactivation for anti-cancer purposes in a wider range of cell lines and experiments. It has been reported that Arf-GAP1 inhibition is associated with the suppression of cell migration, and the potential of the compounds as metastasis inhibitors may also be explored. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2023
- Full Text:
- Date Issued: 2023-10-13
- Authors: Mqwathi, Nomxolisi Vuyokasi
- Date: 2023-10-13
- Subjects: ARF1 , GTPase , Guanosine triphosphatase , Cancer Treatment
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/424666 , vital:72173
- Description: The key regulators of both anterograde and retrograde vesicular traffic, adenosine diphosphate-ribosylation factors (Arfs), also coordinate various signalling pathways and regulate cellular processes required for cell survival and function. In addition to its role in mediating secretory trafficking in the Golgi apparatus, the involvement of Arf1 in signalling pathways that contribute to the formation and progression of cancer has become apparent, and the overexpression and deregulation of Arf1 activity has been associated with cancer cell invasion, proliferation and metastasis. As with other small GTPases, Arf1 must cycle back and forth between an inactive (GDP-bound) and active (GTP-bound) conformation to carry out its function. However, the cycle of Arf1 inactivation and activation is controlled by Arf GTPase activating proteins (Arf-GAPs) that stimulate Arf1 to hydrolyse the bound GTP to GDP and Arf guanine nucleotide exchange factors (Arf-GEFs) that facilitate GDP for GTP exchange on Arf1, respectively. The identification of Arf1 inhibitors that indirectly disrupt Arf1 function by blocking its interaction with Arf-GAPs or Arf-GEFs has generated interest in their use as possible anti-cancer agents. The suppression of Arf1 activation (by targeting Arf-GEFs) has been investigated as a potential cancer therapeutic target and resulted in inhibitor compounds that have micromolar-range activity against cancer cells and targets and promising results in mouse models, but experience problems with bioavailability when used in vivo. This motivates the search for novel Arf1 inhibitors for validation purposes to question whether Arf1 is a viable target for cancer therapy. The purpose of the study was to employ a recently developed colourimetric screening assay to identify inhibitors of Arf1 activation (Arf-GEF inhibitors) and deactivation (Arf-GAP inhibitors), with a focus on evaluating the potential of Arf1 deactivation as an entirely novel anti-cancer target. The proteins required for the assay (Arf1, Arf-GEF and -GAP domains and a reporter protein, GST-GGA3) were expressed in E. coli. and purified using affinity chromatography. The assay could detect the activation of Arf1 by the catalytic Sec7 domain of the three Arf-GEFs chosen for this study, but reproducibility was compromised by the occasional spontaneous activation of Arf1 in the absence of the Arf-GEFs. By contrast, the assay could reproducibly detect Arf1 deactivation by an Arf-GAP domain (Arf-GAP1GAP) and was subsequently used to screen a library of α-helix mimetics. Thirteen hit compounds with IC50 values ranging from 0.53 to 20.95 μM were found to inhibit Arf-GAP1GAP-mediated stimulation of GTP hydrolysis by Arf1-GTP in this assay format, however, they did not effectively suppress the proliferation of three tested cell lines (HeLa, MCF-7 and MCF-12A). Interestingly, the results obtained from fluorescence microscopy studies suggested that the compounds disrupt Golgi structure and Arf1 localisation, presumably by keeping Arf1 in its active conformation by blocking Arf-GAP1 function. This suggests that the compounds affect Arf1 function in cells, and may be used to explore the feasibility of targeting Arf1 deactivation for anti-cancer purposes in a wider range of cell lines and experiments. It has been reported that Arf-GAP1 inhibition is associated with the suppression of cell migration, and the potential of the compounds as metastasis inhibitors may also be explored. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2023
- Full Text:
- Date Issued: 2023-10-13
Liposomal formulations of metallophthalocyanines-nanoparticle conjugates for hypoxic photodynamic therapy and photoelectrocatalysis
- Authors: Nwahara, Nnamdi Ugochinyere
- Date: 2023-10-13
- Subjects: Liposomes , Photochemotherapy , Phthalocyanines , Photoelectrochemistry , Cancer Treatment
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/432159 , vital:72847 , DOI 10.21504/10962/432159
- Description: This thesis investigates new strategies to enhance the efficacy of photodynamic therapy (PDT) under hypoxic conditions using in-vitro cancer cell models. Phthalocyanines are chosen as viable photosensitizer complexes owing to the favourable absorption properties. To this end, this thesis reports on the synthesis and photophysicochemical properties of various zinc and silicon phthalocyanines (Pcs). To afford better photophysicochemical properties, the reported Pcs were conjugated to different nanoparticles (NPs) through chemisorption as well as amide bond formation to yield Pc-NP conjugates. All the studied Pcs showed relatively high triplet and singlet oxygen quantum yields corresponding to their low fluorescence quantum yields. The various mechanisms for hypoxic response include (i) Type I PDT, (ii) PDT coupled with oxygen-independent therapy and (iii) in-situ oxygen generation using catalase-mimicking nanoparticles which serve to supplement in-vitro oxygen concentrations using MPcs or MPc-NPs conjugates. The mechanisms were assessed using electrochemical, computational techniques and catalase mimicking experiments. The as-synthesised Pcs or Pc-NPs were subjected to liposomal loading before PDT studies which led to enhanced biocompatibility and aqueous dispersity. The in-vitro dark cytotoxicity tests and photodynamic therapy activities of the fabricated Pc-liposomes and Pc-NPs-liposomes on either Henrietta Lacks (HeLa) or Michigan Cancer Foundation-7 (MCF-7) breast cancer cells are presented herein. This work further showed that folic acid (FA) functionalization of liposomes could be exploited for active drug delivery and herein led to an almost 3-fold increase in drug uptake vs non-FA functionalised liposomes in accordance with folate receptor (FR) expression levels between HeLa and MCF-7 cells. The in-vitro dark cytotoxicity and photodynamic therapy of selected Pc complexes and conjugates were accessed using MCF-7 and HeLa cell lines. The various mechanisms; (i) Type I PDT, (ii) PDT coupled with oxygen -independent therapy and (iii) in-situ oxygen generation using catalase-mimicking nanoparticles were shown to adequately compensate for the otherwise attenuation of PDT activity under hypoxia. , Thesis (PhD) -- Faculty of Science, Chemistry, 2023
- Full Text:
- Date Issued: 2023-10-13
- Authors: Nwahara, Nnamdi Ugochinyere
- Date: 2023-10-13
- Subjects: Liposomes , Photochemotherapy , Phthalocyanines , Photoelectrochemistry , Cancer Treatment
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/432159 , vital:72847 , DOI 10.21504/10962/432159
- Description: This thesis investigates new strategies to enhance the efficacy of photodynamic therapy (PDT) under hypoxic conditions using in-vitro cancer cell models. Phthalocyanines are chosen as viable photosensitizer complexes owing to the favourable absorption properties. To this end, this thesis reports on the synthesis and photophysicochemical properties of various zinc and silicon phthalocyanines (Pcs). To afford better photophysicochemical properties, the reported Pcs were conjugated to different nanoparticles (NPs) through chemisorption as well as amide bond formation to yield Pc-NP conjugates. All the studied Pcs showed relatively high triplet and singlet oxygen quantum yields corresponding to their low fluorescence quantum yields. The various mechanisms for hypoxic response include (i) Type I PDT, (ii) PDT coupled with oxygen-independent therapy and (iii) in-situ oxygen generation using catalase-mimicking nanoparticles which serve to supplement in-vitro oxygen concentrations using MPcs or MPc-NPs conjugates. The mechanisms were assessed using electrochemical, computational techniques and catalase mimicking experiments. The as-synthesised Pcs or Pc-NPs were subjected to liposomal loading before PDT studies which led to enhanced biocompatibility and aqueous dispersity. The in-vitro dark cytotoxicity tests and photodynamic therapy activities of the fabricated Pc-liposomes and Pc-NPs-liposomes on either Henrietta Lacks (HeLa) or Michigan Cancer Foundation-7 (MCF-7) breast cancer cells are presented herein. This work further showed that folic acid (FA) functionalization of liposomes could be exploited for active drug delivery and herein led to an almost 3-fold increase in drug uptake vs non-FA functionalised liposomes in accordance with folate receptor (FR) expression levels between HeLa and MCF-7 cells. The in-vitro dark cytotoxicity and photodynamic therapy of selected Pc complexes and conjugates were accessed using MCF-7 and HeLa cell lines. The various mechanisms; (i) Type I PDT, (ii) PDT coupled with oxygen -independent therapy and (iii) in-situ oxygen generation using catalase-mimicking nanoparticles were shown to adequately compensate for the otherwise attenuation of PDT activity under hypoxia. , Thesis (PhD) -- Faculty of Science, Chemistry, 2023
- Full Text:
- Date Issued: 2023-10-13
The development of ionic zinc(II) phthalocyanines for sono-photodynamic combination therapy of cervical and breast cancer
- Authors: Nene, Lindokuhle Cindy
- Date: 2023-03-31
- Subjects: Phthalocyanines , Sonochemistry , Photochemotherapy , Cancer Treatment
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/422565 , vital:71958 , DOI 10.21504/10962/422565
- Description: This study focuses on the development of the sono-photodynamic combination therapy (SPDT) activity of phthalocyanines (Pcs) on the cervical and breast cancer cell lines in vitro. The SPDT technique utilizes ultrasound in combination with light to elicit cytotoxic effects for cancer eradication. In this work, a selection of tetra-peripherally substituted Zn(II) cationic and zwitterionic Pcs were prepared. The photophysical parameters of the Pcs were determined including their fluorescence behaviours and efficiency of the triplet excited state population. The effects of the ultrasonic parameters (frequencies (MHz) and power (W.cm-2)) on the stability of the Pcs were evaluated. Four parameters were evaluated: Par I (1 MHz: 1 W.cm-2), Par II (1 MHz: 2 W.cm-2), Par III (3 MHz: 1 W.cm-2) and Par IV (3 MHz: 2 W.cm-2). The stability of the Pcs reduced with the increase in the ultrasonic power (for Par II and Par IV). The Par I showed the least degradation compared to the other parameters and was therefore used for the SPDT treatments. The sonodynamic (SDT), photodynamic (PDT) therapy activities of the Pcs were studied and compared to their SPDT efficacies. The Pcs showed reactive oxygen species generation during the SDT, PDT and SPDT treatments. For the SDT and SPDT, singlet oxygen (1O2) and hydroxyl radicals (•OH) were detected. For PDT, only the 1O2 were detected. The cell cytotoxicity studies for the Pcs showed relatively higher therapeutic efficacies for the SDT treatments compared to the PDT treatments, where the SPDT showed higher therapeutic efficacies compared to both the SDT and PDT monotreatments on both the cell lines in vitro. Overall, the combination treatments were better compared to the monotreatments. The activities of the Pcs were compared by their differences in structures, including the type of R-group, type of quaternizing agent and type of nanoparticle conjugates. , Thesis (PhD) -- Faculty of Science, Chemistry, 2023
- Full Text:
- Date Issued: 2023-03-31
- Authors: Nene, Lindokuhle Cindy
- Date: 2023-03-31
- Subjects: Phthalocyanines , Sonochemistry , Photochemotherapy , Cancer Treatment
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/422565 , vital:71958 , DOI 10.21504/10962/422565
- Description: This study focuses on the development of the sono-photodynamic combination therapy (SPDT) activity of phthalocyanines (Pcs) on the cervical and breast cancer cell lines in vitro. The SPDT technique utilizes ultrasound in combination with light to elicit cytotoxic effects for cancer eradication. In this work, a selection of tetra-peripherally substituted Zn(II) cationic and zwitterionic Pcs were prepared. The photophysical parameters of the Pcs were determined including their fluorescence behaviours and efficiency of the triplet excited state population. The effects of the ultrasonic parameters (frequencies (MHz) and power (W.cm-2)) on the stability of the Pcs were evaluated. Four parameters were evaluated: Par I (1 MHz: 1 W.cm-2), Par II (1 MHz: 2 W.cm-2), Par III (3 MHz: 1 W.cm-2) and Par IV (3 MHz: 2 W.cm-2). The stability of the Pcs reduced with the increase in the ultrasonic power (for Par II and Par IV). The Par I showed the least degradation compared to the other parameters and was therefore used for the SPDT treatments. The sonodynamic (SDT), photodynamic (PDT) therapy activities of the Pcs were studied and compared to their SPDT efficacies. The Pcs showed reactive oxygen species generation during the SDT, PDT and SPDT treatments. For the SDT and SPDT, singlet oxygen (1O2) and hydroxyl radicals (•OH) were detected. For PDT, only the 1O2 were detected. The cell cytotoxicity studies for the Pcs showed relatively higher therapeutic efficacies for the SDT treatments compared to the PDT treatments, where the SPDT showed higher therapeutic efficacies compared to both the SDT and PDT monotreatments on both the cell lines in vitro. Overall, the combination treatments were better compared to the monotreatments. The activities of the Pcs were compared by their differences in structures, including the type of R-group, type of quaternizing agent and type of nanoparticle conjugates. , Thesis (PhD) -- Faculty of Science, Chemistry, 2023
- Full Text:
- Date Issued: 2023-03-31
Fucoidans from South African brown seaweeds: establishing the link between their structure and biological properties (anti-diabetic and anti-cancer activities)
- Authors: Mabate, Blessing
- Date: 2022-10-14
- Subjects: Fucoidan , Diabetes Treatment , Cancer Treatment , Brown algae
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/365677 , vital:65775 , DOI https://doi.org/10.21504/10962/365677
- Description: Type 2 diabetes mellitus (T2DM) and cancer are major non-communicable diseases causing a heavy morbidity-mortality and economic burden globally. The therapeutic efforts in managing these diseases are primarily chemotherapeutic and are associated with demerits, including side effects and toxicity, limiting the prescribed amounts. These dosage limits may cause drug resistance, another major challenge in maintaining quality global health. The pursuit of novel natural bioproducts is a reasonable strategy to add to the arsenal against T2DM and cancer. Fucoidans, sulphated fucose polysaccharides abundant in brown seaweeds, have recently become popular for their biological activities, including anti-diabetic and anti-cancer properties. However, endemic South African brown seaweeds have not been adequately explored. Therefore, this study sought to characterise fucoidans extracted from South African brown seaweeds and elucidate their structure to their biological activities. Also, this study highlighted carbohydrate and glucose metabolism as major target processes in the control efforts of T2DM and cancer using fucoidans. Harvested brown seaweeds were identified as Ecklonia radiata and Sargassum elegans. E. maxima was kindly donated by KelpX. The fucoidans were then extracted using hot water, EDTA assisted, and acid extraction protocols. The integrity of the extracted fucoidan was confirmed through structural analysis using FTIR, NMR and TGA. The fucoidan extracts were then chemically characterised to determine their carbohydrate and monosaccharide composition and sulphate content. The characterised fucoidans were profiled for inhibiting the major amylolytic enzymes, namely α-amylase and α-glucosidase. The mode of inhibition by fucoidans and synergy experiments with the commercial anti-diabetic drug acarbose were also investigated. Furthermore, the fucoidans were screened for potential anti-cancer activities on the human colorectal HCT116 cancer cell line. The cytotoxicity of fucoidans was quantified using the resazurin assay. The effect of fucoidan on HCT116 cell adhesion on the tissue culture plastic was also investigated using the crystal violet-based cell adhesion assay. In addition, cancer antimigration properties of fucoidans were also investigated using 2D wound healing and 3D spheroid-based assays. Furthermore, the long-term survival of HCT116 cells was investigated through the clonogenic assay after treatment with fucoidans. Lastly, glucose uptake and lactate export assays revealed the influence of fucoidan on glucose uptake and the glycolytic flux of HCT116 cells. Fucoidans were successfully extracted with a yield between 2.2% and 14.2% on a dry weight basis. EDTA extracts produced the highest yields than the water and the acid extracts. Ecklonia spp. fucoidans displayed the highest total carbohydrate content, with glucose and galactose being the major monosaccharides. S. elegans and commercial Fucus vesiculosus had lower carbohydrate contents but contained more sulphates than the Ecklonia spp. fucoidans. Furthermore, the extracted fucoidan contained little to no contaminants, including proteins, phenolics and uronic acids. In addition, the extracted fucoidans were determined to be >100 kDa through ultracentrifugation. Mass spectrometry also detected the most abundant peak for all fucoidans to be around 700 Da (m/z). Extracted fucoidans inhibited the activity of α-glucosidase more strongly than the commercial anti-diabetic agent acarbose but were inactive on α-amylase. Fucoidans were also shown to be mixed inhibitors of α-glucosidase. Compellingly, fucoidans synergistically inhibited α-glucosidase in combination with the anti-diabetic agent acarbose, highlighting prospects for combination therapy. Finally, fucoidans demonstrated some anti-proliferative characteristics on HCT116 cancer cells by inhibiting their ability to adhere to the tissue culture plate matrix. Furthermore, some fucoidan extracts inhibited the migration of HCT116 cancer cells from 3D spheroids. Some of our fucoidan extracts also inhibited HCT116 colony formation, demonstrating inhibition of long-term cell survival. The E. maxima water extract also inhibited glucose uptake by HCT116 cells, thereby influencing the glycolytic flux. In conclusion, biologically active fucoidans were successfully extracted from South African brown seaweeds. These fucoidans demonstrated anti-diabetic and anti-cancer properties, revealing their relevance as potential drugs for these diseases. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
- Authors: Mabate, Blessing
- Date: 2022-10-14
- Subjects: Fucoidan , Diabetes Treatment , Cancer Treatment , Brown algae
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/365677 , vital:65775 , DOI https://doi.org/10.21504/10962/365677
- Description: Type 2 diabetes mellitus (T2DM) and cancer are major non-communicable diseases causing a heavy morbidity-mortality and economic burden globally. The therapeutic efforts in managing these diseases are primarily chemotherapeutic and are associated with demerits, including side effects and toxicity, limiting the prescribed amounts. These dosage limits may cause drug resistance, another major challenge in maintaining quality global health. The pursuit of novel natural bioproducts is a reasonable strategy to add to the arsenal against T2DM and cancer. Fucoidans, sulphated fucose polysaccharides abundant in brown seaweeds, have recently become popular for their biological activities, including anti-diabetic and anti-cancer properties. However, endemic South African brown seaweeds have not been adequately explored. Therefore, this study sought to characterise fucoidans extracted from South African brown seaweeds and elucidate their structure to their biological activities. Also, this study highlighted carbohydrate and glucose metabolism as major target processes in the control efforts of T2DM and cancer using fucoidans. Harvested brown seaweeds were identified as Ecklonia radiata and Sargassum elegans. E. maxima was kindly donated by KelpX. The fucoidans were then extracted using hot water, EDTA assisted, and acid extraction protocols. The integrity of the extracted fucoidan was confirmed through structural analysis using FTIR, NMR and TGA. The fucoidan extracts were then chemically characterised to determine their carbohydrate and monosaccharide composition and sulphate content. The characterised fucoidans were profiled for inhibiting the major amylolytic enzymes, namely α-amylase and α-glucosidase. The mode of inhibition by fucoidans and synergy experiments with the commercial anti-diabetic drug acarbose were also investigated. Furthermore, the fucoidans were screened for potential anti-cancer activities on the human colorectal HCT116 cancer cell line. The cytotoxicity of fucoidans was quantified using the resazurin assay. The effect of fucoidan on HCT116 cell adhesion on the tissue culture plastic was also investigated using the crystal violet-based cell adhesion assay. In addition, cancer antimigration properties of fucoidans were also investigated using 2D wound healing and 3D spheroid-based assays. Furthermore, the long-term survival of HCT116 cells was investigated through the clonogenic assay after treatment with fucoidans. Lastly, glucose uptake and lactate export assays revealed the influence of fucoidan on glucose uptake and the glycolytic flux of HCT116 cells. Fucoidans were successfully extracted with a yield between 2.2% and 14.2% on a dry weight basis. EDTA extracts produced the highest yields than the water and the acid extracts. Ecklonia spp. fucoidans displayed the highest total carbohydrate content, with glucose and galactose being the major monosaccharides. S. elegans and commercial Fucus vesiculosus had lower carbohydrate contents but contained more sulphates than the Ecklonia spp. fucoidans. Furthermore, the extracted fucoidan contained little to no contaminants, including proteins, phenolics and uronic acids. In addition, the extracted fucoidans were determined to be >100 kDa through ultracentrifugation. Mass spectrometry also detected the most abundant peak for all fucoidans to be around 700 Da (m/z). Extracted fucoidans inhibited the activity of α-glucosidase more strongly than the commercial anti-diabetic agent acarbose but were inactive on α-amylase. Fucoidans were also shown to be mixed inhibitors of α-glucosidase. Compellingly, fucoidans synergistically inhibited α-glucosidase in combination with the anti-diabetic agent acarbose, highlighting prospects for combination therapy. Finally, fucoidans demonstrated some anti-proliferative characteristics on HCT116 cancer cells by inhibiting their ability to adhere to the tissue culture plate matrix. Furthermore, some fucoidan extracts inhibited the migration of HCT116 cancer cells from 3D spheroids. Some of our fucoidan extracts also inhibited HCT116 colony formation, demonstrating inhibition of long-term cell survival. The E. maxima water extract also inhibited glucose uptake by HCT116 cells, thereby influencing the glycolytic flux. In conclusion, biologically active fucoidans were successfully extracted from South African brown seaweeds. These fucoidans demonstrated anti-diabetic and anti-cancer properties, revealing their relevance as potential drugs for these diseases. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
The Role of HOP in Emerin-Mediated Nuclear Structure
- Authors: Kituyi, Sarah Naulikha
- Date: 2017
- Subjects: Heat shock proteins , Nuclear structure , Nuclear membranes , Cancer Treatment , Molecular chaperones , Cytoskeleton , Cytoplasm
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/59230 , vital:27485 , DOI 10.21504/10962/59230
- Description: A vital component of the integral nuclear membrane is emerin, a Lamin Emerin and Man1 (LEM) domain protein whose concentration determines the levels of partner proteins that together constitute the structure of the nuclear envelope. Deficiencies in any of these proteins causes the failure of the structure and assembly and disassembly of the nuclear envelope, which disrupts chromosome segregation and nuclear compartmentalization that are both associated with disease. Emerin also localizes in the cytoplasm where it is implicated in the structure of the cytoskeleton via interaction with tubulin and actin and thus its deficiency may equally contribute to the collapse of the cytoskeleton. The Hsp70-Hsp90 organising protein (Hop) functions as a cochaperone for entry of client proteins into the Hsp90 folding cycle. Hop is upregulated in cancer and regulates a number of cell biology processes via interactions with proteins independently of Hsp90. In a previous study using global whole cell mass spectrometry, emerin was shown to be the most significantly down regulated protein in Hop depleted cell lysates. In this current study, it was postulated that emerin interacts with Hop, and this interaction regulates the stability, and level of emerin in the nucleus which impacts on the structure of the nuclear envelope. We used HEK293T cell lines stably expressing shRNA against Hop, emerin and a non-targeting control alongside the over expression of Hop in HEK293 cells to determine the effect of Hop levels on emerin expression and vice versa via Western blotting. The effect of Hop on the localization of emerin was assessed via subcellullar fractionation and confocal microscopy, while the impact on the structure of the nucleus was determined by transmission electron microscopy (TEM). We established that the depletion of Hop using shRNA and the over expression of Hop both result in the proteasomal and lysosomal degradation of emerin. Co-immunoprecipitation assays confirmed that Hop and emerin are in a common complex, which was not dependent on the presence of Hsp90. Loss of Hop or emerin led to a deformation of nuclear structure and a statistically significant decrease in nuclear size compared to control cells and was associated with an increase in the levels of nuclear protein, lamin A-C. Loss of emerin and Hop resulted in increased long term cell survival, but only after restriction of the nucleus when the cells had migrated across a transwell membrane. Taken together, the results obtained suggest that Hop acts as a scaffold for the stabilization of emerin and that the effects of Hop depletion on the structure of the nucleus and long term survival are mediated via the depletion of emerin. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2017
- Full Text:
- Date Issued: 2017
- Authors: Kituyi, Sarah Naulikha
- Date: 2017
- Subjects: Heat shock proteins , Nuclear structure , Nuclear membranes , Cancer Treatment , Molecular chaperones , Cytoskeleton , Cytoplasm
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/59230 , vital:27485 , DOI 10.21504/10962/59230
- Description: A vital component of the integral nuclear membrane is emerin, a Lamin Emerin and Man1 (LEM) domain protein whose concentration determines the levels of partner proteins that together constitute the structure of the nuclear envelope. Deficiencies in any of these proteins causes the failure of the structure and assembly and disassembly of the nuclear envelope, which disrupts chromosome segregation and nuclear compartmentalization that are both associated with disease. Emerin also localizes in the cytoplasm where it is implicated in the structure of the cytoskeleton via interaction with tubulin and actin and thus its deficiency may equally contribute to the collapse of the cytoskeleton. The Hsp70-Hsp90 organising protein (Hop) functions as a cochaperone for entry of client proteins into the Hsp90 folding cycle. Hop is upregulated in cancer and regulates a number of cell biology processes via interactions with proteins independently of Hsp90. In a previous study using global whole cell mass spectrometry, emerin was shown to be the most significantly down regulated protein in Hop depleted cell lysates. In this current study, it was postulated that emerin interacts with Hop, and this interaction regulates the stability, and level of emerin in the nucleus which impacts on the structure of the nuclear envelope. We used HEK293T cell lines stably expressing shRNA against Hop, emerin and a non-targeting control alongside the over expression of Hop in HEK293 cells to determine the effect of Hop levels on emerin expression and vice versa via Western blotting. The effect of Hop on the localization of emerin was assessed via subcellullar fractionation and confocal microscopy, while the impact on the structure of the nucleus was determined by transmission electron microscopy (TEM). We established that the depletion of Hop using shRNA and the over expression of Hop both result in the proteasomal and lysosomal degradation of emerin. Co-immunoprecipitation assays confirmed that Hop and emerin are in a common complex, which was not dependent on the presence of Hsp90. Loss of Hop or emerin led to a deformation of nuclear structure and a statistically significant decrease in nuclear size compared to control cells and was associated with an increase in the levels of nuclear protein, lamin A-C. Loss of emerin and Hop resulted in increased long term cell survival, but only after restriction of the nucleus when the cells had migrated across a transwell membrane. Taken together, the results obtained suggest that Hop acts as a scaffold for the stabilization of emerin and that the effects of Hop depletion on the structure of the nucleus and long term survival are mediated via the depletion of emerin. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2017
- Full Text:
- Date Issued: 2017
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