A X All-trans retinoic acid ATRA LS174T

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1 doi /j. issn X ERK1 / R363 A X 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 China 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 P Ras-MEK1 /2-ERK1 / LS174T 3 ATRA LS174T 1 No. 3502Z @ qq. com E- mailyinping2002@ 163. com LS174T MEK /ERK 10% 1% RPMI % 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

2 ERK1 / MTT LS174T 10 min ECL / LS174T ml % RPMI h 1 DMSO 33 ATRA ATRA mol /L ATRA 3 ATRA mol /L 4 PD min 10-5 mol /L AT- ATRA RA 24 h 200 μl Bind- Buffer 5 μl Annexin V 5 μl PI 96 6 ing 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 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 s 30 s s min 2% GelDoc XR ATRA Quantity One Volume = 1 Volume ERK2 / Volume β-actin ERK2 x - ± s 1. 4 Western blot LS174T RPMI h mol /L ATRA 310% FBS 410% FBS mol /L ATRA d RIPA 30 min r /min 10 min BCA Pierce SDS 5 min10% SDS-PAGE 50 μg Bio-Rad 15 v 50 min 10% TBST 1 h 4 ERK ERK p-erk1 / MEK1 / p-mek1 / β-actin TBST 4 10 min Fig h TBST 4 one-way ANOVA P < ATRA LS174T LS174T MTT 4 d ATRA ATRA ATRA 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.

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.

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.

3 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 mol /L ATRA + serum-deprived culture medium3. RPMI1640 medium contained 10% fetal bovine serum4. RPMI1640 medium + 10% fetal bovine serum mol /L ATRA. B. Statistical chart of ERK2 /ACTIN P < 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 PD ATRA LS174T P- ERK1 /2 Fig. 4 PD 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 RPMI % FBSRPMI1640 ERK2 mrna % ERK2 P < / mol /L AT- RA RPMI % ERK2 4 6 P < ATRA LS174T MEK /ERK ERK1 /2 Western blot 10% FBS mol /L ATRA

4 ERK1 / 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 ATRA PD98059 p- 5 ATRA ERK MEK1 /2 PD min 10-5 mol /L ATRA 2 d ERK ATRA ATRA MEK / p-erk ERK kd 8 PD98059 p-erk ATRA ERKs mol /L PD98059 ATRA p-erk 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 % % % MEK1 /2 PD98059 MEK /ERK P < PD98059 ERK ATRA 5 3 retinoic acid A MEK /ERK P53 AT 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

5 ATRA p-erk ATRA MEK /ERK ATRA Prev Res Phila2009 9Jost MHuggett TMKari C 1James Smitha Rodica PBunaciua et al. Retinoic acid induces nuclear accumulation of Raf1 during differentiation of HL-60 cells J. Exp Cell Rese Kun YuBin ZhengMei Han et al. ATRA activates and PDGF- BB represses the SM22α promoter through KLF4 binding toor dissociating from its cis-dna elementsj. Cardiovasc Res Ripple MOKim NSpringett R. Acute mitochondrial inhibition by mitogen-activated protein kinase / extracellular signal-regulated kinase kinase MEK1 /2 inhibitors regulates proliferation J. J Biol Chem Kanki KAkechi YUeda C et al. Biological and clinical implications of retinoic acid-responsive genes in human hepatocellular carcinoma cells J. J Hepatol Shino NakagawaTeruhiko FujiiGoro Yokoyama et al. Cell growth inhibition by all-trans retinoic acid in SKBR-3 breast cancer cellsinvolvement of protein kinase Cα and extracellular signalregulated kinase mitogen-activated protein kinasej. Mol Carcinog Jingbo Qiao Pritha Paul Sora Lee et al. PI3K /AKT and ERK regulate retinoic acid-induced neuroblastoma cellular differentiationj. Biochemi Biophys Res Communi Huai LWang CZhang C et al. Metformin induces differentiation in acute promyelocytic leukemia by activating the MEK / ERK signaling pathway J. Biochem Biophys Res Commun Syed ZCheepala SBGill JN et al. All-trans retinoic acid suppresses Stat3 signaling during skin carcinogenesisj. Cancer et al. Epidermal growth factor receptor-dependent control of keratinocyte survival and Bcl-xL expression through a MEK-dependent pathway J. J Biol Chem Liu YZ Boxer LM Latchman DS. Activation of the Bcl-2 promoter by nerve growth factor is mediated by the p42 /p44 MAPK cascade J. Nucleic Acids Res Ley R Balmanno K Hadfield K et al. Activation of the ERK1 /2 signaling pathway promotes phosphorylation and proteasome-dependent degradation of the BH3-only proteinj. Bim J Biol Chem Martin SchulerGreen DR. Transcriptionapoptosis and p53 catch-22 J. Trends In Genetics Balmanno KCook SJ. Tumour cell survival signalling by the ERK1 /2 pathway J. Cell Death Differ Kojima KKonopleva M Samudio IJ et al. Mitogen-activated protein kinase kinase inhibition enhances nuclear proapoptotic function of p53 in acute myelogenous leukemia cells J. Cancer Res Paul Mrass Michael RendlMichael Mildner et al. Retinoic acid increases the expression of p53 and proapoptotic caspases and sensitizes keratinocytes to apoptosisa possible explanation for tumor preventive action of retinoidsj. Cancer Res Park KJLee SHLee CH et al. Upregulation of Beclin-1 expression and phosphorylation of Bcl-2 and p53 are involved in the JNK-mediated autophagic cell deathj. Biochem Biophys Res Commun Park SHChi GYEom HS et al. Role of autophagy in apoptosis induction by methylene chloride extracts of Mori cortex in NCI- H460 human lung carcinoma cellsj. Int J Oncol Han JHou WGoldstein LA et al. Involvement of protective autophagy in TRAIL resistance of apoptosis-defective tumor cells J. J Biol Chem Kauntz HBousserouel SGossé F et al. Silibinin triggers apoptotic signaling pathways and autophagic survival response in human colon adenocarcinoma cells and their derived metastatic cells J. Apoptosis Zoppoli GCea MSoncini D et al. Potent synergistic interaction between the Nampt inhibitor APO866 and the apoptosis activator TRAIL in human leukemia cellsj. Exp Hematol

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