Contributions
Abstract: PB1608
Type: Publication Only
Background
Acute Lymphoblastic Leukemias (ALLs) are hematologic malignancies characterized by dysregulated cell proliferation and differentiation arrest, resulting in the accumulation of immature lymphoid progenitors (B or T). Burkitt lymphoma (BL) represents a highly aggressive lymphoma, characterized by acute onset and rapid doubling time. The progress in new therapeutic approaches to treat patients with ALL are scarce, the survival rates have not increased substantially in recent years and relapses are frequent. Playing an important role in the regulation of diverse biological processes such as cell proliferation and survival, nuclear factor kappa B (NF-kB) is closely associated with various human malignancies. Parthenolide (PRT), a sesquiterpene lactone, was reported to inhibit the DNA binding of NF-KB. This compound induces apoptotic cell death by multiple pathways, including oxidative stress, endoplasmic reticulum stress, intracellular thiol depletion, caspase activation, and mitochondrial dysfunction, as well as sensitizes cancer cells to chemotherapeutic drugs.
Aims
In this context, this work aimed to evaluate the therapeutic potential of PRT on ALL and BL cell lines and characterize the type of cell death induced and its molecular mechanisms.
Methods
To this end, T-ALL (CEM, JURKAT, and MOLT-4), B-ALL (697 and KOPN-8), and BL cell lines (Raji) were incubated in the absence and presence of PRT in single and fractioned administration schemes. Metabolic activity was assessed by resazurin assay. Cell death was evaluated by optical microscopy (May-Grünwald-Giemsa staining) and by Flow Cytometry (FC; Annexin V/7-AAD staining). The expression of FAS, FAS-L, activated-caspase-3, phosphorylated NF-kB, mitochondrial membrane potential (Δψmit; JC-1) and oxidative stress [superoxide (O2-; DHE), hydrogen peroxide (H2O2, DCFH2DA), and reduced glutathione (GSH)] were also analyzed by FC. The results were statistically analyzed considering a level of significance of 95% (p <0.05).
Results
The results indicate that PRT reduced metabolic activity in a time-, dose- and cell line-dependent manner. KOPN-8, CEM, and RAJI (IC50 50-75μM) were the most sensitive cells and MOLT-4 and JURKAT (IC50 100μM) the less sensitive. Single and fractional administration regimens showed similar results. PRT induced cell death mainly by apoptosis associated with an increase in activated-caspases expression, decrease in Δψmit and an increase in oxidative stress levels (increased ROS and decreased GSH) in all cell lines. The morphological aspects observed in cells treated with PRT, such as blebbing and nuclear fragmentation, confirmed apoptosis induction. In addition, PRT also increased FAS and FAS-L expression levels in all KOPN8, 697 and MOLT4 cell lines. A cytostatic effect was observed in JURKAT (G0/G1 phase arrest) and MOLT-4 (G2/M phase arrest) cells. Finally, PRT induced a decrease in phosphorylated p65 levels (a subunit of NF-KB) confirming the inhibition of NF-kB pathway.
Conclusion
In conclusion, these results suggest that PRT may represent a new potential therapeutic approach in ALL and BL. However, the therapeutic efficacy may depend on disease subtype.
This work was supported by CIMAGO and by the FCT through the fellowship SFRH/BD/51994/2012.
Session topic: 1. Acute lymphoblastic leukemia – Biology & Translational Research
Keyword(s): ALL, Cytotoxicity, Signaling, Therapy
Abstract: PB1608
Type: Publication Only
Background
Acute Lymphoblastic Leukemias (ALLs) are hematologic malignancies characterized by dysregulated cell proliferation and differentiation arrest, resulting in the accumulation of immature lymphoid progenitors (B or T). Burkitt lymphoma (BL) represents a highly aggressive lymphoma, characterized by acute onset and rapid doubling time. The progress in new therapeutic approaches to treat patients with ALL are scarce, the survival rates have not increased substantially in recent years and relapses are frequent. Playing an important role in the regulation of diverse biological processes such as cell proliferation and survival, nuclear factor kappa B (NF-kB) is closely associated with various human malignancies. Parthenolide (PRT), a sesquiterpene lactone, was reported to inhibit the DNA binding of NF-KB. This compound induces apoptotic cell death by multiple pathways, including oxidative stress, endoplasmic reticulum stress, intracellular thiol depletion, caspase activation, and mitochondrial dysfunction, as well as sensitizes cancer cells to chemotherapeutic drugs.
Aims
In this context, this work aimed to evaluate the therapeutic potential of PRT on ALL and BL cell lines and characterize the type of cell death induced and its molecular mechanisms.
Methods
To this end, T-ALL (CEM, JURKAT, and MOLT-4), B-ALL (697 and KOPN-8), and BL cell lines (Raji) were incubated in the absence and presence of PRT in single and fractioned administration schemes. Metabolic activity was assessed by resazurin assay. Cell death was evaluated by optical microscopy (May-Grünwald-Giemsa staining) and by Flow Cytometry (FC; Annexin V/7-AAD staining). The expression of FAS, FAS-L, activated-caspase-3, phosphorylated NF-kB, mitochondrial membrane potential (Δψmit; JC-1) and oxidative stress [superoxide (O2-; DHE), hydrogen peroxide (H2O2, DCFH2DA), and reduced glutathione (GSH)] were also analyzed by FC. The results were statistically analyzed considering a level of significance of 95% (p <0.05).
Results
The results indicate that PRT reduced metabolic activity in a time-, dose- and cell line-dependent manner. KOPN-8, CEM, and RAJI (IC50 50-75μM) were the most sensitive cells and MOLT-4 and JURKAT (IC50 100μM) the less sensitive. Single and fractional administration regimens showed similar results. PRT induced cell death mainly by apoptosis associated with an increase in activated-caspases expression, decrease in Δψmit and an increase in oxidative stress levels (increased ROS and decreased GSH) in all cell lines. The morphological aspects observed in cells treated with PRT, such as blebbing and nuclear fragmentation, confirmed apoptosis induction. In addition, PRT also increased FAS and FAS-L expression levels in all KOPN8, 697 and MOLT4 cell lines. A cytostatic effect was observed in JURKAT (G0/G1 phase arrest) and MOLT-4 (G2/M phase arrest) cells. Finally, PRT induced a decrease in phosphorylated p65 levels (a subunit of NF-KB) confirming the inhibition of NF-kB pathway.
Conclusion
In conclusion, these results suggest that PRT may represent a new potential therapeutic approach in ALL and BL. However, the therapeutic efficacy may depend on disease subtype.
This work was supported by CIMAGO and by the FCT through the fellowship SFRH/BD/51994/2012.
Session topic: 1. Acute lymphoblastic leukemia – Biology & Translational Research
Keyword(s): ALL, Cytotoxicity, Signaling, Therapy