Targeting NF-κB and Induction of Apoptosis by Dehydroxymethylepoxyquinomicin (DHMEQ) in Multiple Myeloma Cells.
Authors: Miyakawa Yoshitaka , Kohmura Kanoko , Saito Kaori , Yoshida Hiroshi , Ikejima Asako , Hattori Yutaka , Ohnishi Yasuyuki , Nakamura Masato , Ikeda Yasuo , Umezawa Kazuo , Kizaki Masahiro 
Published: October 2019

Abstract
We previously designed and synthesized a new NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ) (J Biol Chem, 2002). DHMEQ is a derivative of the weak antibiotics epoxyquinomicin C, which was isolated from the culture broth of Amycolaptosis sp. NF-κB is a critical regulatory protein that activates the transcription of a number of genes, including growth factors, angiogenesis modifiers, cell adhesion molecules and anti-apoptotic factors. As NF-κB has been shown as a good target for the new therapies such as bortezomib, we studied the effects of the new specific NFκB inhibitor, DHMEQ, to myeloma cells. In the present study, we demonstrated that DHMEQ inhibited the proliferation of human myeloma cell lines, RPMI8226 and U266 in dose- and time-dependent manners. Apoptosis was detected using fluorescein-conjugated Annexin-V by FACS. Around 45.3%of RPMI8226 and 45.2% of U266 were in apoptosis 12 hours after treatment with 10 μg/ml DHMEQ. Formation of apoptotic bodies were observed 24 hour-treatment with DHMEQ in both cell lines by Giemsa staining. In contrast, no obvious cell cycle arrest was observed with DHMEQ, indicating DHMEQ directly induces apoptosis without cell cycle arrests in these myeloma cell lines. The activation of caspase-3 in RPMI8226 and U266 cells were detected with the specific antibody against the active form of caspase-3 by FACS. When the myeloma cells were pretreated with 20 μM pan-caspase inhibitor, z-VAD-FMK, DHMEQ-induced apoptosis was inhibited by 62.1% in RPMI8226 and 71.9% in U266 cells, indicating DHMEQ-induced apoptosis was caspase-dependent. The binding activities of nuclear NF-κB protein to the oligonucleotides including NF-κB binding sites was suppressed by 81.9% in RPMI8226 and 69.0% in U266 1 hour after treatment with DHMEQ. NF-κB protein seemed more accumulated in cytoplasm of myeloma cells after treatment with DHMEQ under the confocal microscope, indicating DHMEQ prevents the translocation of NF-κB protein into the nucleus. Bcl-XL is the anti-apoptotic factor and its transcription is regulated by NF-κB. However, the expression level of Bcl-XL protein was not altered 24 hours after treatment with DHMEQ in RPMI8226 and U266. We also studied the effects of DHMEQ to the patient materials. We found that DHMEQ induced apoptosis in CD138-positive plasma cells from the myeloma patients (n=3), demonstrating that DHMEQ is also effective for primary cells. We previsously developed the model of human multiple myeloma by simply injecting U266 cells into the tail vein of the immunodeficient NOG mice. This myeloma model demostrated the massive osteolytic lesions and hind leg paralysis around 7 weeks after transplantation. We did not observe any invasion of U266 cells into other organs except bone marrow. As NF-κB regulates the proliferation of myeloma cells and osteoclasts, we expect DHMEQ will inhibit the tumor growth and prevent pathological fractures by inducing apoptosis in both myeloma cells and osteoclasts in vivo. We are currently evaluating the in vivo efficacies of DHMEQ using this experimental animal model of multiple myeloma. In conclusion, we demonstrated that DHMEQ targets NF-κB and induces apoptosis in myeloma cells through caspase-dependent pathways.