Multiple myeloma (MM) is a highly resistant hematological neoplasia that remains an incurable disease. A leading drug in MM treatment is bortezomib, a selective proteasome inhibitor. Current treatment protocols have extended the overall survival of patients with MM, however, ultimately the disease becomes refractory to all forms of treatments and therefore drugs with new mechanisms of action are urgently needed.
Experimental and clinical observations suggest that thyroid hormones (T3 and T4) modulate neoplastic cells and activate MAPK pathway through binding to αv β3 integrin, commonly overexpressed in cancer. Tetraiodothyroacetic acid (tetrac), a non-agonist T4 analog, selectively blocks T3 and T4 binding to αv β3 receptor site. MM cells interact with αv β3 for invasion/growth and thyroid diseases were associated with increased MM risk. We recently demonstrated the thyroid hormones- αv β3-MAPK axis in myeloma cells. In the current study we further show that T3 and T4 antagonize bortezomib action via MAPK activation and that in the presence of tetrac bortezomib action is significantly enhanced.
Cell lines: MM cell lines (RPMI-8226, ARK, ARP-1, U266 and CAG) are cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS/antibiotics. Before addition of T3 or T4, cells are grown for 48 hours without serum. Bone marrow (BM) aspirates were obtained from patients with MM treated at the Meir Medical Center. Signed institutional review board–approved written informed consent was obtained from all patients. Primary MM cells were separated on Ficoll gradient and were cultured in RPMI 1640. Reagents and chemicals: T3, T4, tetrac, MAPK inhibitor (U0126), autophagy inhibitor (3MA) and pan caspase inhibitor, Z-VAD. Cells were treated with T3 or T4 (1nM-100nM and 1μM) in the presence/absence of tetrac (100nM and 1μM) and/or bortezomib (25nM) and tested by several methods: Cell number. Cell proliferation assay: WST-1 (10% final concentration) is incubated at 37°C for 2 h and read using microELISA reader at 440nm. Cell cycle: Cells are harvested, fixed and stained with DNA propidium iodide (PI) (50 μg/ml) /RNAse A (10mg/ml) and analyzed for DNA content by FACS. Analysis of apoptosis/necrosis: Cells (105) are incubated with 5 μl Annexin V (FITC conjugated)/5 μl PI and analyzed by FACS. Expression of apoptotic genes (real-time PCR). Results were repeated 2–3 times in triplicates and were analyzed using unpaired students t test.
Results and discussion: Results demonstrate that T3 and T4 at near physiological and supra physiological levels, increased myeloma cell viability by 15–50% and cell number by 30%-60%. This increased viability was blocked by U0126, indicating involvement of the MAPK pathway. In parallel a 20–25% reduction in cell death and of pro-apoptotic genes expression was documented following treatment with the hormones. Co-treatment of myeloma cell lines with T3 or T4 reduced bortezomib cytotoxicity and increased cell survival in a MAPK-dependent manner. Pretreatment of MM cell lines and primary cells from MM patients with tetrac, 48 hours before the addition of bortezomib, resulted in a synergistic cytotoxic effect. The effect of tetrac was blocked using a pan-caspase inhibitor (Z-VAD) but not by an autophagy inhibitor (3MA), suggesting apoptosis-related cell death.
We present here novel data demonstrating that T3 and T4 may oppose bortezomib action via MAPK activation. Blocking the thyroid hormones- αv β3 axis using tetrac, promotes bortezomib cytotoxicity, suggesting this approach as a promising adjunct therapy in MM.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.