Interference of HMGB1 Lead to Increased Vulnerability of MM Cells to Dexamethasone and Doxorubicin

Background: Multiple myeloma (MM) is the second mostly diagnosed disease among hematological malignancies after lymphoma. With the novel agents, the survival of MM patients has been improved significantly but still remains incurable because of drug resistance. Studies have found that high-mobility group box 1 protein (HMGB1) was involved in inflammation, angiogenesis, and cancer invasion progression, metastasis, and drug resistance. Our research was aimed at exploring the role of HMGB1 in MM cell proliferation and drug resistance.

Methods: First, semi-quantitative real time-polymerase chain (qRT-PCR) and western blot was used to determine the levels of HMGB1 mRNA and protein expression in MM cell lines (RPMI8226, CAG, and MM.1S) and primary MM samples. Second, MM cells were transfected with HMGB1-knockdown lentivirus and the Cell Counting Kit 8 (CCK8) assay was used to determine the proliferation of MM cells with or without chemotherapeutic drugs dexamethasone (Dex) and doxorubicin (ADM). Then cell apoptosis was detected by flow cytometry. Third, Affymetrix HTA 2.0 Array was used to compare changes in gene expression levels between HMGB1-knockdown cells and the control cells and qRT-PCR was used to verify the array results. Last, Western bolt was performed to analyze changes in signaling pathways after HMGB1 knockdown.

Results: MM cell lines and primary MM samples expressed high levels of HMGB1 mRNA and protein. Although there was no difference in MM cell proliferation between HMGB1-knockdown group and the control group (P>0.05), HMGB1-knockdown significantly enhanced inhibitory effect of chemotherapy with Dex and ADM in comparison with the wildtype HMGB1 control (P<0.05). Flow cytometry results showed that apoptosis of MM cells induced by Dex and ADM was increased when HMGB1 expression was suppressed (P<0.05). Furthermore, gene array analysis on RPMI8226 and CAG cell lines showed that anti-apoptotic genes (bcl-2, HIAP1) and MM survival related genes (DEPTOR, CXCL12) were decreased and pro-apoptotic genes (TNFRSF1B, TRAIL, CXCL10) were increased in knockdown cells compared to the controls. In addition, expression levels of genes that play important roles in signaling pathways such as mTOR, NF-κB, PI3K-AKt, and p38-MAPK were also significantly changed. The gene expression microarray results were verified by qRT-PCR and Western blot demonstrated that phosphorylation of p70S6K (substrate of mTORC1 complex) and AKT-ser473 (substrate of mTORC2 complex) were both elevated in HMGB1-knockdown MM cells compared to that in the control cells.

Conclusions: Our research showed that downregulation of HMGB1 increased sensitivity of MM cells to Dex and ADM through increasing apoptosis and regulating mTOR, NF-κB, PI3K-AKt, and p38-MAPK pathways.