Tumor Biol. DOI 10.1007/s13277-015-3642-5
RESEARCH ARTICLE
Targeting DNA-PKcs increased anticancer drug sensitivity by suppressing DNA damage repair in osteosarcoma cell line MG63 Xin Li 1 & Jiguang Tian 2 & Qiyu Bo 3 & Ka Li 1 & Hongliang Wang 1 & Ting Liu 4 & Jianmin Li 1
Received: 31 March 2015 / Accepted: 3 June 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015
Abstract Many chemotherapy drugs exert anticancer effects through causing DNA damage, such as DNA topoisomerase inhibitor and platinum-containing drugs. DNA damage repair is an important mechanism of drug resistance which is responsible for metastasis and recurrence after chemotherapy. DNAdependent protein kinase (DNA-PK) plays an important role in non-homology end joining (NHEJ) pathway. In this study, we aimed to determine whether DNA-PK catalytic subunit (DNA-PKcs) is expressed in osteosarcoma MG63 cell line and involved in drug resistance induced by DNA repair. We found that DNA-PKcs was expressed in osteosarcoma cell line MG63. The pDNA-PKcsT2609 was more expressed in cells treated with cisplatin (DDP) and etoposide (VP16). Down-regulation of DNA-PKcs produced higher sensitivity of MG63 cells to DDP or VP16 through increasing apoptosis and causing cell cycle arrest in the G1 phase. Our study supported that DNA-PKcs was involved in drug-induced DNA damage repair and related to chemosensitivity of osteosarcoma MG63 cells.
Xin Li and Jiguang Tian contributed equally to this work. * Jianmin Li [email protected] 1
Department of Orthopedics, Qilu Hospital, Shandong University, Shandong, China
2
Emergency Department, Qilu Hospital, Shandong University, Shandong, China
3
Operating room of Qilu Hospital, Shandong University, Shandong, China
4
Department of Obstetrics and Gynecology, Qilu Hospital, Shandong University, Shandong, China
Keywords DNA-PKcs . Osteosarcoma . DNA damage repair . Drug resistance
Introduction Osteosarcoma is one of the most common malignant bone tumors in adolescents. The most effective treatment is aggressive surgery combined with intensified chemotherapy. However, the survival rates have remained at 50– 80 % since the 1970s [1]. Drug resistance is responsible for chemotherapy failure. Some of the anticancer drugs such as cisplatin (cis-diammindichloridoplatin (DDP)) or etoposide (VP16) induce DNA damage and eventually cell death. DNA-dependent protein kinase (DNA-PK) is a serine/threonine protein kinase and expressed in most mammalian cells [2]. DNA-PK is the important component of non-homology end joining (NHEJ) repair, the main repair pathway of DNA double-strand breaks (DSBs). Many studies showed that cells deficient in DNA-PK exhibit hypersensitivity to radio/chemotherapy [3–6]. In this study, we aimed to investigate the expression of DNA-PK catalytic subunit (DNA-PKcs) in osteosarcoma cell line MG63 and determine the changes of sensitivity to DDP or VP16 by targeting DNA-PKcs. We found that the DSBs marker γH2AX was more expressed after pDNAPKcs T2609 expression was inhibited. The sensitivity of MG63 cells to DDP or VP16 was increased when DNAPKcs was down-regulated. Moreover, down-regulation of DNA-PKcs increased apoptosis and cell cycle arrest in osteosarcoma cells treated with anticancer drugs. Our study may provide new ideas for the targeted treatment of osteosarcoma in the future.
Tumor Biol.
Results DNA-PKcs was expressed in osteosarcoma cell line MG63 and involved in DNA damage repair Osteosarcoma cell line MG63 was treated with 3 μg/ml DDP or 4 μg/ml VP16 for 6 h and then was incubated with the normal medium for 10∼20 h for DNA repair. Then, pDNA-PKcsT2609 and γH2AX were detected with confocal microscopy. As a result, pDNA-PKcsT2609 and γH2AX were expressed in a lower level in untreated cells (upper panel, Fig. 1). Ten hours after treatment of chemotherapeutic drugs, the expression of pDNA-PKcsT2609 was dramatically increased and the expression of γH2AX was also increased relatively, but γH2AX was expressed at a low level after another 10 h (Fig. 1). However, after cells were co-incubated with the DNA-PK inhibitor NU7026 at a concentration of 10 μM, pDNA-PKcsT2609 was expressed in a lower level at the 16th hour (6+ 10 h), and γH2AX was expressed in a similar level at the 16th hour (6+10 h) compared with cells incubated without NU7026. But, γH2AX was expressed at a higher level at the 26th hour (6+20 h) (Fig. 1). Additionally, translocation of pDNA-PKcsT2609 from the cytoplasm to nucleus was observed (Fig. 1a, left window). This result indicates that DNA-PKcs is expressed in osteosarcoma MG63 cells and plays an important role in DNA repair.
Fig. 1 DNA-PK was involved in DNA damage repair in MG63 cells. a, b Expression of pDNA-PKcsT2609 and γH2AX in untreated cells (upper panel). MG63 cells were incubated with DDP (3 μg/ml) or VP16 (4 μg/ml) for 6 h and then without drug for 10 or 20 h. Expression of pDNA-PKcsT2609 in cells with NU7026 co-incubated at the 16th (6+10)
Down-regulation of DNA-PKcs made osteosarcoma MG63 cells more sensitive to chemotherapeutic drugs Cells were treated with DDP or VP16 for 48 h in the blank control group, and half maximal inhibitory concentration (IC50) was determined using MTT assay. Meanwhile, DNAPKcs was down-regulated using small interfering RNAs (no 1451 or no 3228, experimental group) or scrambled RNA (negative control group); then, cells were treated with DDP or VP16 at different concentrations; after 48 h, IC50 was determined. After cells were treated with DDP or VP16, IC50 of experimental groups was significantly lower than that of the blank control group or negative control group (p < 0.05) (Fig. 2, Table 1). Down-regulation of DNA-PKcs increased the sensitivity of MG63 osteosarcoma cells to chemotherapeutic drugs. Down-regulation of DNA-PKcs led to increased apoptosis rate induced by chemotherapeutic drugs Cells were transfected with small interfering RNA (siRNAs) (no 1451 or no 3228) (experimental groups) or scrambled RNA (negative control group). After 24 h, normal cells (blank control group) and transfected cells were treated with DDP at 1.5 μg/ml or VP16 at 2 μg/ml for 48 h. We detected the apoptosis rate and the expression of caspase-3/caspase-10. After cells were treated with DDP (Fig. 3a), apoptosis rate
hour (a, bottom panel) or without NU7026 co-incubated (a, middle panel). γH2AX expression in cells with (b, third and fifth panels) or without (b, second and fourth panels) NU7026 co-incubated at the 16th (6+10) hour and 26th (6+20) hour
Tumor Biol.
Fig. 2 Down-regulation of DNA-PKcs led to higher sensitivity of MG63 cells to chemotherapeutic drugs. a Cells were treated with DDP at concentrations of 0, 1, 2, 3, 4, and 5 μg/ml. The MTT assay was performed and IC50 was determined. b Cells were treated with VP16 at
concentrations of 0, 0.75, 1.5, 2.25, 3, 3.75, 4.5, 5.25, and 6 μg/ml. The MTT assay was performed and IC50 was determined. *p
RESEARCH ARTICLE
Targeting DNA-PKcs increased anticancer drug sensitivity by suppressing DNA damage repair in osteosarcoma cell line MG63 Xin Li 1 & Jiguang Tian 2 & Qiyu Bo 3 & Ka Li 1 & Hongliang Wang 1 & Ting Liu 4 & Jianmin Li 1
Received: 31 March 2015 / Accepted: 3 June 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015
Abstract Many chemotherapy drugs exert anticancer effects through causing DNA damage, such as DNA topoisomerase inhibitor and platinum-containing drugs. DNA damage repair is an important mechanism of drug resistance which is responsible for metastasis and recurrence after chemotherapy. DNAdependent protein kinase (DNA-PK) plays an important role in non-homology end joining (NHEJ) pathway. In this study, we aimed to determine whether DNA-PK catalytic subunit (DNA-PKcs) is expressed in osteosarcoma MG63 cell line and involved in drug resistance induced by DNA repair. We found that DNA-PKcs was expressed in osteosarcoma cell line MG63. The pDNA-PKcsT2609 was more expressed in cells treated with cisplatin (DDP) and etoposide (VP16). Down-regulation of DNA-PKcs produced higher sensitivity of MG63 cells to DDP or VP16 through increasing apoptosis and causing cell cycle arrest in the G1 phase. Our study supported that DNA-PKcs was involved in drug-induced DNA damage repair and related to chemosensitivity of osteosarcoma MG63 cells.
Xin Li and Jiguang Tian contributed equally to this work. * Jianmin Li [email protected] 1
Department of Orthopedics, Qilu Hospital, Shandong University, Shandong, China
2
Emergency Department, Qilu Hospital, Shandong University, Shandong, China
3
Operating room of Qilu Hospital, Shandong University, Shandong, China
4
Department of Obstetrics and Gynecology, Qilu Hospital, Shandong University, Shandong, China
Keywords DNA-PKcs . Osteosarcoma . DNA damage repair . Drug resistance
Introduction Osteosarcoma is one of the most common malignant bone tumors in adolescents. The most effective treatment is aggressive surgery combined with intensified chemotherapy. However, the survival rates have remained at 50– 80 % since the 1970s [1]. Drug resistance is responsible for chemotherapy failure. Some of the anticancer drugs such as cisplatin (cis-diammindichloridoplatin (DDP)) or etoposide (VP16) induce DNA damage and eventually cell death. DNA-dependent protein kinase (DNA-PK) is a serine/threonine protein kinase and expressed in most mammalian cells [2]. DNA-PK is the important component of non-homology end joining (NHEJ) repair, the main repair pathway of DNA double-strand breaks (DSBs). Many studies showed that cells deficient in DNA-PK exhibit hypersensitivity to radio/chemotherapy [3–6]. In this study, we aimed to investigate the expression of DNA-PK catalytic subunit (DNA-PKcs) in osteosarcoma cell line MG63 and determine the changes of sensitivity to DDP or VP16 by targeting DNA-PKcs. We found that the DSBs marker γH2AX was more expressed after pDNAPKcs T2609 expression was inhibited. The sensitivity of MG63 cells to DDP or VP16 was increased when DNAPKcs was down-regulated. Moreover, down-regulation of DNA-PKcs increased apoptosis and cell cycle arrest in osteosarcoma cells treated with anticancer drugs. Our study may provide new ideas for the targeted treatment of osteosarcoma in the future.
Tumor Biol.
Results DNA-PKcs was expressed in osteosarcoma cell line MG63 and involved in DNA damage repair Osteosarcoma cell line MG63 was treated with 3 μg/ml DDP or 4 μg/ml VP16 for 6 h and then was incubated with the normal medium for 10∼20 h for DNA repair. Then, pDNA-PKcsT2609 and γH2AX were detected with confocal microscopy. As a result, pDNA-PKcsT2609 and γH2AX were expressed in a lower level in untreated cells (upper panel, Fig. 1). Ten hours after treatment of chemotherapeutic drugs, the expression of pDNA-PKcsT2609 was dramatically increased and the expression of γH2AX was also increased relatively, but γH2AX was expressed at a low level after another 10 h (Fig. 1). However, after cells were co-incubated with the DNA-PK inhibitor NU7026 at a concentration of 10 μM, pDNA-PKcsT2609 was expressed in a lower level at the 16th hour (6+ 10 h), and γH2AX was expressed in a similar level at the 16th hour (6+10 h) compared with cells incubated without NU7026. But, γH2AX was expressed at a higher level at the 26th hour (6+20 h) (Fig. 1). Additionally, translocation of pDNA-PKcsT2609 from the cytoplasm to nucleus was observed (Fig. 1a, left window). This result indicates that DNA-PKcs is expressed in osteosarcoma MG63 cells and plays an important role in DNA repair.
Fig. 1 DNA-PK was involved in DNA damage repair in MG63 cells. a, b Expression of pDNA-PKcsT2609 and γH2AX in untreated cells (upper panel). MG63 cells were incubated with DDP (3 μg/ml) or VP16 (4 μg/ml) for 6 h and then without drug for 10 or 20 h. Expression of pDNA-PKcsT2609 in cells with NU7026 co-incubated at the 16th (6+10)
Down-regulation of DNA-PKcs made osteosarcoma MG63 cells more sensitive to chemotherapeutic drugs Cells were treated with DDP or VP16 for 48 h in the blank control group, and half maximal inhibitory concentration (IC50) was determined using MTT assay. Meanwhile, DNAPKcs was down-regulated using small interfering RNAs (no 1451 or no 3228, experimental group) or scrambled RNA (negative control group); then, cells were treated with DDP or VP16 at different concentrations; after 48 h, IC50 was determined. After cells were treated with DDP or VP16, IC50 of experimental groups was significantly lower than that of the blank control group or negative control group (p < 0.05) (Fig. 2, Table 1). Down-regulation of DNA-PKcs increased the sensitivity of MG63 osteosarcoma cells to chemotherapeutic drugs. Down-regulation of DNA-PKcs led to increased apoptosis rate induced by chemotherapeutic drugs Cells were transfected with small interfering RNA (siRNAs) (no 1451 or no 3228) (experimental groups) or scrambled RNA (negative control group). After 24 h, normal cells (blank control group) and transfected cells were treated with DDP at 1.5 μg/ml or VP16 at 2 μg/ml for 48 h. We detected the apoptosis rate and the expression of caspase-3/caspase-10. After cells were treated with DDP (Fig. 3a), apoptosis rate
hour (a, bottom panel) or without NU7026 co-incubated (a, middle panel). γH2AX expression in cells with (b, third and fifth panels) or without (b, second and fourth panels) NU7026 co-incubated at the 16th (6+10) hour and 26th (6+20) hour
Tumor Biol.
Fig. 2 Down-regulation of DNA-PKcs led to higher sensitivity of MG63 cells to chemotherapeutic drugs. a Cells were treated with DDP at concentrations of 0, 1, 2, 3, 4, and 5 μg/ml. The MTT assay was performed and IC50 was determined. b Cells were treated with VP16 at
concentrations of 0, 0.75, 1.5, 2.25, 3, 3.75, 4.5, 5.25, and 6 μg/ml. The MTT assay was performed and IC50 was determined. *p
