环吡酮胺抑制人胶质瘤细胞SHG44生长及其机制研究
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摘要
该文初步探讨了环吡酮胺(ciclopirox olamine, CPX)抑制人胶质瘤细胞SHG44生长的作用和机制。该研究用梯度浓度的CPX处理人胶质瘤细胞SHG44后,通过MTT实验检测药物半抑制浓度IC50和细胞增殖能力;应用平板克隆实验检测细胞克隆形成能力; Transwell小室和细胞划痕实验检测细胞侵袭和迁移能力;流式细胞术检测细胞内活性氧(reactive oxygen species, ROS)、线粒体内ROS以及细胞凋亡的变化; Seahorse XF96 Flux analysis分析仪检测细胞耗氧率(oxygen consumption rate, OCR);实时荧光定量PCR(q PCR)和Western blot技术检测细胞内m RNA和蛋白的变化。结果表明:SHG44对CPX药物敏感; CPX能使STAT3在m RNA转录水平和蛋白表达水平下降,抑制SHG44的迁移和侵袭,同时,促进线粒体ROS的累积,最终破坏线粒体功能,抑制细胞生长并促进细胞凋亡。该研究结果提示,CPX具有一定的抗胶质瘤细胞SHG44生长的作用,有望成为一种治疗胶质瘤的药物。
The aim of this study was to investigate the effects and molecular mechanism of ciclopirox olamine(CPX) inhibiting the growth of human glioma cell SHG44. In this study, human glioma cell line SHG44 was treated with a gradient concentration of CPX, then the half maximal inhibitory concentration(IC50) and cell proliferation ability of the CPX treated SHG44 cells were determined by MTT assay. In addition, the clone-forming ability was detected to confirm the effect of CPX on SHG44 cell growth. Moreover, the transwell cell migration and invasion assays were performed to study the effects of CPX on the capacity of cell motility and invasiveness.Intracellular reactive oxygen species(ROS), mitochondrial ROS, and apoptosis were detected by flow cytometry.Seahorse XF96 Flux analyzer was used to measure the oxygen consumption rate(OCR), and Real-time PCR(qPCR)as well as Western blot were applied to detect the mRNA and protein levels. Our results showed that SHG44 cells were sensitive to CPX treatment. CPX could reduce both the mRNA and protein expression levels of STAT3, and thus inhibiting the migration and invasion of SHG44 cells. Moreover, CPX treatment significantly enhances the accumulation of mitochondrial ROS, which leads to the impairement of mitochondrial function, and thus inhibiting cell growth and promoting apoptosis. Collectively, our findings demonstrated that CPX could inhibit the growth of SHG44 cells, which may be developed as a drug for the treatment of glioma.
The aim of this study was to investigate the effects and molecular mechanism of ciclopirox olamine(CPX) inhibiting the growth of human glioma cell SHG44. In this study, human glioma cell line SHG44 was treated with a gradient concentration of CPX, then the half maximal inhibitory concentration(IC50) and cell proliferation ability of the CPX treated SHG44 cells were determined by MTT assay. In addition, the clone-forming ability was detected to confirm the effect of CPX on SHG44 cell growth. Moreover, the transwell cell migration and invasion assays were performed to study the effects of CPX on the capacity of cell motility and invasiveness.Intracellular reactive oxygen species(ROS), mitochondrial ROS, and apoptosis were detected by flow cytometry.Seahorse XF96 Flux analyzer was used to measure the oxygen consumption rate(OCR), and Real-time PCR(qPCR)as well as Western blot were applied to detect the mRNA and protein levels. Our results showed that SHG44 cells were sensitive to CPX treatment. CPX could reduce both the mRNA and protein expression levels of STAT3, and thus inhibiting the migration and invasion of SHG44 cells. Moreover, CPX treatment significantly enhances the accumulation of mitochondrial ROS, which leads to the impairement of mitochondrial function, and thus inhibiting cell growth and promoting apoptosis. Collectively, our findings demonstrated that CPX could inhibit the growth of SHG44 cells, which may be developed as a drug for the treatment of glioma.
引文
1 Gupta AK,Skinner AR.Ciclopirox for the treatment ofsuperficial fungal infections:a review.Int J Dermatol 2003;42:3-9.
2 Bohn M,Kraemer KT.Dermatopharmacology ofciclopirox nail lacquer topical solution 8%in the treat-ment of onychomycosis.J Am Acad Dermatol 2000;43:S57-69.
3 Zhou H,Shen T,Luo Y,Luo Y,Liu L,Chen W,et al.The antitumor activity of the fungicide ciclopirox.Int J Cancer 2010;127(10):2467-77.
4 Eberhard Y,McDermott SP,Wang X,Gronda M,Venuqopal A,Wood TE.et al.Chelation of intracellular iron with the antifungal agent ciclopiroxolamine induces cell death in leukemia and myeloma cells.Blood 2009;114(14):3064-73.
5 Hongyu Zhou,Tao Shen,Chaowei Shang,Luo Y,Liu L,Yan J.et al.Ciclopirox induces autophagy through reactive oxygen species-mediated activation of JNK signaling pathway.Oncotarget 2014;5(20):10140-50.
6 Weir SJ,Patton L,Castle K,Rajewski L,Kasper J,Schimmer AD,et al.The repositioning of the anti-fungal agent ciclopirox olamine as a novel therapeutic agent for the treatment of haematologic malignancy.J Clin Pharm Ther 2011;36(2):128-34.
7 Minden MD,Hogge DE,Weir SJ,Kasper J,Wester DA,Patton L,et al.Oral ciclopirox olamine displays biological activity in a phase I study in patients with advanced hematologic malignancies.Am J Hematol 2014;89(4):363-8.
8 Ni Z,Wang B,Dai X,Ding W,Yang T,Li X,et al.HCC cells with high levels of Bcl-2 are resistant to ABT-737 via activation of the ROS-JNK-autophagy pathway.Free Radic Biol Med 2014;70:194-203.
9 Silva CM.Role of STATs as downstream signal transducers in Src family kinase-mediated tumori-genesis.Oncogene 2004;23(48):8017-23.
10 Kisseleva T,Bhattacharya S,Braunstein J,Schindler CW.Signaling through the JAK/STAT pathway,recent advances and future challenges.Gene 2002;285(1/2):1-24.
11 Johnston PA,Grandis JR.STAT3 signaling:anticancer strategies and challenges.Mol Interv 2011;11(1):18-26.
12 Turkson J,Bowman T,Garcia R,Caldenhoven E,De Groot RP,Jove R.Stat3 activation by Src induces specific gene regulation and is required for cell transformation.Mol Cell Biol 1998;18(5):2545-52.
13 Szczepanek K,Lesnefesky EJ,Larner AC.Multi-tasking:nuclear transcription factors with novel roles in the mitochondria.Trends Cell Biol 2012;22(8):429-37.
14 Szczepanek,Chen Q,Derecka M,Salloum FN,Zhang Q,Szelaq M,et al.Mitochondrial-targeted signal transducer and activator of transcription 3(STAT3)protects against ischemiainduced changes in the electron transport chain and the generation of reactive oxygen species.J Biol Chem 2011;286(34):29610-20.
15 Liu Y,Lan L,Huang K,Wang R,Xu C,Shi Y,et al.Inhibition of Lon blocks cell proliferation,enhances chemosensitivity by promoting apoptosis and decreases cellular bioenergetics of bladder cancer:potential roles of Lon as a prognostic marker and therapeutic target in baldder cancer.Oncotarge 2014;5(22):11209-24.
16 Xie D,Wu X,Lan L,Shangguan F,Lin X,Chen F,et al.Downregulation of TFAM inhibits the tumorigenesis of non-small cell lung cancer by activating ROS-mediated JNK/p38MAPK signaling and reducing cellular bioenergetics.Oncotarget 2016;7(10):11609-24.
17 Bromberg JF,Horvath CM,Besser D,Lathem WW,Darnell JEJr.Stat3 activation is required for cellular transformation by v-src.Mol Cell Biol 1998;18(5):2553-8.
18 Jackson C,Ruzevick J,Amin AG,Lim M.Potential role for STAT3 inhibitors in gioblastoma.Neurosurg Clin N Am 2012;23(3):379-89.
19 Poillet-Perez L,Despouy G,Delage-Mourroux R,Boyer-Guittaut M.Interplay between ROS and autophagy in cancer cells,from tumor initiation to cancer therapy.Redox Biol 2015;4(5):184-92.
20 Gupta SC,Hevia D,Patchva S,Park B,Koh W,Aqqarwal BB.Upsides and downsides of reactive oxygen species for cancer:the roles of reactive oxygen species in tumorigenesis,prevention,and therapy.Antioxid Redox Signal 2012;16(11):1295-322.
21 Senft C,Priester M,Polacin M,Schroder K,Seifert V,Koqel D,et al.Inhibition of the JAK-2/STAT3 signaling pathway impedes the migratory and invasive potential of human glioblastoma cells.J Neurooncol 2011;101(3):393-403.
2 Bohn M,Kraemer KT.Dermatopharmacology ofciclopirox nail lacquer topical solution 8%in the treat-ment of onychomycosis.J Am Acad Dermatol 2000;43:S57-69.
3 Zhou H,Shen T,Luo Y,Luo Y,Liu L,Chen W,et al.The antitumor activity of the fungicide ciclopirox.Int J Cancer 2010;127(10):2467-77.
4 Eberhard Y,McDermott SP,Wang X,Gronda M,Venuqopal A,Wood TE.et al.Chelation of intracellular iron with the antifungal agent ciclopiroxolamine induces cell death in leukemia and myeloma cells.Blood 2009;114(14):3064-73.
5 Hongyu Zhou,Tao Shen,Chaowei Shang,Luo Y,Liu L,Yan J.et al.Ciclopirox induces autophagy through reactive oxygen species-mediated activation of JNK signaling pathway.Oncotarget 2014;5(20):10140-50.
6 Weir SJ,Patton L,Castle K,Rajewski L,Kasper J,Schimmer AD,et al.The repositioning of the anti-fungal agent ciclopirox olamine as a novel therapeutic agent for the treatment of haematologic malignancy.J Clin Pharm Ther 2011;36(2):128-34.
7 Minden MD,Hogge DE,Weir SJ,Kasper J,Wester DA,Patton L,et al.Oral ciclopirox olamine displays biological activity in a phase I study in patients with advanced hematologic malignancies.Am J Hematol 2014;89(4):363-8.
8 Ni Z,Wang B,Dai X,Ding W,Yang T,Li X,et al.HCC cells with high levels of Bcl-2 are resistant to ABT-737 via activation of the ROS-JNK-autophagy pathway.Free Radic Biol Med 2014;70:194-203.
9 Silva CM.Role of STATs as downstream signal transducers in Src family kinase-mediated tumori-genesis.Oncogene 2004;23(48):8017-23.
10 Kisseleva T,Bhattacharya S,Braunstein J,Schindler CW.Signaling through the JAK/STAT pathway,recent advances and future challenges.Gene 2002;285(1/2):1-24.
11 Johnston PA,Grandis JR.STAT3 signaling:anticancer strategies and challenges.Mol Interv 2011;11(1):18-26.
12 Turkson J,Bowman T,Garcia R,Caldenhoven E,De Groot RP,Jove R.Stat3 activation by Src induces specific gene regulation and is required for cell transformation.Mol Cell Biol 1998;18(5):2545-52.
13 Szczepanek K,Lesnefesky EJ,Larner AC.Multi-tasking:nuclear transcription factors with novel roles in the mitochondria.Trends Cell Biol 2012;22(8):429-37.
14 Szczepanek,Chen Q,Derecka M,Salloum FN,Zhang Q,Szelaq M,et al.Mitochondrial-targeted signal transducer and activator of transcription 3(STAT3)protects against ischemiainduced changes in the electron transport chain and the generation of reactive oxygen species.J Biol Chem 2011;286(34):29610-20.
15 Liu Y,Lan L,Huang K,Wang R,Xu C,Shi Y,et al.Inhibition of Lon blocks cell proliferation,enhances chemosensitivity by promoting apoptosis and decreases cellular bioenergetics of bladder cancer:potential roles of Lon as a prognostic marker and therapeutic target in baldder cancer.Oncotarge 2014;5(22):11209-24.
16 Xie D,Wu X,Lan L,Shangguan F,Lin X,Chen F,et al.Downregulation of TFAM inhibits the tumorigenesis of non-small cell lung cancer by activating ROS-mediated JNK/p38MAPK signaling and reducing cellular bioenergetics.Oncotarget 2016;7(10):11609-24.
17 Bromberg JF,Horvath CM,Besser D,Lathem WW,Darnell JEJr.Stat3 activation is required for cellular transformation by v-src.Mol Cell Biol 1998;18(5):2553-8.
18 Jackson C,Ruzevick J,Amin AG,Lim M.Potential role for STAT3 inhibitors in gioblastoma.Neurosurg Clin N Am 2012;23(3):379-89.
19 Poillet-Perez L,Despouy G,Delage-Mourroux R,Boyer-Guittaut M.Interplay between ROS and autophagy in cancer cells,from tumor initiation to cancer therapy.Redox Biol 2015;4(5):184-92.
20 Gupta SC,Hevia D,Patchva S,Park B,Koh W,Aqqarwal BB.Upsides and downsides of reactive oxygen species for cancer:the roles of reactive oxygen species in tumorigenesis,prevention,and therapy.Antioxid Redox Signal 2012;16(11):1295-322.
21 Senft C,Priester M,Polacin M,Schroder K,Seifert V,Koqel D,et al.Inhibition of the JAK-2/STAT3 signaling pathway impedes the migratory and invasive potential of human glioblastoma cells.J Neurooncol 2011;101(3):393-403.