Inhibition of Heme Oxygenase-1 Attenuates Bortezomib Resistance in Multiple Myeloma Via Down-Regulating Gas6 Production

Background

Multiple myeloma (MM), one of the most prevalent hematological malignancies, is characterized by the proliferation of abnormal plasma cells. The proteasome inhibitor bortezomib is a frontline drug in the treatment of MM; however, most patients will develop acquired resistance to bortezomib, which has been a critical challenge in clinic. Our previous studies have indicated that inhibition of heme oxygenase-1 (HO-1) can attenuate drug resistance to MM. Moveover, emerging evidence suggests that growth arrest-specific gene 6 (Gas6) may regulate tumor microenvironment and serve as a novel target for cancer therapy. However, whether HO-1 mediated-Gas6 production involved in the development of bortezomib resistance in MM remains unclear.

Aims

The aims to perform the present study were to: 1) assess the association between intracellular HO-1 and Gas6 expression in the bone marrow of CD138⁺ cells of patients with MM; 2) test the role of HO-1 and Gas6 in the development of bortezomib resistance in MM; 3) explore whether HO-1 mediate Gas6 production and the potential mechanism.

Methods

Patients with different stages of MM (n=18) and healthy donors (n=18) at Affiliated Hospital of Guizhou Medical University were included in this study. Baseline clinical characteristic data and bone marrow samples were obtained from study participants. CD138⁺ plasma cells were purified from bone marrow samples. The recombinant lentiviral vector of HO-1 was constructed and subsequent transfected into U266 and RPMI8226 cells. The expressions of mRNA and protein levels were determined by real-time PCR and western blot, respectively. Secreted Gas6 was determined by ELISA. Flow cytometry was used to assess cell apoptosis.

Results

In human study, the HO-1 mRNA and protein level in CD138⁺ cells of patients with MM showed a significant increase compared with those in healthy group, respectively (P<0.01). With the development of MM, HO-1 mRNA and protein level were gradually increased from stage I to III. The similar findings were also observed in the expression of Gas6 mRNA and protein level (P<0.01). Interestingly, spearman correlation analysis showed that higher HO-1 mRNA and protein level were associated with increased Gas6 mRNA and protein level, respectively (P<0.05). To test whether HO-1 mediate Gas6 production, recombinant lentivirus-HO-1 vector and HO-1 siRNA were transfected into U266 and RPMI8226 cells to up/down-regulate HO-1 expression. Compared with control group, Gas6 mRNA and protein expression were significantly increased when U266 and RPMI8226 cells were up-regulated by recombinant lentivirus-HO-1 vector. Additionally, Gas6 mRNA and protein expression were also increased by the use of hemin (HO-1 inducer). In contrast, Gas6 expression presented a decrease in mRNA and protein level with the stimulation of HO-1 siRNA and HO-1 inhibitor ZnPPIX. Similar significantly trend was also observed in the culture medium. Moreover, the effect of HO-1 on bortezomib resistance in MM cells was markedly inhibited by the stimulation with Gas6 siRNA and Gas6 neutralizing antibody (the apoptosis rate in MM cells treated with PBS, Gas6 siRNA and Gas6 neutralizing antibody is 28%, 56% and 42%, respectively; P<0.01). These results indicated that HO-1 up-regulated Gas6 production in the development of bortezomib resistance in MM. Furthermore, to explore the potential mechanism how HO-1 regulates Gas6 production, MAPK, an important pathway in drug resistance, was investigated. U266 or RPMI8226 cells transfected with recombinant lentiviral vector of HO-1 were stimulated with p38MAPK inhibitor SB203580, the MEK inhibitor PD0325901, the JNK inhibitor SP600125, the PI3K inhibitor LY294002, and the STAT3 inhibitor S3I-201, and the HO-1 level of cells and Gas6 levels of cells and culture medium were determined. Findings showed that HO-1 level did not significantly change, while Gas6 expression in cells and Gas6 level in culture medium showed markedly decrease by the treatment with MAPK pathway inhibitor compared with control group. These results indicated that HO-1 mediated Gas6 production via MAPK pathway.

Conclusion

These results demonstrate that inhibition of HO-1 attenuates bortezomib resistance in MM via down-regulating Gas6 production. Targeting these pathological interactions holds promise for improve MM treatment.