Autophagy Inhibition Overcomes Resistance to Cytosine Arabinoside in Acute Myeloid Leukemia Cells through Inducing Autophagic Cell Death and Intrinsic Pathway Apoptosis
Authors: Kim Yundeok , Eom Ju-In , Jeung Hoi-Kyung , Jang Jieun , Kim Jin Seok , Cheong June-Won , Kim Young Sam , Min Yoo Hong 
Published: October 2019

Abstract
Background
A major obstacle to the successful treatment of acute myeloid leukemia (AML) is the development of chemoresistance. Identifying the novel agents overcoming drug resistance is critical for improving AML outcomes. Autophagy is an indispensable lysosomal self-digestion process involved in the degradation of aggregated proteins and damaged organelles. Autophagy has recently been demonstrated as important for conferring resistance to chemotherapy and targeted therapy. The antimalarial drug hydroxychloroquine (HCQ) is able to inhibit autophagy and therefore is being considered for cancer therapeutics. However, the effects of HCQ on chemoresistant myeloid leukemia cells have not been investigated.
Objective
The present study was designed to examine comparatively the effects of HCQ on the induction of cell death of the chemosensitive and chemoresistant acute myeloid leukemia cells, and elucidate its detailed mechanism.
Method
Ara-C (Cytosine arabinoside)-sensitive (U937, AML-2) and Ara-C-resistant (U937/AR, AML-2/AR) human myeloid leukemia cell lines were used to evaluate HCQ-induced cytotoxicity, autophagy, and apoptosis, as well as effects on cell death-related signaling pathways.
Result
U937/AR cell line showed a significantly higher number of autophagic vesicles and higher level of autophagic proteins. We initially found that HCQ caused dose- and time-dependent cell death of myeloid leukemia cells evaluated. HCQ-induced cell death rate was significantly higher in the chemoresistant U937/AR, AML-2/AR compared to chemosensitive U937 and AML-2 cells, respectively. Particularly, in Ara-C-resistant cell lines, HCQ triggers the activation of autophagy based on the results of increased number of autophagosomes, conversion of microtubule-associated protein light chain 3 (LC3)-I to LC3-II, and formation of GFP-LC3-positive punta. However, p62/SQSTM1 level was increased, suggesting that HCQ blocks the degradation of p62/SQSTM1 and autophagy flux. Modest upregulation of beclin-1 and Atg7 (autophagy-related protein 7) was observed. With continued exposure to HCQ, LC3 conversion was followed by nuclear condensation, procaspase-3 and -9 activation, release of cytosolic cytochrome C, and decreased mitochondrial membrane potential, indicating apoptosis via a mitochondria-dependent pathway. Pretreatment of leukemia cells with the autophagy blocker 3-methyladenine or siRNAs against beclin-1 or p62/SQSTM1, reduced HCQ-induced cell death, LC3 conversion, and procaspase-9 cleavage. The pan-caspase inhibitor z-VAD-fmk and the caspase-9 inhibitor z-LEHD-fmk, but not the caspase-8 inhibitor z-IETD-fmk, reduced HCQ-mediated cell death and caspase activation. However, LC3 conversion was unaffected. Additionally, Ara-C and HCQ synergistically induced cell death in U937/AR cells.
Conclusion
Taken together, our data show that HCQ effectively induced cell death in Ara-C-resistant AML cells through activation of autophagy and subsequent intrinsic pathway apoptosis. Our findings suggest HCQ might improve the therapeutic outcome in chemoresistant AML.

Disclosures
No relevant conflicts of interest to declare.