Publicação
Therapeutic Potential of Parthenolide on B-CELL Haematologic Malignancies
| Resumo: | Lymphoid neoplasms comprehend a highly heterogeneous group of diseases, including acute lymphoblastic leukaemia (ALL), chronic lymphocytic leukaemia (CLL) and multiple myeloma (MM), among others. These malignancies exhibit dysregulated NF-kB pathway, crucial for cell survival and proliferation. The inhibition of NF-kB pathway in lymphoid malignancies has emerged as a promising therapeutic option for these diseases. Parthenolide (PRT) is a naturally occurring sesquiterpene lactone that is derived from feverfew plant. Its potential as an anticancer agent has been extensively researched, and it has shown promising results in not only haematological neoplasms, but also in a variety of solid cancers, such as breast, lung, colorectal, prostate and ovarian cancers. PRT has a series of direct targets (e.g. p65, IkB kinase, IGF-1, BRAF) that have already been reported to affect several signalling pathways related to tumorigenesis and progression, mainly inhibiting growth, and inducing apoptosis. In this context, the aim of this study was to assess the therapeutic efficacy of PRT in in vitro models of B-cell neoplasms.To this end, three cell lines were used: 697 (B-ALL), HG-3 (CLL) and U266 (MM). Cells were incubated with PRT in different concentrations, ranging between 0.1-25 µM. Metabolic activity was evaluated after 24, 48 and 72 hours of incubation, using resazurin assay. Subsequent studies were performed using two concentrations, one common to all three cell lines and corresponding to 1 µM and a concentration closer to the IC50 (half maximal inhibition concentration). After a 72-hour incubation, cell death was evaluated by flow cytometry (FCM) using Annexin V/7AAD double staining and by optical microscopy using May Grünwald-Giemsa. Intracellular peroxides, superoxide anion levels, reduced glutathione (GSH) and mitochondrial membrane potential (∆Ψm) were measured by FCM using DCFH2-DA, DHE, MO and JC-1 fluorescent probes, respectively. Cell cycle distribution was also evaluated by FCM using PI/RNase kit. Finally, gene expression analysis was conducted by qPCR to assess expression levels of MYC, BAX, BCL-2, CASP9 and TXNRD1.The results demonstrate that PRT induced a decrease in metabolic activity in a dose-, time- and cell-line-dependent manner. 697 cell line is the most sensitive to PRT with an IC50 of 4.8 µM, at 72h, followed by HG3 cell line with an IC50 of 6.0 µM and U266, the least sensitive, with an IC50 of 22.4 µM. PRT induced cell death, mainly by apoptosis, and these results were confirmed by optical microscopy where cells displayed distinctive morphological features of apoptosis, namely blebbing, which leads to the formation of apoptotic bodies. Furthermore, PRT also induced a decrease in a dose-dependent manner of ∆Ψm in all cell lines, further confirming mitochondrial dysfunctions, which in turn are also highly associated with the intrinsic pathway of apoptosis. To better clarify the mechanisms of PRT in these neoplasms, oxidative stress was assessed in all cell lines. GSH levels and ROS (superoxide anion and/or peroxides) decreased in U266 cell line and increased in the HG3 cell line. In 697, peroxides dropped significantly, superoxide anion marginally increased, and GSH levels were similar to the control. To further assess if PRT possesses an antiproliferative effect, cell cycle was evaluated, and results show that PRT’s IC50 in U266 cell line induced cell cycle arrest in G2/M phase but didn’t seem to affect 697 and HG3. These findings suggest a potential cytotoxic and cytostatic effect of PRT in lymphoid malignancies in vitro.In conclusion, PRT exhibits anticancer activity in our models, paving the way for more studies to be conducted and as such, potentially increasing the treatment landscape for B-cell haematological malignancies. |
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| Autores principais: | Baptista, Mara Amuji da Silveira |
| Assunto: | B-lymphoid malignancies NF-kB Parthenolide Apoptosis Oxidative stress Neoplasias linfoides das células B NF-kB Partenolide Apoptose Stresse oxidativo |
| Ano: | 2024 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso embargado |
| Instituição associada: | Universidade de Coimbra |
| Idioma: | inglês |
| Origem: | Estudo Geral - Universidade de Coimbra |
| Resumo: | Lymphoid neoplasms comprehend a highly heterogeneous group of diseases, including acute lymphoblastic leukaemia (ALL), chronic lymphocytic leukaemia (CLL) and multiple myeloma (MM), among others. These malignancies exhibit dysregulated NF-kB pathway, crucial for cell survival and proliferation. The inhibition of NF-kB pathway in lymphoid malignancies has emerged as a promising therapeutic option for these diseases. Parthenolide (PRT) is a naturally occurring sesquiterpene lactone that is derived from feverfew plant. Its potential as an anticancer agent has been extensively researched, and it has shown promising results in not only haematological neoplasms, but also in a variety of solid cancers, such as breast, lung, colorectal, prostate and ovarian cancers. PRT has a series of direct targets (e.g. p65, IkB kinase, IGF-1, BRAF) that have already been reported to affect several signalling pathways related to tumorigenesis and progression, mainly inhibiting growth, and inducing apoptosis. In this context, the aim of this study was to assess the therapeutic efficacy of PRT in in vitro models of B-cell neoplasms.To this end, three cell lines were used: 697 (B-ALL), HG-3 (CLL) and U266 (MM). Cells were incubated with PRT in different concentrations, ranging between 0.1-25 µM. Metabolic activity was evaluated after 24, 48 and 72 hours of incubation, using resazurin assay. Subsequent studies were performed using two concentrations, one common to all three cell lines and corresponding to 1 µM and a concentration closer to the IC50 (half maximal inhibition concentration). After a 72-hour incubation, cell death was evaluated by flow cytometry (FCM) using Annexin V/7AAD double staining and by optical microscopy using May Grünwald-Giemsa. Intracellular peroxides, superoxide anion levels, reduced glutathione (GSH) and mitochondrial membrane potential (∆Ψm) were measured by FCM using DCFH2-DA, DHE, MO and JC-1 fluorescent probes, respectively. Cell cycle distribution was also evaluated by FCM using PI/RNase kit. Finally, gene expression analysis was conducted by qPCR to assess expression levels of MYC, BAX, BCL-2, CASP9 and TXNRD1.The results demonstrate that PRT induced a decrease in metabolic activity in a dose-, time- and cell-line-dependent manner. 697 cell line is the most sensitive to PRT with an IC50 of 4.8 µM, at 72h, followed by HG3 cell line with an IC50 of 6.0 µM and U266, the least sensitive, with an IC50 of 22.4 µM. PRT induced cell death, mainly by apoptosis, and these results were confirmed by optical microscopy where cells displayed distinctive morphological features of apoptosis, namely blebbing, which leads to the formation of apoptotic bodies. Furthermore, PRT also induced a decrease in a dose-dependent manner of ∆Ψm in all cell lines, further confirming mitochondrial dysfunctions, which in turn are also highly associated with the intrinsic pathway of apoptosis. To better clarify the mechanisms of PRT in these neoplasms, oxidative stress was assessed in all cell lines. GSH levels and ROS (superoxide anion and/or peroxides) decreased in U266 cell line and increased in the HG3 cell line. In 697, peroxides dropped significantly, superoxide anion marginally increased, and GSH levels were similar to the control. To further assess if PRT possesses an antiproliferative effect, cell cycle was evaluated, and results show that PRT’s IC50 in U266 cell line induced cell cycle arrest in G2/M phase but didn’t seem to affect 697 and HG3. These findings suggest a potential cytotoxic and cytostatic effect of PRT in lymphoid malignancies in vitro.In conclusion, PRT exhibits anticancer activity in our models, paving the way for more studies to be conducted and as such, potentially increasing the treatment landscape for B-cell haematological malignancies. |
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