A X All-trans retinoic acid ATRA LS174T

318 2014 30 doi10. 3969 /j. issn. 1000-484X. 2014. 03. 006 1 ERK1 /2 23 2 4 361100 R363 A 1000-484X201403-0318-05 All-trans retinoic acidatra ERK1 /2 MTT ATRA RT-PCR Western blot ATRA LS174T MEK1 /2 /ERK1 /2 ATRA PD98059MEK1 /2 LS174T MTT ATRA LS174T RT-PCR Western blot ATRA ERK mrna ERK PD98059 ATRA p-erk ATRA ATRA MEK / ERK ERK All-trans retinoic acid ATRAinhibit proliferation of colon cancer cell lines by extracellular regulated protein kinases 1 and 2 ERK1 /2signal pathway CHEN YanLIU Zheng-JinYIN PingJIN Guang-Hui. Department of PathologyThird Hospital of XiamenFujian ProvinceXiamen 361100China AbstractObjectiveTo explore whether all-trans retinoic acid ATRAinhibits proliferation of colon cancer cell by acting ERK1 /2 signal pathway. MethodsATRA on the proliferation of colon cancer cell lines were detected by MTT assayrt-pcr and Western blot assay were used to detect MEK1 /2 / ERK1 /2 expression levels of LS174T cells treated by ATRA. LS174T cells apoptosis levels were measured by flow cytometry after combined treatment of ATRA and PD98059 MEK1 /2 specific inhibitor. ResultsMTT growth curve showed that ATRA could inhibit the proliferation of colon cancer cell line LS174T. RT-PCR and Western blot results showed that ATRA could increase expression levels of ERK mrna and proteinand could promote ERK phosphorylation. Combination of PD98059 and ATRA lowered some p-erk expressions in colon cancer cellsbut meanwhile enhanced ATRA-induced apoptosis. ConclusionATRA can inhibit proliferation of colon cancer celland its induction of apoptosis of tumor may be conducted by multiple signal pathways including MEK / ERK. Key wordsall-trans retinoic acid ATRA ERKColon cancerapoptosis All-trans retinoic acidatra MAPK Raf1 JNK P38 1 1 2 Ras-MEK1 /2-ERK1 /2 1. 1 LS174T 3 ATRA LS174T 1 No. 3502Z20124051 2 361004 3 4 361004 1977 - E-mail743089817@ qq. com 1963 - E- mailyinping2002@ 163. com LS174T MEK /ERK 10% 1% RPMI1640 37 5% CO 2 2 d 1 ERK1 ERK2 MEK1 /2 p- MEK1 /2 β-actin Santa p- ERK1 /2 CST Merck MTT DMSO Sigma FERMENTAS Annexin-V BI- OMIGA

ERK1 /2 3 319 1. 2 MTT LS174T 10 min ECL 5 10 4 / 96 6 1. 5 LS174T 1 21 10 5 ml - 1 6 10% RPMI1640 24 h 1 DMSO 33 ATRA ATRA 2 10-5 mol /L ATRA 3 ATRA 10-7 10-6 10-5 mol /L 4 PD98059 30 min 10-5 mol /L AT- ATRA RA 24 h 200 μl Bind- Buffer 5 μl Annexin V 5 μl PI 96 6 ing 1 2 4 6 d 15 min 300 μl Binding Buffer 1 h 3 MTT5 mg /ml 20 μl 37 4 h DMSO 150 μl 1. 6 MTT RT-PCR FACS Western 10 min 570 nm blot SPSS 13. 0 OD570 nm x - ± s 1. 3 RT-PCR Trizol RNA RNA Fermantas RevertAid x - ± s First Strand cdna Synthesis Kit cdna cd- 2 NA - 20 β-actin5'-caaagacctgtacgccaa- CAC3'-CATACTCCTGCTTGCTGATCCERK25'- TGATCACACAGGGTTCCT 3'-TGTGATGGGGATCCA AGAAT 213 bp PCR 94 5 min 94 30 s 30 s 72 10 s 35 72 7 min 2% GelDoc XR ATRA Quantity One 4. 4-4. 6 Volume = 1 Volume ERK2 / Volume β-actin ERK2 x - ± s 1. 4 Western blot LS174T RPMI1640 24 h 4 1 2 10-5 mol /L ATRA 310% FBS 410% FBS + 10-5 mol /L ATRA 2 4 6 d RIPA 30 min 4 12 000 r /min 10 min BCA Pierce SDS 5 min10% SDS-PAGE 50 μg Bio-Rad 15 v 50 min 10% TBST 1 h 4 ERK1 1 5 000 ERK2 1 6 000 p-erk1 /2 1 1 000 MEK1 /2 1 1 000 p-mek1 /2 1 500 β-actin 1 5 000 TBST 4 10 min Fig. 1 1 10 000 1 10 00025 1 h TBST 4 one-way ANOVA P < 0. 05 2. 1 ATRA LS174T LS174T MTT 4 d ATRA ATRA ATRA 1 2 4 6 d 1 LS174T ATRA inhibits the growth of LS174T colon cells by MTT assay

320 2014 30 2 Fig. 2 LS174T ERK2 RT-PCR Volume ERK2 /Volume β-actin ERK2's RT-PCR results of LS174T cells and column chart according to ratio of volume ERK2 / Volume β-actin NoteA. ERK2 mrna levels are determined by RT-PCR. M. Marker LS174T cells are cultured in four groups1. Serum-deprived RPMI1640 medium 2. 10-5 mol /L ATRA + serum-deprived culture medium3. RPMI1640 medium contained 10% fetal bovine serum4. RPMI1640 medium + 10% fetal bovine serum + 10-5 mol /L ATRA. B. Statistical chart of ERK2 /ACTIN P < 0. 05. 3 Fig. 3 ATRA LS174T MEK /ERK P-ERK1 /2 ATRA stimulates protein expression of MEK /ERK P-ERK1 /2 in LS174T cell to increase NoteAfter starved 24 hours LS174T cells are divided into 4 group P-ERK1 /2 in LS174T colon cancer cells to increase as above. Western blot analysis of MEK1 /2p-MEK1 /2ERK1ER2p-ERK1 /2 during different treatment. β-actin is used as a lane loading control. 4 PD98059 + ATRA LS174T P- ERK1 /2 Fig. 4 PD98059 + ATRA decreases expression of PERK1 / 2 in LS174T colon cancer cells NoteControl. Cells grow in culture medium contained 10% fetal bovine serum without any drug based on it different drugs are added. LS174T 2. 2 ATRA ERK mrna MEK1 /2 p-mek1 /2 ERK1 ERK2 p-erk LS174T RT-PCR RPMI1640 10% FBSRPMI1640 ERK2 mrna 4 6 10% ERK2 P < 0. 052 1 /3 10-5 mol /L AT- RA RPMI1640 10% ERK2 4 6 P < 0. 052 2. 3 ATRA LS174T MEK /ERK ERK1 /2 Western blot 10% FBS 4 6 10-5 mol /L ATRA

ERK1 /2 3 321 5 Fig. 5 PI LS174T LS174T cells apoptosis figure by PI staining flow cytometry analysis MEK 1 /2 p-mek1 /2 ERK1 ERK2 p-erk 3A RARE 4 p-erk ATRA MAPK RPMI1640 ATRA JNK ATRA MEK 1 /2 p-mek1 /2 ERK1 ERK2 p- ERK 3B ATRA 2 SKBR-3 2. 4 ATRA PD98059 p- 5 ATRA ERK MEK1 /2 PD98059 30 min 10-5 mol /L ATRA 2 d ERK ATRA ATRA MEK / p-erk ERK 44 42 kd 8 PD98059 p-erk ATRA ERKs 5 10-5 mol /L PD98059 ATRA p-erk 4 2. 5 ATRA PD98059 S LS174T Annexin V PI LS174T ATRA LS174T LS174T ATRA 10-5 mol /L ATRA 48 h UR + LR25. 17% MEK /ERK 18. 1% P < ATRA 0. 05ATRA PD98059 ATRA MEK /ERK 26. 53% 30. 33% 32. 47% MEK1 /2 PD98059 MEK /ERK P < 0. 01 PD98059 ERK ATRA 5 3 retinoic acid A MEK /ERK P53 AT- 12-14 ATRA RA ATRA P53 caspase3 15 PD98059 ATRA RAR X RXR RAR RARE P38 Krüppel-like factor 4 KLF4 ATRA ERK 6 ATRA MEK /ERK 7 ATRA p-erk ATRA PD98059 ERK Bcl-2 Bcl-2 Bcl-Xl Bad Bim 9-11 PD98059 ATRA P53 RXR PD98059

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