The human AML-M2 cell line, Kasumi-1 cells (15) were cryopreserved in the center laboratory of Hematopoietic Stem Cell Transplantation Center of Guizhou Province (Guiyang, China). Cells were maintained in Roswell Park Memorial Institute-1640 (RPMI-1640) medium containing 10% fetal bovine serum, 100 U/ml penicillin and 100 g/ml streptomycin at 37°C in an incubator containing 5% CO₂ and humidified air. The medium and antibiotics were from Sijiqing (Hangzhou Tianhang Biological Technology Co., Ltd., Zhejiang, China). The study was approved by the ethics committee of Hospital affiliated to Guiyang Medical College (Guiyang, China).

Daunorubicin (DNR), N-acetyl-L-cysteine (NAC) and BAPTA-AM were respectively purchased from Sigma (St. Louis, MI, USA), Beyotime Institute of Biotechnology (Haimen, Jiangsu, China) and Dojindo Molecular Technologies (Kumamoto, Japan). Cytoplasmic and mitochondrial protein extraction kit was purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Antibodies for western blot analysis were obtained from Cell Signaling Technology (Beverly, MA, USA) and secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

According to the instructions, DNR was dissolved with ddH₂O to prepare a 1 g/ml stock solution. Based on the preliminary results, the stock solution was further diluted into working solutions at the final concentrations of 5, 7.5, 10 and 12.5 μg/ml by RPMI-1640 medium. NAC was dissolved with RPMI-1640 to prepare a 1 mol/l stock solution that was diluted into working solutions at the final concentration of 10 mmo/l. BAPTA-AM was dissolved with dimethyl sulfoxide (DMSO) (Solarbio Science & Technology Co., Ltd., Beijing, China) to form a 1 mol/l stock solution, which was then diluted into working solutions with Hank’s balanced salt solution (HBSS) (Life Technologies, Foster City, CA, USA) at the final concentration of 10 μmol/l. Corresponding blank controls were set for different treatments.

HO-1 (NM_002133.2)-targeted siRNA, pRNAi-siHO-1-GFP and pRNAi-GFP (lentiviral vector carrying with/without HO-1 siRNA), were designed and purchased from Biomics Biotechnologies (Nantong) Co., Ltd. (Jiangsu, China). According to the results of preliminary experiments, the most effective one (5′-AAGCUUUCUGGU GGCGACAGU-3′) was chosen in our study. Kasumi-1 cells in the log phase were planted at the concentration of 1×10⁶/well in 6-well plate after overnight culture and were then transfected with pRNAi-GFP(with/without siHO-1)at multiplicities of infection of 8 in serum-free medium. Polybrene (5 μg/ml) (Sigma) was added to improve transfection efficiency as enhancing reagent. After 10 h, the medium was changed for complete medium, and the cells without intervention were used as blank control group. After transfection for 24, 48 and 72 h, infection efficiency of lentivirus was observed by fluorescence microscopy (Olympus, Tokyo, Japan) and flow cytometry (FCM) with FL-1 channel.

Kasumi-1 cells in the log phase were inoculated into 96-well plates with the density of 1×10⁴/well, and treated with different interventions. Serum-free RPMI-1640 was added for the blank control group until the total volume of 200 μl in each well. Six replicates were set and the cells were cultured for 24, 48 and 72 h. At the end of culture, the medium in each well was replaced with 200 μl of fresh medium containing MTT (Solarbio). After incubation at 37°C for 4 h, cells were centrifuged (1,000 r/min, 10 min) and the supernatant was discarded. The reaction was terminated by adding 200 μl/well of DMSO, and the resultant solution was subjected to vibrate-shaking for 10 min to fully dissolve the crystals. The absorbance at 570 nm (A₅₇₀) of each sample well was measured using an automated 96-well plate reader. All assays were performed in triplicate. The inhibition rate was calculated using the following equation:

Total RNA was extracted using TRIzol^® Reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). RNA was reverse transcribed into cDNA using a reverse transcription kit (Promega Corp., Madison, WI, USA), which was stored at −20°C. Real-time PCR was performed using a real-time PCR thermocycler (Applied Biosystems, Foster City, CA, USA) and quantified using SYBR^® Green PCR Master Mix (Applied Biosystems) using 1 μl cDNA in a final reaction volume of 20 μl. The PCR reactions were cycled 45 times after initial denaturation (94°C, 1 min) with the following parameters: denaturation at 94°C, 10 sec; annealing at 58°C, 15 sec (caspase-3, caspase-8, GAPDH) at 60°C, 10 sec (HO-1, caspase-9), and extension at 72°C, 10 sec. At the end of PCR, the temperature was increased from 60 to 95°C at a rate of 2°C/min, and the fluorescence was measured every 15 sec to plot the melting curve. GAPDH was used as the internal control. SDS 2.2.1 software (Applied Biosystems) was used to perform relative quantification of the target genes using the ΔΔCt method. The primers (Takara Bio Inc., Dalian, China), shown in Table I, were designed using Primer Express 3.0 Software (Applied Biosystems).

Cells were washed three times with cold phosphate-buffered saline (PBS; 10 mmo/l HEPES, 140 mmo/l NaCl and 2.5 mmo/l CaCl₂, pH 7.4) and then lysed in Tris-buffered saline (TBS; 50 mmo/l Tris-HCl, pH 8.0, 150 mmo/l NaCl, 100 μg/ml phenylmethylsulfonyl fluoride and 1% Triton X-100) for 30 min on ice. Then the cells were divided into two Eppendorf tubes, one for cytoplasmic and mitochondrial protein extraction and the other one for total protein extraction following the manufacturer’s instructions. Protein (60 mg) was fractioned by SDS-PAGE using 10% gels and was transferred electrophoretically to hydrophilic polyvinylidene fluoride membranes (GE Healthcare Life Sciences, Piscataway, NJ, USA). The membrane was blocked with 5% skimmed milk in TBS overnight and incubated with polyclonal antibodies of HO-1 (1:800), cleaved-caspase-3 (1:800), caspase-8 (1:800), caspase-9 (1:800), cleaved-PARP (1:800), caspase-12 (1:800), Cyto C (1:800) and β-actin (1:1,200) for 2 h, washed in TBS-T (0.1% Tween-20 in TBS) and incubated with secondary antibodies [anti-rabbit IgG (1:1,500) and anti-mouse IgG (1:1,500)] at room temperature for 1 h. The signals were visualized by exposing the membrane to X-ray film (Carestream Health, Inc., Xiamen, China), and quantified using Image J 1.44p (National Institutes of Health, Bethesda, MD, USA).

Cell death was determined using flow cytometric analysis, subsequent to the cells being double-stained with Annexin V-fluorescein isothiocyanate (FITC) and 7-aminoactinomycin D (7-AAD). The cells (10⁵/sample) were incubated for 24 h after different treatments, and then they were harvested and washed twice with cold PBS. Cell pellets were harvested and resuspended with 500 μl of 50 mg/ml 7-AAD and Annexin V both/respectively containing RNase. After being stained at room temperature for 15 min in dark, the samples were analyzed by flow cytometry using the BD FACSCalibur™ flow cytometer with BD CellQuest™ software (Becton-Dickinson and Company, Franklin Lakes, NJ, USA). Parameters were confirmed by no-treated Kasumi-1 cells with Annexin V or 7-AAD staining, respectively, and data were collected in FL2 channel for Annexin V and FL3 channel for 7-AAD.

The measurement of intracellular ROS was based on the ROS-mediated conversion of non-fluorescent dichlorofluorescin diacetate (DCFH-DA) into DCFH. Briefly, after different treatments, cells were then centrifuged and resuspended in 1X PBS containing a final concentration of 10 μmol/l DCFH-DA (Beyotime) and incubated at 37°C for 30 min. Cells were subsequently washed with 1X PBS and analyzed using the FL2 channel in a flow cytometer.

Cells in culture medium were loaded with fluo-3/AM (final concentration, 10 μmol/l), an intracellular Ca²⁺ indicator (Dojindo), at 37°C for 30 min. The cells were washed and resuspended in culture medium. Intracellular Ca²⁺ was measured using a flow cytometer. A stimulus was applied to the cells 1 min after measuring the fluorescence intensity of resting cells, then continuous recording was started again until another stimulus was applied.

Cells were resuspended in 500 μl of JC-1 probe staining working solution (Beyotime), incubated at 37°C for 20 min, and washed with JC-1 staining working solution (1X) three times. Thereafter FCM was used to detect the intensities of green and red fluorescences of JC-1 to determine the MTP changes. JC-1 showed an aggregation structure with red fluorescence and a monomer structure with green fluorescence when MTP was reduced. Since there existed inter-interference between green (JC-1) and GFP fluorescence in FL1 channel, the decrease of red fluorescence in FL2 channel was chosen to reflect the change of MTP in this study.

Data are expressed as mean ± standard deviation (mean ± SD). Analysis of variance (ANOVA) and Mann-Whitney test were performed with SPSS 16.0 (SPSS, Inc., Chicago, IL, USA). A value of P<0.05 was considered to indicate a statistically significant difference.

After transfection for 24, 48 and 72 h, the GFP expression in Kasumi-1 cells was observed using a fluorescence microscopy. The efficiency of transfection (average proportion of GFP-expressing cells to the total cell count) was >80% after transfection for 72 h (Fig. 1A). FCM analysis showed that the transfection efficiency rose to 73.70% at 48 h and peaked at 72 h (Fig. 1B). Then the cells were harvested after transfection for 72 h to detect expression of mRNA and protein of HO-1 by real-time PCR and western blot analysis. The rates of silenced HO-1 mRNA and protein expression were 77.95 and 75.95%, respectively (Fig. 1C and D).

Targeted HO-1 silencing enhanced the chemotherapy sensitivity of Kasumi-1 cells to DNR.

The inhibitory effects of various concentrations of DNR (5, 7.5, 10 and 12.5 μg/ml) on Kasumi-1 cell growth for different time intervals (24, 48 and 72 h) were investigated. DNR exerted moderate inhibitory effects on cell proliferation in a time- and dose-dependent manner (Fig. 2A). IC₅₀ (the concentration necessary to produce 50% inhibition of cell growth) value of DNR was 7.0 μg/ml. Using Kasumi-1 cells alone and Kasumi-1 cells with vehicle treatment as the control, the cells were exposed to DNR (7.0 μg/ml) and pRNAi-siHO-1-GFP alone or together for 24, 48 and 72 h. The inhibition rate of Kasumi-1 cells with HO-1 siRNA plus DNR treatment was significantly higher than that with HO-1 siRNA or DNR treatment alone (Fig. 2B). Besides, HO-1 siRNA and vehicle itself did not obviously affect the survival of Kasumi-1 cells.

To further identify the inhibitory effect of DNR on Kasumi-1 cells, FCM was used to detect the apoptotic rate after the cells were treated as mentioned above for 24 h (Fig. 3A and B). The apoptotic rate of DNR treatment alone was (39.66±1.66)%, while it increased to (75.42±1.79)% together with pRNAi-siHO-1-GFP, which was significantly higher than those of Kasumi-1 cell group, vehicle group and pRNAi-siHO-1-GFP group [(8.45±0.89)%, (9.88±0.50)% and (9.80±0.81)% respectively]. MTT assay further confirmed that pRNAi-siHO-1-GFP or vehicle alone did not induce apoptosis, so the possibilities that pRNAi-siHO-1-GFP or vehicle led to cell death were excluded. At the same time, the cells after DNR (7.0 μg/ml) treatment only or that combined with pRNAi-siHO-1-GFP were collected. Real-time PCR and western blot analysis were used to detect the mRNA and protein expressions of caspase-3 (cleaved-caspase-3), caspase-8 and caspase-9, which were 11.4-, 5.5- and 8.2-fold compared with those of DNR treatment only, respectively (Fig. 3C–E), suggesting that HO-1 siRNA promoted apoptosis through caspase activation.

In order to further discuss the role of HO-1 siRNA in enhancing apoptosis through caspases activation, various concentrations (5, 7.5, 10 and 12.5 μg/ml) of DNR were used to treat Kasumi-1 cells with/without pRNAi-siHO-1-GFP transfection. Western blot analysis was used to detect the expressions of HO-1 and apoptosis-related genes (cleaved-caspase-3, caspase-8 and caspase-9). Protein expressions of cleaved-caspase-3, caspase-8 and caspase-9 increased with rising concentration of DNR dose-dependently. After combined pRNAi-siHO-1-GFP and DNR treatment, the protein expression of HO-1 was lower than that of DNR treatment alone (Fig. 4A), while the expressions of apoptosis-related proteins were higher (Fig. 4C–E). Therefore, HO-1 siRNA was able to facilitate cell apoptosis through activation of caspase activation.

To further discuss the possible mechanisms involved in the activation of cleaved-caspase-3, caspase-8 and caspase-9, two classical pathways, i.e., endoplasmic reticulum apoptotic pathway and mitochondrial apoptotic pathway, were taken into consideration. Caspase-12, a characteristic protein mediating the endoplasmic reticulum apoptotic pathway, as well as Cyto C which was activated and released from mitochondria into cytoplasm in the mitochondrial apoptotic pathway. Nuclear poly(ADP-ribose) polymerase (PARP) is one of the main cleavage targets of caspase-3. Cleavage of PARP facilitates cellular disassembly as a marker of cell apoptosis. In this study, western blot analysis was used to determine the protein expression of caspase-12, cytoplasm-Cyto C and cleaved-PARP after treatment with 7.0 μg/ml DNR with/without HO-1 siRNA. The results indicated that HO-1 siRNA could promote the expression of caspase-12, cytoplasm-Cyto C and cleaved-PARP (Fig. 5) in Kasumi-1 cells when treated with DNR.

Ca²⁺ homeostasis is mainly maintained through endoplasmic reticulum to which most Ca²⁺ activities are related. The changes of intracellular [Ca²⁺] give rise to endoplasmic reticulum stress, while over-stress may lead to cell apoptosis (16). In addition, Ca²⁺ is also a key link of endoplasmic reticulum and mitochondrial function. Considerable Ca²⁺ releases due to endoplasmic reticulum stress are bound to be absorbed by mitochondria because of membrane potential gradient, and mitochondrial calcium overload is one of the known important apoptosis-inducing factors. On endoplasmic reticulum, the release of Ca²⁺ enhances the sensitivity of mitochondria, which changes the permeability and depolarizes mitochondrial membrane, thus resulting in cell apoptosis (17,18). Therefore, the activations of endoplasmic reticulum and mitochondrial apoptotic pathways can be reflected by intracellular Ca²⁺ concentration. Fluo3-AM is a fluorescent dye that can penetrate the cell membrane and can be cleaved in Fluo 3 form by esterase. Fluo 3 in combination with calcium ions can generate strong and detectable fluorescence. In this study, Fluo3-AM probe was used to detect Ca²⁺ accumulation of Kasumi-1 cells after different treatments. After combined treatment with pRNAi-GFP-siHO-1 and DNR (7.0 μg/ml) for 24 h, the intracellular Ca²⁺ accumulation was higher than those of the other groups (Fig. 6A). Besides, Ca²⁺ accumulation in Kasumi-1 cells increased with prolonged infection with pRNAi-siHO-1-GFP and peaked at 24 h (23.38%) (Fig. 6B).

During cell apoptosis, there is also a close relationship between intracellular Ca²⁺ accumulation and ROS generation, and the latter often precedes the former. On one hand, intracellular ROS production can activate the mitochondrial apoptotic pathway and augment the accumulation of intracellular Ca²⁺ simultaneously. On the other hand, the accumulation of intra-cellular Ca²⁺ can activate multiple signal pathways as a second messenger, thus increasing the generation of ROS. Excessive ROS leads to cell oxidative damage, thus promoting cell apoptosis (19,20). In our study, the increase of cytoplasmic Cyto C suggested that HO-1 siRNA facilitated cell apoptosis through the mitochondrial apoptotic pathway. To further explore the relationship between ROS generation and HO-1 siRNA-mediated cell apoptosis, DCFH-DA probe was used to detect the intracellular ROS generation of Kasumi-1 cells after different treatments. After combined treatment with pRNAi-GFP-siHO-1 and DNR (7.0 μg/ml) for 24 h, the ROS generation was higher than those of other groups (Fig. 7A). Besides, ROS generation in Kasumi-1 cells increased with prolonged infection with pRNAi-siHO-1-GFP and peaked at 24 h (21.90%) (Fig. 7B).

In this study, intracellular Ca²⁺ accumulation and ROS generation were involved in cell apoptosis mediated by HO-1 siRNA. Accumulation of these two substances might open the mitochondrial permeability transition pore (MPTP) (21), which reduced MTP and subsequently activated pro-apoptotic genes, thus inducing cell apoptosis (22). Reduction of MTP is an early event in activation of the mitochondrial apoptotic pathway (23). In order to evaluate the changes of MTP in HO-1 siRNA-mediated cell apoptosis, JC-1 probe was used to detect the green and red fluorescence intensities in Kasumi-1 cells. Compared with other groups, red fluorescence remarkably decreased in the group treated by DNR (7.0 μg/ml) together with pRNAi-siHO-1-GFP (p<0.05) (Table II and Fig. 8).

Intensity of red fluorescence was significantly lower in cells transfected by pRNAi-siHO-1-GFP and treated by DNR together than those in the cells undergoing pRNAi-siHO-1-GFP or DNR treatment alone.

HO-1 siRNA contributed to cell apoptosis through endoplasmic reticulum and mitochondrial apoptotic pathways. Activation of caspase-12 and Ca²⁺ accumulation were involved in the endoplasmic reticulum apoptotic pathway, while ROS generation, reduction of MTP and release of Cyto C were involved in activation of the mitochondrial apoptotic pathway. To elucidate the relationship between the two pathways in cell apoptosis mediated by HO-1 siRNA, antioxidant NAC or (and) Ca²⁺-chelating agent BAPTA-AM were used alone or in combination to pretreat Kasumi-1 cells infected by pRNAi-siHO-1-GFP, and then DNR (7.0 μg/ml) was added. Subsequently, Ca²⁺ accumulation, ROS generation and cell apoptosis were detected by FCM. Meanwhile, the protein expressions of caspase-12 and cytoplasm-Cyto C were determined by western blot analysis to discuss the influences of inhibited ROS generation or (and) Ca²⁺ accumulation on the endoplasmic reticulum and mitochondrial apoptotic pathways.

To further clarify the relationship between endoplasmic reticulum and mitochondrial apoptotic pathways in HO-1 siRNA-mediated apoptosis, Kasumi-1 cells were treated as follows: 1, Kasumi-1 cells were treated by DNR (7.0 μg/ml) alone (control group). 2, Kasumi-1 cells were treated with pRNAi-siHO-1-GFP plus DNR (7.0 μg/ml). 3, After pretreatment with 10 mmol/l NAC, Kasumi-1 cells were treated with pRNAi-siHO-1-GFP plus DNR (7.0 μg/ml). 4, After pretreatment with 10 μmol/l BAPTA-AM, Kasumi-1 cells were treated with pRNAi-siHO-1-GFP plus DNR (7.0 μg/ml). 5, After combined pretreatment with 10 μmol/l BAPTA-AM and 10 mmo/l NAC, Kasumi-1 cells were treated with pRNAi-siHO-1-GFP plus DNR (7.0 μg/ml). The protein expression of caspase-12 and cytoplasmic Cyto C after different treatments was detected by western blot analysis. The protein expressions of caspase-12 and cytoplasmic Cyto C were significantly reduced in Kasumi-1 cells pretreated with NAC or (and) BAPTA-AM than those of other groups (p<0.05) (Fig. 9).

In order to further explore the relationship between Ca²⁺ accumulation and ROS generation in cell apoptosis mediated by HO-1 siRNA, Kasumi-1 cells were treated as described above. Intracellular Ca²⁺ accumulation and ROS generation after different treatments were detected by FCM to unravel the relationship between them in Kasumi-1 cell apoptosis mediated by HO-1 siRNA. The control group was used to confirm the analysis parameters of FL2 channel on FCM. Both NAC and BAPTA-AM could effectively restrain the increases of intracellular Ca²⁺ and ROS caused by HO-1 siRNA (Fig. 9C and D).

NAC or (and) BAPTA-AM reduced cell apoptosis mediated by HO-1 siRNA. In order to confirm the influences of Ca²⁺ accumulation and ROS generation on the cell apoptosis mediated by HO-1 siRNA, apoptotic analysis was performed after cells were treated as described above. NAC and BAPTA-AM significantly alleviated HO-1 siRNA-mediated apoptosis (p<0.05) (Fig. 9E and F). Therefore, there may be potential Ca²⁺-ROS-interaction between endoplasmic reticulum and mitochondrial apoptotic pathways mediated by HO-1 siRNA to regulate cell apoptosis.