A Novel Putative Mechanism of Anti-Myeloma Activity Targeted Against Heat Shock Protein 27 by Derivative of Artemisinin, Artesunate.
Authors: Chizuka Aki , Uchida Hideo , Xian MingJi , Shimizu Takayuki , Ikeda Yasuo , Kizaki Masahiro 
Published: April 2020

Abstract
Recent introduction of molecular targeted drugs such as bortezomib and IMIDs in the clinical settings has achieved the improved treatment outcome of multiple myeloma (MM). However, MM is still an incurable disease and these drugs also possess serious adverse reactions, therefore, safe and more effective therapy should be established. Artesunate (ART) is a semi-synthetic derivative of artemisinin and is widely used for the treatment of malaria as a salvage therapy. Recent in vitro studies showed that ART also has an anti-tumor activity against several cancer cell lines. We thus investigated whether ART could possess anti-myeloma activity and demonstrated that apoptosis of myeloma cells is strongly induced by ART. Interestingly, we also found that this activity is mediated through heat shock protein (Hsp) 27-dependent pathway. Several MM cell lines (IM9, OPM2, RPMI8226, U266) were treated with various concentrations of ART for 48 hours, and MTT assays were performed to assess the anti-myeloma activity. IM9, OPM2, RPMI8226 cells showed the striking reduction of viability (up to 40% at 1μM and up to 90% at 10μM) in a dose- and time-dependent manner, whereas ART has less inhibitory effect on U266 cell line. ART induced G1 arrest of IM9 cells, and apoptosis was confirmed by decreased mitochondrial membrane potential, and flow cytometric analysis using AnnexinV/PI staining. Colony assay showed that ART has no growth inhibitory effect on normal CD34-positive bone marrow cells even at a concentration of 10μM. Immunoblot analyses demonstrated the activation of caspases-3 and -9, and the decreased expression of pro-apoptotic protein Bid. Interestingly, heat shock protein (Hsp) 27 was downregulated in myeloma cell lines (IM9, RPMI8226) which are sensitive to the ART treatment. In contrast, downregulation of Hsp 27 was not observed in U266 cells which are resistant to ART. Other anti-apoptotic Hsps (Hsp70, 90), as well as Bcl-2 family proteins (Bcl-2, Bax, Bad, Bcl-xL), Akt, MDM2, p53 and MAPKs (SAPK/JNK, p38, ERK1/2) were not affected by ART. Quantitative RT-PCR analysis showed that ART did not influence the mRNA expression of Hsp27, suggesting that ART could downregulate the Hsp27 protein at a posttranscriptional or posttranslational levels. Overexpression of Hsp27 cDNA by transfection method in ART-sensitive myeloma cell lines demonstrated that these cell lines acquired resistance to ART and apoptosis was not induced at a concentration which ART is effective to the parental cells. Our preliminary data using knockdown procedure of Hsp27 mRNA by RNAi-expressing lentivirus showed the growth suppression of myeloma cells, which apparently suggested that Hsp27 could confer a critical function in the proliferation of myeloma cells and that downregulation of Hsp27 could result in induction of apoptosis in MM. Hsp27 has an important role in anti-apoptotic effect against various stresses to the cells. Our data implies a novel mechanism that downregulation of Hsp27 by ART could induce misfolding of several client proteins indispensable for the cell growth, which results in the induction of apoptosis in MM cells. In conclusion, ART could become a candidate of safe and effective therapeutic drug for MM, and Hsp 27 might be a potential molecular target for the treatment of MM.