Tris DBA Palladium Overcomes Hypoxia Mediated Drug Resistance in Multiple Myeloma

Introduction:

Despite recent progress in novel and targeted therapies, multiple myeloma (MM) remains a therapeutically challenging incurable disease. The regulation of important cellular processes and its link to cancer presented Src as an attractive target for MM. Src is a non-receptor protein tyrosine kinase which regulates multiple fundamental cellular processes including cell growth, migration, survival and differentiation. Activated Src in cancer lead to studies with Src as a target for anti-cancer drugs, and numerous Src inhibitors have become available to test the importance of Src in tumor initiation and progression. In MM, it has been described that in cell lines and MM patient-derived tumors, c-Src is constitutively activated, which plays an important role in drug resistance mechanisms. Tris dibenzylideneacetone dipalladium (Tris DBA), a small-molecule palladium complex, was shown to reduce Src/NMT-1 complex in melanoma cells, as well as inhibit downstream signaling including mitogen-activated protein kinase (MAPK kinase) and  phosphoinositol-3-kinase (PI3K). We suggest a novel strategy to improve the treatment of MM and overcome the drug resistance for the current therapeutic agents by specific inhibition of Src in MM cells by an organopalladium compound, Tris DBA.

Methods:

Tris DBA was prepared by Dr. Arbiser. MM cell lines (MM.1S, MM.1R, H929, RPMI-8826, and OPM2) and PBMCs were cultured with Tris DBA (0–10 µM) for 24h. MM cells were analyzed for cell proliferation by MTT assay; cell cycle by DNA staining with PI and analyzed by flow cytometry; apoptosis was analyzed by Annexin V/PI staining and analyzed by flow cytometry; and cell signaling associated with proliferation, cell cycle, and apoptosis was analyzed by western blotting. In addition, cell proliferation assay of Tris DBA with or without combination of proteasome inhibitors (PIs) bortezomib or carfilzomib for 24h was analyzed on the proliferation of MM cells in normoxic or hypoxic conditions. Moreover, we tested the effect of combination treatment on cell cycle and apoptosis signaling under normoxic conditions.  We then evaluated the effect of Tris DBA on HIF1α expression, migration and drug resistance under normoxic or hypoxic conditions.

Results:

The Src inhibitor Tris DBA reduced the proliferation of MM cell lines with an IC50 of about 1.5 - 3 µM after 24h treatment as a single agent, while none of the normal PBMC controls showed effect on their proliferation in the same dose range. These results were consistent with the decreased expression of proliferation signaling proteins from MAPK pathways (pERK), as well as PI3K (pS6R). Src inhibition led to the induction of a sub-G1 peak, which indicated accumulating apoptotic cells shown by DNA staining with PI. Apoptosis was then analyzed by Annexin/PI and confirmed by cleavage of caspase-3 and PARP.

We found that Tris DBA synergized with bortezomib and carfilzomib by inhibiting proliferation of MM cells and reducing cell cycle protein signaling more than either of the drugs alone. Moreover, the Tris DBA/Bortezomib or Tris DBA/Carfilzomib combination therapies significantly increased apoptosis by caspase-3 cleavage more than treatment with either proteasome inhibitor individually.

Tris DBA inhibited HIF1α expression in both normoxic and hypoxic conditions. HIF1α is an important target for hypoxia-driven drug resistance. Our studies confirmed hypoxia promoted faster chemotaxis of MM cells towards the chemo-attractants found in stromal cell conditioned media, and that Tris DBA treatment could overcome this hypoxia-induced effect. In addition, the development of hypoxia-induced drug resistance to individual bortezomib or carfilzomib treatment was overcome with combination treatment of Tris DBA under hypoxic conditions.

Conclusions:

Tris DBA reduces proliferation and induces G1 arrest and apoptosis in MM cells. Tris DBA synergized with PIs reducing proliferation and cell cycle signaling, as well as increasing apoptosis more than each drug alone. Tris DBA overcame hypoxia-induced effects such as enhanced chemotaxis or drug resistance to PIs by inhibition of HIF1α expression. Moreover, we found that Tris DBA is an effective anti-myeloma agent alone or in combination with other targeted drugs and that it reverses hypoxia-induced drug resistance in myeloma.  These results suggest the use of Tris DBA as a new therapeutic agent in relapsed refractory myeloma.