| ObjectiveTo comparatively investigate the difference of capsaicin-inducing apoptosis of multi-drug resistant leukemia K562/ADM cells and its parental drug-sensitive K562 cells, and whether the capsaicin-induced apoptosis of drug-sensitive and -resistant leukemia cells occured via endoplasmic reticulum stress-related pathway. Furthermore, the cytototic or apoptotic effect of capsaicin to leukemia stem cells (LSC) was observed.MethodsK562 cells and K562/ADM cells were used as target cells. The cells were treated with capsaicin, and the cell proliferating activity was assessed by MTT colorimetric assay. Double staining of FITC-Annexin V and propidium iodide (PI) was used to identify the apoptotic cells. The cellular morphologies and ultrastructure were observed by light microscope and electronic microscope, respectively. Real-time RT-PCR was employed to analyze the mRNA expression of glucose regulated protein 78 (GRP78), C/EBP homology protein (CHOP), X box binding protein-1 (XBP-1) and caspase-12 genes. The expression of GRP78 protein was examined by Western blotting. Flow cytometry (FCM) was used to detect the proportion of LSCs (CD34+CD38-CD123+), the apoptotic ratio of LSCs (Annexin V+), and the expression of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) in leukemia cells.Results1 Capsaicin inhibited the proliferation and induced apoptosis of drug-sensitive leukemia K562 cells Capsaicin markedly inhibited the proliferation of K562 cells at a dose- and time-dependent manner (P<0.01). The 50% inhibitory concentration (IC50) was (86.58±3.11)μmol/L, (79.91±4.03)μmol/L and (49.01±0.98)μmol/L at 24,48 and 72 h, respectively. When K562 cells were induced with 50μmol/L capsaicin for 48 h, the apoptotic K562 cells appeared with shrinkage, chromatin condensation, margination, nuclear fragmentation, intact cell membrane and organelles and apoptotic bodies. The endoplasmic reticulum exhibited obvious expansion and degranulation, and the mitochondria illustrated inner and outer membranes fusion, reduced and confused cristae, swelling and vacuolization. When the cells were treated with 20μmol/L and 50μmol/L capsaicin for 48 h, the apoptotic rate of K562 cells increased from (1.28±0.01)% to (35.28±1.15)% and (53.10±2.03)% by FITC-Annexin V/PI double staining detection. Exposed the cells to 20μmol/L and 50μmol/L capsaisin, the expression of GRP78 mRNA was 1.7 and 3.8 times of that the control cells at 24 h, and then slightly reduced at 48 h. The GRP78 protein expression of the capsaisin-treated cells was 1.2 and 1.4 times more than that of the control at 24 h, and reduced to near normal levels at 48 h. After administration with 25μmol/L,50μmol/L and 100μmol/L capsaicin for 24 h, compared with the control cells the treated cells had 1.1,2.8 and 7.5 times more expression for CHOP mRNA,6.1,3.2 and 1.2 times for caspase-12 mRNA, and 1.7,1.9 and 5.0 times for XBP-1 mRNA, respectively. Then, the expression of the above genes decreased to near control level at 72 h. K562 cells were treated with 25μmol/L,50μmol/L and 100μmol/L capsaicin for 48 h, the proportion of LSC(CD34+CD38-CD123+ cells) in the capsaicin-treated K562 cell population increased from control(0.55±0.01)% to (2.45±0.03)%, (10.40±0.10)% and (17.30±0.80)%, respectively. And the proportion of apoptotic (Annexin V+) cells in LSCs were (0.35±0.01)%, (4.95±0.01)%, (12.26±0.23)% and (16.55±0.79)%, respectively. Furthermore, the numbers of P-gp+, BCRP+ and P-gp+BCRP+ cells in K562 cell population also increased.2 Capsaicin inhibited the proliferation and induced apoptosis of drug-resistant leukemia K562/ADM cellsCapsaicin also significantly inhibited the growth of K562/ADM cells as with as K562 cells at a concentration- and time-dependent manner (P<0.05) and the IC50 was (123.00±1.45)μmol/L, (89.16±1.08)μmol/L and (32.71±0.68)μmol/L for 24,48 and 72hours, respectively. K562/ADM cells treated with 50μmol/L capsaicin for 48 h, the apoptotic cells showed the morphological changes as shrinkage, chromatin condensation, margination, nuclear fragmentation, intact cell membrane and organelles and apoptotic bodies. The endoplasmic reticulum exhibited obvious expansion and degranulation, and the mitochondria illustrated inner and outer membranes fusion, reduced and confused cristae, swelling and vacuolization. Treatment with 20μmol/L and 50μmol/L capsaicin for 48 h, the apoptotic rate of K562/ADM cells detected with FITC-Annexin V/PI staining increased from control(0.91±0.09)% to (33.43±2.01)% and (42.39±0.43)%, respectively. In the cells exposed to 20μmol/L and 50μmol/L capsaisin, the expression of GRP78 mRNA increased to 1.2 and 1.4 times of the control cells at 24h, and then there was a slight decrease at 48h, and the expression of GRP78 protein also increased by 1.5 and 2.2 times at 24h and nearly reduced to normal levels at 48h. At the same time, K562/ADM cells treated with 25μmol /L,50μmol/L and 100μmol/L capsaicin for 24 h, the expression of CHOP mRNA showed a mild increase, and there were 3.2-, 3.1- and 1.6-time increases for caspase-12, and 1.5-,1.7- and 1.8-time for XBP-1. Treated for 72 hours, the expression of all the above genes was down-regulated slightly. After administration with 25μmol/L,50μmol/L and 100μmol/L for 48 hours, the proportion of LSCs in K562/ADM cell population increased from control(4.25±0.05)% to (10.95±0.85)%, (14.95±0.75)% and (18.40±0.50)%, respectively. The apoptotic (Annexin V+) cells in LSCs were (0.45±0.07)%, (7.65±0.15)%, (11.45±1.08)% and (14.95±0.98)%, respectively. Furthermore, the content of P-gp+ cells in capsaicin-treated K562/ADM cells decreased slightly, that of the BCRP+ cells increased from control(2.06±0.03)% to (2.83±0.01)%, (3.58±0.09)% and (8.59±0.14)%, and P-gp+BCRP+ cells from (2.05±0.02)% to (2.74±0.01)%, (3.48±0.07)% and (8.55±0.15)%, respectively.ConclusionCapsaicin significantly inhibits the proliferation of drug-sensitive leukemia K562 cells as well as multi-drug resistant K562/ADM cells and induces to apoptosis. Capsaicin may initiate the leukemia cells to apoptosis via the endoplasmic reticulum stress response-related pathway by regulating the expression of GRP78, CHOP, XBP-1 and caspase-12. Capsaicin is able to induce a part of the leukemia stem cells in K562 cells and K562/ADM cells to undergo apoptosis. Drug-sensitive leukemia K562 cells are slight more sensitive to capsaicin than multi-drug resistant K562/ADM cells. |
PDF Full Text Request