Study Of Anti-multiple Myeloma Activities And The Effective Mechanisms Of Natural Small Molecules CADPE And ZZZ-1

Objctives:Multiple myeloma (MM) is a hematologic neoplastic plasma cell disorder characterized by clonal malignant proliferation, apoptosis loss and accumulation of malignant plasma cells in bone marrow, resulting in the bone destruction, monoclonal proteins secretion into the blood or urine, and associated organ dysfunction. In spite of the extended overall survival rate by the application of novel chemotherapy regimens, the availability of novel agents such as bortezomib and the application of autologous stem-cell transplantation, multiple myeloma is still incurable. Cancer cells exhibit aerobic glycolytic phenotype with decreased oxidative phosphorylation, termed as "Warburg effect"; this metabolic reprogramming facilitates the synthesis of biological macromolecules to support the rapid proliferation of cancer cells. Our previous studies have demonstrated the potent in vitro and in vivo anticancer activities of two natural small molecular compounds CADPE and ZZZ-1 in colorectal cancers and glioblastoma. This study firstly determined whether CADPE and ZZZ-1 could affect energy metabolism of multiple myeloma, then the compounds were tested the effectivity against multiple myeloma cells in vitro; lastly, the potential mechanisms underlying the anti-multiple myeloma activities of CADPE and ZZZ-1 were studied.Methods:Human multiple myeloma cells U266 and RPMI8226 were chosen as research objects. First, we examined the effects of CADPE and ZZZ-1 on energy metabolism, including glucose consumption, lactate levels and ATP levels. Then, cell viability was evaluated by MTT, cell cycle and apoptosis were assessed by flow cytometry, followed by the measurement on protein levels of cell cycle regulators by western blot. Finally, the inhibitors of MAPK, pan-caspase, AIF, paraptosis and autophagy were used to examine the potential mechanisms underlying the anti-multiple myeloma activities of these two agents.Results:1) Treatment with CADPE or ZZZ-1 resulted in a sharp increase in glucose consumption and ATP levels with a marked decrease in lactate levels in human multiple myeloma cell lines U266 and RPMI8226, indicating that CADPE and ZZZ-1 were able to compromise the metabolic phenorype of cancer cells; 2) CADPE and ZZZ-1 significantly decreased cell viability in a dose-and time-dependent manner; 3) CADPE arrested U266 cells at G2/M phase, arrested RPMI8226 cells at S phase, and accordingly downregulated the levels of c-Myc, cyclin Dl, CDK4, CDK6, cyclin A, p-Rb and upregulated Rb protein level in U266 cells; ZZZ-1 arrested both U266 and RPMI8226 cells at G0/G1 phase. Consistently, ZZZ-1 downregulated the levels of c-Myc, cyclin D1, cyclin D2, CDK4, CDK6 and p-Rb, and upregulated Rb, without an obvious effect on cyclin A in RPMI8226 cells; 4) CADPE and ZZZ-1 induced U266 and RPMI8226 cells into apoptosis, and downregulated the levels of the important anti-apoptosis proteins Notch-1, p-Stat3 and Bcl-2, but upregulated pro-apoptosis protein Noxa levels, while had no effects on the expression of Stat3, Bax, survivin; apoptosis induced by CADPE couldn’t be reversed by MAPK inhibitors, pan-caspase inhibitor, or AIF inhibitor; while apoptosis induced by ZZZ-1 could only be partially reversed by pan-caspase inhibitors; 5) Decreased cell viability by CADPE or ZZZ-1 couldn’t be reversed by the inhibitors of paraptosis or autophagy.Conclusions:CADPE and ZZZ-1 significantly decreased cell viability of multiple myeloma cells, arrested cell cycle, induced apoptosis, possibly through the regulation of energy metabolism, downregulation of the levels of c-Myc, Notch-1 and p-Stat3 and their downstream proteins.