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Letters to the Editor
in MM cells preferentially at pH6.4 in parallel with the reduction of Sp1 protein by RM-A.
Because an extracellular acidification makes RM-A per- meate cell membrane to induce apoptosis, it is plausible that an acidic milieu created by the OC-MM cell interac- tion rather induces cytotoxic activity by RM-A against MM cells as well as acid-producing OC. To clarify whether RM-A affects MM cell viability in the presence of OC, we next examined the cytotoxic effects of RM-A on MM cells in co-cultures with OC on bone slices gen- erated from rabbit BM cells. RM-A at 1 mM was able to decrease the MM cell viability in the co-cultures with OC, although RM-A at this concentration did not affect MM cell viability when MM cells were cultured alone (Figure 3A, left). When the bisphosphonate zoledronic acid was added to deplete mature OC, viable INA6 cells were decreased in number in co-cultures with OC to the levels observed in the cultures of INA6 cells alone. RM-A reduced the viability of INA6 cells more potently than zoledronic acid in the presence of OC, although RM-A and zoledronic acid similarly reduced the numbers of TRAP-positive multinucleated OC (Figure 3A, right), sug- gesting that the anti-MM effects of RM-A is not merely due to depletion of mature OC. Blockade of acid release by the proton pump inhibitor concanamycin A abolished the cytotoxic effects of RM-A on MM cells in the co-cul- tures with OC. These results suggest that RM-A not only impairs OC but also disrupts the OC-MM cell interac- tion.
We next examined the combinatory effects of RM-A with the proteasome inhibitor bortezomib. Although bortezomib was able to induce MM cell death, the cyto- toxic effects of bortezomib on MM cells were mitigated in co-cultures with OC (Figure 3B), indicating drug resist- ance by OC. However, RM-A impaired the viability of MM cells cultured in the presence of OC; and further potentiated the cytotoxic effects on MM cells in combi- nation with bortezomib, suggesting that RM-A over- comes the drug resistance induced by OC. Finally, we validated the combinatory therapeutic effects of RM-A and bortezomib in vivo, using human MM cell-bearing SCID-rab models. Treatment with RM-A suppressed bone destruction and MM tumor growth; importantly, the suppressive effects of RM-A on MM tumor growth and bone destruction was further enhanced in combina- tion with bortezomib, as shown in X-ray and mCT images and the levels of human soluble IL-6 receptor in mouse sera, a marker of MM tumor burden (Figure 3D). In histological analyses, MM cells were tightly packed in the BM cavity of the rabbit bones while bone trabeculae decreased in size with the appearance of multinucleated OC on the surfaces of the remaining bone (Figure 3E). RM-A, but not bortezomib, markedly reduced the num- ber of OC in the SCID-rab mouse MM lesions (Figure 3E). However, treatment with RM-A and bortezomib co- operatively reduced MM tumors along with the disap- pearance of TRAP-positive large OC on the bone surface.
These results collectively suggest that the acidic microenvironment produced by the MM-OC interaction enhances MM tumor progression but can trigger the cytotoxic effects of RM-A on MM cells as well as acid- producing OC. Given that an acidic condition makes MM cells resistant to chemotherapeutic agents, RM-A could be a candidate to target MM cells at acidic bone lesions, and augment the therapeutic efficacy of currently avail- able anti-MM agents which are active at non-acidic sites.
Keiichiro Watanabe,1,2* Ariunzaya Bat-Erdene,3* Hirofumi Tenshin,1,2* Qu Cui,4 Jumpei Teramachi,5
Masahiro Hiasa,2 Asuka Oda,1 Takeshi Harada,1
Hirokazu Miki,6 Kimiko Sogabe,1 Masahiro Oura,1
Ryohei Sumitani,1 Yukari Mitsui,1 Itsuro Endo,1 Eiji Tanaka,2 Makoto Kawatani,7 Hiroyuki Osada,7 Toshio Matsumoto8 and Masahiro Abe1
1Department of Hematology, Endocrinology and Metabolism, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan; 2Department of Orthodontics and Dentofacial Orthopedics, Institute of Biomedical Sciences, Tokushima
3 UniversityGraduateSchool,Tokushima,Japan; Departmentof
Immunology, School of Bio-Medicine, Mongolian National University of Medical Sciences, Ulaanbaatar, Mongolia; 4Department of Hematology, Beijing Tiantan Hospital, Capital Medical University, Beijing, China; 5Department of Tissue Regeneration, Institute of Biomedical Sciences, Tokushima University Graduate School, Japan; 6Division of Transfusion Medicine and Cell Therapy, Tokushima University Hospital, Tokushima, Japan; 7RIKEN Center for Sustainable Resource Science, Chemical Biology Research Group, Saitama, Japan and 8Fujii Memorial Institute of Medical Sciences, Tokushima University, Tokushima, Japan
*KW, AB and HT contributed equally as co-first authors.
Correspondence:
MASAHIRO ABE - masabe@tokushima-u.ac.jp
doi:10.3324/haematol.2019.244418
Disclosures: MA received research funding from Chugai Pharmaceutical, Sanofi KK, Pfizer Seiyaku KK, Kyowa Hakko Kirin, MSD KK, Astellas Pharma, Takeda Pharmaceutical, Teijin Pharma and Ono Pharmaceutical, and honoraria from Daiichi Sankyo Company. The other authors declare no competing financial interests.
Contributions: KW, AB, HT, MK, HO and MA designed the research and conceived the project. Animal experiments were performed by KW, QC, HT, TH, HM, KS, MO, RS and JT; cell cultures by KW, AB, HT, AO, TH, KS, MO and RS; immunoblotting by AB, JT, HT, AO and MH; and bone analyses by KW, QC, YM, MH, IE, and ET. KW, AB, HT, MK, HO, TM and MA analyzed and discussed the data, and wrote the manuscript.
Funding: this work was supported in part by JSPS KAKENHI grant ns. JP15K20536, JP16K11504, JP17KK0169, JP17K09956, JP17H05104, JP18K08329, JP18K16118, and JP18H06294, and the Research Clusters program of Tokushima University. The funders had no role in study design, data collection and analysis, the decision to publish or the preparation of the manuscript. The authors would like to thank Dr. Toshihiko Nogawa (RIKEN) for preparing reveromycin A.
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