Multiple myeloma cells antagonize hypoxia induced cell death through upregulation bcl-2 and bcl-xl

Background. Hypoxia, a condition of decreased availability of oxygen, is major challenge for anticancer therapy. In solid tumors, hypoxia is considered as a double-edged sword: on the one hand, cells can be irreversibly injured and die under conditions of oxygen deficiency; on the other hand, cells that become adaptive to hypoxia are more resistant to apoptosis and less responsive to cancer therapy, resulting in relapse of the disease and transformation into more aggressive phenotypes. However, the oxygen level in bone marrow (BM) and the role of hypoxia in the pathogenesis of hematological malignancies such as multiple myeloma (MM) is still unclear. By staining the exogenous and endogenous hypoxia markers pimonidazole and HIF-1a in the bone marrow of naive and 5T33MM mice, we demonstrated that MM cells exist in a strong hypoxic niche. Aims. To investigate the mechanism of MM cells escaping from hypoxia induced cell death and anticancer therapy. Materials and Methods. The 5T33MM murine model, which mimicks the human MM disease closely, originated spontaneously in elderly C57BLKaLwRij mice. The 5T33vv MM cells were propagated by i.v. transfer of the diseased bone marrow into young recipients. The 5T33vv MM cells were isolated from 5T33MM bearing mice. Human and murine MM cell lines RPMI-8226, LP-1, 5T33vt were maintained in RPMI-1640 medium with 10% serum, supplemented glutamine (2 mM), and antibiotics (penicillin 100 U/mL and streptomycin 50 µg/mL). Hypoxic (1%, 0% O2) conditions were established by culturing MM cells in a sealed chamber. Apoptosis was measured by flow cytometric analysis of Annexin V-APC/PI staining. Real-Time PCR and Western-blot were performed to the expression at both RNA and protein levels. Chromatin Immunoprecipitation (ChIP) was used to immunoprecipitate HIF1a-DNA complexes and to allow for the rapid PCR detection of HIF1a-regulated genes. Results. we found that hypoxia (1% O2 and 0% O2) can increase the expression of Bcl-2 and Bcl-xL at both mRNA and protein levels by qRT-PCR and Western blot. Stabilization of HIF1a enhances Bcl-2/Bcl-xL expression at both mRNA and protein levels in normoxic condition. In contrast, inhibition of the accumulation of HIF1a decreases Bcl-2/Bcl-xL expression in hypoxic condition. Furthermore, ChIP analysis provided proof that HIF1a can bind to the hypoxia response element (HRE) at Bcl-2 and Bcl-xL promoters. Antagonizing Bcl-2 and Bcl-xL with BH3 mimetic small-molecule inhibitor with high affinity to Bcl-2 and Bcl-xL, overcomes Bcl-2/Bcl-xL mediated MM cell apoptotic resistance to hypoxic stress both in vitro and in vivo, by increasing the expression of cleaved proapoptotic proteins caspase-3, 9 and PARP. BH3 mimetic small-molecule inhibitor in combination with a HIF1a inhibitor can synergistically induce MM cell apoptosis in hypoxic condition. Conclusions. (1) Bcl-2 and Bcl-xL are target genes of HIF1a: hypoxia-induced Bcl-2 and Bcl-xL gene expression is regulated via a HIF1a-mediated mechanism. (2) Hypoxia up-regulated Bcl-2 and Bcl-xL expression is associated with the resistance of MM cells to hypoxia induced cell death. Antagonizing Bcl-2 and Bcl-xL with BH3 mimetic small-molecule inhibitor or in combination with HIF1a inhibitor, synergistically overcomes Bcl-2/Bcl-xL mediated resistance to hypoxic stress.

Publication year:2010

Keywords:hypoxia, myeloma, HIF-1a, Myeloma cell lines