Abstract
Chronic myeloid leukemia (CML) is a myeloproliferative pathology, originating from the hematopoietic cancer stem cells (hCSCs) due to the Bcl-Abl Philadelphia chromosome transformation. However, targeting these hCSCs as an effective anti-CML strategy is relatively less explored. Ovatodiolide (Ova) is a natural diterpenoid isolate of Anisomeles indica with broad anticancer activity. In this study, we investigated the anti-hCSCs potential of Ova against CD34+/CD38−, CD34+/CD38+, and unsorted K562 cell lines using flow cytometry, western blot, RT-PCR, genomic mapping, and tumorsphere formation assays. We demonstrated that compared to unsorted K562 and CD34+/CD38+, CD34+/CD38− cells were significantly enriched with Oct4, Sox2, CD133, Bcr-Abl, p-CrkL and p-Stat5 protein and/or mRNA. Furthermore, we showed that Ova alone or by enhancing the therapeutic potential of Imatinib, reduced the viability of CML cell lines, dose-dependently, irrespective of the cancer stemness, as well as markedly inhibit the Bcr-Abl, p-CrkL, Stat5, and MDR protein expression levels in CD34+ cells. Mechanistic investigations revealed a significant up-regulation of hsa-miR-155, which resulted in the reduction of dysregulating the PIK3CA expression in Ova-treated K562 CD34+/CD38− cells. Additionally, Ova alone or in combination with Imatinib suppressed the hCSC traits of the CD34+/CD38− cells, resulting in loss of their ability to form tumorspheres, enhanced apoptosis, increase in the Bax/Bcl-2 ratio, and dysregulation of the PI3K/AKT/mTOR signaling pathway. Together, these results demonstrate the PI3K/AKT/mTOR signaling-mediated anti-hCSC effect of Ova in CML, as well as suggest a likely role for Ova as a small molecule PI3K/mTOR dual inhibitor, thus, extending its potential benefit to other mTOR-mediated pathologies.
Highlights
Chronic myeloid leukemia (CML) accounts for about 15% of all leukemia in the developing world [1], and though it is not restricted to any age, CML is predominantly an adult disease, accounting for 20% of all leukemia in adults, with an estimated incidence of 1.6 per 100,000 in U.S alone, and 5430 diagnosed new cases in 2012 [2]
We identified that CD34+/CD38− CML cells harboring the Bcr-Abl fusion gene, were enriched with Oct4, Sox2 and CD133 (Figure 1)
The identification of the over-expression of these CSC-specific markers in the CD34+/CD38− CML cells, has been associated with tumor aggressiveness, metastasis, resistance to treatment and tumor recurrence [1, 3], defining an enhanced hematopoietic cancer stem cells (hCSCs)-like phenotype. This is consistent with the seminal findings of initial studies by Bonnet and Dick’s team on leukemia wherein flow cytometry-based sorting was used to isolate a subpopulation of leukemic cells with the CD34+/CD38− cell surface phenotype identified in bone marrow samples of acute www.impactjournals.com/oncotarget myeloid leukemia (AML) patients and the inoculation of immune-compromised mice with 5,000 of these CD34+/ CD38− cells resulted in the development of leukemia in the mice models, this small subset of leukemia cells with inherently greater stem cell self-renewal potential, compared to other leukemia or normal adult bone marrow cells, was referred to as CSCs [23, 24]
Summary
Chronic myeloid leukemia (CML) accounts for about 15% of all leukemia in the developing world [1], and though it is not restricted to any age, CML is predominantly an adult disease, accounting for 20% of all leukemia in adults, with an estimated incidence of 1.6 per 100,000 in U.S alone, and 5430 diagnosed new cases in 2012 [2]. The small molecule inhibitor of Bcr-Abl kinase, Imatinib mesylate (IM), with demonstrated induction of complete cytogenetic and hematologic response in most chronic phase CML patients is the current standard of care for Ph+ CML [6], IM does not provide definitive cure to CML patients because of the development of resistance to IM by the Bcr-Abl fusion gene, as well as the primary insensitivity of hCSCs to IM [7]. Together, targeting Brc-Abl/STAT5 axis may represent a more effective approach for CML interventions
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