一种新的植物提取物抗鼻咽癌转移的实验研究
摘要
目的：
临床上,鼻咽癌具有高转移、高复发和低分化等特点,鼻咽癌高转移为鼻咽癌有效治疗带来了极大的困难。为寻求有效、毒性小、纯度高的抗鼻咽癌药物,本文通过高效液相色谱法分离鼻咽癌中医药治疗常用经验方(益气解毒颗粒)组分,分别将每一组分处理鼻咽癌细胞(CNE1细胞),分析其抗鼻咽癌药效,筛选最佳药效组分。探讨该组分对鼻咽癌细胞运动转移的影响。为开发新的抗鼻咽癌转移药提供实验依据。
方法：
1.选择以植物药黄连为主药的益气解毒颗粒,该药为鼻咽癌中医药治疗常用经验方,在抗鼻咽癌放疗后的转移方面有较好疗效。采用高效液相色谱法分离益气解毒颗粒组分,纯化、干燥,得到各组分的纯净物。
2.采用细胞增殖抑制实验(MTT)分析上述组分的抗鼻咽癌疗效,筛选抗鼻咽癌有效组分。
3.用液相色谱—质谱联用、核磁共振法等方法分析有效组分的分子量、化学结构。
4.通过MTT法分析该组分对CNE1细胞增殖的影响,通过测定药物处理后细胞培养液中LDH的浓度,筛选有效组分的非毒性浓度；通过异硫酸氢荧光素-鬼笔环肽研究组分对CNE1细胞微丝骨架和细胞伪足形成的影响；通过划痕和Transwell实验研究其对CNE1细胞迁移能力的影响。
5.利用SPSS15.0统计软件分析数据。
结果：
1.MTT结果显示,组分7为所有组分抗鼻咽癌效果最佳组分,确定该组分为下步研究的实验材料。进一步研究表明,该组分在高浓度(20μmol/1以上)时对CNE1细胞的增殖生长有明显的抑制作用,而在20μmol/L以下对CNE1细胞生长没有明显影响。0-20μmol/1为该组分对CNE1细胞的无毒性浓度。
2.通过核磁共振、液相色谱-质谱联用等方法分析获得该组分的分子量为418、分子式为C21H22O9,化学结构(略)。
3.LDH结果显示,YQJD7和BBR比较,处理CNE1细胞后培养基中LDH浓度低,细胞死亡率低,毒性作用小。
4.异硫酸氢荧光素.鬼笔环肽细胞微丝骨架染色结果显示,未加药的细胞体积较大,形态不规则,能观察到完整的细胞微丝骨架和丰富的细胞伪足。加药后的细胞体积小,偏圆,极性小,细胞微丝骨架和伪足形成不明显。
5.划痕实验结果显示,加药组的细胞创伤边界的缩窄程度较未加药组明显减小；Transwell迁移实验结果显示,加药组的穿膜细胞数明显少于未加药组,加药后CNE1细胞的迁移能力显著减弱。
结论：
1.通过高效液相色谱法成功分离、纯化获得一新抗鼻咽癌转移的植物药提取物,为鼻咽癌治疗提供了一新的手段。
2.提取物对鼻咽癌CNE1细胞的增殖有明显的抑制作用,毒性较小,对CNE1细胞的形态、细胞微丝骨架和细胞伪足形成等有较明显的影响,抑制CNE1细胞的迁移能力。
3.鼻咽癌中医药治疗常用经验方益气解毒颗粒抗鼻咽癌转移临床疗效可能与该组分抑制鼻咽癌细胞伪足形成和细胞迁移有关。
Objective:
Nasopharyngeal carcinoma (NPC) has a high metastasis, high recurrence and low differentiate. These set a difficult question for NPC therapy. To search for a novel anti- metastasis drug for NPC which has characteristics including anticancer effect, low toxicity, high purity. We use HPLC to extract a component of Qi-Boosting Toxin-Resolving Granule, which has been to treat NPC patients with radiotherapy and has a well therapeutic effect. The extracts were confirmed their anti-metastasis effect. It will provide a new strategy and method to develop new drugs for anti-NPC. At the same time, we provide evidences for developing a new drug in vitro.
Method:
1. A complex, Qi-Boosting Toxin-Resolving Granule containing Coptis chinensis, which generally was used to treat NPC patients and has a well clinic effect, was separated using HPLC.
2. The extracts were used to treated NPC cell (CNE1) and get an effective component. This component structure was determined by Nuclear Magnetic Resonance (NMR), Liquid Chromatography-Mass Spectrometry (LC-MS).
3. The abilities of proliferation of the component on CNE1 cells were analyzed by MTT assay. To observe the toxicity of the component, LDH was measured in media of cells with drug treatment. We used FITC-phalloidin to analyze the drug affect on cytoskeleton. The abilities of migration were observed by scratch assay and transwell assay.
4. SPSS 15.0 was used to analyze the data. Results:
1. MTT assay data showed that Component 7 was an effective component, could inhibit the proliferation of CNE1 cells at the concentration greater than 20μmol/L (P<0.05), and no effect less than 20μmol/l.0-20μmol/l is the non-cytotoxic concentration (NCC) of the component.
2. The chemical formula of Component 7 is C21H22O9, and the molecular weight is 418.
3. LDH results showed that the concentration of LDH in the culture medium was lower after Component 7 treated CNE1 cells compared with berberine.
4. FITC-phalloidin staining results showed that Component 7 could inhibit the growth of cytoskeleton and filopodia of CNE1.
5. Scratch assay and transwell results showed that Component 7 could dramatically inhibit the migration of CNE1.
Conclusions:
1. We successfully extracted and separated components of Qi-Boosting Toxin-Resolving Granule by use HPLC and got a novel component for NPC therapy. This provides a novel strategy NPC therapy.
2. The effect component could inhibit the morphology, cytoskeleton, and migration ability of CNE1 cells at non-cytotoxic concentration.
3. Clinic anti-metastasis of Qi-Boosting Toxin-Resolving Granule may be determined by this effective component.
临床上,鼻咽癌具有高转移、高复发和低分化等特点,鼻咽癌高转移为鼻咽癌有效治疗带来了极大的困难。为寻求有效、毒性小、纯度高的抗鼻咽癌药物,本文通过高效液相色谱法分离鼻咽癌中医药治疗常用经验方(益气解毒颗粒)组分,分别将每一组分处理鼻咽癌细胞(CNE1细胞),分析其抗鼻咽癌药效,筛选最佳药效组分。探讨该组分对鼻咽癌细胞运动转移的影响。为开发新的抗鼻咽癌转移药提供实验依据。
方法：
1.选择以植物药黄连为主药的益气解毒颗粒,该药为鼻咽癌中医药治疗常用经验方,在抗鼻咽癌放疗后的转移方面有较好疗效。采用高效液相色谱法分离益气解毒颗粒组分,纯化、干燥,得到各组分的纯净物。
2.采用细胞增殖抑制实验(MTT)分析上述组分的抗鼻咽癌疗效,筛选抗鼻咽癌有效组分。
3.用液相色谱—质谱联用、核磁共振法等方法分析有效组分的分子量、化学结构。
4.通过MTT法分析该组分对CNE1细胞增殖的影响,通过测定药物处理后细胞培养液中LDH的浓度,筛选有效组分的非毒性浓度；通过异硫酸氢荧光素-鬼笔环肽研究组分对CNE1细胞微丝骨架和细胞伪足形成的影响；通过划痕和Transwell实验研究其对CNE1细胞迁移能力的影响。
5.利用SPSS15.0统计软件分析数据。
结果：
1.MTT结果显示,组分7为所有组分抗鼻咽癌效果最佳组分,确定该组分为下步研究的实验材料。进一步研究表明,该组分在高浓度(20μmol/1以上)时对CNE1细胞的增殖生长有明显的抑制作用,而在20μmol/L以下对CNE1细胞生长没有明显影响。0-20μmol/1为该组分对CNE1细胞的无毒性浓度。
2.通过核磁共振、液相色谱-质谱联用等方法分析获得该组分的分子量为418、分子式为C21H22O9,化学结构(略)。
3.LDH结果显示,YQJD7和BBR比较,处理CNE1细胞后培养基中LDH浓度低,细胞死亡率低,毒性作用小。
4.异硫酸氢荧光素.鬼笔环肽细胞微丝骨架染色结果显示,未加药的细胞体积较大,形态不规则,能观察到完整的细胞微丝骨架和丰富的细胞伪足。加药后的细胞体积小,偏圆,极性小,细胞微丝骨架和伪足形成不明显。
5.划痕实验结果显示,加药组的细胞创伤边界的缩窄程度较未加药组明显减小；Transwell迁移实验结果显示,加药组的穿膜细胞数明显少于未加药组,加药后CNE1细胞的迁移能力显著减弱。
结论：
1.通过高效液相色谱法成功分离、纯化获得一新抗鼻咽癌转移的植物药提取物,为鼻咽癌治疗提供了一新的手段。
2.提取物对鼻咽癌CNE1细胞的增殖有明显的抑制作用,毒性较小,对CNE1细胞的形态、细胞微丝骨架和细胞伪足形成等有较明显的影响,抑制CNE1细胞的迁移能力。
3.鼻咽癌中医药治疗常用经验方益气解毒颗粒抗鼻咽癌转移临床疗效可能与该组分抑制鼻咽癌细胞伪足形成和细胞迁移有关。
Objective:
Nasopharyngeal carcinoma (NPC) has a high metastasis, high recurrence and low differentiate. These set a difficult question for NPC therapy. To search for a novel anti- metastasis drug for NPC which has characteristics including anticancer effect, low toxicity, high purity. We use HPLC to extract a component of Qi-Boosting Toxin-Resolving Granule, which has been to treat NPC patients with radiotherapy and has a well therapeutic effect. The extracts were confirmed their anti-metastasis effect. It will provide a new strategy and method to develop new drugs for anti-NPC. At the same time, we provide evidences for developing a new drug in vitro.
Method:
1. A complex, Qi-Boosting Toxin-Resolving Granule containing Coptis chinensis, which generally was used to treat NPC patients and has a well clinic effect, was separated using HPLC.
2. The extracts were used to treated NPC cell (CNE1) and get an effective component. This component structure was determined by Nuclear Magnetic Resonance (NMR), Liquid Chromatography-Mass Spectrometry (LC-MS).
3. The abilities of proliferation of the component on CNE1 cells were analyzed by MTT assay. To observe the toxicity of the component, LDH was measured in media of cells with drug treatment. We used FITC-phalloidin to analyze the drug affect on cytoskeleton. The abilities of migration were observed by scratch assay and transwell assay.
4. SPSS 15.0 was used to analyze the data. Results:
1. MTT assay data showed that Component 7 was an effective component, could inhibit the proliferation of CNE1 cells at the concentration greater than 20μmol/L (P<0.05), and no effect less than 20μmol/l.0-20μmol/l is the non-cytotoxic concentration (NCC) of the component.
2. The chemical formula of Component 7 is C21H22O9, and the molecular weight is 418.
3. LDH results showed that the concentration of LDH in the culture medium was lower after Component 7 treated CNE1 cells compared with berberine.
4. FITC-phalloidin staining results showed that Component 7 could inhibit the growth of cytoskeleton and filopodia of CNE1.
5. Scratch assay and transwell results showed that Component 7 could dramatically inhibit the migration of CNE1.
Conclusions:
1. We successfully extracted and separated components of Qi-Boosting Toxin-Resolving Granule by use HPLC and got a novel component for NPC therapy. This provides a novel strategy NPC therapy.
2. The effect component could inhibit the morphology, cytoskeleton, and migration ability of CNE1 cells at non-cytotoxic concentration.
3. Clinic anti-metastasis of Qi-Boosting Toxin-Resolving Granule may be determined by this effective component.
引文
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[1]Bolhassani A, Rafati S. Heat-shock proteins as powerful weapons in vaccine development. Expert Rev Vaccines,2008,7(8):1185-1199.
[2]Feige U, Polla NS. Hsp70--a multi-gene, multi-structure, multi-function family with potential clinical applications. Experentia,1994,50(11-12):979-986.
[3]Sherman M, Multhoff G. Heat shock proteins in cancer. Ann N Y Acad Sci,2007, 1113:192-201.
[4]Liu M, Aneja R, Liu C, et al. Inhibition of the mitotic kinesin Eg5 up-regulates Hsp70 through the phosphatidylinositol 3-kinase/Akt pathway in multiple myeloma cells. J Biol Chem,2006,281(26):18090-18097.
[5]Seo HR, Chung DY, Lee YJ, et al. Heat shock protein 25 or inducible heat shock protein 70 activates heat shock factor 1:dephosphorylation on serine 307 through inhibition of ERK1/2 phosphorylation. J Biol Chem,2006,281(25): 17220-17227.
[6]Widlak W.The heat shock response and HSP70 gene expression in male germ cells. Postepy Biochem,2006,52(3):289-295.
[7]Zhao M, Tang D, Lechpammer S, et al. Double-stranded RNA-dependent protein kinase (pkr) is essential for thermotolerance, accumulation of HSP70, and stabilization of ARE-containing HSP70 mRNA during stress. J Biol Chem,2002, 277(46):44539-44547.
[8]Sherman MY, Gabai V, O'Callaghan C, et al. Molecular chaperones regulate p53 and suppress senescence programs. FEBS Lett,2007,581(29):5732.
[9]Chae HD, Yun J, Shi DY.Transcription repression of a CCAAT-binding transcription factor CBF/HSP70 by p53. Exp Mol Med,2005,37(5):488-491.
[10]Esser C, Scheffher M, Hohfeld J.The chaperone-associated ubiquitin ligase CHIP is able to target p53 for proteasomal degradation. J Biol Chem,2005,280(29): 27443-27448.
[11]Frese S, Schaper M, Kuster JR, et al. Cell death induced by down-regulation of heat shock protein 70 in lung cancer cell lines is p53-independent and does not require DNA cleavage. J Thorac Cardiovasc Surg,2003,126(3):748-754.
[12]Abarzua F, Sakaguchi M, Tanimoto R, et al. Heat shock proteins play a crucial role in tumor-specific apoptosis by REIC/Dkk-3. Int J Mol Med,2007,20(1): 37-43.
[13]Rohde M, Daugaard M, Jensen MH, et al. Members of the heat-shock protein 70 family promote cancer cell growth by distinct mechanisms. Genes Dev,2005, 19(5):570-582.
[14]Bivik C, Rosdahl I, Ollinger K.Hsp70 protects against UVB induced apoptosis by preventing release of cathepsins and cytochrome c in human melanocytes. Carcinogenesis,2007,28(3):537-544.
[15]Han X, Wang F, Yao W.Heat shock proteins and p53 play a critical role in K+ channel-mediated tumor cell proliferation and apoptosis. Apoptosis,2007,12(10): 1837-1846.
[16]Lasunskaia EB, Fridlianskaia Ⅱ, Darieva ZA, et al.Transfection of NSO myeloma fusion partner cells with HSP70 gene results in higher hybridoma yield by improving cellular resistance to apoptosis. Biotechnol Bioeng,2003,81(4): 496-504.
[17]Phillips PA, Dudeja V, McCarroll JA, et al. Triptolide induces pancreatic cancer cell death via inhibition of heat shock protein 70. Cancer Res,2007,67(19): 9407-9416.
[18]Clemons NJ, Anderson RL.TRAIL-induced apoptosis is enhanced by heat shock protein 70 expression. Cell Stress Chaperones,2006,11(4):343-355.
[19]Afanasyeva EA, Komarova EY, Larsson LG, et al. Drug-induced Myc-mediated apoptosis of cancer cells is inhibited by stress protein Hsp70.Int J Cancer,2007, 121(12):2615-2621.
[20]Ray S, Lu Y, Kaufmann SH, et al. Genomic mechanisms of p210BCR-ABL signaling:induction of heat shock protein 70 through the GATA response element confers resistance to paclitaxel-induced apoptosis. J Biol Chem,2004,279(34): 35604-35615.
[21]Pocaly M, Lagarde V, Etienne G, et al. Overexpression of the heat-shock protein 70 is associated to imatinib resistance in chronic myeloid leukemia. Leukemia,2007,21(1):93-101.
[22]Fei Guo, Kathy Rocha, Purva Bali. Abrogation of heat shock protein 70 induction as a strategy to increase antileukemia activity of heat shock protein 90 inhibitor 17-Allylamino-Demethoxy geldanamycin. Cancer Research,2005,65(22): 10536-10544.
[23]Chauhan D, Li G, Auclair D, et al. Identification of genes regulated by 2-methoxyestradiol (2ME2) in multiple myeloma cells using oligonucleotide arrays. Blood,2003,101(9):3606-3614.
[24]Narayanan NK, Narayanan BA, et al. Docosahexaenoic acid in combination with celecoxib modulates HSP70 and p53 proteins in prostate cancer cells. Int J Cancer, 2006,119(7):1586-1598.
[25]Hu W, Wu W, Yeung SC,et al.Increased expression of heat shock protein 70 in adherent ovarian cancer and mesothelioma following treatment with manumycin, a farnesyl transferase inhibitor. Anticancer Res,2002,22(2A):665-672.
[26]Hashemi SM, Hassan ZM, Soudi S.Evaluation of anti-tumor effects of tumor cell lysate enriched by HSP-70 against fibrosarcoma tumor in BALB/c mice. Int Immunopharmacol,2007,7(7):920-927.
[27]Zhao ZG, Ma QZ, Xu CX. Abrogation of heat-shock protein (HSP) 70 expression induced cell growth inhibition and apoptosis in human androgen-independent prostate cancer cell line PC-3m. Asian J Androl,2004,6(4):319-324.
[28]Van Molle W, Van Roy M, Van Bogaert T, et al. Protection of zinc against tumor necrosis factor induced lethal inflammation depends on heat shock protein 70 and allows safe antitumor therapy. Cancer Res,2007,67(15):7301-7307.
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[1]Bolhassani A, Rafati S. Heat-shock proteins as powerful weapons in vaccine development. Expert Rev Vaccines,2008,7(8):1185-1199.
[2]Feige U, Polla NS. Hsp70--a multi-gene, multi-structure, multi-function family with potential clinical applications. Experentia,1994,50(11-12):979-986.
[3]Sherman M, Multhoff G. Heat shock proteins in cancer. Ann N Y Acad Sci,2007, 1113:192-201.
[4]Liu M, Aneja R, Liu C, et al. Inhibition of the mitotic kinesin Eg5 up-regulates Hsp70 through the phosphatidylinositol 3-kinase/Akt pathway in multiple myeloma cells. J Biol Chem,2006,281(26):18090-18097.
[5]Seo HR, Chung DY, Lee YJ, et al. Heat shock protein 25 or inducible heat shock protein 70 activates heat shock factor 1:dephosphorylation on serine 307 through inhibition of ERK1/2 phosphorylation. J Biol Chem,2006,281(25): 17220-17227.
[6]Widlak W.The heat shock response and HSP70 gene expression in male germ cells. Postepy Biochem,2006,52(3):289-295.
[7]Zhao M, Tang D, Lechpammer S, et al. Double-stranded RNA-dependent protein kinase (pkr) is essential for thermotolerance, accumulation of HSP70, and stabilization of ARE-containing HSP70 mRNA during stress. J Biol Chem,2002, 277(46):44539-44547.
[8]Sherman MY, Gabai V, O'Callaghan C, et al. Molecular chaperones regulate p53 and suppress senescence programs. FEBS Lett,2007,581(29):5732.
[9]Chae HD, Yun J, Shi DY.Transcription repression of a CCAAT-binding transcription factor CBF/HSP70 by p53. Exp Mol Med,2005,37(5):488-491.
[10]Esser C, Scheffher M, Hohfeld J.The chaperone-associated ubiquitin ligase CHIP is able to target p53 for proteasomal degradation. J Biol Chem,2005,280(29): 27443-27448.
[11]Frese S, Schaper M, Kuster JR, et al. Cell death induced by down-regulation of heat shock protein 70 in lung cancer cell lines is p53-independent and does not require DNA cleavage. J Thorac Cardiovasc Surg,2003,126(3):748-754.
[12]Abarzua F, Sakaguchi M, Tanimoto R, et al. Heat shock proteins play a crucial role in tumor-specific apoptosis by REIC/Dkk-3. Int J Mol Med,2007,20(1): 37-43.
[13]Rohde M, Daugaard M, Jensen MH, et al. Members of the heat-shock protein 70 family promote cancer cell growth by distinct mechanisms. Genes Dev,2005, 19(5):570-582.
[14]Bivik C, Rosdahl I, Ollinger K.Hsp70 protects against UVB induced apoptosis by preventing release of cathepsins and cytochrome c in human melanocytes. Carcinogenesis,2007,28(3):537-544.
[15]Han X, Wang F, Yao W.Heat shock proteins and p53 play a critical role in K+ channel-mediated tumor cell proliferation and apoptosis. Apoptosis,2007,12(10): 1837-1846.
[16]Lasunskaia EB, Fridlianskaia Ⅱ, Darieva ZA, et al.Transfection of NSO myeloma fusion partner cells with HSP70 gene results in higher hybridoma yield by improving cellular resistance to apoptosis. Biotechnol Bioeng,2003,81(4): 496-504.
[17]Phillips PA, Dudeja V, McCarroll JA, et al. Triptolide induces pancreatic cancer cell death via inhibition of heat shock protein 70. Cancer Res,2007,67(19): 9407-9416.
[18]Clemons NJ, Anderson RL.TRAIL-induced apoptosis is enhanced by heat shock protein 70 expression. Cell Stress Chaperones,2006,11(4):343-355.
[19]Afanasyeva EA, Komarova EY, Larsson LG, et al. Drug-induced Myc-mediated apoptosis of cancer cells is inhibited by stress protein Hsp70.Int J Cancer,2007, 121(12):2615-2621.
[20]Ray S, Lu Y, Kaufmann SH, et al. Genomic mechanisms of p210BCR-ABL signaling:induction of heat shock protein 70 through the GATA response element confers resistance to paclitaxel-induced apoptosis. J Biol Chem,2004,279(34): 35604-35615.
[21]Pocaly M, Lagarde V, Etienne G, et al. Overexpression of the heat-shock protein 70 is associated to imatinib resistance in chronic myeloid leukemia. Leukemia,2007,21(1):93-101.
[22]Fei Guo, Kathy Rocha, Purva Bali. Abrogation of heat shock protein 70 induction as a strategy to increase antileukemia activity of heat shock protein 90 inhibitor 17-Allylamino-Demethoxy geldanamycin. Cancer Research,2005,65(22): 10536-10544.
[23]Chauhan D, Li G, Auclair D, et al. Identification of genes regulated by 2-methoxyestradiol (2ME2) in multiple myeloma cells using oligonucleotide arrays. Blood,2003,101(9):3606-3614.
[24]Narayanan NK, Narayanan BA, et al. Docosahexaenoic acid in combination with celecoxib modulates HSP70 and p53 proteins in prostate cancer cells. Int J Cancer, 2006,119(7):1586-1598.
[25]Hu W, Wu W, Yeung SC,et al.Increased expression of heat shock protein 70 in adherent ovarian cancer and mesothelioma following treatment with manumycin, a farnesyl transferase inhibitor. Anticancer Res,2002,22(2A):665-672.
[26]Hashemi SM, Hassan ZM, Soudi S.Evaluation of anti-tumor effects of tumor cell lysate enriched by HSP-70 against fibrosarcoma tumor in BALB/c mice. Int Immunopharmacol,2007,7(7):920-927.
[27]Zhao ZG, Ma QZ, Xu CX. Abrogation of heat-shock protein (HSP) 70 expression induced cell growth inhibition and apoptosis in human androgen-independent prostate cancer cell line PC-3m. Asian J Androl,2004,6(4):319-324.
[28]Van Molle W, Van Roy M, Van Bogaert T, et al. Protection of zinc against tumor necrosis factor induced lethal inflammation depends on heat shock protein 70 and allows safe antitumor therapy. Cancer Res,2007,67(15):7301-7307.