Targeting Redox Signalling Pathways as Anti-cancer strategy
Responsible: Lucia Coppo
Introduction
Increased generation of reactive oxygen species (ROS) and an upregulation of antioxidant systems is associated with abnormal cancer cell growth.
A promising, recently emerging, anti-cancer strategy exploit this characteristic of cancer cells by either increasing the generation of ROS or decreasing the capacity of the antioxidant systems. In fact, cancer cells would be more sensitive to further ROS accumulation than normal cells as they are already at the limit of their antioxidant capacity (1).
If the increase of ROS reaches a certain threshold cancer cells cannot upregulate further the antioxidant enzymes level, so a cytotoxic effect due to “overload” of ROS will selectively lead to malignant cells death (2).
Apatone (3) (vitamin C: vitamin K3, 100:1), is an investigational drug under clinical trials for cancer treatment. Extracellular Vitamin C (VC) is quickly oxidized to dehydroascorbic acid (DHA) and taken up by cells via glucose transporters, over-expressed in many cancer cells. It has been shown that Apatone kills cancer cells by inducing hydrogen peroxide via a redox cycling reaction, and in our previous study we investigated more in depth the mechanism.
Inside the cells, DHA is reduced by NADPH via GSH and glutaredoxin as well as by thioredoxin (Trx) and the selenoenzyme thioredoxin reductase (TrxR). We found that both the Trx and GSH/glutaredoxin systems were oxidized by Apatone, which resulted in the loss of GSH, increased glutathionylation, and highly oxidized Trx1.
Thioredoxin and GSH/glutaredoxin systems are antioxidants but also electron donors for ribonucleotide reductase. Apatone depleted the electron donors and also caused partial inactivation of the ribonucleotide reductase, an enzyme essential for deoxyribonucleotide synthesis required for DNA replication and repair. The ensuing oxidative and replicative stress in cancer cells activated cell death programs including lipid peroxidation and activation of apoptosis-inducing factor and cell death.
Despite the very promising results in vitro, VC can only reach low concentration in blood if orally administered, around 100µM (4), a concentration that in our preliminary data sensitize the cells but did not lead to highly effective reduction of cell viability.
In accordance with the principles of “drug re-proposal” (5) and the “Combined anticancer therapy” (6,7), we would like to combine Apatone with other known inhibitors of antioxidant systems looking for a synergistic or additive effect, to screen for possible new therapeutic approach against prostate cancer and pancreatic cancer.
A promising, recently emerging, anti-cancer strategy exploit this characteristic of cancer cells by either increasing the generation of ROS or decreasing the capacity of the antioxidant systems. In fact, cancer cells would be more sensitive to further ROS accumulation than normal cells as they are already at the limit of their antioxidant capacity (1).
If the increase of ROS reaches a certain threshold cancer cells cannot upregulate further the antioxidant enzymes level, so a cytotoxic effect due to “overload” of ROS will selectively lead to malignant cells death (2).
Apatone (3) (vitamin C: vitamin K3, 100:1), is an investigational drug under clinical trials for cancer treatment. Extracellular Vitamin C (VC) is quickly oxidized to dehydroascorbic acid (DHA) and taken up by cells via glucose transporters, over-expressed in many cancer cells. It has been shown that Apatone kills cancer cells by inducing hydrogen peroxide via a redox cycling reaction, and in our previous study we investigated more in depth the mechanism.
Inside the cells, DHA is reduced by NADPH via GSH and glutaredoxin as well as by thioredoxin (Trx) and the selenoenzyme thioredoxin reductase (TrxR). We found that both the Trx and GSH/glutaredoxin systems were oxidized by Apatone, which resulted in the loss of GSH, increased glutathionylation, and highly oxidized Trx1.
Thioredoxin and GSH/glutaredoxin systems are antioxidants but also electron donors for ribonucleotide reductase. Apatone depleted the electron donors and also caused partial inactivation of the ribonucleotide reductase, an enzyme essential for deoxyribonucleotide synthesis required for DNA replication and repair. The ensuing oxidative and replicative stress in cancer cells activated cell death programs including lipid peroxidation and activation of apoptosis-inducing factor and cell death.
Despite the very promising results in vitro, VC can only reach low concentration in blood if orally administered, around 100µM (4), a concentration that in our preliminary data sensitize the cells but did not lead to highly effective reduction of cell viability.
In accordance with the principles of “drug re-proposal” (5) and the “Combined anticancer therapy” (6,7), we would like to combine Apatone with other known inhibitors of antioxidant systems looking for a synergistic or additive effect, to screen for possible new therapeutic approach against prostate cancer and pancreatic cancer.
Project details
| MBB/Biochemistry | |
| Basic Science study | |
| 1 | |
| Not decided | |
| All data have already been collected and only need to be analyzed | |
| Ethical permit is not required |
Supervisor/Contact
Lucia Coppo
Lucia.coppo@ki.se
Contact 2
Aims
The project aims to find the optimal pre-incubation time and concentration with Apatone, followed by exposure to range of doses of different drugs like Auranofin, Aurothioglucose, Ebselen and Doxorubicin, which are all known to increase oxidative stress in cells. We will test two different gold-conpounds because they have different localization in cells: while auranofin acts mostly at mitochondrial level, therefore inhibiting Thioredoxin Reductase2 , ATG remain in the cytosol where it binds Thioredoxin Reductase 18.
Our final target will be to combine the VC:VK with a lower therapeutic dosage of chemioterapeutic drug to prevent the toxic effects on normal cells while simultaneously producing cytotoxic effects on cancer cells.
If the IC50 and the pre-incubation optimization with VC:VK will be performed in the minimum time planned (best work scenario) the student will be involved in the second step of the project that consist in study the mechanism of cell death. The experiments will include Western Blot to check the redox state of key proteins such as Thioredoxin and Peroxiredoxins and measurement of glutathione (reduced and oxidized form).
Our final target will be to combine the VC:VK with a lower therapeutic dosage of chemioterapeutic drug to prevent the toxic effects on normal cells while simultaneously producing cytotoxic effects on cancer cells.
If the IC50 and the pre-incubation optimization with VC:VK will be performed in the minimum time planned (best work scenario) the student will be involved in the second step of the project that consist in study the mechanism of cell death. The experiments will include Western Blot to check the redox state of key proteins such as Thioredoxin and Peroxiredoxins and measurement of glutathione (reduced and oxidized form).
Project time schedule
Experimental plan-Combined drugs:
• Apatone (VC:VK) max concentration 100µM VC: 1µM VK, which is the maximal concentration that can be reached by oral administration. Oral administration renders good compliance which could be a big advantage over i.v. infusion.
• Auranofin (AF) (8( Auranofin is a drug that is approved for the treatment of rheumatoid arthritis but is being investigated for potential therapeutic application in a number of other diseases including cancer, neurodegenerative disorders, HIV/AIDS, parasitic infections and bacterial infections. The main mechanism of action of auranofin is through the inhibition of reduction/oxidation (redox) enzymes that are essential for maintaining intracellular levels of reactive oxygen species.
• Aurothioglucose (ATG), a gold compound used clinically to treat rheumatoid arthritis, has recently been found to be a potent PKCiota-Par6 interaction inhibitor, with an IC50 approximately 1 μM. Disruption of this interaction disrupts a rac1 signaling pathway that is required for transformed growth in non-small-cell lung cancer. ATG is also known to be uptake by cells and inhibit Thioredoxin reductase activity (9).
• Ebselen (Ebs), is a synthetic organoselenium drug molecule with anti-inflammatory, anti-oxidant and cytoprotective activity. It acts as a mimic of glutathione peroxidase. It is being investigated as a possible treatment for reperfusion injury and stroke (10), hearing loss (11) and tinnitus, and bipolar disorder (12). Additionally, ebselen may be effective against bacterial infections (13,14).
• Doxorubicin (DOX), is a chemotherapy drug and is also known by its brand name Adriamycin. It is a treatment for many different types of cancer, breast cancer, advanced ovarian cancer in women who have failed a first-line platinum-based chemotherapy regimen, used in combination with bortezomib for the treatment of progressive multiple myeloma in patients who have received at least one prior therapy and who have already undergone or are unsuitable for bone marrow transplant.
Experimental plan- Treatment : Different cell lines, PANC-1 and PC3, representative of aggressive tumors, will be treated at different concentration of VC:VK and for different time points.
Experimental plan- Viability assays: Viability test will be performed after 72h from the drug addition
• MTT tetrazolium assay (15):
MTT which is positively charged and it is uptake by viable eukaryotic cells. Viable cells with active metabolism convert MTT into a purple coloured formazan product with an absorbance maximum near 570 nm. When cells die, they lose the ability to convert MTT into formazan. The conversion seems to be exerted by mitochondrial enzymes therefore MTT is considered measuring mitochondrial activity.
The formazan product of the MTT tetrazolium accumulates as an insoluble precipitate inside cells as well as being deposited near the cell surface and in the culture medium. The formazan must be solubilized prior to recording absorbance readings. The amount of signal generated is dependent on several parameters including: the concentration of MTT, the length of the incubation period, the number of viable cells and their metabolic activity.
The MTT assay was developed as a non-radioactive alternative to tritiated thymidine incorporation into DNA for measuring cell proliferation.
• Neutral red16: It is based on the ability of viable cells to incorporate and bind the supravital dye neutral red. This weakly cationic dye penetrates cell membranes by nonionic passive diffusion and concentrates in the lysosomes, where it binds by electrostatic hydrophobic bonds to anionic and/or phosphate groups of the lysosomal matrix. The dye is then extracted from the viable cells using an acidified ethanol solution, and the absorbance of the solubilized dye is quantified using a spectrophotometer.
• Apatone (VC:VK) max concentration 100µM VC: 1µM VK, which is the maximal concentration that can be reached by oral administration. Oral administration renders good compliance which could be a big advantage over i.v. infusion.
• Auranofin (AF) (8( Auranofin is a drug that is approved for the treatment of rheumatoid arthritis but is being investigated for potential therapeutic application in a number of other diseases including cancer, neurodegenerative disorders, HIV/AIDS, parasitic infections and bacterial infections. The main mechanism of action of auranofin is through the inhibition of reduction/oxidation (redox) enzymes that are essential for maintaining intracellular levels of reactive oxygen species.
• Aurothioglucose (ATG), a gold compound used clinically to treat rheumatoid arthritis, has recently been found to be a potent PKCiota-Par6 interaction inhibitor, with an IC50 approximately 1 μM. Disruption of this interaction disrupts a rac1 signaling pathway that is required for transformed growth in non-small-cell lung cancer. ATG is also known to be uptake by cells and inhibit Thioredoxin reductase activity (9).
• Ebselen (Ebs), is a synthetic organoselenium drug molecule with anti-inflammatory, anti-oxidant and cytoprotective activity. It acts as a mimic of glutathione peroxidase. It is being investigated as a possible treatment for reperfusion injury and stroke (10), hearing loss (11) and tinnitus, and bipolar disorder (12). Additionally, ebselen may be effective against bacterial infections (13,14).
• Doxorubicin (DOX), is a chemotherapy drug and is also known by its brand name Adriamycin. It is a treatment for many different types of cancer, breast cancer, advanced ovarian cancer in women who have failed a first-line platinum-based chemotherapy regimen, used in combination with bortezomib for the treatment of progressive multiple myeloma in patients who have received at least one prior therapy and who have already undergone or are unsuitable for bone marrow transplant.
Experimental plan- Treatment : Different cell lines, PANC-1 and PC3, representative of aggressive tumors, will be treated at different concentration of VC:VK and for different time points.
Experimental plan- Viability assays: Viability test will be performed after 72h from the drug addition
• MTT tetrazolium assay (15):
MTT which is positively charged and it is uptake by viable eukaryotic cells. Viable cells with active metabolism convert MTT into a purple coloured formazan product with an absorbance maximum near 570 nm. When cells die, they lose the ability to convert MTT into formazan. The conversion seems to be exerted by mitochondrial enzymes therefore MTT is considered measuring mitochondrial activity.
The formazan product of the MTT tetrazolium accumulates as an insoluble precipitate inside cells as well as being deposited near the cell surface and in the culture medium. The formazan must be solubilized prior to recording absorbance readings. The amount of signal generated is dependent on several parameters including: the concentration of MTT, the length of the incubation period, the number of viable cells and their metabolic activity.
The MTT assay was developed as a non-radioactive alternative to tritiated thymidine incorporation into DNA for measuring cell proliferation.
• Neutral red16: It is based on the ability of viable cells to incorporate and bind the supravital dye neutral red. This weakly cationic dye penetrates cell membranes by nonionic passive diffusion and concentrates in the lysosomes, where it binds by electrostatic hydrophobic bonds to anionic and/or phosphate groups of the lysosomal matrix. The dye is then extracted from the viable cells using an acidified ethanol solution, and the absorbance of the solubilized dye is quantified using a spectrophotometer.
Teaching/Supervision activities
Dr. Lucia Coppo will be responsible for supervision of the student. The student will participate in weekly seminars in the division of Biochemistry, MBB and will have access to the Biomedicum seminars.
Miscellaneous
1. Trachootham, D., Alexandre, J. & Huang, P. Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach? Nat. Rev. Drug Discov. 8, 579–91 (2009).
2. Benhar, M., Shytaj, I. L., Stamler, J. S. & Savarino, A. Dual targeting of the thioredoxin and glutathione systems in cancer and HIV. J. Clin. Invest. 126, 1630–1639 (2016).
3. Tareen, B. et al. A 12 week, open label, phase I/IIa study using apatone for the treatment of prostate cancer patients who have failed standard therapy. Int. J. Med. Sci. 5, 62–7 (2008).
4. Medicine, I. of. Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium, and Carotenoids. (National Academies Press, 2000). doi:10.17226/9810
5. Chong, C. R. & Sullivan, D. J. New uses for old drugs. Nature 448, 645–646 (2007).
6. Piccolo, M. T., Menale, C. & Crispi, S. Combined anticancer therapies: an overview of the latest applications. Anticancer. Agents Med. Chem. 15, 408–22 (2015).
7. Bayat Mokhtari, R. et al. Combination therapy in combating cancer. Oncotarget 8, 38022–38043 (2017).
8. Wang, H. et al. Auranofin radiosensitizes tumor cells through targeting thioredoxin reductase and resulting overproduction of reactive oxygen species. Oncotarget 8, 35728–35742 (2017).
9. Du, Y., Zhang, H., Lu, J. & Holmgren, A. Glutathione and glutaredoxin act as a backup of human thioredoxin reductase 1 to reduce thioredoxin 1 preventing cell death by aurothioglucose. J. Biol. Chem. 287, 38210–9 (2012).
10. Yamaguchi, T. et al. Ebselen in acute ischemic stroke: a placebo-controlled, double-blind clinical trial. Ebselen Study Group. Stroke 29, 12–7 (1998).
11. Kil, J. et al. Safety and efficacy of ebselen for the prevention of noise-induced hearing loss: a randomised, double-blind, placebo-controlled, phase 2 trial. Lancet 390, 969–979 (2017).
12. Singh, N. et al. A safe lithium mimetic for bipolar disorder. Nat. Commun. 4, 1332 (2013).
13. Zou, L. et al. Synergistic antibacterial effect of silver and ebselen against multidrug‐resistant Gram‐negative bacterial infections. EMBO Mol. Med. 9, 1165–1178 (2017).
14. Thangamani, S., Younis, W. & Seleem, M. N. Repurposing ebselen for treatment of multidrug-resistant staphylococcal infections. Sci. Rep. 5, 11596 (2015).
15. Riss, T. L. et al. Cell Viability Assays. Assay Guidance Manual (Eli Lilly & Company and the National Center for Advancing Translational Sciences, 2004).
16. Repetto, G., del Peso, A. & Zurita, J. L. Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nat. Protoc. 3, 1125–1131 (2008).
2. Benhar, M., Shytaj, I. L., Stamler, J. S. & Savarino, A. Dual targeting of the thioredoxin and glutathione systems in cancer and HIV. J. Clin. Invest. 126, 1630–1639 (2016).
3. Tareen, B. et al. A 12 week, open label, phase I/IIa study using apatone for the treatment of prostate cancer patients who have failed standard therapy. Int. J. Med. Sci. 5, 62–7 (2008).
4. Medicine, I. of. Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium, and Carotenoids. (National Academies Press, 2000). doi:10.17226/9810
5. Chong, C. R. & Sullivan, D. J. New uses for old drugs. Nature 448, 645–646 (2007).
6. Piccolo, M. T., Menale, C. & Crispi, S. Combined anticancer therapies: an overview of the latest applications. Anticancer. Agents Med. Chem. 15, 408–22 (2015).
7. Bayat Mokhtari, R. et al. Combination therapy in combating cancer. Oncotarget 8, 38022–38043 (2017).
8. Wang, H. et al. Auranofin radiosensitizes tumor cells through targeting thioredoxin reductase and resulting overproduction of reactive oxygen species. Oncotarget 8, 35728–35742 (2017).
9. Du, Y., Zhang, H., Lu, J. & Holmgren, A. Glutathione and glutaredoxin act as a backup of human thioredoxin reductase 1 to reduce thioredoxin 1 preventing cell death by aurothioglucose. J. Biol. Chem. 287, 38210–9 (2012).
10. Yamaguchi, T. et al. Ebselen in acute ischemic stroke: a placebo-controlled, double-blind clinical trial. Ebselen Study Group. Stroke 29, 12–7 (1998).
11. Kil, J. et al. Safety and efficacy of ebselen for the prevention of noise-induced hearing loss: a randomised, double-blind, placebo-controlled, phase 2 trial. Lancet 390, 969–979 (2017).
12. Singh, N. et al. A safe lithium mimetic for bipolar disorder. Nat. Commun. 4, 1332 (2013).
13. Zou, L. et al. Synergistic antibacterial effect of silver and ebselen against multidrug‐resistant Gram‐negative bacterial infections. EMBO Mol. Med. 9, 1165–1178 (2017).
14. Thangamani, S., Younis, W. & Seleem, M. N. Repurposing ebselen for treatment of multidrug-resistant staphylococcal infections. Sci. Rep. 5, 11596 (2015).
15. Riss, T. L. et al. Cell Viability Assays. Assay Guidance Manual (Eli Lilly & Company and the National Center for Advancing Translational Sciences, 2004).
16. Repetto, G., del Peso, A. & Zurita, J. L. Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nat. Protoc. 3, 1125–1131 (2008).