HO-1 Regulated Autophagy and Apoptosis In CML Cells Through Mitochondrial-beclin1 Pathway
Authors: Wang Jishi , Wei Sixi , Wang Yating , Chai Qixiang 
Published: October 2019

Aim

To explore the mechanisms underlying the HO-1-regulated apoptosis and autophagy in human chronic myeloid leukemia (CML) K562 cell line in vitro.

Methods

K562 cells were devided into three groupsFK562(untreated),K562-H(infected by lenti-GFP-HO-1) and K562-siHO-1(infected by lenti-GFP-siHO-1). The protein expression of HO-1 were determined by Western blot, and its mRNA levels were obtained by real-time PCR.  Cell viability was assessed by MTT assay. Morphological changes of apoptosis and autophagy were observed by the application of transmission electron microscope. Annexin V-FITC/PI dual staining assays were used to measure apoptosis. Autophagy was analyzed by Western blot and immunofluorescence staining with an anti-LC3 antibody. DCFH-DA was used as the cell-permeate indicator for intracellular ROS measurement. Protein levels of active caspase 3, Beclin1,cyto-C and Bcl2 were determined by Western blot.

Results

The Expression of HO-1 on mRNA and protein level increased in K562-H group while reduced in K562-siHO-1, the differences showed statistically significance compared to K562-untreated group(p<0.05). Imatinib (0.20-1.60μmol/L) exerted inhibitory effects on cell proliferation in a concentration-dependent manner with IC(50) value of 1.0μmol/L, 0.7μmol/L and 0.4μmol/L, while viability of 63.41±1.46%, 41.24±2.01%, 29.49±1.87% in K562-H, K562 and K562-siHO-1(p<0.05), respectively. Exposure to imatinib (0.40 μmol/L) simultaneously induced mitochondrial-mediated apoptosis and Beclin1-dependent autophagy in all groups. Autophagy flux was found in K562-siHO-1 group while autophagosome formation decreased in K562-H group, which was significantly different from K562 group. The expressions of LC3 II and cyto-C were higher in K562-siHO-1 group than other two groups (p<0.05). Both apoptosis and autophagy caused cell death in a time-dependent manner. However, apoptosis played a more effective role in cell death. After exposure to imatinib (0.40 μmol/L), both intracellular ROS generation and the protein expression of Beclin1 and caspase9 were increased, while Bcl2 protein level were reduced in a time-dependent manner. The combination of NAC (2.50 mmol/L) with imatinib (0.40 μmol/L) contributed to the blockade of ROS generation which abrogated apoptosis, autophagy and Beclin1 expression. 3-MA (3.0mmol/L) and HO-1 silencing resulting in the inhibition of autophagy enhanced the effect of apoptosis which was induced by imatinib (respectively 70.65±2.71% and 69.27±3.13%), while this phenomenon was accompanied by increased ROS generation (1.3 and 1.6 folds higher than K562 group).

Conclusions

Imatinib and HO-1 siRNA simultaneously induced apoptosis and autophagy in K562 cells, which was associated with the increasing of intracellular ROS generation, cyto-C and Beclin1 protein expression. Beclin1, an autophagy related protein induced by HO-1 silencing, could further induce apoptosis in K562 cells through the inhibition of Bcl2. The responsive cytoprotection to chemotherapeutics caused by autophagy decreased while sustaining up-regulation of HO-1 in K562 cells. However, the underlying mechanisms remained to be well elucidated.

Disclosures:

No relevant conflicts of interest to declare.