Abstract
Patients with metastatic melanoma or multiple myeloma have a dismal prognosis because these aggressive malignancies resist conventional treatment. A promising new oncologic approach uses molecularly targeted therapeutics that overcomes apoptotic resistance and, at the same time, achieves tumor selectivity. The unexpected selectivity of proteasome inhibition for inducing apoptosis in cancer cells, but not in normal cells, prompted us to define the mechanism of action for this class of drugs, including Food and Drug Administration-approved bortezomib. In this report, five melanoma cell lines and a myeloma cell line are treated with three different proteasome inhibitors (MG-132, lactacystin, and bortezomib), and the mechanism underlying the apoptotic pathway is defined. Following exposure to proteasome inhibitors, effective killing of human melanoma and myeloma cells, but not of normal proliferating melanocytes, was shown to involve p53-independent induction of the BH3-only protein NOXA. Induction of NOXA at the protein level was preceded by enhanced transcription of NOXA mRNA. Engagement of mitochondrial-based apoptotic pathway involved release of cytochrome c, second mitochondria-derived activator of caspases, and apoptosis-inducing factor, accompanied by a proteolytic cascade with processing of caspases 9, 3, and 8 and poly(ADP)-ribose polymerase. Blocking NOXA induction using an antisense (but not control) oligonucleotide reduced the apoptotic response by 30% to 50%, indicating a NOXA-dependent component in the overall killing of melanoma cells. These results provide a novel mechanism for overcoming the apoptotic resistance of tumor cells, and validate agents triggering NOXA induction as potential selective cancer therapeutics for life-threatening malignancies such as melanoma and multiple myeloma.
Highlights
Malignancies such as melanoma and multiple myeloma are characterized by aberrant cellular responses to signals governing proliferation and apoptosis
Bortezomib triggered a dose-dependent increase in apoptosis of all proliferating melanoma cell lines tested ranging from 30% to 70% dead cells at a 10 Amol/L concentration of the proteasome inhibitor after 24 hours of continuous exposure (Fig. 1B, right)
The results indicate that proteasome inhibitors induce NOXA in malignant melanoma cell lines and trigger NOXA induction in a multiple myeloma cell line, indicating that NOXA induction is not limited to melanoma cells and occurs in cells from the disease for which bortezomib was originally approved by the Food and Drug Administration (FDA)
Summary
Malignancies such as melanoma and multiple myeloma are characterized by aberrant cellular responses to signals governing proliferation and apoptosis. Approach uses agents targeting the proteasome, a universal and broadly active cellular complex responsible for regulating protein degradation and maintenance of normal cell function [1, 2]. As substrates for the proteasome include regulatory proteins involved in cell cycle progression, apoptosis, and angiogenesis, targeting the proteasome represents an attractive therapeutic approach for cancer treatment [3]. The unexpected selectivity of proteasome inhibitors for cancer cells versus normal cells has challenged investigators to delineate the molecular mechanism responsible for induction of apoptosis in neoplastic cells [5]. We discovered that proteasome inhibitors selectively induce the proapoptotic BH3-only protein NOXA in melanoma and myeloma cells, but not in normal melanocytes, providing new insight into the molecular basis for differential apoptotic responses of neoplastic versus normal cells
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