低频磁场通过调控MDSCs的功能抑制结肠癌的生长
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摘要
磁场生物效应是当前生物医学工程学界的研究热点.磁场对人类自身的影响与健康效应已引起人们的普遍关注,涉及对中枢神经系统、血液和免疫系统、血管与内分泌系统等效应的研究,其中引人注目的是低频磁场在肿瘤治疗方面的应用.前期研究发现,低频磁场能够明显抑制肝癌、肺癌和黑色素瘤细胞的增殖和调控免疫细胞来发挥抑瘤作用.本研究将进一步探究低频磁场(0.4 T, 7.5 Hz)对结肠癌细胞的生长和髓系来源的抑制性细胞(MDSCs)的影响.本研究建立了小鼠结肠癌移植瘤模型,通过流式细胞术、免疫组化、实时定量PCR、蛋白质印记等技术研究了低频磁场对结肠癌和MDSCs的影响.结果发现,低频磁场明显抑制CT26结肠癌移植瘤模型的肿瘤生长,降低了肿瘤组织中的Ki-67阳性细胞数.低频磁场降低了肿瘤组织中MDSCs的细胞比例并减弱了MDSCs的抑制功能.体外实验发现低频磁场通过抑制了Gp91~(phox)和P47~(phox)的表达降低了MDSCs的活性氧(ROS)产生,抑制了MDSCs的功能,进而发挥对结肠癌治疗作用.
The biological effect of magnetic field is a hot topic in biomedical engineering. The influence of magnetic fields on human health has caused the widespread concern, involving the study of magnetic field in central nervous system, blood and immune system, vascular and endocrine system, among which the most attractive application is usage of low frequency magnetic fields(LF-MF) in the tumor treatment. Our previous studies showed that LF-MF significantly inhibited the proliferation of liver cancer, lung cancer and melanoma cells, and regulated the immune cells to exert anti-tumor effect. We have found that LF-MF exposure was associated with activation of macrophages and dendritic cells, enhanced profiles of CD4+Tand CD8+T lymphocytes, the balance of Th17/Treg in tumor-bearing mice. In cancer, myeloid-derived suppressor cells(MDSCs)demonstrating one of the key players engaged in tumor immunosuppression. This study will further explore the role of LFMF in the growth of colon cancer and function of myeloid-derived suppressor cells(MDSCs). In this study, mouse colon cancer xenografts models were established and the tumor-bearing mice were exposed to a LF-MF(0.4 T, 7.5 Hz) for 35 days.The tumor growth and tumor weight were measured. Immunohistochemical staining for Ki-67 in tumor tissue was performed. Flow cytometry analyzed the proportion of MDSCs in the bone marrow, spleen, tumor, and peripheral blood of mice. Magnetic beads separated MDSCs from tumor tissues and MDSCs suppression assays were performed. Q-PCR was performed to detect the mRNA expression of Arg-1, i NOS-2, Gp91~(phox), P47~(phox), and S100 A8/A9 in MDSCs from tumor tissues. The reactive oxygen species(ROS)level in MDSCs was analyzed by flow cytometry. Western blot was used to detect the protein level of Gp91~(phox), P47~(phox)and GAPDH. Results showed that LF-MF inhibited the tumor growth in the CT26 colon cancer xenograft model, and decreased the number of Ki-67 positive cells in the tumor tissues. LF-MF reduced the proportion of MDSCs in tumor tissues and attenuated the inhibitory function of MDSCs. In vitro, LF-MF had no effect on cell viability and cell apoptosis of MDSCs. In addition, LF-MF reduced the production of reactive oxygen species in MDSCs by inhibiting the expression of Gp91~(phox)and P47~(phox). With the reduced the production of reactive oxygen species,the inhibitory function of MDSCs was attenuated by LF-MF. Together, our present study demonstrated that LF-MF obviously suppressed the development of colon cancer via regulating the expansion and function of MDSCs. LF-MF suppressed the inhibitory function of MDSCs by reducing the production of reactive oxygen species with the decreased expression of Gp91~(phox)and P47~(phox). Our studies reveal a novel mechanism behind the anti-tumor effect of LF-MFs on colon cancer and provide a potentially useful adjunct therapy for treatment of colon cancer.
The biological effect of magnetic field is a hot topic in biomedical engineering. The influence of magnetic fields on human health has caused the widespread concern, involving the study of magnetic field in central nervous system, blood and immune system, vascular and endocrine system, among which the most attractive application is usage of low frequency magnetic fields(LF-MF) in the tumor treatment. Our previous studies showed that LF-MF significantly inhibited the proliferation of liver cancer, lung cancer and melanoma cells, and regulated the immune cells to exert anti-tumor effect. We have found that LF-MF exposure was associated with activation of macrophages and dendritic cells, enhanced profiles of CD4+Tand CD8+T lymphocytes, the balance of Th17/Treg in tumor-bearing mice. In cancer, myeloid-derived suppressor cells(MDSCs)demonstrating one of the key players engaged in tumor immunosuppression. This study will further explore the role of LFMF in the growth of colon cancer and function of myeloid-derived suppressor cells(MDSCs). In this study, mouse colon cancer xenografts models were established and the tumor-bearing mice were exposed to a LF-MF(0.4 T, 7.5 Hz) for 35 days.The tumor growth and tumor weight were measured. Immunohistochemical staining for Ki-67 in tumor tissue was performed. Flow cytometry analyzed the proportion of MDSCs in the bone marrow, spleen, tumor, and peripheral blood of mice. Magnetic beads separated MDSCs from tumor tissues and MDSCs suppression assays were performed. Q-PCR was performed to detect the mRNA expression of Arg-1, i NOS-2, Gp91~(phox), P47~(phox), and S100 A8/A9 in MDSCs from tumor tissues. The reactive oxygen species(ROS)level in MDSCs was analyzed by flow cytometry. Western blot was used to detect the protein level of Gp91~(phox), P47~(phox)and GAPDH. Results showed that LF-MF inhibited the tumor growth in the CT26 colon cancer xenograft model, and decreased the number of Ki-67 positive cells in the tumor tissues. LF-MF reduced the proportion of MDSCs in tumor tissues and attenuated the inhibitory function of MDSCs. In vitro, LF-MF had no effect on cell viability and cell apoptosis of MDSCs. In addition, LF-MF reduced the production of reactive oxygen species in MDSCs by inhibiting the expression of Gp91~(phox)and P47~(phox). With the reduced the production of reactive oxygen species,the inhibitory function of MDSCs was attenuated by LF-MF. Together, our present study demonstrated that LF-MF obviously suppressed the development of colon cancer via regulating the expansion and function of MDSCs. LF-MF suppressed the inhibitory function of MDSCs by reducing the production of reactive oxygen species with the decreased expression of Gp91~(phox)and P47~(phox). Our studies reveal a novel mechanism behind the anti-tumor effect of LF-MFs on colon cancer and provide a potentially useful adjunct therapy for treatment of colon cancer.
引文
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4 Juutilainen J.Do electromagnetic fields enhance the effects of environmental carcinogens?Radiat Prot Dosimetry,2008,132:228-231
5 Juutilainen J,Kumlin T,Naarala J.Do extremely low frequency magnetic fields enhance the effects of environmental carcinogens?A meta-analysis of experimental studies.Int J Radiat Biol,2006,82:1-12
6 Chung M K,Yu W J,Kim Y B,et al.Lack of a co-promotion effect of 60 Hz circularly polarized magnetic fields on spontaneous development of lymphoma in AKR mice.Bioelectromagnetics,2010,31:130-139
7 Mariucci G,Villarini M,Moretti M,et al.Brain DNA damage and 70-kDa heat shock protein expression in cd1 mice exposed to extremely low frequency magnetic fields.Int J Radiat Biol,2010,86:701-710
8 Li J,Ma Y,Li N,et al.Natural static magnetic field-induced apoptosis in liver cancer cell.ElectroMagn Biol Med,2014,33:47-50
9 Koh E K,Ryu B K,Jeong D Y,et al.A 60-Hz sinusoidal magnetic field induces apoptosis of prostate cancer cells through reactive oxygen species.Int J Radiat Biol,2008,84:945-955
10 Zhang X,Liu X,Pan L,et al.Magnetic fields at extremely low-frequency(50 Hz,0.8 m T)can induce the uptake of intracellular calcium levels in osteoblasts.Biochem BioPhys Res Commun,2010,396:662-666
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12 Bellossi A,Desplaces A.Effect of a 9 MT pulsed magnetic field on C3H/BI female mice with mammary carcinoma.A comparison between the
12 Hz and the 460 Hz frequencies.In Vivo,1991,5:39-40
13 Strieth S,Strelczyk D,Eichhorn M E,et al.Static magnetic fields induce blood flow decrease and platelet adherence in tumor microvessels.Cancer Biol Ther,2008,7:814-819
14 Gellrich D,Becker S,Strieth S.Static magnetic fields increase tumor microvessel leakiness and improve antitumoral efficacy in combination with paclitaxel.Cancer Lett,2014,343:107-114
15 Delle Monache S,Angelucci A,SanitàP,et al.Inhibition of angiogenesis mediated by extremely low-frequency magnetic fields(ELF-MFS).PLoSONE,2013,8:e79309
16 El-Bialy N S,Rageh M M.Extremely low-frequency magnetic field enhances the therapeutic efficacy of low-dose cisplatin in the treatment of ehrlich carcinoma.Biomed Res Int,2013,2013:189352
17 Wang T,Nie Y,Zhao S,et al.Involvement of midkine expression in the inhibitory effects of low-frequency magnetic fields on cancer cells.Bioelectromagnetics,2011,32:443-452
18 Nie Y,Chen Y,Mou Y,et al.Low frequency magnetic fields enhance antitumor immune response against mouse H22 hepatocellular carcinoma.PLoS ONE,2013,8:e72411
19 Nie Y,Du L,Mou Y,et al.Effect of low frequency magnetic fields on melanoma:Tumor inhibition and immune modulation.BMC Cancer,2013,13:582
20 Tang R,Xu Y,Ma F,et al.Extremely low frequency magnetic fields regulate differentiation of regulatory T cells:Potential role for ROS-mediated inhibition on akt.Bioelectromagnetics,2016,37:89-98
21 Gabrilovich D I,Nagaraj S.Myeloid-derived suppressor cells as regulators of the immune system.Nat Rev Immunol,2009,9:162-174
22 Nagaraj S,Gupta K,Pisarev V,et al.Altered recognition of antigen is a mechanism of CD8+T cell tolerance in cancer.Nat Med,2007,13:828-835
23 Lu T,Ramakrishnan R,Altiok S,et al.Tumor-infiltrating myeloid cells induce tumor cell resistance to cytotoxic T cells in mice.J Clin Invest,2011,121:4015-4029
24 Jiang Y,Li X,Yang W,et al.PKM2 regulates chromosome segregation and mitosis progression of tumor cells.Mol Cell,2014,53:75-87
25 Ren J,Ding L,Zhang D,et al.Carcinoma-associated fibroblasts promote the stemness and chemoresistance of colorectal cancer by transferring exosomal LncRNA H19.Theranostics,2018,8:3932-3948
26 Xu Y,Zhao W,Wu D,et al.Isolation of myeloid-derived suppressor cells subsets from spleens of orthotopic liver cancer-bearing mice by fluorescent-activated and magnetic-activated cell sorting:Similarities and differences.Int J Clin Exp Pathol,2014,7:7545-7553
27 Ju Kim M,Shin K S,Chung Y B,et al.Immunohistochemical study of P47phoxand GP91phoxdistributions in rat brain.Brain Res,2005,1040:178-186
28 Dolcetti L,Peranzoni E,Ugel S,et al.Hierarchy of immunosuppressive strength among myeloid-derived suppressor cell subsets is determined by GM-CSF.Eur J Immunol,2010,40:22-35
29 Pallett L J,Gill U S,Quaglia A,et al.Metabolic regulation of hepatitis b immunopathology by myeloid-derived suppressor cells.Nat Med,2015,21:591-600
30 Dong G,You M,Fan H,et al.17β-estradiol contributes to the accumulation of myeloid-derived suppressor cells in blood by promoting TNF-αsecretion.Acta Biochim Biophys Sin,2015,47:620-629
31 Corzo C A,Condamine T,Lu L,et al.HIF-1αregulates function and differentiation of myeloid-derived suppressor cells in the tumor microenvironment.J Exp Med,2010,207:2439-2453
32 Guo C,Hu F,Yi H,et al.Myeloid-derived suppressor cells have a proinflammatory role in the pathogenesis of autoimmune arthritis.Ann Rheum Dis,2016,75:278-285
33 Holmgaard R B,Zamarin D,Li Y,et al.Tumor-expressed IDO recruits and activates MDSCs in a treg-dependent manner.Cell Rep,2015,13:412-424
34 Wang H,Zhang X.Magnetic fields and reactive oxygen species.Int J Mol Sci,2017,18,doi:10.3390/ijms18102175
35 Shine M B,Guruprasad K N,Anand A.Effect of stationary magnetic field strengths of 150 and 200 mT on reactive oxygen species production in soybean.Bioelectromagnetics,2012,33:428-437
36 Sun L,Chen L,Bai L,et al.Reactive oxygen species mediates 50-Hz magnetic field-induced EGF receptor clustering via acid sphingomyelinase activation.Int J Radiat Biol,2018,94:1-7
37 Duong C N,Kim J Y.Exposure to electromagnetic field attenuates oxygen-glucose deprivation-induced microglial cell death by reducing intracellular Ca2+and ROS.Int J Radiat Biol,2016,92:195-201
38 Poniedzia?ek B,Rzymski P,Karczewski J,et al.Reactive oxygen species(ROS)production in human peripheral blood neutrophils exposed in vitro to static magnetic field.ElectroMagn Biol Med,2013,32:560-568
2 Lagroye I,Percherancier Y,Juutilainen J,et al.ELF magnetic fields:Animal studies,mechanisms of action.Prog Biophys Mol Biol,2011,107:369-3733 Humans IWGotEoCRt.Non-ionizing radiation,part 1:Static and extremely low-frequency(ELF)electric and magnetic fields.IARC Monogr Eval Carcinog Risks Hum,2002,80:1-395
4 Juutilainen J.Do electromagnetic fields enhance the effects of environmental carcinogens?Radiat Prot Dosimetry,2008,132:228-231
5 Juutilainen J,Kumlin T,Naarala J.Do extremely low frequency magnetic fields enhance the effects of environmental carcinogens?A meta-analysis of experimental studies.Int J Radiat Biol,2006,82:1-12
6 Chung M K,Yu W J,Kim Y B,et al.Lack of a co-promotion effect of 60 Hz circularly polarized magnetic fields on spontaneous development of lymphoma in AKR mice.Bioelectromagnetics,2010,31:130-139
7 Mariucci G,Villarini M,Moretti M,et al.Brain DNA damage and 70-kDa heat shock protein expression in cd1 mice exposed to extremely low frequency magnetic fields.Int J Radiat Biol,2010,86:701-710
8 Li J,Ma Y,Li N,et al.Natural static magnetic field-induced apoptosis in liver cancer cell.ElectroMagn Biol Med,2014,33:47-50
9 Koh E K,Ryu B K,Jeong D Y,et al.A 60-Hz sinusoidal magnetic field induces apoptosis of prostate cancer cells through reactive oxygen species.Int J Radiat Biol,2008,84:945-955
10 Zhang X,Liu X,Pan L,et al.Magnetic fields at extremely low-frequency(50 Hz,0.8 m T)can induce the uptake of intracellular calcium levels in osteoblasts.Biochem BioPhys Res Commun,2010,396:662-666
11 de Seze R,Tuffet S,Moreau J M,et al.Effects of 100 mt time varying magnetic fields on the growth of tumors in mice.Bioelectromagnetics,2000,21:107-111
12 Bellossi A,Desplaces A.Effect of a 9 MT pulsed magnetic field on C3H/BI female mice with mammary carcinoma.A comparison between the
12 Hz and the 460 Hz frequencies.In Vivo,1991,5:39-40
13 Strieth S,Strelczyk D,Eichhorn M E,et al.Static magnetic fields induce blood flow decrease and platelet adherence in tumor microvessels.Cancer Biol Ther,2008,7:814-819
14 Gellrich D,Becker S,Strieth S.Static magnetic fields increase tumor microvessel leakiness and improve antitumoral efficacy in combination with paclitaxel.Cancer Lett,2014,343:107-114
15 Delle Monache S,Angelucci A,SanitàP,et al.Inhibition of angiogenesis mediated by extremely low-frequency magnetic fields(ELF-MFS).PLoSONE,2013,8:e79309
16 El-Bialy N S,Rageh M M.Extremely low-frequency magnetic field enhances the therapeutic efficacy of low-dose cisplatin in the treatment of ehrlich carcinoma.Biomed Res Int,2013,2013:189352
17 Wang T,Nie Y,Zhao S,et al.Involvement of midkine expression in the inhibitory effects of low-frequency magnetic fields on cancer cells.Bioelectromagnetics,2011,32:443-452
18 Nie Y,Chen Y,Mou Y,et al.Low frequency magnetic fields enhance antitumor immune response against mouse H22 hepatocellular carcinoma.PLoS ONE,2013,8:e72411
19 Nie Y,Du L,Mou Y,et al.Effect of low frequency magnetic fields on melanoma:Tumor inhibition and immune modulation.BMC Cancer,2013,13:582
20 Tang R,Xu Y,Ma F,et al.Extremely low frequency magnetic fields regulate differentiation of regulatory T cells:Potential role for ROS-mediated inhibition on akt.Bioelectromagnetics,2016,37:89-98
21 Gabrilovich D I,Nagaraj S.Myeloid-derived suppressor cells as regulators of the immune system.Nat Rev Immunol,2009,9:162-174
22 Nagaraj S,Gupta K,Pisarev V,et al.Altered recognition of antigen is a mechanism of CD8+T cell tolerance in cancer.Nat Med,2007,13:828-835
23 Lu T,Ramakrishnan R,Altiok S,et al.Tumor-infiltrating myeloid cells induce tumor cell resistance to cytotoxic T cells in mice.J Clin Invest,2011,121:4015-4029
24 Jiang Y,Li X,Yang W,et al.PKM2 regulates chromosome segregation and mitosis progression of tumor cells.Mol Cell,2014,53:75-87
25 Ren J,Ding L,Zhang D,et al.Carcinoma-associated fibroblasts promote the stemness and chemoresistance of colorectal cancer by transferring exosomal LncRNA H19.Theranostics,2018,8:3932-3948
26 Xu Y,Zhao W,Wu D,et al.Isolation of myeloid-derived suppressor cells subsets from spleens of orthotopic liver cancer-bearing mice by fluorescent-activated and magnetic-activated cell sorting:Similarities and differences.Int J Clin Exp Pathol,2014,7:7545-7553
27 Ju Kim M,Shin K S,Chung Y B,et al.Immunohistochemical study of P47phoxand GP91phoxdistributions in rat brain.Brain Res,2005,1040:178-186
28 Dolcetti L,Peranzoni E,Ugel S,et al.Hierarchy of immunosuppressive strength among myeloid-derived suppressor cell subsets is determined by GM-CSF.Eur J Immunol,2010,40:22-35
29 Pallett L J,Gill U S,Quaglia A,et al.Metabolic regulation of hepatitis b immunopathology by myeloid-derived suppressor cells.Nat Med,2015,21:591-600
30 Dong G,You M,Fan H,et al.17β-estradiol contributes to the accumulation of myeloid-derived suppressor cells in blood by promoting TNF-αsecretion.Acta Biochim Biophys Sin,2015,47:620-629
31 Corzo C A,Condamine T,Lu L,et al.HIF-1αregulates function and differentiation of myeloid-derived suppressor cells in the tumor microenvironment.J Exp Med,2010,207:2439-2453
32 Guo C,Hu F,Yi H,et al.Myeloid-derived suppressor cells have a proinflammatory role in the pathogenesis of autoimmune arthritis.Ann Rheum Dis,2016,75:278-285
33 Holmgaard R B,Zamarin D,Li Y,et al.Tumor-expressed IDO recruits and activates MDSCs in a treg-dependent manner.Cell Rep,2015,13:412-424
34 Wang H,Zhang X.Magnetic fields and reactive oxygen species.Int J Mol Sci,2017,18,doi:10.3390/ijms18102175
35 Shine M B,Guruprasad K N,Anand A.Effect of stationary magnetic field strengths of 150 and 200 mT on reactive oxygen species production in soybean.Bioelectromagnetics,2012,33:428-437
36 Sun L,Chen L,Bai L,et al.Reactive oxygen species mediates 50-Hz magnetic field-induced EGF receptor clustering via acid sphingomyelinase activation.Int J Radiat Biol,2018,94:1-7
37 Duong C N,Kim J Y.Exposure to electromagnetic field attenuates oxygen-glucose deprivation-induced microglial cell death by reducing intracellular Ca2+and ROS.Int J Radiat Biol,2016,92:195-201
38 Poniedzia?ek B,Rzymski P,Karczewski J,et al.Reactive oxygen species(ROS)production in human peripheral blood neutrophils exposed in vitro to static magnetic field.ElectroMagn Biol Med,2013,32:560-568