“超声增敏ADM”治疗乳腺癌的作用及机制
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摘要
第一部分超声活化ADM产生活性氧的检测
目的：研究低频超声波是否不改变阿霉素(ADM)光谱特性,以及能否活化ADM产生活性氧。
方法：利用活性氧探针并借助荧光光谱分析超声活化ADM后活性氧的产生,并进一步利用荧光染色法测定人乳腺癌耐阿霉素细胞MCF-7/ADM细胞内活性氧的水平。另外,比较超声干预前、超声干预后的ADM的吸收光谱曲线。
结果：超声干预前、超声干预后的ADM的吸收光谱曲线没有差异；而超声干预后的ADM溶液使得FCLA曲线峰值提高,但加入SOD以及NaN3后峰值降低；超声干预后MCF-7/ADM细胞、MCF-7/S细胞的DCF平均荧光强度分别为28.4±6.5、36.7±4.2明显高于相应的超声干预前的DCF平均荧光强度(16.5±1.8,19.3±2.9)(P<0.05)。
结论：经过上述的三个实验,我们发现,一定波长强度的超声能产生单线态氧以及超氧阴离子自由基(实验2),并提高乳腺癌细胞内ROS的水平(实验3),而且并不改变ADM的分子结构(实验1)。
第二部分超声增敏ADM、逆转化疗耐药的检测
目的：研究低频超声波是否能增敏ADM化疗、以及逆转耐阿霉素的人乳腺癌细胞的化疗耐药。
方法：以人乳腺癌细胞MCF-7/S、人乳腺癌耐阿霉素细胞MCF-7/ADM为研究对象,通过绘制细胞生长曲线、检测细胞内阿霉素浓度、检测耐药蛋白MDR1、P-gP的表达,以及检测细胞凋亡来验证低频超声波增敏ADM化疗、逆转化疗耐药的价值及机制。
结果：“超声+阿霉素”("US+ADM")对乳腺癌细胞(MCF-7/S、MCF-7/ADM细胞)均有明显的抑制效应。‘'US+ADM"耐药细胞的IC50值为12.96,耐药逆转倍数3.76。分别测定细胞内阿霉素浓度发现：超声照射后MCF-7/S和MCF-7/ADM细胞内的阿霉素浓度明显上升。RT-PCR分析发现："US+ADM"处理组MDR1 mRNA. MRP1 mRNA的表达水平明显降低,低于单纯ADM组和空白对照组(P<0.05)；Western blot检测发现：‘'US+ADM"处理组的P-gp蛋白、MRP1蛋白的表达水平明显低于单纯ADM组和空白对照组(P<0.05);AO/EB荧光染色后细胞形态学观察显示：超声干预后细胞凋亡明显增加,与对照组相比有显著的统计学差异(P<0.05)。
结论：“超声+阿霉素”能明显抑制细胞表面耐药标志物P-gp、MRP1的表达。“US+ADM”提高了MCF-7/ADM及敏感株MCF-7/S的细胞内阿霉素的浓度。超声对MCF-7/ADM及MCF-7/S的细胞增殖具有一定的抑制作用；并且具有逆转耐药的生物学效应。
第三部分超声增敏ADM化疗对鼠乳腺癌移植瘤的作用
目的：观察"US+ADM"对小鼠乳腺癌的治疗效果。
方法：在BALB/c小鼠右腋皮下注射MA782细胞,制作鼠乳腺癌移植瘤,成功建模鼠40只,随机分成单纯超声组、US+ADM组、单纯ADM组以及空白对照组,每组10只；予以相应干预后观察移植瘤生长情况以及小鼠的存活情况,绘制移植瘤生长曲线以及小鼠的生存曲线,从而探讨超声增敏ADM化疗对鼠乳腺癌的治疗效果。
结果：US+ADM组的小鼠移植瘤生长受到显著抑制,与单纯ADM组比较,差异有统计学意义(P<0.01);US+ADM组的小鼠整体存活时间较单纯ADM组延长,(P<0.05)。单纯ADM组则表现出一定的抗肿瘤作用,移植瘤生长较慢；而单纯超声干预几乎没有抑制作用。
结论：超声增敏ADM化疗对小鼠乳腺癌移植瘤的生长有抑制作用,能够延长乳腺癌小鼠的生存时间。
PART ONE Detecting oxygen species arising from the activation of ADM by ultrasonic
Objectives:To investigate whether ultrasound can activate ADM and produce oxygen species without change the spectral characteristics of ADM.
Methods:Using ROS probe to explore the wavelength intensity of ADM, and generation of reactive oxygen species, and using fluorescence staining method to explore the level of intracellular reactive oxygen species of cells. In addition, by analyzing the absorption spectrum curve of ADM to see the change of the molecular structure of ADM.
Results:There are no difference in the fluorescence spectra curve between the ultrasound before and the ultrasound after. The the intervention of ultrasound after, the ADM solution make the curve peak increased, but after the flowing test, adding SOD, and NaN3 reduced the peak; ultrasound intervention of MCF-7/ADM cells and MCF-7/S cells DCF mean fluorescence intensity were 28.4±6.5,36.7±4.2,which were significantly lower than the corresponding ultrasound before the intervention (16.5±1.8,19.3±2.9) (P <0.05).
Conclusions:After the above three experiments, we found that a certain wavelength intensity ultrasound can produce singlet oxygen 1O2 and the superoxide anion radical O2—(Experiment 2), and increase the level of ROS in breast cancer cells (experiment 3), without changing the molecular structure of ADM (Experiment 1).
PART TWO Detection of Ultrasound-assistant ADM chemotherapy and the reversion of drug resistance
Objective:To study the effection of Ultrasound (US) assistant ADM therapy and the reversion of drug resistance
Methods:A human breast cancer cells MCF-7/S and adriamycin-resistant human breast cancer cells MCF-7/ADM were on this study. By cell growth curve, detection MCF-7/ADM resistance protein MDR1, P-gP expression, measure the concentration of doxorubicin in breast cancer cells, test the apoptosis of cells and value the reversion of drug resistance and mechanisms of US assistant ADM therapy.
Results:"US+ADM" on MCF-7 cells were inhibited and MCF-7/ADM; the drug concentration increased, the inhibitory effect is investigated. After the ultrasound intervention,the IC50 of MCF-7/ADM cells was 12.96, the drug resistance reversation was 3.76 multiple. Doxorubicin within cells of Ultrasound treated MCF-7/S and MCF-7/ADM were significantly increased. RT-PCR analysis showed that:MDR1 mRNA, MRP1 mRNA expression of "US+ADM" group were significantly lower than ADM alone group and the control grou(P<0.05). On "ultrasound+ADM" treatment group,the P-gp protein, MRP1 protein expression were significantly lower than those of ADM group and the control group(P<0.05). AO/EB staining showed cell morphology:ultrasound after the intervention significantly increased apoptosis, compared with the control group (P<0.05).
Conclusion:"US+ADM" could inhibit resistant cell surface markers P-gp, MRP expression on adriamycin resistant human breast cancer cell lines. And increase doxorubicin concentration of MCF-7/ADM cells and sensitive strains of MCF-7 cells. Ultrasound could inhibit the cell proliferation and reverse the drug resistance of ADM.
PART THREE Effect of ultrasound assistant ADM chemotherapy on the mouse mammary cancer
Objective:To observe the ultrasound assistant ADM therapy on the mouse mammary cancer
Methods:BALB/c mice were injected subcutaneously in the right axillary MA782 cells of mouse breast cancer, and a total of 40 mice was randomly divided into 4 groups: simple ultrasound group, ultrasound+ADM group, simple ADM group and control group; tumor growth curves and the survival curves of mice were drawn to explore the effect of ultrasound assistant ADM chemotherapy on the mouse mammary cancer.
Results:The "US+ADM" group of mice tumor growth was significantly inhibited, compared with ADM alone, There was significant difference (p<0.01), while the ultrasound-assisted ADM group overall survival time of mice, compared with ADM alone had significant differences (p<0.05). ADM alone group showed some anti-tumor effects, tumor growth slow, and simple inhibition of ultrasonic little intervention.
Conclusion:Tumor growth of "US+ADM" group were inhibited, the survival time of the mouse of mammary cancer were prolonged.
目的：研究低频超声波是否不改变阿霉素(ADM)光谱特性,以及能否活化ADM产生活性氧。
方法：利用活性氧探针并借助荧光光谱分析超声活化ADM后活性氧的产生,并进一步利用荧光染色法测定人乳腺癌耐阿霉素细胞MCF-7/ADM细胞内活性氧的水平。另外,比较超声干预前、超声干预后的ADM的吸收光谱曲线。
结果：超声干预前、超声干预后的ADM的吸收光谱曲线没有差异；而超声干预后的ADM溶液使得FCLA曲线峰值提高,但加入SOD以及NaN3后峰值降低；超声干预后MCF-7/ADM细胞、MCF-7/S细胞的DCF平均荧光强度分别为28.4±6.5、36.7±4.2明显高于相应的超声干预前的DCF平均荧光强度(16.5±1.8,19.3±2.9)(P<0.05)。
结论：经过上述的三个实验,我们发现,一定波长强度的超声能产生单线态氧以及超氧阴离子自由基(实验2),并提高乳腺癌细胞内ROS的水平(实验3),而且并不改变ADM的分子结构(实验1)。
第二部分超声增敏ADM、逆转化疗耐药的检测
目的：研究低频超声波是否能增敏ADM化疗、以及逆转耐阿霉素的人乳腺癌细胞的化疗耐药。
方法：以人乳腺癌细胞MCF-7/S、人乳腺癌耐阿霉素细胞MCF-7/ADM为研究对象,通过绘制细胞生长曲线、检测细胞内阿霉素浓度、检测耐药蛋白MDR1、P-gP的表达,以及检测细胞凋亡来验证低频超声波增敏ADM化疗、逆转化疗耐药的价值及机制。
结果：“超声+阿霉素”("US+ADM")对乳腺癌细胞(MCF-7/S、MCF-7/ADM细胞)均有明显的抑制效应。‘'US+ADM"耐药细胞的IC50值为12.96,耐药逆转倍数3.76。分别测定细胞内阿霉素浓度发现：超声照射后MCF-7/S和MCF-7/ADM细胞内的阿霉素浓度明显上升。RT-PCR分析发现："US+ADM"处理组MDR1 mRNA. MRP1 mRNA的表达水平明显降低,低于单纯ADM组和空白对照组(P<0.05)；Western blot检测发现：‘'US+ADM"处理组的P-gp蛋白、MRP1蛋白的表达水平明显低于单纯ADM组和空白对照组(P<0.05);AO/EB荧光染色后细胞形态学观察显示：超声干预后细胞凋亡明显增加,与对照组相比有显著的统计学差异(P<0.05)。
结论：“超声+阿霉素”能明显抑制细胞表面耐药标志物P-gp、MRP1的表达。“US+ADM”提高了MCF-7/ADM及敏感株MCF-7/S的细胞内阿霉素的浓度。超声对MCF-7/ADM及MCF-7/S的细胞增殖具有一定的抑制作用；并且具有逆转耐药的生物学效应。
第三部分超声增敏ADM化疗对鼠乳腺癌移植瘤的作用
目的：观察"US+ADM"对小鼠乳腺癌的治疗效果。
方法：在BALB/c小鼠右腋皮下注射MA782细胞,制作鼠乳腺癌移植瘤,成功建模鼠40只,随机分成单纯超声组、US+ADM组、单纯ADM组以及空白对照组,每组10只；予以相应干预后观察移植瘤生长情况以及小鼠的存活情况,绘制移植瘤生长曲线以及小鼠的生存曲线,从而探讨超声增敏ADM化疗对鼠乳腺癌的治疗效果。
结果：US+ADM组的小鼠移植瘤生长受到显著抑制,与单纯ADM组比较,差异有统计学意义(P<0.01);US+ADM组的小鼠整体存活时间较单纯ADM组延长,(P<0.05)。单纯ADM组则表现出一定的抗肿瘤作用,移植瘤生长较慢；而单纯超声干预几乎没有抑制作用。
结论：超声增敏ADM化疗对小鼠乳腺癌移植瘤的生长有抑制作用,能够延长乳腺癌小鼠的生存时间。
PART ONE Detecting oxygen species arising from the activation of ADM by ultrasonic
Objectives:To investigate whether ultrasound can activate ADM and produce oxygen species without change the spectral characteristics of ADM.
Methods:Using ROS probe to explore the wavelength intensity of ADM, and generation of reactive oxygen species, and using fluorescence staining method to explore the level of intracellular reactive oxygen species of cells. In addition, by analyzing the absorption spectrum curve of ADM to see the change of the molecular structure of ADM.
Results:There are no difference in the fluorescence spectra curve between the ultrasound before and the ultrasound after. The the intervention of ultrasound after, the ADM solution make the curve peak increased, but after the flowing test, adding SOD, and NaN3 reduced the peak; ultrasound intervention of MCF-7/ADM cells and MCF-7/S cells DCF mean fluorescence intensity were 28.4±6.5,36.7±4.2,which were significantly lower than the corresponding ultrasound before the intervention (16.5±1.8,19.3±2.9) (P <0.05).
Conclusions:After the above three experiments, we found that a certain wavelength intensity ultrasound can produce singlet oxygen 1O2 and the superoxide anion radical O2—(Experiment 2), and increase the level of ROS in breast cancer cells (experiment 3), without changing the molecular structure of ADM (Experiment 1).
PART TWO Detection of Ultrasound-assistant ADM chemotherapy and the reversion of drug resistance
Objective:To study the effection of Ultrasound (US) assistant ADM therapy and the reversion of drug resistance
Methods:A human breast cancer cells MCF-7/S and adriamycin-resistant human breast cancer cells MCF-7/ADM were on this study. By cell growth curve, detection MCF-7/ADM resistance protein MDR1, P-gP expression, measure the concentration of doxorubicin in breast cancer cells, test the apoptosis of cells and value the reversion of drug resistance and mechanisms of US assistant ADM therapy.
Results:"US+ADM" on MCF-7 cells were inhibited and MCF-7/ADM; the drug concentration increased, the inhibitory effect is investigated. After the ultrasound intervention,the IC50 of MCF-7/ADM cells was 12.96, the drug resistance reversation was 3.76 multiple. Doxorubicin within cells of Ultrasound treated MCF-7/S and MCF-7/ADM were significantly increased. RT-PCR analysis showed that:MDR1 mRNA, MRP1 mRNA expression of "US+ADM" group were significantly lower than ADM alone group and the control grou(P<0.05). On "ultrasound+ADM" treatment group,the P-gp protein, MRP1 protein expression were significantly lower than those of ADM group and the control group(P<0.05). AO/EB staining showed cell morphology:ultrasound after the intervention significantly increased apoptosis, compared with the control group (P<0.05).
Conclusion:"US+ADM" could inhibit resistant cell surface markers P-gp, MRP expression on adriamycin resistant human breast cancer cell lines. And increase doxorubicin concentration of MCF-7/ADM cells and sensitive strains of MCF-7 cells. Ultrasound could inhibit the cell proliferation and reverse the drug resistance of ADM.
PART THREE Effect of ultrasound assistant ADM chemotherapy on the mouse mammary cancer
Objective:To observe the ultrasound assistant ADM therapy on the mouse mammary cancer
Methods:BALB/c mice were injected subcutaneously in the right axillary MA782 cells of mouse breast cancer, and a total of 40 mice was randomly divided into 4 groups: simple ultrasound group, ultrasound+ADM group, simple ADM group and control group; tumor growth curves and the survival curves of mice were drawn to explore the effect of ultrasound assistant ADM chemotherapy on the mouse mammary cancer.
Results:The "US+ADM" group of mice tumor growth was significantly inhibited, compared with ADM alone, There was significant difference (p<0.01), while the ultrasound-assisted ADM group overall survival time of mice, compared with ADM alone had significant differences (p<0.05). ADM alone group showed some anti-tumor effects, tumor growth slow, and simple inhibition of ultrasonic little intervention.
Conclusion:Tumor growth of "US+ADM" group were inhibited, the survival time of the mouse of mammary cancer were prolonged.
引文
[1]赵云罡,徐建兴.线粒体,活性氧和细胞凋亡.生物化学与生物物理进展.2001：28(2)168-170
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[5]Korbelik M, Stott B, Sun J.Photodynamic therapy-generated vaccines:relevance of tumour cell death expression. Br J Cancer 2007;97:1381-7.
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[8]Chen WR, Huang Z, Korbelik M, Nordquist RE, Liu H. Photoimmunotherapy for cancer treatment. J Environ Pathol Toxicol Oncol 2006;25:281-91.
[9]Tang W, Q Liu, X Wang, et al. Ultrasound exposure sure in the presence of hematoporphyrin induced loss of membrane integral proteins and inactivity of cell proliferation associated enzymes in sarcoma 180 cells in vitro[J]. Ultrasonics Sonochemistry,2008,15(5):747-754.
[10]Liu Q, X Li, L Xiao, et al. Study of the synergistic effect on hepatoma 22 tumor cells by focused ultrasound activation of hematoporphyrin[J]. Journal of Ultrasound in Medicine,2008,27(1):57-64.
[11]Hiraoka W, H Honda, L B Feril, Jr, et al. Comparison between sonodynamic effect and photodynamic effect with photosensitizers on free radical formation and cell killing[J]. Ultrasonics Sonochemistry,2006,13(6):535-542.
[1]Yumita N,Sassaki K,et al.Sonodynamically induced antitumor effect of a gallium-prophyrin comples,ATX-70[J].Jpn J Cancer Res,1996,87(3):310-318.
[2]Jin ZH,Miyoshi N,et al.Combination effect of photodynamic and sonodynamic therapy on experimental skin squamous cell carcinoma in C3H/HeN mice.J Dermatology,2000,27(5):294-306.
[3]Noboru T,Hiroshi U,et al.Study of the mechanisms of drug excitation in sonodynamic therapy:direct measurement of the acoustic cavitation-induced luminescence of photosensttizers[J].J Med Ultrasonics,2000,27(1):33-34.
[4]Itoh K, Yamada A, Mine T, Noguchi M.Recent advances in cancer vaccines:an overview. Jpn J Clin Oncol.2009 Feb;39(2):73-80.
[5]Korbelik M, Stott B, Sun J.Photodynamic therapy-generated vaccines:relevance of tumour cell death expression. Br J Cancer 2007;97:1381-7.
[6]Korbelik M, Sun J.Photodynamic therapy-generated vaccine for cancer therapy. Cancer Immunol Immunother 2006;55:900-9.
[7]Gollnick SO, Vaughan L, Henderson BW. Generation of Effective Antitumor Vaccines Using Photodynamic Therapy. Cancer Res 2002;62:1604-8.
[8]Chen WR, Huang Z, Korbelik M, Nordquist RE, Liu H. Photoimmunotherapy for cancer treatment. J Environ Pathol Toxicol Oncol 2006;25:281-91.
[9]Chen WR, Carubelli R, Liu H, Nordquist RE. Laser immunotherapy:a novel treatment modality for metastatic tumors. Mol Biotechnol 2003;25:37-44.
[10]Rosenthal I, Sostaric JZ, Riesz P.Sonodynamic therapy--a review of the synergistic effects of drugs and ultrasound. Ultrason Sonochem.2004; 11:349-63.
[11]Xiaopeng Ma, Huixuan Pan, Jilin Yi.Combination Sonodynamic therapy with immunoadjuvant may be a promising new modality for cancer treatment. Medical Hypotheses.2009;72(4):418-20.
[12]Zeng X, Liao AJ, Tang HL, Yi L, Xie N, Su Q. Screening human gastric carcinoma-associatedantigens by serologic proteome analysis. Ai Zheng.2007;26(10):1080-4.
[13]Liu Q, X Li, L Xiao, et al. Sonodynamically induced antitumor effect of hematoporphyrin on Hepatoma 22 [J]. Ultrasonics Sonochemistry,2008,15(6): 943-948.
[14]Tang W, Q Liu, X Wang, et al. Involvement of caspase 8 in apoptosis induced by ultrasound-activated hematoporphyrin in 180 cells in vitro[J]. Journal of Ultrasound in Medicine,2008,27(4):645-656
[15]Chen WR, Huang Z, Korbelik M, Nordquist RE, Liu H. Photoimmunotherapy for cancer treatment. J Environ Pathol Toxicol Oncol 2006;25:281-91.
[16]Chen WR, Carubelli R, Liu H, Nordquist RE. Laser immunotherapy:a novel treatment modality for metastatic tumors. Mol Biotechnol 2003;25:37-44.
[17]Rosenthal I, Sostaric JZ, Riesz P.Sonodynamic therapy--a review of the synergistic effects of drugs and ultrasound. Ultrason Sonochem.2004; 11:349-63.
[18]Xiaopeng Ma, Huixuan Pan, Jilin Yi.Combination Sonodynamic therapy with immunoadjuvant may be a promising new modality for cancer treatment. Medical Hypotheses.2009;72(4):418-20.
[19]Zeng X, Liao AJ, Tang HL, Yi L, Xie N, Su Q. Screening human gastric carcinoma-associatedantigens by serologic proteome analysis. Ai Zheng.2007;26(10):1080-4.
[1]Yumita N,Sassaki K,et al.Sonodynamically induced antitumor effect of a gallium-prophyrin comples,ATX-70[J].Jpn J Cancer Res,1996,87(3):310-318.
[2]Jin ZH,Miyoshi N,et al.Combination effect of photodynamic and sonodynamic therapy on experimental skin squamous cell carcinoma in C3H/HeN mice.J Dermatology,2000,27(5):294-306.
[3]Noboru T,Hiroshi U,et al.Study of the mechanisms of drug excitation in sonodynamic therapy:direct measurement of the acoustic cavitation-induced luminescence of photosensttizers[J].J Med Ultrasonics,2000,27(l):33-34.
[4]Tang W, Q Liu, X Wang, et al. Ultrasound exposure sure in the presence of hematoporphyrin induced loss of membrane integral proteins and inactivity of cell proliferation associated enzymes in sarcoma 180 cells in vitro[J]. Ultrasonics Sonochemistry,2008,15(5):747-754.
[5]Liu Q, X Li, L Xiao, et al. Study of the synergistic effect on hepatoma 22 tumor cells by focused ultrasound activation of hematoporphyrin[J]. Journal of Ultrasound in Medicine,2008,27(1):57-64.
[6]Hiraoka W, H Honda, L B Feril, Jr, et al. Comparison between sonodynamic effect and photodynamic effect with photosensitizers on free radical formation and cell killing[J]. Ultrasonics Sonochemistry,2006,13(6):535-542.
[7]Liu Q, X Li, L Xiao, et al. Sonodynamically induced antitumor effect of hematoporphyrin on Hepatoma 22 [J]. Ultrasonics Sonochemistry,2008,15(6): 943-948.
[8]Tang W, Q Liu, X Wang, et al. Involvement of caspase 8 in apoptosis induced by ultrasound-activated hematoporphyrin in 180 cells in vitro[J]. Journal of Ultrasound in Medicine,2008,27(4):645-656
[9]Chen WR, Huang Z, Korbelik M, Nordquist RE, Liu H. Photoimmunotherapy for cancer treatment. J Environ Pathol Toxicol Oncol 2006;25:281-91.
[10]Chen WR, Carubelli R, Liu H, Nordquist RE. Laser immunotherapy:a novel treatment modality for metastatic tumors. Mol Biotechnol 2003;25:37-44.
[11]Rosenthal I, Sostaric JZ, Riesz P.Sonodynamic therapy--a review of the synergistic effects of drugs and ultrasound. Ultrason Sonochem.2004;11:349-63.
[12]Xiaopeng Ma, Huixuan Pan, Jilin Yi.Combination Sonodynamic therapy with immunoadjuvant may be a promising new modality for cancer treatment. Medical Hypotheses.2009;72(4):418-20.
[13]Zeng X, Liao AJ, Tang HL, Yi L, Xie N, Su Q. Screening human gastric carcinoma-associatedantigens by serologic proteome analysis. Ai Zheng.2007;26(10):1080-4.
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