Tumor Biol. DOI 10.1007/s13277-014-2648-8
RESEARCH ARTICLE
GDC-0152 induces apoptosis through down-regulation of IAPs in human leukemia cells and inhibition of PI3K/Akt signaling pathway Rong Hu & Jia Li & Zhuogang Liu & Miao Miao & Kun Yao
Received: 7 July 2014 / Accepted: 18 September 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract The inhibitor of apoptosis proteins (IAPs) is closely related to leukemia apoptosis. The present study was undertaken to determine the molecular mechanisms by which GDC0152, an IAP inhibitor, induces apoptosis in human leukemia cells (K562 and HL60 cells). GDC-0152 inhibited the proliferation of K562 and HL60 cells in a dose- and time-dependent manner, which was largely attributed to intrinsic apoptosis. GDC-0152 down-regulated the IAPs including X-linked inhibitor of apoptosis protein (XIAP), cellular inhibitor of apoptosis protein-1 (cIAP1), and cellular inhibitor of apoptosis protein-2 (cIAP2) expression and induced the activation of caspase-9 and caspase-3. GDC-0152-induced cell proliferation inhibition in K562 cells was prevented by pan-caspase inhibitor. GDC-0152 also inhibited PI3K and Akt expression in K562 and HL60 cells. Taken together, these findings suggest that GDC-0152 results in human leukemia apoptosis through caspase-dependent mechanisms involving downregulation of IAPs and inhibition of PI3K/Akt signaling. Keywords GDC-0152 . IAPs . Leukemia . PI3K/Akt signaling pathway
Introduction Leukemia is one of the most aggressive hematologic malignancies. So far, except for acute promyelocytic leukemia and chronic myeloid leukemia, there are still no effective drugs for most of types of leukemia [1]. Multiple factors such as inhibitor of apoptosis proteins (IAPs) contribute to the infectivity of R. Hu : J. Li : Z. Liu (*) : M. Miao : K. Yao Department of Hematology, Shengjing Hospital, China Medical University, Shenyang 110004, China e-mail: [email protected]
treatment for leukemia. IAPs including X-linked inhibitor of apoptosis protein (XIAP), cellular inhibitor of apoptosis protein-1 (cIAP1), and cellular inhibitor of apoptosis protein-2 (cIAP2) are frequently overexpressed in leukemia cells, where they serve as regulators of leukemia cell survival and are often indicators of poor prognosis [2–4]. IAPs regulate apoptosis, in part, through the inhibition of activated caspases which are essential for apoptosis to occur [5, 6]. As the only cellular protein that has evolved to potently inhibit the enzymatic activity of mammalian caspases and promotes resistance to apoptosis [7], XIAP is also one of the negative factors which could promote proliferation of leukemia cells and promote resistance [4, 8]. The cIAP1 and cIAP2 also inhibit cell apoptosis in leukemia through binding caspases such as caspase-3 [9]. Given its important role in apoptosis and its frequently elevated expression in leukemia cells, IAPs have a great possibility to become a promising therapeutic target in leukemia. Some IAP inhibitors have been found to inhibit the proliferation and induce apoptosis of leukemia cells during past several years [8, 10]. The compound (S)-1-((S)-2-cyclohexyl-2-((S)2-(methylamino)propanamido)acetyl)-N-(4-phenyl-1,2,3thiadiazol-5-yl)pyrrolidine-2-carboxamide (GDC-0152) has been designed and synthesized as an IAP inhibitor [10, 11]. It is a potent pan inhibitor of IAP proteins, with binding affinities to the third baculoviral IAP repeat domains of XIAP, cIAP1, cIAP2, and the baculoviral IAP repeat domain of melanoma IAP in the low nanomolar range. There has been a considerable interest in the anti-tumor activities of GDC0152, and previous studies have shown that GDC-0152 induces growth inhibition and cell apoptosis in human solid tumors [10]. However, the effect of GDC-0152 on human leukemia cells has not been studied, and the precise mechanisms have not been elucidated. The present study was therefore undertaken to determine the effect of GDC-0152 on human leukemia cells and its molecular mechanisms. Our data
Tumor Biol.
demonstrated that GDC-0152 induces apoptosis of human leukemia cells through caspase-dependent mechanisms involving down-regulation of XIAP.
Materials and methods
staining buffer. The fluorescence intensity was analyzed with a FACScan flow cytometry. A drop of K562 cells was dropped on the slide and covered by a cover glass, then was observed under fluorescence microscope (single filter, ×40). The cells with high MMP are greenish yellow and the cells with low MMP are green.
Main reagents
Western blot analysis
Cell Counting Kit-8 (CCK-8) and Annexin V-FITC/ Propidium Iodide (PI) were purchased from KeyGen Biotech (China), JC-1 was purchased from Beyotime (China), and Z-VAD-FMK, GDC-0152, and antibodies were purchased from Sigma-Aldrich (USA).
Western blot analysis was determined as previously [8]. Cells were harvested at various times after GDC-0152 treatment. Media were aspirated from cultures. The cells were washed twice with 1× PBS and drained thoroughly. The cells were then lysed by adding 1× SDS gel-loading buffer (100 μl per well of a 6-well plate). The lysate was immediately scraped into a microfuge tube and kept on ice. The lysate was sonicated for 15 s to shear DNA and reduce ample viscosity. Cell debris was removed by centrifugation at 10,000g for 10 min at 4 °C. The resulting supernatants were resolved on a 10 % SDS-PAGE under denatured reducing conditions and transferred to nitrocellulose membranes. The membranes were blocked with 5 % nonfat dried milk at room temperature for 30 min and incubated with different primary antibodies. The membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies. The membranes were incubated with 10 ml LumiGLO with gentle agitation for 1 min at room temperature. A membrane of excess developing solution was drained, not let to dry, wrapped in a plastic wrap, and exposed to X-ray film. An initial 10-s exposure should indicate the proper exposure time. The signals of Western blot results were quantified by ImageJ software.
Cell culture K562 (chronic myeloid leukemia cell line) and HL60 (acute promyelocytic leukemia cell line) cells were kept in our laboratory. The cells were incubated in RPMI 1640 media, 10 % fetal bovine serum, and 1 % penicillin-streptomycin at 37 °C in humidified environment of 5 % CO2 and subcultured every 3–4 days to maintain exponential growth. Cells were seeded in 24-well plates at a density of 5×105 viable cells/well in 500 μl growth medium and incubated for 24 h. Measurement of cell viability, proliferation, and cell apoptosis Cell proliferation in vitro was determined using the CCK-8 as previously described [12]. After exposure to GDC-0152 of various concentrations (12.5, 25, 50, and 100 ng/ml) for various times (12, 24, and 48 h), 10 μl CCK-8 solution was added to each well with 100 μl cells. The cells were then incubated for 2 h at 37 °C. The absorbance of each well was measured at 450 nm with ELISA reader. The results were duplicated by four times. Data were expressed as a percentage of control measured in the absence of GDC-0152. Apoptotic cells were evaluated and quantified by the Annexin V-FITC/PI staining and flow cytometry. The 5×105 cells in 500 μl PBS were stained with 5 μl annexin V-FITC and 5 μl PI at room temperature for 10 min. The cells were then analyzed by flow cytometry with 488 nm for excitation. Apoptotic cells were counted as Annexin V-positive and PInegative cells. Measurement of mitochondrial membrane potential (MMP) The fluorescent probe, JC-1, was employed to measure the MMP of K562 cells. Briefly, after being exposed to GDC0152 of 25 ng/ml for 24 h, K562 cells were cultured in 24-well plates and incubated with JC-1 staining solution (5 μg/ml) for 20 min at 37 °C. Cells were then rinsed twice with JC-1
Statistical analysis The data are expressed as means±SEM and the difference between two groups was evaluated using Student’s t test. A probability level of 0.05 is used to establish significance and indicated as P
RESEARCH ARTICLE
GDC-0152 induces apoptosis through down-regulation of IAPs in human leukemia cells and inhibition of PI3K/Akt signaling pathway Rong Hu & Jia Li & Zhuogang Liu & Miao Miao & Kun Yao
Received: 7 July 2014 / Accepted: 18 September 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract The inhibitor of apoptosis proteins (IAPs) is closely related to leukemia apoptosis. The present study was undertaken to determine the molecular mechanisms by which GDC0152, an IAP inhibitor, induces apoptosis in human leukemia cells (K562 and HL60 cells). GDC-0152 inhibited the proliferation of K562 and HL60 cells in a dose- and time-dependent manner, which was largely attributed to intrinsic apoptosis. GDC-0152 down-regulated the IAPs including X-linked inhibitor of apoptosis protein (XIAP), cellular inhibitor of apoptosis protein-1 (cIAP1), and cellular inhibitor of apoptosis protein-2 (cIAP2) expression and induced the activation of caspase-9 and caspase-3. GDC-0152-induced cell proliferation inhibition in K562 cells was prevented by pan-caspase inhibitor. GDC-0152 also inhibited PI3K and Akt expression in K562 and HL60 cells. Taken together, these findings suggest that GDC-0152 results in human leukemia apoptosis through caspase-dependent mechanisms involving downregulation of IAPs and inhibition of PI3K/Akt signaling. Keywords GDC-0152 . IAPs . Leukemia . PI3K/Akt signaling pathway
Introduction Leukemia is one of the most aggressive hematologic malignancies. So far, except for acute promyelocytic leukemia and chronic myeloid leukemia, there are still no effective drugs for most of types of leukemia [1]. Multiple factors such as inhibitor of apoptosis proteins (IAPs) contribute to the infectivity of R. Hu : J. Li : Z. Liu (*) : M. Miao : K. Yao Department of Hematology, Shengjing Hospital, China Medical University, Shenyang 110004, China e-mail: [email protected]
treatment for leukemia. IAPs including X-linked inhibitor of apoptosis protein (XIAP), cellular inhibitor of apoptosis protein-1 (cIAP1), and cellular inhibitor of apoptosis protein-2 (cIAP2) are frequently overexpressed in leukemia cells, where they serve as regulators of leukemia cell survival and are often indicators of poor prognosis [2–4]. IAPs regulate apoptosis, in part, through the inhibition of activated caspases which are essential for apoptosis to occur [5, 6]. As the only cellular protein that has evolved to potently inhibit the enzymatic activity of mammalian caspases and promotes resistance to apoptosis [7], XIAP is also one of the negative factors which could promote proliferation of leukemia cells and promote resistance [4, 8]. The cIAP1 and cIAP2 also inhibit cell apoptosis in leukemia through binding caspases such as caspase-3 [9]. Given its important role in apoptosis and its frequently elevated expression in leukemia cells, IAPs have a great possibility to become a promising therapeutic target in leukemia. Some IAP inhibitors have been found to inhibit the proliferation and induce apoptosis of leukemia cells during past several years [8, 10]. The compound (S)-1-((S)-2-cyclohexyl-2-((S)2-(methylamino)propanamido)acetyl)-N-(4-phenyl-1,2,3thiadiazol-5-yl)pyrrolidine-2-carboxamide (GDC-0152) has been designed and synthesized as an IAP inhibitor [10, 11]. It is a potent pan inhibitor of IAP proteins, with binding affinities to the third baculoviral IAP repeat domains of XIAP, cIAP1, cIAP2, and the baculoviral IAP repeat domain of melanoma IAP in the low nanomolar range. There has been a considerable interest in the anti-tumor activities of GDC0152, and previous studies have shown that GDC-0152 induces growth inhibition and cell apoptosis in human solid tumors [10]. However, the effect of GDC-0152 on human leukemia cells has not been studied, and the precise mechanisms have not been elucidated. The present study was therefore undertaken to determine the effect of GDC-0152 on human leukemia cells and its molecular mechanisms. Our data
Tumor Biol.
demonstrated that GDC-0152 induces apoptosis of human leukemia cells through caspase-dependent mechanisms involving down-regulation of XIAP.
Materials and methods
staining buffer. The fluorescence intensity was analyzed with a FACScan flow cytometry. A drop of K562 cells was dropped on the slide and covered by a cover glass, then was observed under fluorescence microscope (single filter, ×40). The cells with high MMP are greenish yellow and the cells with low MMP are green.
Main reagents
Western blot analysis
Cell Counting Kit-8 (CCK-8) and Annexin V-FITC/ Propidium Iodide (PI) were purchased from KeyGen Biotech (China), JC-1 was purchased from Beyotime (China), and Z-VAD-FMK, GDC-0152, and antibodies were purchased from Sigma-Aldrich (USA).
Western blot analysis was determined as previously [8]. Cells were harvested at various times after GDC-0152 treatment. Media were aspirated from cultures. The cells were washed twice with 1× PBS and drained thoroughly. The cells were then lysed by adding 1× SDS gel-loading buffer (100 μl per well of a 6-well plate). The lysate was immediately scraped into a microfuge tube and kept on ice. The lysate was sonicated for 15 s to shear DNA and reduce ample viscosity. Cell debris was removed by centrifugation at 10,000g for 10 min at 4 °C. The resulting supernatants were resolved on a 10 % SDS-PAGE under denatured reducing conditions and transferred to nitrocellulose membranes. The membranes were blocked with 5 % nonfat dried milk at room temperature for 30 min and incubated with different primary antibodies. The membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies. The membranes were incubated with 10 ml LumiGLO with gentle agitation for 1 min at room temperature. A membrane of excess developing solution was drained, not let to dry, wrapped in a plastic wrap, and exposed to X-ray film. An initial 10-s exposure should indicate the proper exposure time. The signals of Western blot results were quantified by ImageJ software.
Cell culture K562 (chronic myeloid leukemia cell line) and HL60 (acute promyelocytic leukemia cell line) cells were kept in our laboratory. The cells were incubated in RPMI 1640 media, 10 % fetal bovine serum, and 1 % penicillin-streptomycin at 37 °C in humidified environment of 5 % CO2 and subcultured every 3–4 days to maintain exponential growth. Cells were seeded in 24-well plates at a density of 5×105 viable cells/well in 500 μl growth medium and incubated for 24 h. Measurement of cell viability, proliferation, and cell apoptosis Cell proliferation in vitro was determined using the CCK-8 as previously described [12]. After exposure to GDC-0152 of various concentrations (12.5, 25, 50, and 100 ng/ml) for various times (12, 24, and 48 h), 10 μl CCK-8 solution was added to each well with 100 μl cells. The cells were then incubated for 2 h at 37 °C. The absorbance of each well was measured at 450 nm with ELISA reader. The results were duplicated by four times. Data were expressed as a percentage of control measured in the absence of GDC-0152. Apoptotic cells were evaluated and quantified by the Annexin V-FITC/PI staining and flow cytometry. The 5×105 cells in 500 μl PBS were stained with 5 μl annexin V-FITC and 5 μl PI at room temperature for 10 min. The cells were then analyzed by flow cytometry with 488 nm for excitation. Apoptotic cells were counted as Annexin V-positive and PInegative cells. Measurement of mitochondrial membrane potential (MMP) The fluorescent probe, JC-1, was employed to measure the MMP of K562 cells. Briefly, after being exposed to GDC0152 of 25 ng/ml for 24 h, K562 cells were cultured in 24-well plates and incubated with JC-1 staining solution (5 μg/ml) for 20 min at 37 °C. Cells were then rinsed twice with JC-1
Statistical analysis The data are expressed as means±SEM and the difference between two groups was evaluated using Student’s t test. A probability level of 0.05 is used to establish significance and indicated as P
