Abstract
Multiple myeloma (MM) is characterized by the clonal proliferation of malignant plasma cells that accumulate preferentially in the bone marrow. In spite of high-dose chemotherapy and novel targeted therapies, myeloma remains to be an incurable disease due to emergence of drug resistance. Therefore, identification of mechanisms involved in drug resistance are essential to develop new and more effective targeted therapies. Heat shock proteins (HSPs) are a super family of highly conserved proteins, which are induced in plant, yeast, bacterial and mammalian cells in response to an array of physiological and environmental stress cues. Among heat shock protein families, HSP70 is one of the most highly conserved and is the only protein expressed in response to cellular stress. HSPs have been implicated in multidrug resistance, as they have been repeatedly demonstrated to inhibit apoptosis induced by a number of chemotherapeutic agents (Chant et al., 1996). We have shown that adhesion of myeloma cells to either bone marrow stromal cells or FN enhances HSP70 expression and secretion as determined by real-time RT-PCR and ELISA, respectively. Inhibition of the HSP70 expression using either KNK437 (HSF-1 inhibitor) or RNAi to HSP70, decreased 8226 cell adhesion to stromal cells as well as to FN as early as two hours, and this adhesion was mediated through α4β1 and α5β1 integrins. Treatment of 8226 cells with KNK437 or RNAi HSP70, induce apoptosis at 24 hours in a dose dependent manner. Interestingly, this effect was independent of adhesion (FN 55% apoptosis vs suspension 42% apoptosis) and is mediated by caspase-3 and PARP cleavage. Further more, treatment of 8226 cells with HSP70 inhibitors reversed CAM-DR to melphalan. To investigate whether HSP70 inhibition can cause apoptosis in Melphalan-resistant myeloma cells, we treated 8226/S and 8226/LR5 cells with either KNK437 alone or in combination with Melphalan. Our results show that KNK437 not only caused more apoptosis in 8226/LR5 (55% with 100 μM) cells than in the sensitive parental cells (42%), but also sensitized 8226/LR5 cells to Melphalan (64%), even though intracellular protein and RNA expression of heat shock protein 27, 70 and 90 was not affected in either Melphalan-sensitive or -resistant cells. These results suggest that 8226/LR5 cells depend on HSP70 for survival more than parental 8226 cells Similarly, pretreatment of 8226 cells with either KNK437, or RNAi against HSP70, enhanced the proteasome inhibitor, Bortezomib- induced apoptosis (Bortezomib 10 nM 8 %, KNK437 25 μM 14 % Combination, 30 %). This apoptosis was mediated by Caspase 3 and was correlated with reduced HSP70 expression. 8226 myeloma cells treated with Bortezomib (10 nM) caused increased RNA and protein expression of HSP70, HSP27 and HSP90 as early as 4 and 8 hrs, respectively. Further studies elucidating the mechanism/s by which HSP70 inhibition sensitizes Melphalan or bortezomib induced apoptosis are currently under investigation. Our preclinical studies provide the basis for potential need for the development of anti HSP70 inhibitors for clinical studies in myeloma.
Involvement of poly(ADP-ribose) polymerase and activation of caspase-3-like protease in heat shock-induced apoptosis in tobacco suspension cells
