(P 181) MITOCHONDRIAL TRANSFER FROM MESENCHYMAL STEM CELSS MEDIATED BY HMGB1 COULD BE INVOLVED IN DRUG RESISTANCE IN OSTEOSARCOMA CELLS.

Objective: In malignant neoplasms, it has been reported that mesenchymal stem cells (MSCs) migrate into tumor tissue after chemo- or radiation therapies and contribute to the survival of malignant tumor cells, but the detailed mechanisms are not fully elucidated. High mobility group box-1 (HMGB1), a nuclear protein is oxidized and released outside the nucleus due to cell damage, and is involved in inflammation and tissue repair in damaged tissues. We report here that the oxidized form of HMGB1 released from osteosarcoma cells by doxorubicin (DOX) and cisplatin (CDDP) induces mitochondria transfer (MT) from MSCs into osteosarcoma cells and contributes to drug resistance.

Methods: We used human osteosarcoma (OS) cell lines, SaOS2 and U2OS. HMGB1 concentration was measured by ELISA and expressions of MT-related genes were assessed by RT-PCR and western blot. MT was observed under the fluorescence microscope with migrated red-fluorescent (Mito Deep red) MSCs’ mitochondria migration into green-fluorescent (PKH67) OS cells. Mitochondrial superoxide (O−2) was assessed with mitoROS580 staining. The effect of MT for chemotherapy was evaluated with forced MT from MSCs to OS cells performed by centrifuge.

Results: MT from MSCs into OS cells was observed in the co-cultured system with MSCs and OS cells (Fig.1). After the treatment of DOX and CDDP, we observed marked increase of the HMGB1 concentration in the supernatant (Fig.2). MT was enhanced by DOX and CDDP administration and was suppressed by anti-HMGB1 antibodies (Fig.3). Mitochondrial superoxide induced by chemotherapy in OS cells was decrease by co-culture of MSCs. MT from MSCs induced DXR resistance in SaOS2 cells. MT correlates with the expression levels of MT-related genes Miro1, Cx43 in MSCs, and these MT-related genes were dominantly elevated at both the protein and RNA levels when MSCs were cultured in a supernatant of OS cells treated with DOX, and expression was suppressed by HMGB1 antibody treatment. These genes were induced only in MSCs treated with oxidized HMGB1 but not in those treated with non-oxidized HMGB1, and were suppressed by HMGB1 antibody and the inhibitor of NF-κb, JSH23 at downstream of HMGB1.

Conclusion: Upon these results, the HMGB1 released from OS cells damaged by chemotherapy induces MT from MSCs to OS cells, and mitochondrial ROS levels in OS cells was reduced in the co-culture system with MSCs. MT might contribute to the resistance against anticancer therapy in OS cells. Chemotherapy enhanced mitochondria transfer from MSCs and anti-HMGB1 ab suppressed this phenomenon. Oxidized HMGB1-targeting therapy such as neutralization therapy using antibodies might overcome the drug resistance and contribute to improving the prognosis in OS patients.