Mechanism Of Endogenous HMBGB1-mediated Autophagic Response For The Chemosensitivity In Leukemia Cells

Acute leukemia (AL) is a malignant disease of the bone marrow and blood, and is the most common cancer in children. Chemotherapy is the main treatments for childhood leukemia. Recently, chemoresistance has become a major obstacle to the successful treatment of AL. But the mechanism response to chemoresistance in leukemia cells is not clearly defined. High mobility group box1(HMGB1), a highly conserved nuclear protein, is important in the regulation of stabilizing the structure and function of chromatin and gene transcription. Our former studies have shown that exogenous HMGB1mediated autophagy induced the chemoresistance of leukemia cells. However, whether endogenous HMGB1regulates autophagy and influences chemotherapy sensitivity in leukemia cells remains unknown.Firstly, HMGB1mRNA and protein expression levels in bone marrow mononuclear cells (BMMCs) were measured by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and western blotting in a cohort of41children with acute leukemia (acute lymphoblastic leukemia (ALL):30, acute non-lymphoblastic leukemia (ANLL):11). Expression levels of HMGB1mRNA in primary/relapsing group were much higher than complete continuous remission (CCR) group, which was positively correlated with clinical status in childhood leukemia. Meanwhile, we investigated HMGB1mRNA and protein levels in different cell lines by RT-PCR and western blotting analysis. Expression of HMGB1was higher in three leukemia cell lines (K562, HL-60and Jurkat), whereas noticeably lower in non-cancer cell lines. HMGB1was also found to be higher in all other non-blood cancer cell-lines, suggesting that HMGB1may play an important role in tumorigenesis. To further characterize the role of endogenous HMGB1in the chemosensitivity of leukemia cells, Jurkat and K562cells were treated with several chemotherapeutic drugs (ADM, VCR and Ara-C). Up-regulated HMGB1expression in leukemia cells by gene transfection decreased chemotherapy sensitivity in methylthiazolyltertrazolium (MTT) analysis. Notably, suppressing HMGB1expression by RNA interference (RNAi) increased leukemia cell chemosensitivity.Secondly, we demonstrated that endogenous HMGB1-mediated autophagy influenced the chemosensitivity of leukemia cells. Expression of LC3-II was increased in K562cells which were treated with different chemotherapy drugs and starvation (Hank's balanced salt solution, HBSS), in comparison with the control group. The upregulation of HMGB1expression by gene transfection in leukemia cells increased the expressions of microtubule-associated protein1light chain3-â…¡ (LC3-â…¡), Beclinl and autophagosomes significantly. In contrast, the suppression of HMGB1expression by RNAi significantly decreased these expressions and pepstatin A (PA)/E64D could not increase LC3-â…¡ expression. Meanwhile, the inhibition of autophagy by using3-methyladenine (3-MA), an autophagy inhibitor, decreased the expression of LC3-II and the formation of autophagosomes and it reversed HMGB1-mediated drug resistance.At last, the phosphatidylinositol3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway was found to be functionally connected with HMGBl. HMGB1gene transfection increased the LC3-II level and inhibited phosphorylation of Akt and p70S6K levels. Knockdown of HMGB1expression blocked the association between mTOR and raptor in the setting of enhanced autophagy. When class I PI3K was inhibited by PI3K-I shRNA, it decreased the PI3K-â…  expression level. But the suppression of HMGB1expression had no further effects on LC3-â…¡, suggesting that HMGB1was an upstream regulator of PI3K-I.In conclusion, endogenous HMGB1was involved in the pathogenesis and chemosensitivity in pediatric AL. Endogenous HMGB1was an intrinsic regulator of autophagy in leukemia cells and it enhances leukemia cell chemoresistance likely through the PI3K/Akt/mTOR pathway. These findings supplied further evidence of the role of endogenous HMGB1in autophagy and chemoresistance of leukemia cells, which will help to establish more efficient chemotherapies for leukemia by means of affecting autophagy, and consequently modifying the chemoresistance of leukemia cells.