1-硝基-2-酰基蒽醌-缬氨酸通过抑制ERK1/2激活和ERCC1表达诱导结肠癌细胞死亡
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摘要
化学方法合成是新药研发的一种重要途径。结合抗肿瘤药物的作用机制以及蒽醌类衍生物的构效关系,设计合成了一类新的蒽醌类衍生物1-硝基-2-酰基蒽醌-缬氨酸(简称C3),发现其具有很好的抗肿瘤活性。为了确定蒽醌类衍生物C3对结肠癌HCT116和HT29细胞的作用及其分子机制,首先通过MTT比色法检测C3对结肠癌HCT116和HT29细胞活性的影响。结果显示,C3对这两种结肠癌细胞具有明显的抑制作用,呈时间和剂量依赖性。60μg/mL的C3处理HCT116和HT29细胞48 h,细胞活性分别是50.67%和59.77%,达到了半抑制浓度;同时,其细胞形态和细胞核发生明显变化。进一步采用Western印迹和qRT-PCR技术,检测C3对DNA切除修复交叉互补1(excision repair cross-complementation group 1,ERCC1)转录水平和蛋白质水平表达及其稳定性的影响。结果表明,C3降低了ERCC1转录水平和蛋白质水平的表达,并且减弱了ERCC1转录水平和蛋白质水平的稳定性。最后,用U0126(MEK1/2抑制剂)和C3联合作用结肠癌HCT116和HT29细胞,通过Western印迹检测ERCC1蛋白质水平的表达。结果表明,C3通过降低p-ERK1/2的蛋白质水平的表达,从而抑制ERCC1的表达。上述结果证明,C3通过细胞外调节蛋白激酶(extracellular regulated protein kinases, ERK1/2)信号通路,降低了ERCC1转录水平和蛋白质水平的稳定性,使ERCC1转录水平和蛋白质水平表达发生下调,进而抑制结肠癌HCT116和HT29细胞的活性。
Chemical synthesis is an important way to develop new drugs. A new class of anthraquinone derivatives, 1-nitro-2-acylanthraquinone-valine(C3), was designed and synthesized by combining the mechanism of antitumor drugs and the structure-activity relationship of anthraquinone derivatives. It was found that C3 has good antitumor activity. Therefore, we aim to determine the effect of anthraquinone derivative C3 on human colon cancer HCT116 and HT29 cells and its molecular mechanism. Firstly, the effects of C3 on the activity of colon cancer HCT116 and HT29 cells were detected by MTT colorimetry. The results showed that C3 could inhibit these two kinds of colon cancer cells in a time-and dose-dependent manner. Moreover, HCT116 and HT29 cells were treated with C3 at 60 μg/mL for 48 hours, and the cell viability was 50.67% and 59.77% respectively, which reached a semi-inhibitory concentration. At the same time, cellular and nuclear morphology changed significantly. Western blotting and qRT-PCR were used to detect the effects of C3 on the expression of ERCC1(excision repair cross-complementation group 1) and stability of ERCC1 protein levels. The results showed that both the transcriptional and protein levels were decreased. Finally, HCT116 and HT29 cells were treated with U0126(MEK1/2 inhibitor) and C3. The abundance of ERCC1 proteins was detected by Western blotting. The results showed that C3 inhibited the expression of ERCC1 by reducing p-ERK1/2 proteins. These results suggest that C3 reduces ERCC1 transcription levels and ERCC1 protein levels through the ERK1/2(extracellular regulated protein kinase) signaling pathway, and down-regulates the expression of ERCC1 and its protein levels, thereby inhibiting the activity of HCT116 and HT29 cells.
Chemical synthesis is an important way to develop new drugs. A new class of anthraquinone derivatives, 1-nitro-2-acylanthraquinone-valine(C3), was designed and synthesized by combining the mechanism of antitumor drugs and the structure-activity relationship of anthraquinone derivatives. It was found that C3 has good antitumor activity. Therefore, we aim to determine the effect of anthraquinone derivative C3 on human colon cancer HCT116 and HT29 cells and its molecular mechanism. Firstly, the effects of C3 on the activity of colon cancer HCT116 and HT29 cells were detected by MTT colorimetry. The results showed that C3 could inhibit these two kinds of colon cancer cells in a time-and dose-dependent manner. Moreover, HCT116 and HT29 cells were treated with C3 at 60 μg/mL for 48 hours, and the cell viability was 50.67% and 59.77% respectively, which reached a semi-inhibitory concentration. At the same time, cellular and nuclear morphology changed significantly. Western blotting and qRT-PCR were used to detect the effects of C3 on the expression of ERCC1(excision repair cross-complementation group 1) and stability of ERCC1 protein levels. The results showed that both the transcriptional and protein levels were decreased. Finally, HCT116 and HT29 cells were treated with U0126(MEK1/2 inhibitor) and C3. The abundance of ERCC1 proteins was detected by Western blotting. The results showed that C3 inhibited the expression of ERCC1 by reducing p-ERK1/2 proteins. These results suggest that C3 reduces ERCC1 transcription levels and ERCC1 protein levels through the ERK1/2(extracellular regulated protein kinase) signaling pathway, and down-regulates the expression of ERCC1 and its protein levels, thereby inhibiting the activity of HCT116 and HT29 cells.
引文
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[7] Li Y, Lu X, Qi H, et al. Ursolic acid induces apoptosis through mitochondrial intrinsic pathway and suppression of ERK1/2 MAPK in HeLa cells[J]. J Pharmacol Sci, 2014, 125(2): 202-210
[8] Shi H, Bi H, Sun X, et al. Tubeimoside-1 inhibits the proliferation and metastasis by promoting miR-126-5p expression in non-small cell lung cancer cells[J]. Oncol Lett, 2018, 16(3): 3126-3134
[9] Liao T, Wei WJ, Wen D, et al. Verteporfin inhibits papillary thyroid cancer cells proliferation and cell cycle through ERK1/2 signaling pathway[J]. J Cancer, 2018, 9(8): 1329-1336
[10] Chen G, Qiu H, Ke SD, et al. Emodin regulating excision repair cross-complementation group 1 through fibroblast growth factor receptor 2 signaling[J]. World J Gastroenterol, 2013, 19(16): 2481-2491
[11] Liu J, Zhang L, Mao P, et al. Functional characterization of a novel transcript of ERCC1 in chemotherapy resistance of ovarian cancer[J]. Oncotarget, 2017, 8(49): 85759-85771
[12] Su Y, Orelli B, Madireddy A, et al. Multiple DNA binding domains mediate the function of the ERCC1-XPF protein in nucleotide excision repair[J]. J Biol Chem, 2012, 287(26): 21846-21855
[13] Takahata C, Masuda Y, Takedachi A, et al. Repair synthesis step involving ERCC1-XPF participates in DNA repair of the Top1-DNA damage complex[J]. Carcinogenesis, 2015, 36(8): 841-851
[14] Ko JC, Su YJ, Lin ST, et al. Emodin enhances cisplatin-induced cytotoxicity via down-regulation of ERCC1 and inactivation of ERK1/2[J]. Lung Cancer, 2010, 69(2): 155-164
[15] ?zenver N, Saeed M, Demirezer L?, et al. Aloe-emodin as drug candidate for cancer therapy[J]. Oncotarget, 2018, 9(25): 17770-17796
[16] Tabolacci C, Cordella M, Turcano L, et al. Aloe-emodin exerts a potent anticancer and immunomodulatory activity on BRAF-mutated human melanoma cells[J]. Eur J Pharmacol, 2015, 762: 283-292
[17] Subramaniam A, Loo SY, Rajendran P, et al. An anthraquinone derivative, emodin sensitizes hepatocellular carcinoma cells to TRAIL induced apoptosis through the induction of death receptors and downregulation of cell survival proteins[J]. Apoptosis, 2013, 18(10): 1175-1187
[18] Guo J, Xiao B, Liu Q, et al. Suppression of C-myc expression associates with anti-proliferation of aloe-emodin on gastric cancer cells[J]. Cancer Invest, 2008, 26(4): 369-374
[19] 李玉英, 牛敏, 张立伟, 等. 新的蒽醌类衍生物 1-硝基-2-酰基蒽醌-苯丙氨酸通过抑制NF-κB/p65 信号通路降低乳腺癌 MCF-7 细胞的迁移能力[J]. 中国生物化学与分子生物学报(Li YY, Niu M, Zhang LW, et al. A novel anthraquinone derivative 1-nitro-2-acylanthraquinone- phenylalanine inhibits human breast cancer MCF-7 cell migration by blocking NF-κB/p65 pathway[J] Chin J Biochem Mol Biol), 2017, 33(3): 269-276
[20] Garcia-Vilas JA, Quesada AR, Medina MA. Damnacanthal, a noni anthraquinone, inhibits c-Met and is a potent antitumor compound against Hep G2 human hepatocellular carcinoma cells[J]. Sci Rep, 2015, 5: 8021
[21] Hong JY, Chung HJ, Bae SY, et al. Induction of cell cycle arrest and apoptosis by physcion, an anthraquinone isolated from rhubarb (rhizomes of rheum tanguticum), in MDA-MB-231 human breast cancer cells[J]. J Cancer Prev, 2014, 19(4): 273-278
[22] You L, Dong X, Yin X, et al. Rhein induces cell death in hepaRG cells through cell cycle arrest and apoptotic pathway[J]. Int J Mol Sci, 2018, 19(4).pii:E 1060
[23] Song X, Zhou X, Qin Y, et al. Emodin inhibits epithelial-mesenchymal transition and metastasis of triple negative breast cancer via antagonism of CC-chemokine ligand 5 secreted from adipocytes[J]. Int J Mol Med, 2018, 42(1): 579-588
[24] Lim W, Yang C, Bazer FW, et al. Chrysophanol induces apoptosis of choriocarcinoma through regulation of ROS and the AKT and ERK1/2 pathways[J]. J Cell Physiol, 2017, 232(2): 331-339
[25] Aziz MY, Omar AR, Subramani T, et al. Damnacanthal is a potent inducer of apoptosis with anticancer activity by stimulating p53 and p21 genes in MCF-7 breast cancer cells[J].Oncol Lett,2014,7(5):1479- 1484
[26] Reef S, Zalckvar E, Shifman O, et al. A short mitochondrial form of p19ARF induces autophagy and caspase-independent cell death[J]. Mol Cell, 2006, 22(4): 463-475
[27] Tsai MS, Weng SH, Kuo YH, et al. Synergistic effect of curcumin and cisplatin via down-regulation of thymidine phosphorylase and excision repair cross-complementary 1 (ERCC1)[J]. Mol Pharmacol, 2011, 80(1): 136-146
[28] Ko JC, Su YJ, Lin ST, et al. Suppression of ERCC1 and Rad51 expression through ERK1/2 inactivation is essential in emodin-mediated cytotoxicity in human non-small cell lung cancer cells[J]. Biochem Pharmacol, 2010, 79(4): 655-664
[29] Ko JC, Tsai MS, Kuo YH, et al. Modulation of Rad51, ERCC1, and thymidine phosphorylase by emodin result in synergistic cytotoxic effect in combination with capecitabine[J]. Biochem Pharmacol, 2011, 81(5): 680-690
[30] Faridounnia M, Wienk H, Kova■i■ L, et al. The cerebro-oculo-facio-skeletal syndrome point mutation F231L in the ERCC1 DNA repair protein causes dissociation of the ERCC1-XPF complex[J]. J Biol Chem, 2015, 290(33): 20541-20555
[31] Perez-Oliva AB, Lachaud C, Szyniarowski P, et al. USP45 deubiquitylase controls ERCC1-XPF endonuclease-mediated DNA damage responses[J]. EMBO J, 2015, 34(3): 326-343
[32] Fu JM, Zhou J, Shi J, et al. Emodin affects ERCC1 expression in breast cancer cells[J]. J Transl Med, 2012, 10 Suppl 1: S7
[2] Patil S, Kuman MM, Palvai S, et al. Impairing powerhouse in colon cancer cells by hydrazide-hydrazone- based small molecule[J]. ACS Omega, 2018, 3(2): 1470-1481
[3] Chen X, Lu K, Timko NJ, et al. IL-33 notably inhibits the growth of colon cancer cells[J]. Oncol Lett, 2018, 16(1): 769-774
[4] Palvai S, Kuman MM, Sengupta P, et al. Hyaluronic acid layered chimeric nanoparticles: targeting MAPK-PI3K signaling hub in colon cancer cells[J]. ACS Omega, 2017, 2(11): 7868-7880
[5] Bi Y, Wang G, Liu X, et al. Low-after-high glucose down-regulated Cx43 in H9c2 cells by autophagy activation via cross-regulation by the PI3K / Akt /mTOR and MEK / ERK1/2 signal pathways[J]. Endocrine, 2017, 56(2): 336-345
[6] Dang JH, Jin ZJ, Liu XJ, et al. Metformin in combination with cisplatin inhibits cell viability and induces apoptosis of human ovarian cancer cells by inactivating ERK 1/2[J]. Oncol Lett, 2017, 14(6): 7557-7564
[7] Li Y, Lu X, Qi H, et al. Ursolic acid induces apoptosis through mitochondrial intrinsic pathway and suppression of ERK1/2 MAPK in HeLa cells[J]. J Pharmacol Sci, 2014, 125(2): 202-210
[8] Shi H, Bi H, Sun X, et al. Tubeimoside-1 inhibits the proliferation and metastasis by promoting miR-126-5p expression in non-small cell lung cancer cells[J]. Oncol Lett, 2018, 16(3): 3126-3134
[9] Liao T, Wei WJ, Wen D, et al. Verteporfin inhibits papillary thyroid cancer cells proliferation and cell cycle through ERK1/2 signaling pathway[J]. J Cancer, 2018, 9(8): 1329-1336
[10] Chen G, Qiu H, Ke SD, et al. Emodin regulating excision repair cross-complementation group 1 through fibroblast growth factor receptor 2 signaling[J]. World J Gastroenterol, 2013, 19(16): 2481-2491
[11] Liu J, Zhang L, Mao P, et al. Functional characterization of a novel transcript of ERCC1 in chemotherapy resistance of ovarian cancer[J]. Oncotarget, 2017, 8(49): 85759-85771
[12] Su Y, Orelli B, Madireddy A, et al. Multiple DNA binding domains mediate the function of the ERCC1-XPF protein in nucleotide excision repair[J]. J Biol Chem, 2012, 287(26): 21846-21855
[13] Takahata C, Masuda Y, Takedachi A, et al. Repair synthesis step involving ERCC1-XPF participates in DNA repair of the Top1-DNA damage complex[J]. Carcinogenesis, 2015, 36(8): 841-851
[14] Ko JC, Su YJ, Lin ST, et al. Emodin enhances cisplatin-induced cytotoxicity via down-regulation of ERCC1 and inactivation of ERK1/2[J]. Lung Cancer, 2010, 69(2): 155-164
[15] ?zenver N, Saeed M, Demirezer L?, et al. Aloe-emodin as drug candidate for cancer therapy[J]. Oncotarget, 2018, 9(25): 17770-17796
[16] Tabolacci C, Cordella M, Turcano L, et al. Aloe-emodin exerts a potent anticancer and immunomodulatory activity on BRAF-mutated human melanoma cells[J]. Eur J Pharmacol, 2015, 762: 283-292
[17] Subramaniam A, Loo SY, Rajendran P, et al. An anthraquinone derivative, emodin sensitizes hepatocellular carcinoma cells to TRAIL induced apoptosis through the induction of death receptors and downregulation of cell survival proteins[J]. Apoptosis, 2013, 18(10): 1175-1187
[18] Guo J, Xiao B, Liu Q, et al. Suppression of C-myc expression associates with anti-proliferation of aloe-emodin on gastric cancer cells[J]. Cancer Invest, 2008, 26(4): 369-374
[19] 李玉英, 牛敏, 张立伟, 等. 新的蒽醌类衍生物 1-硝基-2-酰基蒽醌-苯丙氨酸通过抑制NF-κB/p65 信号通路降低乳腺癌 MCF-7 细胞的迁移能力[J]. 中国生物化学与分子生物学报(Li YY, Niu M, Zhang LW, et al. A novel anthraquinone derivative 1-nitro-2-acylanthraquinone- phenylalanine inhibits human breast cancer MCF-7 cell migration by blocking NF-κB/p65 pathway[J] Chin J Biochem Mol Biol), 2017, 33(3): 269-276
[20] Garcia-Vilas JA, Quesada AR, Medina MA. Damnacanthal, a noni anthraquinone, inhibits c-Met and is a potent antitumor compound against Hep G2 human hepatocellular carcinoma cells[J]. Sci Rep, 2015, 5: 8021
[21] Hong JY, Chung HJ, Bae SY, et al. Induction of cell cycle arrest and apoptosis by physcion, an anthraquinone isolated from rhubarb (rhizomes of rheum tanguticum), in MDA-MB-231 human breast cancer cells[J]. J Cancer Prev, 2014, 19(4): 273-278
[22] You L, Dong X, Yin X, et al. Rhein induces cell death in hepaRG cells through cell cycle arrest and apoptotic pathway[J]. Int J Mol Sci, 2018, 19(4).pii:E 1060
[23] Song X, Zhou X, Qin Y, et al. Emodin inhibits epithelial-mesenchymal transition and metastasis of triple negative breast cancer via antagonism of CC-chemokine ligand 5 secreted from adipocytes[J]. Int J Mol Med, 2018, 42(1): 579-588
[24] Lim W, Yang C, Bazer FW, et al. Chrysophanol induces apoptosis of choriocarcinoma through regulation of ROS and the AKT and ERK1/2 pathways[J]. J Cell Physiol, 2017, 232(2): 331-339
[25] Aziz MY, Omar AR, Subramani T, et al. Damnacanthal is a potent inducer of apoptosis with anticancer activity by stimulating p53 and p21 genes in MCF-7 breast cancer cells[J].Oncol Lett,2014,7(5):1479- 1484
[26] Reef S, Zalckvar E, Shifman O, et al. A short mitochondrial form of p19ARF induces autophagy and caspase-independent cell death[J]. Mol Cell, 2006, 22(4): 463-475
[27] Tsai MS, Weng SH, Kuo YH, et al. Synergistic effect of curcumin and cisplatin via down-regulation of thymidine phosphorylase and excision repair cross-complementary 1 (ERCC1)[J]. Mol Pharmacol, 2011, 80(1): 136-146
[28] Ko JC, Su YJ, Lin ST, et al. Suppression of ERCC1 and Rad51 expression through ERK1/2 inactivation is essential in emodin-mediated cytotoxicity in human non-small cell lung cancer cells[J]. Biochem Pharmacol, 2010, 79(4): 655-664
[29] Ko JC, Tsai MS, Kuo YH, et al. Modulation of Rad51, ERCC1, and thymidine phosphorylase by emodin result in synergistic cytotoxic effect in combination with capecitabine[J]. Biochem Pharmacol, 2011, 81(5): 680-690
[30] Faridounnia M, Wienk H, Kova■i■ L, et al. The cerebro-oculo-facio-skeletal syndrome point mutation F231L in the ERCC1 DNA repair protein causes dissociation of the ERCC1-XPF complex[J]. J Biol Chem, 2015, 290(33): 20541-20555
[31] Perez-Oliva AB, Lachaud C, Szyniarowski P, et al. USP45 deubiquitylase controls ERCC1-XPF endonuclease-mediated DNA damage responses[J]. EMBO J, 2015, 34(3): 326-343
[32] Fu JM, Zhou J, Shi J, et al. Emodin affects ERCC1 expression in breast cancer cells[J]. J Transl Med, 2012, 10 Suppl 1: S7