2016 31 1 29 DMAS PERK NSCLC PC9 /GR 1 2* 3 1. 310053 2. 310006 3. 310006 β β'- β dimethylacryl shikonin DMAS PKR ER PKR-like ER kinaseperk PC9 /GR DMAS PC9 /GR CCK-8 Western blot 78 glucose regulated protein 78 GRP78 C /EBP C /EBP homologous protein CHOP unfolded protein response UPR PERK GSK2606414 Annexin-V-PI Western blot caspase-3 caspase-8 caspase-9 poly ADP-ribose polymerase PARP DMAS 0 5 10 15 μmol /L PC9 /GR P < 0. 05 DMAS PC9 /GR caspase-3 caspase-8 caspase-9 PARP P < 0. 05 GRP78 CHOP 0 2 4 8 P < 0. 05 PERK 2 eukaryotic initiation factor-2α eif2α 4 activating transcription factor 4 ATF4 P < 0. 05 UPR 1α inositol-requiring enzyme 1α IRE1α X 1 X-box binding protein 1 spliced XBP1s ATF6 P > 0. 05 DMAS GSK2606414 P < 0. 05 PERK P < 0. 05 caspase-3 caspase-8 caspase-9 PARP P < 0. 05 PERK-eIF2α DMAS PC9 /GR / / R734. 2 R730. 53 2015-08-11 81303114 1990-. * E-mail alexisczh@ sohu. com 檭檭檭檭檭檭檭檭檭檭檭檭檭檭檭檭檭檭檭檭檭檭檭檭檭檭檭檭檭檭檭檭檭檭檭檭檭檭檭檭檭檭檭檭檭檭 0 1 2 3 4 2015 ESMO NED no evidence of disease 3 States National Cancer Institute of CanadaJ. J Natl Cancer Inst 2000 92 3 205-216. A 1001-1692 2016 01-0029-06 1Therasse P Arbuck SG Eisenhauer EA et al. New guidelines to evaluate the response to treatment in solid tumors. European Organization for Research and Treatment of Cancer National Cancer Institute of the United 2Suarez J. Analysis of clinical and economic implications 2 MDT of Esmo 2014 Clinical Practice Guidelines for metastatic 4 MDT colorectal cancer treatmentj. Value Health 2015 18 3 MDT 7 A446. 3Van Cutsem E Cervantes A Nordlinger B et al. Metastatic colorectal cancer ESMO Clinical Practice Guide- lines for diagnosis treatment and follow-up J. Ann On- col 2014 25 Suppl 3 iii1-9. 4. J. 2014 17 11 1060-1061.
30 Journal of Practical Oncology Vol. 31 No. 1 2016 DOI:10.13267/j.cnki.syzlzz.2016.01.008 Effect of DMAS combined with PERK inhibitor on apoptosis of human NSCLC PC9 /GR cells Yu Yu 1 Chen Zhe 2 Ruan Shanming 3 1. The First Clinical Medicine College Zhejiang Chinese Medical UniversityHangzhou 310053 China 2. Central Laboratory Zhejiang Provincial Hospital of TCM Hangzhou 310006 China 3. Department of OncologyZhejiang Provincial Hospital of TCM Hangzhou 310006 China Abstract Objective To investigate the effect of β β'-dimethylacryl shikonin DMAS combined with PKR-like ER kinase PERK inhibitor on the apoptosis of gefitinib resistant non-small-cell lung cancer NSCLC PC9 /GR cells and its molecular mechanism. Methods In DMAS treated PC9 /GR cellscell proliferation was measured by CCK-8 method and the levels of glucose regulated protein 78 GRP78 C /EBP homologous protein CHOP and proteins related to the unfolded protein response UPR signaling pathway were examined by Western blot. After treated with PERK inhibitor GSK2606414Annexin-V-PI flow cytometric analysis was used to study the cell apoptosis rate of PC9 /GR cells. The cleavage of apoptosis related proteinsincluding caspase-3caspase-8caspase-9and poly ADP-ribose polymerase PARP were analyzed by Western blot. Results DMAS inhibited the proliferation of PC9 / GR cells in a dose-dependent manner 0 5 10 15 μmol /L P < 0. 05. DMAS also dose-dependently induced PC9 /GR apoptosisas well as the cleavage of caspase-3 caspase-8 caspase-9 and PARP all P < 0. 05 endoplasmic reticulum stress markers GRP78 and CHOP were upregulated in a time-dependent manner 0 2 4 8 h P < 0. 05. Increased phosphorylation of PERK and eukaryotic initiation factor-2α eif2α were observedtogether with elevated expression of its substrate activating transcription factor 4 ATF4 all P < 0. 05. However the level of other URP related proteins were not changedincluding inositol-requiring enzyme 1α IRE1α X-box binding protein 1 spliced XBP1s and ATF6 all P > 0. 05. Compared with DMAS or GSK2606414 single treatmentdmas and GSK2606414 combined therapy led to increased cell apoptosis P < 0. 05 reduced PERK phosphorylation P < 0. 05 increased cleavage of caspase-3caspase-8caspase-9 and increased phosphorylation of PARP P < 0. 05. Conclusion PERK-eIF2α pathway plays a protective role in the pro-apoptotic process of DMAS in PC9 /GR cells. Key words carcinoma non-small-cell lung /drug therapy SHIKONIN /therapeutic use apoptosis endoplasmic reticulum stress protein kinases / -κb nuclear factor κb NF- κb non-small-cell lung cancer NSCLC 80% 8 DMAS 1 PC9 /GR DMAS PC9 /GR 2 β β'- β β'- dimethylacryl shikonin 6 4 Notch-1 7 1 1. 1 NSCLC PC9 DMAS 3 PC9 /GR DMAS 1. 2 β β'- 1 epidermal DMEM growth factor receptor EGFR 4 2 EDTA reactive oxygen species ROS 5 3 Hyclone DMAS
2016 31 1 31 PKR ER PKR-like 37 5% CO 2 10 μmol /L ER kinase PERK 2 eukaryotic initiation factor-2α eif2α P-eIF2α 4 GSK2606414 24 DMAS 10 μmol /L GSK2606414 DMAS activating transcription factor 4 ATF4 1α FACS Canto TM Ⅱ inositol-requiring enzyme 1α IRE1α X 1 X-box binding protein 1 spliced XBP1s poly ADP- PC9 /GR 25 cm 2 ribose polymerase PARP cleaved caspase-3 cleaved caspase-8 cleaved caspase-9 β-actin 0 5 10 15 μmol /L DMAS 24 Santa Cruz Biotechnology Cell Counting Kit-8 FITC Annexin V Appoptosis Detection Kit BD FACS Canto TM Ⅱ BD SDS-PAGE GSK2606414 30 10 μmol /L DMAS 4 4 10 μmol /L DMAS 10 μmol /L GSK2606414 10 μmol /L BIO-RAD DMAS 10 μmol /L GSK2606414 1. 3 PC9 PC9 /GR PC9 PC9 / Annexin V-FITC 5 μl propidium iodidepi 30 400 μl 1 Binding Buffer GR 37 5% CO 2 DMEM PBS 1 RI- 10% FBS 20 mmol /L NaHCO 3 20 mmol /L PA 30 13 300 r /min HEPES 100 U /ml 100 mg /L 80% ~ 90% 2 SDS 100 5 1. 4 CCK-8 SDS-PAGE NSCLC PC9 PC9 /GR 6 10 4 PVDF 96 37 5% CO 2 0 5 10 15 20 μmol /L Thermo VARIOSKAN FLASH 450 9 PARP 2 78 glucose nm 30 regulated protein 78 GRP78 C /EBP C /EBP homologous % = - / proteinchop PERK P-PERK eif2α P-eIF2α - 100% 3 1. 5 Annexin-V-PI PERK P-eIF2α ATF4 2 NSCLC PC9 /GR 10 10 5 ~ 15 10 5 / 12 37 5% CO 2 0 5 10 15 μmol /L DMAS 24 0. 1 ml Chemi Dox 100 μl 1 Binding Buffer 5 μl FACS Canto TM Ⅱ NSCLC PC9 /GR 1. 6 Western blot NSCLC 70% ~ 80% 1 Cell Signaling Technology P-PERK ATF6 2 10 μmol /L DMAS 0 2 4 8 3 10 μmol /L 24 15 DC 5% /BSA TBST 2 PVDF DMAS PC9 PC9 /GR 24 5% 1 4 10 μl CCK-8 1-3 cleaved caspase-3 cleaved caspase-8 cleaved caspase- ATF4 IRE1α XBP1s ATF6 3 P- 3 10 / PVDF 2 3 5 / ECL A B XRS 1. 7 SPSS 17. 0 ANOVA P < 0. 05 2 10 10 5 ~ 15 10 5 / 12 2. 1 DMAS PC9 PC9 /GR DMAS
32 Journal of Practical Oncology Vol. 31 No. 1 2016 PC9 PC9 /GR 4. 9 ± 1. 8 % 17. 6 ± DMAS 0 5 10 15 20 μmol /L 24 4. 0 % 24. 7 ± 6. 0 % 33. 0 ± 5. 0 % P < 0. 05 PC9 19. 1% 2A Western blot 26. 1% 33. 4% 53. 3% PC9 /GR caspase-3 caspase-8 caspase-9 13. 4% 28. 5% 37. 3% 57. 8% PARP P P < 0. 052B DMAS PC9 / < 0. 05 DMAS 2 GR caspase P > 0. 05 DMAS PC9 2. 3 DMAS DMAS 10 μmol /L PC9 /GR PC9 /GR 2 4 8 Western blot 1 GRP78 CHOP P < 0. 053A PERK eif2α 2 4 8 1 DMAS PC9 PC9 /GR DMAS PC9 /GR PERK- 2. 2 DMAS PC9 /GR eif2α IRE1-XBP1s ATF6 DMAS 0 5 10 15 μmol /L PC9 /GR 24 P < 0. 05 PERK eif2α P > 0. 05 PERKeIF2α ATF4 4 P < 0. 053B IRE1 α ATF6 IRE1 α XBP1s P > 0. 053C 2 DMAS PC9 /GR A B A GRP78 CHOP B PERK eif2α ATF4 PERK eif2α C IRE1α XBP1s ATF6 3 10 μmol /L DMAS PC9 /GR
2016 31 1 33 2. 4 DMAS GSK2606414 caspase-3 caspase-8 caspase-9 PARP PERK P < 0. 05 GSK2606414 10 μmol /L PERK eif2α 63. 9% DMAS ATF4 P < 0. 054A DMAS 10 μmol /L DMAS 23. 8% P < 0. 01 4B GSK2606414 DMAS PC9 / GSK2606414 P < GR DMAS 0. 05 PERK PERK-eIF2α A 10 μmol /L GSK2606414 30 10 μmol /LDMAS 4 Western blot PERK eif2α ATF4 B C 10 μmol /L DMAS 10 μmol /L GSK2606414 10 μmol /L DMAS 10 μmol /L GSK2606414 PC9 /GR 24 B Western blot caspase-3 caspase-8 caspase-9 PARP C 3 4 PERK DMAS PC9 /GR GRP78 PERK eif2a GRP78 eif2a P-eIF2a 3 ATF4 IRE1 PERK ATF6 3 DMAS PC9 /GR PERK-eIF2α PERK-eIF2α IRE1-XBP1 ATF6 unfolded protein response UPR 8 DMAS PERK-eIF2α IRE1-XBP1 ATF6 ER associated PERK protein degradation ERAD GSK2606414 DMAS PC9 /GR PERK-eIF2α DMAS PC9 /GR DMAS 9 PERK-eIF2α ROS 1 eif2a 10 2 11 12 3 PERK E2 PERK-eIF2α PERK /eif2α PERK 2 nuclear factor erythroid 2-related factor 2 IRE1-XBP1 ATF6 3 Nrf2 Kelch -1 Kelch-like
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