APOPTOSIS IN HUMAN OSTEOSARCOMA CELL LINE MG63 INDUCED BY PYROPHEOPHORBIDE-a METHYL ESTER-MEDIATED PHOTODYNAMIC THERAPY_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

TAOYong , HUANGQiu ,  OUYunsheng , YINHang , CHENYanyang , TUPinghua

Department of Orthopedics, the First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, P.R.China;

Corresponding author：

OUYunsheng, Email: ouyunsheng2001@163.com

Keywords：

Pyropheophorbide-a methyl ester; Photodynamic therapy; Osteosarcoma; Apoptosis

DOI：

10.7507/1002-1892.20160136

Video：

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Objective To explore the effect of pyropheophorbide-a methyl ester-mediated photodynamic therapy (MPPa-PDT) on the apoptosis in human osteosarcoma cell line MG63 and the underlying mechanism. Methods Human osteosarcoma MG63 cells in logarithmic growth phase were divided into 4 groups: blank control group (control group), the MPPa treatment group (MPPa group), the light irradiation group (LED group), and MPPa-PDT treatment group (MPPa-PDT group). MPPa-PDT group and MPPa group were incubated with MPPa (0.75 μmol/ L) for 20 hours in dark condition; control group and LED group were incubated with equal volume of fresh medium for 20 hours in the same condition. After washing with PBS and replacement with fresh culture medium, LED group and MPPa-PDT group cells were exposed to light (4.8 J/cm²) for 120 seconds. After light exposure, all groups were cultured in dark condition again. Then cellular morphology changes were observed by an inverted phase contrast microscopy, endoplasmic reticulum morphology changes were observed by transmission electron microscopy, cellular apoptosis was detected by Hoechst33258 nuclear staining, cell apoptotic rate and the levels of Ca in cells were analyzed by flow cytometry, the expression of p-PERK, C/EBP homologous protein (CHOP), cleaved-Caspase-12 were assayed by Western blot. Results In MPPa-PDT group, the retracted and round cells were observed; Hoechst33258 nuclear staining showed nuclear condensation, fragmentation, and other typical apoptotic morphological changes; the cell apoptotic rate (48.76%±3.54%) was significantly higher than that of control group (5.04%±0.41%), MPPa group (5.33%±0.38%), and LED group (6.48%±0.46%) (P < 0.05); the levels of Ca²⁺ in cells (485.29±58.77) was also significantly higher than that of control group (97.24±4.77), MPPa group (97.95±6.30), and LED group (101.17±5.26) (P < 0.05); swelling endoplasmic reticulum was observed under transmission electron microscope; the expressions of p-PERK, CHOP, and cleaved-Caspase-12 gradually increased at 1, 3, and 6 hours after treatment respectively, which were significantly higher than those of the other groups (P < 0.05). There was no typical apoptotic morphological changes and endoplasmic reticulum morphological changes in control group, MPPa group, and LED group, and there was no significant difference in the above indexes among 3 groups (P > 0.05). Conclusion MPPa-PDT can significantly induce apoptosis in MG63 cells. The endoplasmic reticulum stress pathway is involved in the MPPa-PDT induced apoptosis.

Citation： TAOYong, HUANGQiu, OUYunsheng, YINHang, CHENYanyang, TUPinghua. APOPTOSIS IN HUMAN OSTEOSARCOMA CELL LINE MG63 INDUCED BY PYROPHEOPHORBIDE-a METHYL ESTER-MEDIATED PHOTODYNAMIC THERAPY. Chinese Journal of Reparative and Reconstructive Surgery, 2016, 30(6): 669-674. doi: 10.7507/1002-1892.20160136 Copy

1.	姚伟涛, 蔡启卿, 高嵩涛, 等. 骨骺牵开保骺术在儿童股骨骨肉瘤保肢术中的应用. 中国修复重建外科杂志, 2013, 27(4): 423-427.
2.	Wu Y, Zhao J, He M. Correlation between TGF-β1 gene 29 T > C single nucleotide polymorphism and clinicopathological characteristics of osteosarcoma. Tumor Biol, 2015, 36(7): 5149-5156.
3.	李黎波, 李文敏, 项蕾红, 等. 光动力疗法在中国的应用与临床研究. 中国激光医学杂志, 2012, 21(5): 278-307.
4.	Kidane B, Hirpara D, Yasufuku K. Photodynamic therapy in non-gastrointestinal thoracic malignancies. Int J Mol Sci, 2016, 17(1): 749-760.
5.	Kozarek RA. Photodynamic therapy in esophageal cancer. Gastrointestinal Intervention, 2015, 4(2): 95-98.
6.	Savoia P, Deboli T, Previgliano A, et al. Usefulness of photodynamic therapy as a possible therapeutic alternative in the treatment of basal cell carcinoma. Int J Mol Sci, 2015, 16(10): 23300-23317.
7.	Tu P, Huang Q, Ou Y, et al. Aloe-emodin-mediated photodynamic therapy induces autophagy and apoptosis in human osteosarcoma cell line MG-63 through the ROS/JNK signaling pathway. Oncology Reports, 2016, 35(6): 3209-3215.
8.	廖明德, 赵劲民, 陈石海, 等. 皮肤毛细血管瘤模型鸡冠的激光和光动力学治疗研究. 中国修复重建外科杂志, 2008, 22(12): 1437-1440.
9.	Zheng Y, Yin G, Le V, et al. Photodynamic-therapy activates immune response by disrupting immunity homeostasis of tumor cells, which generates vaccine for cancer therapy. Int J Biol Sci, 2016, 12(1): 120-132.
10.	李晓龙, 刘斌. 光敏剂研究进展. 中国激光医学杂志, 2015, 24(6): 327-333.
11.	Li KT, Zhang J, Duan QQ, et al. Apoptosis triggered by pyropheophorbide-α methyl ester-mediated photodynamic therapy in a giant cell tumor in bone. Laser Physics, 2014, 24(6): 066002.
12.	黄秋, 涂平华, 欧云生, 等. MPPa-PDT诱导人骨巨细胞瘤细胞自噬. 第三军医大学学报, 2015, 37(17): 1755-1760.
13.	Tian YY, Hu XY, Leung WN, et al. Investigation of photodynamic effect caused by MPPa-PDT on breast cancer. Laser Physics Letters, 2012, 9(10): 754-758.
14.	Tian Y, Leung W, Yue K, et al. Cell death induced by MPPa-PDT in prostate carcinoma in vitro and in vivo. Biochem Biophys Res Commun, 2006, 348(2): 413-420.
15.	Tan Y, Xu CS, Xia XS, et al. Preliminary studies on LED-activated pyropheophorbide-a methyl ester killing cisplatin-resistant ovarian carcinoma cells. Laser Physics, 2009, 19(5): 1045-1049.
16.	Li W, Tan G, Cheng J, et al. A novel photosensitizer 31,131-pheny-lhydrazine-Mppa (BPHM) and its in vitro photodynamic therapy against HeLa Cells. Molecules, 2016, 21(5): 558-570.
17.	Huang Q, Ou YS, Tao Y, et al. Apoptosis and autophagy induced by pyropheophorbide-α methyl ester-mediated photodynamic therapy in human osteosarcoma MG-63 cells. Apoptosis, 2016, 21(6): 749-760.
18.	李悦, 曹良启, 彭和平, 等. 腹腔镜联合光动力治疗晚期胰腺癌的近期临床疗效分析. 临床医学工程, 2011, 18(4): 514-516.
19.	秦怀海, 刘智, 张楠, 等. 显微手术联合光动力治疗恶性脑胶质瘤. 中华神经外科杂志, 2008, 24(3): 200-201.
20.	蔡郑东, 胡硕, 龚海洋, 等. PSD-007介导的光动力疗法治疗小鼠骨肉瘤的实验研究. 中华骨科杂志, 2011, 31(6): 692-698.
21.	Munshi S, Dahl R. Cytoprotective small molecule modulators of endoplasmic reticulum stress. Bioorganic & Medicinal Chemistry, 2016, 24(11): 2382-2388.
22.	Verfaillie T, Garg AD, Agostinis P. Targeting ER stress induced apoptosis and inflammation in cancer. Cancer Lett, 2013, 332(2): 249-264.
23.	Sidhu A, Miller JR, Tripathi A, et al. Borrelidin induces the unfolded protein response in oral cancer cells and Chop-dependent apoptosis. ACS Med Chem Lett, 2015, 6(11): 1122-1127.
24.	Xu S, Zhao X, Zhao Q, et al. Wogonin prevents rat dorsal root ganglion neurons death via inhibiting tunicamycin-induced ER stress in vitro. Cell Mol Neurobiol, 2015, 35(3): 389-398.
25.	Buytaert E, Dewaele M, Agostinis P. Molecular effectors of multiple cell death pathways initiated by photodynamic therapy. Biochim Biophys Acta, 2007, 1776(1): 86-107.

1. 姚伟涛, 蔡启卿, 高嵩涛, 等. 骨骺牵开保骺术在儿童股骨骨肉瘤保肢术中的应用. 中国修复重建外科杂志, 2013, 27(4): 423-427.
2. Wu Y, Zhao J, He M. Correlation between TGF-β1 gene 29 T > C single nucleotide polymorphism and clinicopathological characteristics of osteosarcoma. Tumor Biol, 2015, 36(7): 5149-5156.
3. 李黎波, 李文敏, 项蕾红, 等. 光动力疗法在中国的应用与临床研究. 中国激光医学杂志, 2012, 21(5): 278-307.
4. Kidane B, Hirpara D, Yasufuku K. Photodynamic therapy in non-gastrointestinal thoracic malignancies. Int J Mol Sci, 2016, 17(1): 749-760.
5. Kozarek RA. Photodynamic therapy in esophageal cancer. Gastrointestinal Intervention, 2015, 4(2): 95-98.
6. Savoia P, Deboli T, Previgliano A, et al. Usefulness of photodynamic therapy as a possible therapeutic alternative in the treatment of basal cell carcinoma. Int J Mol Sci, 2015, 16(10): 23300-23317.
7. Tu P, Huang Q, Ou Y, et al. Aloe-emodin-mediated photodynamic therapy induces autophagy and apoptosis in human osteosarcoma cell line MG-63 through the ROS/JNK signaling pathway. Oncology Reports, 2016, 35(6): 3209-3215.
8. 廖明德, 赵劲民, 陈石海, 等. 皮肤毛细血管瘤模型鸡冠的激光和光动力学治疗研究. 中国修复重建外科杂志, 2008, 22(12): 1437-1440.
9. Zheng Y, Yin G, Le V, et al. Photodynamic-therapy activates immune response by disrupting immunity homeostasis of tumor cells, which generates vaccine for cancer therapy. Int J Biol Sci, 2016, 12(1): 120-132.
10. 李晓龙, 刘斌. 光敏剂研究进展. 中国激光医学杂志, 2015, 24(6): 327-333.
11. Li KT, Zhang J, Duan QQ, et al. Apoptosis triggered by pyropheophorbide-α methyl ester-mediated photodynamic therapy in a giant cell tumor in bone. Laser Physics, 2014, 24(6): 066002.
12. 黄秋, 涂平华, 欧云生, 等. MPPa-PDT诱导人骨巨细胞瘤细胞自噬. 第三军医大学学报, 2015, 37(17): 1755-1760.
13. Tian YY, Hu XY, Leung WN, et al. Investigation of photodynamic effect caused by MPPa-PDT on breast cancer. Laser Physics Letters, 2012, 9(10): 754-758.
14. Tian Y, Leung W, Yue K, et al. Cell death induced by MPPa-PDT in prostate carcinoma in vitro and in vivo. Biochem Biophys Res Commun, 2006, 348(2): 413-420.
15. Tan Y, Xu CS, Xia XS, et al. Preliminary studies on LED-activated pyropheophorbide-a methyl ester killing cisplatin-resistant ovarian carcinoma cells. Laser Physics, 2009, 19(5): 1045-1049.
16. Li W, Tan G, Cheng J, et al. A novel photosensitizer 31,131-pheny-lhydrazine-Mppa (BPHM) and its in vitro photodynamic therapy against HeLa Cells. Molecules, 2016, 21(5): 558-570.
17. Huang Q, Ou YS, Tao Y, et al. Apoptosis and autophagy induced by pyropheophorbide-α methyl ester-mediated photodynamic therapy in human osteosarcoma MG-63 cells. Apoptosis, 2016, 21(6): 749-760.
18. 李悦, 曹良启, 彭和平, 等. 腹腔镜联合光动力治疗晚期胰腺癌的近期临床疗效分析. 临床医学工程, 2011, 18(4): 514-516.
19. 秦怀海, 刘智, 张楠, 等. 显微手术联合光动力治疗恶性脑胶质瘤. 中华神经外科杂志, 2008, 24(3): 200-201.
20. 蔡郑东, 胡硕, 龚海洋, 等. PSD-007介导的光动力疗法治疗小鼠骨肉瘤的实验研究. 中华骨科杂志, 2011, 31(6): 692-698.
21. Munshi S, Dahl R. Cytoprotective small molecule modulators of endoplasmic reticulum stress. Bioorganic & Medicinal Chemistry, 2016, 24(11): 2382-2388.
22. Verfaillie T, Garg AD, Agostinis P. Targeting ER stress induced apoptosis and inflammation in cancer. Cancer Lett, 2013, 332(2): 249-264.
23. Sidhu A, Miller JR, Tripathi A, et al. Borrelidin induces the unfolded protein response in oral cancer cells and Chop-dependent apoptosis. ACS Med Chem Lett, 2015, 6(11): 1122-1127.
24. Xu S, Zhao X, Zhao Q, et al. Wogonin prevents rat dorsal root ganglion neurons death via inhibiting tunicamycin-induced ER stress in vitro. Cell Mol Neurobiol, 2015, 35(3): 389-398.
25. Buytaert E, Dewaele M, Agostinis P. Molecular effectors of multiple cell death pathways initiated by photodynamic therapy. Biochim Biophys Acta, 2007, 1776(1): 86-107.

Chinese Journal of Reparative and Reconstructive Surgery

APOPTOSIS IN HUMAN OSTEOSARCOMA CELL LINE MG63 INDUCED BY PYROPHEOPHORBIDE-a METHYL ESTER-MEDIATED PHOTODYNAMIC THERAPY

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