CCK-8. Western blot 78 glucose regulated protein 78 GRP78 C /EBP. C /EBP homologous protein CHOP. poly ADP-ribose polymerase PARP

Transcript

1 DMAS PERK NSCLC PC9 /GR 1 2* 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 GSK Annexin-V-PI Western blot caspase-3 caspase-8 caspase-9 poly ADP-ribose polymerase PARP DMAS μmol /L PC9 /GR P < DMAS PC9 /GR caspase-3 caspase-8 caspase-9 PARP P < GRP78 CHOP P < PERK 2 eukaryotic initiation factor-2α eif2α 4 activating transcription factor 4 ATF4 P < UPR 1α inositol-requiring enzyme 1α IRE1α X 1 X-box binding protein 1 spliced XBP1s ATF6 P > DMAS GSK P < PERK P < caspase-3 caspase-8 caspase-9 PARP P < PERK-eIF2α DMAS PC9 /GR / / R R * 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 ESMO NED no evidence of disease 3 States National Cancer Institute of CanadaJ. J Natl Cancer Inst A Therasse 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 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 Suppl 3 iii J

2 30 Journal of Practical Oncology Vol. 31 No DOI: /j.cnki.syzlzz 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 China 2. Central Laboratory Zhejiang Provincial Hospital of TCM Hangzhou China 3. Department of OncologyZhejiang Provincial Hospital of TCM Hangzhou 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 GSK Annexin-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 μmol /L P < DMAS also dose-dependently induced PC9 /GR apoptosisas well as the cleavage of caspase-3 caspase-8 caspase-9 and PARP all P < endoplasmic reticulum stress markers GRP78 and CHOP were upregulated in a time-dependent manner h P < 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 < 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 > Compared with DMAS or GSK single treatmentdmas and GSK combined therapy led to increased cell apoptosis P < reduced PERK phosphorylation P < increased cleavage of caspase-3caspase-8caspase-9 and increased phosphorylation of PARP P < 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 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

3 PKR ER PKR-like 37 5% CO 2 10 μmol /L ER kinase PERK 2 eukaryotic initiation factor-2α eif2α P-eIF2α 4 GSK DMAS 10 μmol /L GSK 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 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 GSK μmol /L DMAS μmol /L DMAS 10 μmol /L GSK μmol /L BIO-RAD DMAS 10 μmol /L GSK PC9 PC9 /GR PC9 PC9 / Annexin V-FITC 5 μl propidium iodidepi μl 1 Binding Buffer GR 37 5% CO 2 DMEM PBS 1 RI- 10% FBS 20 mmol /L NaHCO 3 20 mmol /L PA r /min HEPES 100 U /ml 100 mg /L 80% ~ 90% 2 SDS CCK-8 SDS-PAGE NSCLC PC9 PC9 /GR PVDF % CO μmol /L Thermo VARIOSKAN FLASH PARP 2 78 glucose nm 30 regulated protein 78 GRP78 C /EBP C /EBP homologous % = - / proteinchop PERK P-PERK eif2α P-eIF2α - 100% Annexin-V-PI PERK P-eIF2α ATF4 2 NSCLC PC9 /GR ~ / % CO μmol /L DMAS 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 ATF μmol /L DMAS μmol /L DC 5% /BSA TBST 2 PVDF DMAS PC9 PC9 /GR 24 5% μl CCK cleaved caspase-3 cleaved caspase-8 cleaved caspase- ATF4 IRE1α XBP1s ATF6 3 P / PVDF / ECL A B XRS 1. 7 SPSS ANOVA P < ~ / DMAS PC9 PC9 /GR DMAS

4 32 Journal of Practical Oncology Vol. 31 No PC9 PC9 /GR 4. 9 ± 1. 8 % ± DMAS μmol /L % ± 6. 0 % ± 5. 0 % P < PC % 2A Western blot 26. 1% 33. 4% 53. 3% PC9 /GR caspase-3 caspase-8 caspase % 28. 5% 37. 3% 57. 8% PARP P P < B DMAS PC9 / < DMAS 2 GR caspase P > DMAS PC DMAS DMAS 10 μmol /L PC9 /GR PC9 /GR Western blot 1 GRP78 CHOP P < A PERK eif2α DMAS PC9 PC9 /GR DMAS PC9 /GR PERK DMAS PC9 /GR eif2α IRE1-XBP1s ATF6 DMAS μmol /L PC9 /GR 24 P < PERK eif2α P > PERKeIF2α ATF4 4 P < B IRE1 α ATF6 IRE1 α XBP1s P > C 2 DMAS PC9 /GR A B A GRP78 CHOP B PERK eif2α ATF4 PERK eif2α C IRE1α XBP1s ATF μmol /L DMAS PC9 /GR

5 DMAS GSK caspase-3 caspase-8 caspase-9 PARP PERK P < GSK μmol /L PERK eif2α 63. 9% DMAS ATF4 P < A DMAS 10 μmol /L DMAS 23. 8% P < B GSK DMAS PC9 / GSK P < GR DMAS PERK PERK-eIF2α A 10 μmol /L GSK μmol /LDMAS 4 Western blot PERK eif2α ATF4 B C 10 μmol /L DMAS 10 μmol /L GSK μmol /L DMAS 10 μmol /L GSK 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 GSK DMAS PC9 /GR PERK-eIF2α DMAS PC9 /GR DMAS 9 PERK-eIF2α ROS 1 eif2a PERK E2 PERK-eIF2α PERK /eif2α PERK 2 nuclear factor erythroid 2-related factor 2 IRE1-XBP1 ATF6 3 Nrf2 Kelch -1 Kelch-like

6 34 Journal of Practical Oncology Vol. 31 No ECH-associated protein-1 Keap-1 - E2 2 - antioxidant-response elementare Keap1-Nrf2-ARE ROS DNA pathway in vitro and in vivoj. Biochem Pharmacol PERK-eIF2α DMAS 8. PC9 /GR J DMAS 9. 10Chevet EHetz CSamali Aet al. Endoplasmic reticu- 1. lum stress-activated cell reprogramming in oncogenesis NSCLC J. C. Cancer Discov and extrinsic activators of the unfolded protein response J in cancer Mechanisms and targets for therapy J. Se- 3. min Cancer Biol J Zhao QKretschmer NBauer Ret al. Shikonin and its derivatives inhibit the epidermal growth factor receptor signaling and synergistically kill glioblastoma cells in combination with erlotinibj. Int J Cancer Kwak SY Jeong YKKim BYet al. Beta beta-dimethylacrylshikonin sensitizes human colon cancer cells to ionizing radiation through the upregulation of reactive oxygen species J. Oncol Lett Xiong YMa XYZhang Zet al. Apoptosis induced by β β-dimethylacrylshikonin is associated with Bcl-2 and NF-κB in human breast carcinoma MCF-7 cells J. Oncol Lett Shao ZJZhang YYFan YYet al. β β-dimethylacrylshikonin exerts antitumor activity via Notch-1 signaling J Tameire FVerginadis IIKoumenis C. Cell intrinsic 12Kouroku YFujita EIUeno Tet al. ER stress PERK /eif2alpha phosphorylation mediates the polyglutamine-induced LC3 conversionan essential step for autophagy formationj. Cell Death Differ Bobrovnikova-Marjon EGrigoriadou CPytel Det al. PERK promotes cancer cell proliferation and tumor growth by limiting oxidative DNA damagej. Oncogene Tenkerian CKrishnamoorthy JMounir Zet al. mtorc2 balances Akt activation and eif2alpha serine 51 phosphorylation to promote survival under stress. J. Mol Cancer Res 檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿檿 DOI: /j.cnki.syzlzz http / /www. syzlzz. com