Abstract
Introduction
Some macrolide antibiotics exert effects other than anti-bacterial activity on the growth and viability of certain cancer cells. The presence of cytoplasmic vacuoles is one the salient features of autophagy, a cellular event believed to recycle cellular ingredients under nutrient-starved conditions. Such vacuoles (autophagosomes) fuse with lysozomes, generating autolysozomes toward later stages of autophagy, digesting organelles and degenerated proteins. Our own and others’ findings that a macrolide antibiotic clarithromicin (CAM) occasionally shows anti-myeloma effects when combined with thalidomide and/or dexamethasone prompted us to examine CAM for its effects on myeloma cells in vitro.
Methods
Four myeloma cell lines (12PE, KHM-11, KMM-1 and U266) and primary myeloma cells purified by CD138-conjugated immune-magnetic beads (Miltenvi Biotec, Auburn, CA) were utilized. Clarithromicin was obtained from Taisho-Toyama pharmaceuticals (Tokyo, JAPAN). Morphology was analyzed either by May-Giemza staining or electron microscopy. Autolysozome was stained with Lysotracker (Invitrogen, Carlsbad, CA) and analyzed using fluorescent microscopy. Antibody to LC3 was obtained from Dr. T. Yoshimori (Department of Cellular Regulation, Research Institute for Microbial Diseases, Osaka University).
Results and discussion
CAM induced vacuoles in the cytoplasm of both myeloma cell lines and primary myeloma cells at concentrations ranging from 10 to 50 mg/ml at a dose-dependent manner after ~18 hours treatment. Electron microscopy revealed that those vacuoles morphologically resemble autolysozomes. To further confirm the identity of autolysozomes, cells were stained with Lysotracker, which specifically stains acid lysozome. After the treatment with CAM, the accumulation of vacuoles in the cytoplasm, stained with Lysotacker, was observed. Since initiation of autophagy depends on PI3-kinase, we investigated whether CAM induced AKT phosphorylation. AKT phosphorylation was readily observed, and moreover, the emergence of vacuoles stainable with Lysotracker was inhibited when the cells were pretreated with PI3-kinase inhibitors, 3MA or LY294002, strongly suggesting that vacuolation is indeed mediated with PI3-kinase. To further confirm that autopahgy is induced by CAM, the process of LC3-I to LC3-II, a hallmark of autophagy, was examined. We found that the induction of LC3-II by CAM occurred at a dose-dependent manner. Taken together, these findings strongly suggest that CAM induces autolysozome accumulation through activating PI3-kinase. Finally, we examined whether CAM induced apoptosis when combined with thalidomide. Three myeloma cells lines, which abundantly expressed Bcl-2, showed no growth inhibition, while KHM-11, which was defective in Bcl-2, showed marked apoptosis and growth inhibition with the combination of CAM and thalidomide, suggesting that CAM might potentially augment anti-myeloma activity of thalidomide although the mechanisms are to be determined. Taken these observations together, the manipulation of certain autophagy processes with reagents such as macrolides (i.e., CAM) might represent a new therapeutic approach in the treatment of myeloma. We hypothesize that CAM dually functions in the event of autophagy, i.e., it initiates autophagy while it suppresses autophagy at later stages. Further study under the hypothesis is currently underway.
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