Resistance of lung cancer cells grown as multicellular tumour spheroids to Zinc sulfophthalocyanine photosensitization
- Manoto, Sello Lebohang, Houreld, Nicolette Nadene, Abrahamse, Heidi
- Authors: Manoto, Sello Lebohang , Houreld, Nicolette Nadene , Abrahamse, Heidi
- Date: 2016
- Subjects: Apoptosis , Resistance , Cytotoxicity
- Language: English
- Type: Conference proceedings
- Identifier: http://hdl.handle.net/10210/91933 , uj:20164 , Citation: Manoto, S.L., Houreld, N.N. & Abrahamse, H. 2016. Resistance of lung cancer cells grown as multicellular tumour spheroids to Zinc sulfophthalocyanine photosensitization.
- Description: Abstract: Photodynamic therapy (PDT) is phototherapeutic modality used in the treatment of neoplastic and non-neoplastic diseases. The photochemical interaction of light, photosensitizer (PS) and molecular oxygen produces singlet oxygen which induces cell death. Zinc sulfophthalocyanine (ZnPcSmix) has been shown to be effective in A549 monolayers, multicellular tumor spheroids (MCTSs) (250 μm) and not on MCTSs with a size of 500 μm. A549 cells used in this study were grown as MCTSs to a size of 500 μm in order to determine their susceptibility to PDT. ZnPcSmix distribution in MCTSs and nuclear morphology was determined using a fluorescent microscope. Changes in cellular responses were evaluated using cell morphology, viability, proliferation, cytotoxicity, cell death analysis and mitochondrial membrane potential. Untreated MCTSs, showed no changes in cellular morphology, proliferation, cytotoxicity and nuclear morphology. Photoactivated ZnPcSmix also showed no changes in cellular morphology and nuclear morphology. However, photoactivated ZnPcSmix resulted in a significant dose dependant decrease in viability and proliferation as well as an increase in cell membrane damage in MCTSs over time. ZnPcSmix photosensitization induces apoptotic cell death in MCTSs with a size of 500 μm and more resistantance when compared to monolayer cells and MCTSs
- Full Text:
- Authors: Manoto, Sello Lebohang , Houreld, Nicolette Nadene , Abrahamse, Heidi
- Date: 2016
- Subjects: Apoptosis , Resistance , Cytotoxicity
- Language: English
- Type: Conference proceedings
- Identifier: http://hdl.handle.net/10210/91933 , uj:20164 , Citation: Manoto, S.L., Houreld, N.N. & Abrahamse, H. 2016. Resistance of lung cancer cells grown as multicellular tumour spheroids to Zinc sulfophthalocyanine photosensitization.
- Description: Abstract: Photodynamic therapy (PDT) is phototherapeutic modality used in the treatment of neoplastic and non-neoplastic diseases. The photochemical interaction of light, photosensitizer (PS) and molecular oxygen produces singlet oxygen which induces cell death. Zinc sulfophthalocyanine (ZnPcSmix) has been shown to be effective in A549 monolayers, multicellular tumor spheroids (MCTSs) (250 μm) and not on MCTSs with a size of 500 μm. A549 cells used in this study were grown as MCTSs to a size of 500 μm in order to determine their susceptibility to PDT. ZnPcSmix distribution in MCTSs and nuclear morphology was determined using a fluorescent microscope. Changes in cellular responses were evaluated using cell morphology, viability, proliferation, cytotoxicity, cell death analysis and mitochondrial membrane potential. Untreated MCTSs, showed no changes in cellular morphology, proliferation, cytotoxicity and nuclear morphology. Photoactivated ZnPcSmix also showed no changes in cellular morphology and nuclear morphology. However, photoactivated ZnPcSmix resulted in a significant dose dependant decrease in viability and proliferation as well as an increase in cell membrane damage in MCTSs over time. ZnPcSmix photosensitization induces apoptotic cell death in MCTSs with a size of 500 μm and more resistantance when compared to monolayer cells and MCTSs
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Phototoxic effects of Zn sulfophthalocyanine on lung cancer cells (A549) grown as a monolayer and three dimensional multicellular tumour spheroids
- Authors: Manoto, Sello Lebohang
- Date: 2015-07-16
- Subjects: Lungs - Cancer - Photochemotherapy , Drugs - Toxicology
- Type: Thesis
- Identifier: uj:13794 , http://hdl.handle.net/10210/14059
- Description: D.Tech. (Biomedical Technology) , Photodynamic therapy (PDT) is an alternative treatment modality for malignant tumours based on the photodamage to tumour cells through a photochemical reaction (Ahn et al., 2013). PDT utilizes a light sensitive photosensitizer (PS) that selectively localizes in tumour cells and is excited by light of a specific wavelength in the presence of molecular oxygen. The excited PS leads to the generation of singlet oxygen or other reactive oxygen species(ROS) which induces cytotoxic damage to cellular organelles and eventually cell death. Singlet oxygen has a very short life and its generation is controlled by the presence of the PS and the laser light (Senge and Radomski, 2013).The subcellular localization site of the PS plays a vital role in determining the effectiveness and the extent of cellular damage as well as the mechanism involved in cell death. Lung cancer is the leading cause of cancer death worldwide in both males and females, with an estimated 1.4 million deaths each year (American Cancer Society, 2011). Therapeutic modalities used in the treatment of lung cancer such as chemotherapy, radiotherapy and immunotherapy have rarely yielded a good prognosis and effective treatment remains a challenging problem to date. An alternative treatment modality with minimal complications such as PDT needs to be explored. Most in vitro PDT experiments are conducted on monolayer cultures and the cellular environment of these cultures does not correspond to that of in vivo studies. Multicellular tumour spheroids (MCTSs) serves as an important model in cancer research for the evaluation of therapeutic interventions since they mimic different aspects of the human tumour tissue environment.
- Full Text:
- Authors: Manoto, Sello Lebohang
- Date: 2015-07-16
- Subjects: Lungs - Cancer - Photochemotherapy , Drugs - Toxicology
- Type: Thesis
- Identifier: uj:13794 , http://hdl.handle.net/10210/14059
- Description: D.Tech. (Biomedical Technology) , Photodynamic therapy (PDT) is an alternative treatment modality for malignant tumours based on the photodamage to tumour cells through a photochemical reaction (Ahn et al., 2013). PDT utilizes a light sensitive photosensitizer (PS) that selectively localizes in tumour cells and is excited by light of a specific wavelength in the presence of molecular oxygen. The excited PS leads to the generation of singlet oxygen or other reactive oxygen species(ROS) which induces cytotoxic damage to cellular organelles and eventually cell death. Singlet oxygen has a very short life and its generation is controlled by the presence of the PS and the laser light (Senge and Radomski, 2013).The subcellular localization site of the PS plays a vital role in determining the effectiveness and the extent of cellular damage as well as the mechanism involved in cell death. Lung cancer is the leading cause of cancer death worldwide in both males and females, with an estimated 1.4 million deaths each year (American Cancer Society, 2011). Therapeutic modalities used in the treatment of lung cancer such as chemotherapy, radiotherapy and immunotherapy have rarely yielded a good prognosis and effective treatment remains a challenging problem to date. An alternative treatment modality with minimal complications such as PDT needs to be explored. Most in vitro PDT experiments are conducted on monolayer cultures and the cellular environment of these cultures does not correspond to that of in vivo studies. Multicellular tumour spheroids (MCTSs) serves as an important model in cancer research for the evaluation of therapeutic interventions since they mimic different aspects of the human tumour tissue environment.
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The effect of ZN sulfophthalocyanine and laser irradiation (636 nm) on a lung cancer cell line (A549)
- Authors: Manoto, Sello Lebohang
- Date: 2009-11-16T06:27:09Z
- Subjects: Cancer - Photochemotherapy , Lungs
- Type: Thesis
- Identifier: uj:8640 , http://hdl.handle.net/10210/2998
- Description: M. Tech. , Photodynamic therapy (PDT) is a type of phototherapy which is based on the interaction of photosensitizer with visible light in the presence of oxygen resulting in the production of reactive oxygen species (Pfaffel-Schubart et al., 2008). PDT has rapidly matured in the past 6 years and is an accepted standard treatment for various cancerous diseases. The main advantages of PDT include significant drug selectivity in tumour cells, absence of toxicity in the dark, possibility to treat only cancer cells and the ability to retreat a tumour in order to improve the prognosis (Pfaffel-Schubart et al., 2008). Photofrins are the most studied photosensitizers and their disadvantages is the inability of these photosensitizers to localize specifically in tumour cells and are retained in normal cells for prolonged periods (Nowis et al., 2005). This factor has stimulated the development of second generation photosensitizers with improved physical, chemical and spectral properties (Sharman et al., 1999). Phthalocyanine compounds are second generation photosensitizers which have a potential as photosensitizers in PDT treatment of many cancers. Traditional treatments of cancer show limited efficiency and other treatment modalities need to be explored. PDT has shown promising results in the treatment of many cancers using phthalocyanine compounds as photosensitizers but little is known about Zn sulfophthalocyanine on lung cancer cells. This study investigated the effects of Zn sulfophthalocyanine photosensitizer and laser irradiation on a lung cancer cell line (A549). Lung cancer cells were cultured in RPMI medium 1640 supplemented with 10% foetal bovine serum and antibiotics and incubated at 37 °C with 5% CO2 and 85% humidity. Cells were divided into 4 groups. Group 1 was an unirradiated control not treated with a photosensitizer. Group 2 was photosensitized at a concentration of 15.8 μM but not irradiated. Group 3 was irradiated but not photosensitized while Group 4 was irradiated and photosensitized at a concentration of 15.8 μM. Laser irradiations were done using a diode laser emitting 636 nm with an output of 110 mW at 5.3 J/cm2. Cell morphology was evaluated using the light inverted microscope. Cell viability was assessed using adenosine triphosphate (ATP) luminescence assay and Trypan blue exclusion test while cell proliferation was measured using the alamarBlue assay. Cytotoxicity was evaluated by assessing membrane permeability for lactate dehydrogenase and DNA damage was evaluated by comet assay. The mode of cell death was assessed by annexin V-FITC apoptosis detection kit using flow cytometry. In addition, expression of Bcl-2/Bax was monitored by western blot analysis and to determine the levels of induced Heat shock protein 70, Hsp 70 ELISA was used. Cells treated with photosensitizer at 15.8 μM and irradiated with 5.3 J/cm2 showed changes in cell morphology, decrease in cellular viability, proliferation and Hsp 70 expression and an increase in cytotoxicity and DNA damage. This indicated that the metalated phthalocyanine was effective in inducing cell death and the analysis of cell death by annexin FITC-V revealed an apoptotic cell death pattern. Furthermore, loss of the antiapoptotic Bcl-2 protein suggested that there was no inhibition of apoptosis while the absence of proapoptotic Bax suggested that other proapoptotic protein might have played a role in the induction of apoptosis. Untreated cells, irradiated cells and cells treated with photosentitizer alone showed no changes in morphology, increase in cellular viability, proliferation, expression of Bcl2/Bax, and Hsp 70 and a decrease in DNA damage and cell membrane damage. However, treatment of cell with photosensitizer alone caused DNA damage. These results indicate that irradiation or photosensitizer alone has no effect on the A549 cells but photosensitizer alone induces DNA damage.
- Full Text:
- Authors: Manoto, Sello Lebohang
- Date: 2009-11-16T06:27:09Z
- Subjects: Cancer - Photochemotherapy , Lungs
- Type: Thesis
- Identifier: uj:8640 , http://hdl.handle.net/10210/2998
- Description: M. Tech. , Photodynamic therapy (PDT) is a type of phototherapy which is based on the interaction of photosensitizer with visible light in the presence of oxygen resulting in the production of reactive oxygen species (Pfaffel-Schubart et al., 2008). PDT has rapidly matured in the past 6 years and is an accepted standard treatment for various cancerous diseases. The main advantages of PDT include significant drug selectivity in tumour cells, absence of toxicity in the dark, possibility to treat only cancer cells and the ability to retreat a tumour in order to improve the prognosis (Pfaffel-Schubart et al., 2008). Photofrins are the most studied photosensitizers and their disadvantages is the inability of these photosensitizers to localize specifically in tumour cells and are retained in normal cells for prolonged periods (Nowis et al., 2005). This factor has stimulated the development of second generation photosensitizers with improved physical, chemical and spectral properties (Sharman et al., 1999). Phthalocyanine compounds are second generation photosensitizers which have a potential as photosensitizers in PDT treatment of many cancers. Traditional treatments of cancer show limited efficiency and other treatment modalities need to be explored. PDT has shown promising results in the treatment of many cancers using phthalocyanine compounds as photosensitizers but little is known about Zn sulfophthalocyanine on lung cancer cells. This study investigated the effects of Zn sulfophthalocyanine photosensitizer and laser irradiation on a lung cancer cell line (A549). Lung cancer cells were cultured in RPMI medium 1640 supplemented with 10% foetal bovine serum and antibiotics and incubated at 37 °C with 5% CO2 and 85% humidity. Cells were divided into 4 groups. Group 1 was an unirradiated control not treated with a photosensitizer. Group 2 was photosensitized at a concentration of 15.8 μM but not irradiated. Group 3 was irradiated but not photosensitized while Group 4 was irradiated and photosensitized at a concentration of 15.8 μM. Laser irradiations were done using a diode laser emitting 636 nm with an output of 110 mW at 5.3 J/cm2. Cell morphology was evaluated using the light inverted microscope. Cell viability was assessed using adenosine triphosphate (ATP) luminescence assay and Trypan blue exclusion test while cell proliferation was measured using the alamarBlue assay. Cytotoxicity was evaluated by assessing membrane permeability for lactate dehydrogenase and DNA damage was evaluated by comet assay. The mode of cell death was assessed by annexin V-FITC apoptosis detection kit using flow cytometry. In addition, expression of Bcl-2/Bax was monitored by western blot analysis and to determine the levels of induced Heat shock protein 70, Hsp 70 ELISA was used. Cells treated with photosensitizer at 15.8 μM and irradiated with 5.3 J/cm2 showed changes in cell morphology, decrease in cellular viability, proliferation and Hsp 70 expression and an increase in cytotoxicity and DNA damage. This indicated that the metalated phthalocyanine was effective in inducing cell death and the analysis of cell death by annexin FITC-V revealed an apoptotic cell death pattern. Furthermore, loss of the antiapoptotic Bcl-2 protein suggested that there was no inhibition of apoptosis while the absence of proapoptotic Bax suggested that other proapoptotic protein might have played a role in the induction of apoptosis. Untreated cells, irradiated cells and cells treated with photosentitizer alone showed no changes in morphology, increase in cellular viability, proliferation, expression of Bcl2/Bax, and Hsp 70 and a decrease in DNA damage and cell membrane damage. However, treatment of cell with photosensitizer alone caused DNA damage. These results indicate that irradiation or photosensitizer alone has no effect on the A549 cells but photosensitizer alone induces DNA damage.
- Full Text:
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