Induction of Autophagy to Myeloma Cells by Thalidomide and Clarithromycin.
Authors: Nakamura Miki , Kamimoto Takahiro , Yoshimori Tamotsu , Okuno Yutaka , Takeya Motohiro , Mitsuya Hiroaki , Hata Hiroyuki 
Published: April 2020

Abstract
Introduction
While therapeutic benefit of thalidomide (Thal) for myeloma has been well recognized, its mechanism of action is not yet fully understood. It has also been reported that clarithromycin (CAM) occasionally exserts anti-myeloma effects in combination with Thal. The aim of the present study is to clarify the mechanism(s) of anti-myeloma effects of Thal and/or CAM.
Methods
Myeloma cell lines were cultured either with Thal and/or CAM at a concentration of 50 mg/ml for 24 hours with or without serum, and the morphology of the cells was examined using light or electron microscopy. Myeloma cells from patients were purified by CD138-immunomagnetic beads at a concentration of 10 mg/ml of Thal or CAM for 24 hours and subsequently analysed as cell lines. A PI-3 kinase inhibitor, 3-methyladenine, was utilized as an autophagy inhibitor. Processing of LC3-I to LC3-II, a key feature of autophagy, was detected by western blot utilising anti-LC3 antibody. A CMV enhancer-driven plasmid containing GFP-tagged LC3 cDNA was introduced to 12PE cells by electroporation.
Results
Significant induction of vacuoles containing cytoplasmic organelle was seen when myeloma cell lines and fresh myeloma cells were treated with CAM. Induction of vacuoles was more prominent under a serum-free condition. Interestingly, the vacuole formation was substantially enhanced when treated with Thal and CAM, accompanying cell death. Since nucli of cells with vacuole formation appeared intact, the changes were thought to be different from apoptosis. The vacuolization was significantly inhibited in the presence of 3-methyladenine, an autophagy inhibitor, regardless of the presence of serum, suggesting that these vacuole formation is a feature of autophagy. Electron microscopic analysis confirmed the vacuoles are characteristic of autophagy. Western blot analysis revealed that processing of LC3-I to LC3-II, a feature of autophagy, was not induced by Thal alone, whereas CAM alone weakly induced the processing. Interestingly, LC3-II was significantly induced with combination of Thal and CAM. When GFP-LC3-transfected 12PE cells were treated with combination of Thal and CAM, significant accumulation of GFP-LC3-labelled cytoplasmic spots occurred. These results strongly suggest that Thal and CAM cooperatively induce autophagy in myeloma cells.
Conclusion
Autophagy represents a mechanism of recycling micro-organelles when exposed to various stresses. It is believed that proteasome rapidly degrades unnecessary proteins while some proteins are degraded by autophagosomes. Although autophagy is necessary for survival of cells under unfavourable conditions such as nutrient deficiency, it is suggested autophagy may induce cell death in some situations. The present study suggests that CAM may mediate autophagy as a single agent and autophagy induction is enforced by Thal. Given the fact that combination of Thal and CAM is occasionally effective for refractory myeloma, our findings suggest that the combination of Thal and CAM could produce cell death through autophagy. The present data should provide insights in and merit further investigation of the mechanism of anti-myeloma activity of Thal and CAM.