p53 dependent and independent therapy: Developing resazurin as a novel targeted therapy in acute myeloid leukemia
Prosjekt
- Prosjektnummer
- 911930
- Ansvarlig person
- Trung Quang Ha
- Institusjon
- Helse Bergen HF
- Prosjektkategori
- Doktorgradsstipend
- Helsekategori
- Blood, Cancer
- Forskningsaktivitet
- 5. Treatment Developement
Rapporter
Resazurin, a dye used in cell viability assays, has been shown to have cytotoxic effects on leukemic cells. However, its molecular mechanisms of action are still elusive. Here we investigated the pharmacological and biological effect of resazurin and its reduced form resorufin in acute myeloid leukemia (AML). Treatment with resazurin or resorufin alone induced cell death in AML cell lines cultured both at normoxia and hypoxia. Also, resazurin caused differentiation of MOLM-13 and MV4-11 cells. Furthermore, resazurin inhibited proliferation and induced apoptosis in AML patient cells. We found that both resazurin and resorufin inhibited CK2 in vitro and in intact cells, and that altered CK2 expression modulated the cellular response to resazurin. Moreover, an attenuation of protein level of phosphorylated Akt (S129) and STAT5 (Y694) after resazurin treatment was observed. To evaluate the efficacy of resazurin in vivo, we developed a subcutaneous and an orthotopic NOD/SCID IL2rγ null (NSG) xenograft murine model of MV4-11. Treatment with resazurin significantly decreased tumor growth and prolonged survival of treated mice in comparison with control mice (p=0.0002). We also investigated the activities of resazurin in AML cell lines with deletion or mutations of TP53. TP53 mutations were found in more than half of AML cases with complex karyotype and are associated with older age, genomic complexity, conventional chemotherapy resistance and poor outcome. Leukemic cells having low level or absence of p53 protein were sensitive to resazurin. Treatment with resazurin, cultured both at normoxia and hypoxia, induced cell death in AML cell lines with deletion or mutations of TP53 (HL-60, NB4, THP-1 and U937). Treatment with resazurin induced p53-independent downregulation of the expression level of c-Myc and Mdm2 and upregulation of p21. Moreover, the anti-apoptotic proteins Mcl-1, Bcl-xL, XIAP and a cleavage of caspase-9 and caspase-3 after treatment with resazurin were observed.
Valproic acid (VPA), a well-tolerated anticonvulsant drug, has been shown to induce apoptosis, inhibit histone deacetylases’ activity, activate tumour suppressor p53, downregulate c-Myc and increase p21 expression levels in AML cells. We investigated the effect of combination therapy of resazurin and VPA in four different AML cell lines (MV4-11, MOLM-13, NB4 and HL-60) and AML patient cells in vitro. We found that the combination therapy of resazurin and VPA induced cell death synergistically in AML cell lines as analysed by the Chou-Talalay method. 6 of 13 AML patient samples tested revealed synergistic effect of the combination therapy on viability as analysed by Bliss Independence calculation. Resazurin and VPA co-treatment resulted in massive upregulation of p53 and p53 targets (Mdm2, p21, Puma) compared with either agent alone in wild-type TP53 AML cell line MOLM-13. Furthermore, the combinational treatment caused super additive downregulation of the expression levels of c-Myc, Mdm2, anti-apoptotic protein Mcl-1 and substantial upregulation of p21 as compared to each agent alone in deleted TP53 AML cell line HL-60.
Our results suggest that resazurin/resorufin may represent an experimental therapeutics in AML. Moreover, the combination therapy of resazurin and VPA needs to be further elucidated in vivo for developing a potential new combination therapy in the treatment of AML.
The project is a preclinical research. Therefore, it has no user participation.
Combination therapy of resazurin and valproic acid (VPA) induced synergistic apoptosis in AML cell lines and patient cells. The combination caused substantial upregulation of p53 and p53 targets in wild-type TP53 AML cell line or massive downregulation of c-Myc, Mdm2 and Mcl-1 in mutated TP53 AML cell line in comparison with each agent alone. The combination of resazurin and VPA needs to be further elucidated for developing a potential new combination therapy in the treatment of AML.Acute myeloid leukemia (AML) is a malignant disease of hematopoietic tissues represented by the accumulation of clonal abnormal (leukemic) myeloid progenitors and hematopoietic stem cells, accompanied by impaired production of normal blood cells. AML is the most common acute leukemia accounting for nearly 30% of all adult leukemias with three year survival below 10% in patients over 60 years of age. Hence, there is an unmet need for low toxic therapy in elderly AML patients with co-morbidities. Resazurin, the principle component of Alamar Blue, has been widely used in cell viability assays through the conversion from its blue color to the pink color of its fluorescent product, resorufin. Resazurin has been shown to induce apoptosis in AML cells. We have previously found that treatment with resazurin induced p53-independent downregulation of c-Myc and Mdm2 oncoproteins and upregulation of p21 in AML cells. Valproic acid (VPA) is a well-tolerated anticonvulsant drug which has been clinically utilized for many years. VPA has been shown to induce apoptosis in AML cells with a limited toxicity profile in elderly AML patients. VPA has also been shown to inhibit histone deacetylases’ activity which in turn induces the acetylation and activation of tumour suppressor p53. Besides, it was found that VPA downregulated c-Myc and increased p21 expression levels in AML cells. We hypothesized that co-treatment of resazurin and VPA would additively inhibit AML cells. In this study, we investigated the effect of combination therapy of resazurin and VPA in four different AML cell lines (MV4-11, MOLM-13, NB4 and HL-60) and AML patient cells in vitro. We found that the combination therapy of resazurin and VPA induced cell death synergistically in AML cell lines as analysed by the Chou-Talalay method. AML cells from heterogenous AML patients displayed various sensitivities to the combination treatment. Analysis of 11 AML patient samples for synergism by Bliss Independence calculation showed that 5 patient samples revealed synergistic effect of the combination therapy on viability of the AML patient cells. Cell death was assessed by Annexin V – To-pro-3 labelling or nuclear staining. Next, we investigated molecular mechanisms causing the synergistic anti-leukemic activity of resazurin and VPA in AML. Resazurin and VPA co-treatment resulted in massive upregulation of p53 and p53 targets (Mdm2, p21, Puma) compared with either agent alone in wild-type TP53 AML cell line MOLM-13. Moreover, the combinational treatment caused super additive downregulation of the expression levels of c-Myc, Mdm2, anti-apoptotic protein Mcl-1 and substantial upregulation of p21 as compared to each agent alone in deleted TP53 AML cell line HL-60. To preliminarily evaluate the effect of resazurin in vivo, MOLM-13 cells or HL-60 cells were orthotopically injected into NOD/SCID IL2 receptor gamma null (NSG) mice. Treatment of mice (n=4) with 75 mg/kg resazurin and 300 mg/kg VPA once a day, four consecutive days per week in two continuous weeks did not significantly prolong survival as compared to control mice (n=4). These achieved results suggest that the combination therapy of resazurin and VPA needs to be further elucidated in vivo for developing a potential new combination therapy in the treatment of AML.
Resazurin/resorufin induced apoptosis in AML cell lines and patient cells. They downregulated the protein level of phosphorylated CK2 alpha in MV4-11 cells. Treatment with resazurin prolonged mice survival and eliminated the engraftment of MV4-11 cells. Our results suggest that resazurin/resorufin may be a potential new targeted therapy in AML.Acute myeloid leukemia (AML) is a malignant disease of hematopoietic tissues represented by the accumulation of clonal abnormal (leukemic) myeloid progenitors and hematopoietic stem cells, accompanied by impaired production of normal blood cells. AML is the most common acute leukemia accounting for nearly 30% of all adult leukemias with three year survival below 10% in patients over 60 years of age. Hence, there is an unmet need for low toxic therapy in older patients with co-morbidities. Resazurin which is the principle component of Alamar Blue has been used in cell viability assays through the conversion from its blue color to the pink color of its fluorescent product, resorufin. Resazurin has been shown to have cytotoxic effects on leukemic cells but its molecular mechanisms of action are still elusive. In this study, we investigated the efficacy of resazurin in five different AML cell lines (MV4-11, MOLM-13, OCI-AML-3, NB4 and HL-60) and AML patient cells. We found that resazurin treatment induced apoptosis in AML cell lines at both normal and hypoxic conditions and patient cells. Apoptosis was assessed by Annexin V – To-pro-3 labeling and nuclear staining. Interestingly, also, treatment with resorufin alone caused apoptosis in MV4-11 (AML FAB M5) cells suggesting that resorufin may be the biologically active form responsible for reducing the viability of AML cells. Resorufin has been proved to be a protein kinase CK2 inhibitor. Next, we investigated the molecular mechanisms causing the anti-leukemic activity of resazurin in AML. We found that resazurin or resorufin treatment downregulated the protein level of phosphorylated CK2 alpha (Thr360 + Ser362), phosphorylated AKT (Ser129 and Ser473), phosphorylated STAT5 (Tyr694) and STAT5 in MV4-11 cells both in vitro and in vivo. To preliminarily evaluate the effect of resazurin in vivo, MV4-11 cells were subcutaneously injected into NOD/SCID IL2 receptor gamma null (NSG) mice after evaluation of toxicity and determination of maximum tolerated dose. Eight mice were divided into control (n = 4) and treatment (n = 4) groups and tumour volume was examined. Measurement of tumour volume presented that resazurin treatment significantly decreased tumour growth in the treated mice. Thereafter, MV4-11 cells were orthotopically transplanted into NSG mice. Twenty mice were divided into control (n = 10) and treatment (n = 10) groups and survival as well as human engraftment were investigated. Treatment with resazurin significantly prolonged survival and effectively eliminated the engraftment of MV4-11 cells in the bone marrow of mice. We conclude that inhibition of protein kinase CK2 by resazurin/resorufin may be a potential new targeted therapy in the treatment of AML.
AML is the most common acute leukemia and with three year survival below 10% in patients over 60 years of age. Resazurin is a redox indicator used for cell viability assay has been shown to have cytotoxic effects on leukemic cells . We discovered that resazurin induces p53-independent apoptosis and down-regulation of c-Myc in AML cells.Acute myeloid leukemia (AML) is a malignant disease of hematopoietic tissues represented by the accumulation of clonal abnormal (leukemic) blast cells, primarily found in the bone marrow, accompanied by impaired production of normal blood cells. AML is the most common acute leukemia accounting for nearly 30% of all adult leukemias with three year survival below 10% in patients over 60 years of age. Hence, there is an immediate requirement for low toxic therapy in older patients with co-morbidities. Resazurin is a redox indicator used for cell viability assay has been shown to have cytotoxic effects on leukemic cells and involve in the production of reactive oxygen species (ROS). However, molecular mechanisms of resazurin action are still elusive. The tumor suppressor protein p53 has been considered as the “guardian of the genome” because of its essential roles in maintaining genetic stability, inducing cellular differentiation and delaying the development of cancer. We discovered that resazurin induces apoptosis in AML cell lines with different status of p53 such as wild-type, null and mutated p53. Moreover, treatment of AML cells with resazurin showed a p53-independent down-regulation of c-Myc oncoprotein. This attenuation of c-Myc leads to the up-regulation of p21 which in turn inhibits cell cycle progression. Resazurin also induces p53-independent down-regulation of the E3 ubiquitin ligase Mdm2 which functions as a main negative regulator of p53 through an autoregulatory feedback loop permits both proteins to mutually control the cellular levels of each other. Furthermore, combinational therapy of resazurin and Nutlin-3, a small molecule Mdm2 antagonist, was found to cause apoptosis synergistically in different AML cell lines. These achieved results suggest that the effect and molecular mechanisms of resazurin need to be further elucidated for developing a potential new targeted therapy in the treatment of AML.
Deltagere
- Vibeke Andresen Medveileder
- Bjørn Tore Gjertsen Hovedveileder
- Trung Quang Ha Ph.d.-kandidat
- Trung Ha Ph.d.-kandidat
eRapport er utarbeidet av Sølvi Lerfald og Reidar Thorstensen, Regionalt kompetansesenter for klinisk forskning, Helse Vest RHF, og videreutvikles av de fire RHF-ene i fellesskap, med støtte fra Helse Vest IKT
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