Parthenolide and DMAPT Induce Cell Death in Chronic Myeloid Leukemia Cells Via Generation of Reactive Oxygen Species

Abstract

Chronic Myeloid Leukemia (CML) is a malignant hematologic disease characterized by the presence of a chromosomal alteration known as the Philadelphia Chromosome (Ph). Ph originates in Leukemia Stem Cells (LSC) and is caused by the reciprocal translocation between chromosome 9 and 22, giving rise to the Bcr-Abl oncogene, which produces a constitutively active tyrosine kinase -the BCR-ABL oncoprotein. This CML feature has made it possible the development of Tyrosine Kinase Inhibitors (TKI's) Imatinib, Dasatinib, and Nilotinib, which are able to inhibit BCR-ABL and take patients into molecular remission. However TKI's are not able to eliminate the population of LSC, which cause patients to relapse if treatment is discontinued. This has made it necessary search for alternative ways to eliminate LSC in CML patients. Parthenolide (PTL), a sesquiterpene lactone extracted from the feverfew plant (Thanacethum partenium), which has been used in folk medicine to treat inflammatory symptoms, has shown activity against LSC in Acute Myeloid Leukemia (AML). Recently Guzman and collaborators have developed an analog of PTL, named DMAPT, which is orally bioavailable. Both molecules have shown to be active inhibitors of NFkB and induce high levels of Reactive Oxygen Species (ROS) in LSC of AML. In the present study we asked whether CML cells (LSC, Progenitors and CML cell lines) are sensitive to PTL and DMAPT. Cell lines K562, Kcl-22 and HL60 (as an AML control) were cultured in liquid suspension cultures with increasing concentrations of PTL or DMAPT at a cell density of 300,000 cells per ml. At 24 hours of exposure, PTL and DMAPT showed significant induction of apoptosis at 7.5 µM and 10 µM, respectively. To determine whether NFkB activity is inhibited by PTL and DMAPT, K562, Kcl-22, HL60 cell lines, as well as CD34+ cells from a CML patient in Chronic phase where exposed to 7.5 µM PTL or 10 µM DMAPT. After 6 hours, protein lysis was induced and Western blots were performed with protein extracts from 250,000 cells per lane. Membranes where probed for Phosphorilated-p65, total p65 and b-actin as a loading control. We observed a decrease in the levels of phosphorylated p65 in cells treated with PTL and DMAPT, compared with untreated cells, while levels of total p65 remained constant. We had previosly used DCFDA stain and HMOX expression, to observe ROS induction in CML cells exposed to PTL. To asses if ROS induction is necessary for CML cell death we pretreated cells with N-acetyl cysteine (NAC), precursor of glutathione, for 1 hour before treating cells with 7.5 µM PTL or 10 µM DMAPT, and evaluated ROS levels by staining with Cell rox deep red, Mitosox-PE, and also assessing cell viability with YoPro-1 and 7AAD stain. After 6 hours of PTL treatment, we found that NAC pretreatment diminished ROS levels in cytoplasm and mitochondrial dysfunction when cells were treated with PTL. Pretreatment with NAC was able to rescue the majority of cells from apoptosis induced by PTL. Similar results were observed in cultures treated with DMAPT. Taken together, our results indicate that PTL and DMAPT were able to induce cell death via apoptosis in CML cells. This process seems to involve inhibition of p65 phosphorylation and an increase in ROS levels, which we found to be necessary for cell death to occur. Disclosures No relevant conflicts of interest to declare.

Anticancer activities of parthenolide in primary effusion lymphoma preclinical models

“…Parthenolide has well-documented proapoptotic activities against a variety of cancers including hematological malignancies. 8,11,34,35,37,37,44 Similarly, it induced apoptosis in PEL ascites, evident by the increase in TUNEL-positive cells and PARP cleavage.…”

“…Thus, drugs such as parthenolide targeting survival pathways and activating apoptosis are promising strategies for cancer treatment. Parthenolide has well‐documented proapoptotic activities against a variety of cancers including hematological malignancies 8,11,34,35,37,37,44 . Similarly, it induced apoptosis in PEL ascites, evident by the increase in TUNEL‐positive cells and PARP cleavage.…”

“…1,2 It has been extensively studied for its anti-inflammatory properties mediated by nuclear factor-κB (NF-κB) pathway inhibition. [1][2][3] Besides its immunomodulatory effect, parthenolide is reported to exhibit antioxidant and antitumor properties in a wide range of human cancer models, such as colorectal cancer, 4 melanoma, 5 Breast, 6 gastric, 7 ALL, 8 AML, 9,10 CML, 11 BCL, and others 8,9 while sparing normal cells. 12 However, there are no reports studying the effects of parthenolide on primary effusion lymphoma (PEL) models.…”