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1、Cellular PhysiologyCell Physiol Biochem 2017;43:465-480DDOOII: 1100.1.1115599/0/00004084084074373 2017 The Author(s2).0P1u7bTlihseheAdubthyoSr(. sK)arger AG, Baseland Biochemistry PPuubblliisshheeddoonnlilninee: S: eSpeptetemmbbeerr1199, 22001177465lished by S. Karger AG, Baseln Human LungZAhccaenpg

2、teedt: aMl.:aCy N26T,N2-0117Enhances ChemothroAdenocarcinomaThis article is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 Interna-tional License (CC BY-NC-ND) (). Usage and distributionIRU FRPPHUFLDO SXUSRVHV DV ZHOO DV DQ GLVWULEXWLRQ RI PRGLHG PDWHULDO UHTXLUHV ZU

3、LWWHQ SHUPLVVLRQ Original PaperCNTN-1 Enhances ChemoinHuman Lung Adenocarcinoma Through Induction of Epithelial-Mesenchymal Transition by Targeting the PI3K/Akt PathwayRuijie Zhanga Shenghua Suna Fuyun Jib Chun Liua Hua Lina Lihua Xiea Honghui Yanga Wenxiang Tanga Yan Zhoua JianXuc Pei Lid,eaDepartm

4、ent of Respiratory Medicine, the Third Xiangya Hospital, Central South University, Changsha, Hunan, bInstitute of Human Respiratory Disease, ;LQTLDR +RVSLWDO 7KLUG 0LOLWDU 0HGLFDO 8QLYHUVLW &KRQJTLQJ cHSDUWPHQW RI 3DWKRORJ ;LQTLDR +RVSLWDO 7KLUG 0LOLWDU 0HGLFDO 8QLYHUVLW &KRQJTLQJ dDepartment of Ort

5、hopedic Surgery, No. 89 hospital of PLA, Wei, Sh, eDepartment of2UWKRSHGLF 6XUJHU 6RXWKZHVW +RVSLWDO 7KLUG 0LOLWDU 0HGLFDO 8QLYHUVLW &KRQJTLQJ Key WordsCNTN-1 EMT ChemoAbstract PI3K/Akt NSCLCBackground/Aims: Chemohas been a major obstacle to the effective treatment oflung cancer. Previously, we foun

6、d that contactin-1 (CNTN-1) is related to cisplatinin lung adenocarcinoma. Here, we aimed to investigate the underlying mechanismthe role of CNTN-1 in cisplatinin lung adenocarcinoma. Methods: EMT-associatedphenotypes, including alterations in cellular morphology and marker (E-cadherin, N-cadherinan

7、d Vimentin) expression, were compared between A549 cells and A549/DDP cells (a cisplatin- resistant cell line of lung adenocarcinoma with abnormal CNTN-1 expression) by using real- time time PCR and Western blotting. Other methods, including CNTN-1 overexpression in A549 cells and CNTN-1 knockdown i

8、n A549/DDP cells, were also used to investigate therole of CNTN-1 in mediating the EMT phenotype and thr resulting cisplatinandmalignant progression of cancer cells in vitro and in vivo. Results: A549/DDP cells exhibited anEMT phenotype and aggravated malignant behaviors. CNTN-1 knockdown in A549/DD

9、P cells partly reversed the EMT phenotype, increased drug sensitivity, and attenuated the malignant progression whereas CNTN-1 overexpression in A549 cells resulted in the opposite trend. Furthermore, the PI3K/Akt pathway was involved in the effects of CNTN-1 on EMT progressionLQ $ 3 FHOOV YHULHG E

10、WKH HQRJUDIW PRXVH PRGHO : CNTN-1 promotescisplatinin humsplatin-resistant lung adenocarcinoma through inducingthe EMT process by activating the PI3K/Akt signaling pathway. CNTN-1 may be a potentialtherapeutic target to reverse chemoin cisplatin-resistant lung adenocarcinoma. 2017 The Author(s)Publi

11、shed by S. Karger AG, Basel) -L 6 6XQ DQG 3 /L FRQWULEXWHG HTXDOO WR WKLV ZRUN Pei Li, Shenghua Sun and Fuyun JiDept Orthopedic Surgery, No. 89 hospital of PLA and Dept Orthopedic Surgery, Southwest Hospital, Third Military Medical University; Dept Respiratory Medicine, the Third Xiangya Hospital, C

12、entral South University; Institute of Human Respiratory LVHDVH ;LQTLDR +RVSLWDO 7KLUG 0LOLWDU 0HGLFDO 8QLYHUVLW &KLQD lipeizrjDownloaded by:Southern Medical University 183.62.37.111 - 10/17/2017 6:52:55 AMCellular Physiology and BiochemistryCell Physiol Biochem 2017;43:465-480466Zhang et al.: CNTN-1

13、 Enhances Chemo Adenocarcinomathrough Inducing EMT in Human LungIntroductionLung cancer, particularly non-small cell lung cancer (NSCLC), remains a challenging disease with high mortality worldwide 1-2. Despite the tremendous mountainous progress in multidisciplinary therapy, the overall 5-year surv

14、ival rate is still poor 3. One of the major obstacles restricting therapeutic eficiency is intrinsic or acuiredto chemotherapeutic agents in clinical practice 4-8. To explore the mechanism of acuiredmultidrug(MDR) to cisplatin, a number of studies have been carried out, andthe accumulating data have

15、 displayed that an increase in drug eflux, DN damage repair,activation of a detoxiication systemthe chemotherapeutic regimen, and apoptosisare the main mechanisms of MDR 8-11, which have helped us to promotetreatment eficiency for patients with cancer to a certain extent.Recently, several studies ha

16、ve suggested arelationship between epithelial-mesenchymal transition (EMT) progression and chemoin multiple cancer cells12-14, which shed light on advng the treatment eficiency for cancer patients. EMT is acritical step during embryo development, wound healing, tissue ibrosis, tumor migration, and i

17、nvasion 15-16. s a dynamic and reversible process, the EMT progression, characteried by an alteration in cellular morphology, enhancement in the mobile ability, formation of acancer stem cell (CSC) phenotype, and acuisition of anoiis, was detected incertain drug-resistant cancer cells such as hepato

18、cellular carcinoma 17, colorectal cancer 18, nasopharyngeal carcinoma 19, and breast cancer 20. Concomitantly, downregulation of the epithelial marer (E-cadherin) and upregulation of mesenchymal marers (vimentin, N-cadherin, ibronectin) and other related transcription factors (snail, slug, and twist

19、) were also found in drug-resistant cancer cells with an EMT phenotype 12. dditionally, the complex EMT process was reported to be related to the PI3K/ t signaling pathway 21-24,which also participated in the cisplatinof many cancer cells, including colorectalcancer 25, lung cancer 26, and ovarian c

20、ancer 27. However, whether the EMT process iscorrelated with cisplatinin NSCLC is still unclear.Contactin-1 (CNTN-1), a neuronal cell adhesion molecular, has been shown to beinvolved in nervous system development 28-29. In recent years, the abnormal expressionof CNTN-1 was reported to bely correlate

21、d with carcinogenesis and tumor progression 11, 30-34. Furthermore, the elevated expression of CNTN-1 was demonstrated to facilitate metastasis of MKN45 gastric cancer cells via activation of the EMT process 35. dditionally, in our previous study, silencing CNTN-1 promoted the sensitivity to chemoth

22、erapeutic drugs and attenuated metastasis and the invasion ability of 549/DDP (a MDR cell line of NSCLC) cells 36. The studies mentioned above suggest that CNTN-1 may contribute to the EMTprocess and resulting chemoin 549/DDP cells as a mediator.To test the hypothesis, 549/DDP cells and xenograft mo

23、use ms established withthe corresponding cells were utilied and the data demonstrated that the EMT phenotypeand malignant progression of 549/DDP cells was regulated by CNTN-1 via activation of the PI3K/ t signaling pathway. CNTN-1 may be a potential therapeutic target to reversethe chemoand malignan

24、t progression in cisplatin-resistant lung adenocarcinoma.Materials and MethodsEthics statement ll experiments were approved by the Experimental nimal Ethics Committee of iniao Hospital afiliated with the Third Military Medical niversity S K ( ) 2012-0011.Cell lines and culture conditionsThe human lu

25、ng adenocarcinoma cell line ( 549) was purchased from the Institute of iochemistryand iology, 10% fetal bovi cademy of Sciences (Shanghai,) and cultured in RPMI-1640 medium containingrum (F S, ibco, S ) under standard conditions (37C, 20% O2, and 5% CO2). TheDownloaded by:Southern Medical University

26、 183.62.37.111 - 10/17/2017 6:52:55 AMDOI: 10.1159/000480473Published online: September 19, 2017 2017 The Author(s). Published by S. Karger AG, BaselCellular Physiology and BiochemistryCell Physiol Biochem 2017;43:465-480467Zhang et al.: CNTN-1 Enhances Chemo Adenocarcinomathrough Inducing EMT in Hu

27、man Lungcisplatin-resistant cell line ( 549/DDP) was established and veriied as described in our previous studies 36-37.Cell transfectionThe recombinant lentiviral vectors LV5-CNTN-1 and LV3-CNTN-1 ( enePharma, Shanghai,) wereused to overexpress CNTN-1 in 549 cells ( 549-CNTN-1, the full seuence of

28、the recombinant lentiviralvector LV5-CNTN-1 was obtained from) andsilence CNTN-1 in 549/DDP cells ( 549/DDP-shCNTN-1, the shRN human CNTN-1-targeting seuence was 5- C T CT T T -3), respectively. Cells transfected with negative vectors ( 549- CNTN-1-NC and 549/DDP-shCNTN-1-NC) were used as controls.

29、ccording to the operation manual, the transfected cells were selected via puromycin and cultured for future use.Cell morphology observationCell morphology was irst observed using a light microscopy (Olympus 51, apan). dditionally, cancer cells were viewed under a luorescence microscope (Olympus I 71

30、, apan) after staining with lexa Fluor 488 Phalloidin (Invitrogen) according to the manufacturers instructions.Anoikisassay riely, after 5105 cells were seeded in a 6-well plate coated with 12 mg/mL poly-2-hydroxyethyl methacrylate (poly-HEM , Sigma) to prevent cell attachment and suspension-culture

31、d for 48 hrs, the cell clones suspended in the medium were captured under a light microscope (Olympus 51, apan) and harvested to examine the apoptotic ratio of cells by low cytometry using the annexin V-FITC/PI stainingmethod ( eyotime,).Cell apoptosis analysis fter cells were seeded in petri dishes

32、 (10-cm diameter, 3104 cells per dish) and grown to 70-80% conluence, the cells were incubated with 2 g/mL cisplatin for 48 hrs and collected. Then, low cytometry assay was performed to detect apoptotic cells using the annexin V-FITC/PI staining method ( eyotime,).In vitro drug sensitivity assay rie

33、ly, after cells were incubated with different concentrations of cisplatin (10, 5, 2.5, 1.25, 0.625, and 0 ug/mL) for 48 hrs, 100 l fresh culture medium and 10 l Cell Counting Kit-8 (CCK-8) solution ( eyotime,) weded to each well and incubated for 1 hr. Then, the absorbance value at the wavelength of

34、 450nm was measured. Cells incubated without cisplatin were treated as negative controls. The 50% inhibitory concentration (IC50) was calculated using raphPad Prism 5.0 software.Migration and invasion assayThe migration ability of cancer cells was evaluated by a wound-healing assay. riely, after cel

35、ls were seeded in a 6-well plate (5105 cells per well) and cultured overnight, the conluent cell monolayer was seuentially scratched in a straight line with a 200-l pipette tip, washed with sterile P S and cultured in fresh medium for 20 hrs. Migrated cells were photographed at 0 hr and 20 hrs under

36、 a light microscope (Olympus 51, apan). The cell metastasis ability in each group was calculated using Image-Pro Plus software (Version 5.1, Media Cybernetics, Inc.). The cell invasion ability was evaluated using modiied oyden chambers with ilter inserts (pore sie of 8 m, Corning Inc., Life Science)

37、 according to our previous method 36. Invasive cells were observed under a light microscope (Olympus 51, apan), and calculated using Image-Pro Plus software (Version 5.1, Media Cybernetics, Inc.).RNA isolation and real-time PCR analysis fter the total RN of cancer cells was extracted using TRIol rea

38、gent (Invitrogen, S ) according to the manufacturers instructions, irst-strand cDN was synthesied from total RN (1 ug) using a First Strand cDN Synthesis Kit (Roche). Then, real-time PCR was performed on a reaction system containing cDN , S R reen Mix (TO O O), and primers (Table 1). lyceraldehyde-3

39、-phosphate dehydrogenase ( PDH) was used as an internal control. The relative expression of target genes was expressed as 2-Ct.Downloaded by:Southern Medical University 183.62.37.111 - 10/17/2017 6:52:55 AMDOI: 10.1159/000480473Published online: September 19, 2017 2017 The Author(s). Published by S.

40、 Karger AG, BaselCellular Physiology and BiochemistryCell Physiol Biochem 2017;43:465-480468Zhang et al.: CNTN-1 Enhances Chemo Adenocarcinomathrough Inducing EMT in Human LungTable 1. Primers of target genesTable 2. Primary and secondary tochemistry)bodies used in this study ( : estern blotting, IH

41、C: Immunohis-Western blot analysis fter the total protein was extracted from cancer cells using RIP lysis solution ( eyotime,),protein expression was analyed by estern blotting assay as described previously 36. The primarybodies and secondarybodies used in this experiment are listed in Table 2. Prot

42、ein bands werevisualied using a SuperSignal est Pico Trial Kit (Thermo) and analyed using Image software (National Institutes of Health, S ).Xenograft tumorigenicity experimentHealthy SCID mice (Four to ive-wee-old female, n 20) were purchased from the nimal Laboratoryof iniao Hospital and housed in

43、 a climate-control SPF (Speciic Pathogen) facility. The mice weredivided into two groups that were subcutaneously inoculated with 549/DDP-shCNTN-1 cells and 549/ DDP-shCNTN-1-NC cells into the right lan (100 L, 1106 cells per animal), respectively. Tumors were monitored on the day of tumor formation

44、 and measured every three days for a total of 48 days. Tumor volume (mm3) was calculated according to the formula ab2/2 (alargest diameter bsmallest diameter) 38. s previously described 16, when the tumor volume reached 150 mm3, one subgroup of nude mice in each group was injected with a cisplatin s

45、olution (cisplatin concentration: 10 mg per 2 mL in normal saline (NS) injection dosage: 0.2 mg cisplatin per 10 g animal weight (20 mg/g) via intraperitoneal injection every three days, while the other subgroup of mice was used as controls and injected with NS. fter 48 days, saline-treated xenograf

46、t tumors ( 549/DDP-shCNTN-1-NC and 549/DDP-shCNTN-1) were isolated to detect protein expression of CNTN-1 and EMT biomarers by estern blot and immunohistochemistry, respectively. Meanwhile, the lung and liver tissues were separated to observe tumor metastasis in vivo by HE staining.Hematoxylin and e

47、osin (HE) staining and immunohistochemistryTissue samples including xenograft tumors, lung tissues, and liver tissues were seuentially ixed with 4% paraformaldehyde for 24 hrs, embedded in parafin, and sectioned. Then, HE staining and immunostainingDownloaded by:Southern Medical University 183.62.37

48、.111 - 10/17/2017 6:52:55 AMDOI: 10.1159/000480473Published online: September 19, 2017 2017 The Author(s). Published by S. Karger AG, BaselCellular Physiology and BiochemistryCell Physiol Biochem 2017;43:465-480469Zhang et al.: CNTN-1 Enhances Chemo Adenocarcinomathrough Inducing EMT in Human Lungwe

49、re performed on the 4-m-thic sections of xenograft tumors as previously described 36. The primaryand secondarybodies used for immunohistochemistry are shown in Table 2. dditionally, HE stainingwas performed on the 4-m-thic sections of liver and lung tissues to simply observe the metastasis of the xe

50、nografted tumors in vivo. ll sections were observed under a light microscope (Olympus 51, apan).Statistical analysis ll numerical data in this study were presented as the meanSD and analyed using SPSS 13.0 software. fter a homogeneity test for variance, the signiicant difference between two groups w

51、as analyed using an independent-samples t test, whereas the signiicant difference between multiple groups was analyed by one-way analysis of variance ( NOV ) followed by LSD post hoc test. p-value of less than 0.05 was regarded as statistically signiicant.ResultsCisplatin-resistant lung adenocarcino

52、ma cells displayed an EMT phenotypeConsistent with our previous report 36, 549/DDP cells exhibited moreto cisplatin than 549 cells (Fig. 1 ), veriied by the apoptosis analysis, which showed that 549/DDP cells displayed a lower percentage of apoptotic cells than 549 cells when exposed to cisplatin (F

53、ig. 1 ). To evaluate the association of EMT development withcisplatin, EMT-related cellular morphological changes and expression of EMT-related marers (E-cadherin, N-cadherin and vimentin) were compared between 549 cells and 549/DDP cells. Interestingly, 549/DDP cells displayed an elongated and irre

54、gular ibroblastic morphology with growth separately from one another, whereas 549 cells exhibited an epithelial morphology characteried by a round shape and growth in clusters (Fig. 1C), veriied by the cytoseleton F-actin staining (Fig. 1D). Compared with that of 549 cells, both the expression of N-

55、cadherin and vimentin (mesenchymal marers) was upregulated, while the expression of E-cadherin (epithelial marer) was downregulated in 549/DDP cells both at the mRN and protein levels (Fig. 1E-F). These results indicate that cisplatin-resistant lung adenocarcinoma cells acuire an EMT phenotype. ddit

56、ionally, the association of the EMT phenotype with malignant progression was also analyed in 549/DDP and 549 cells. The wound-healing assay and transwell invasion assay showed that the migration and invasion of 549/DDP cells was signiicantlygreater than that of 549 cells (Fig. 2 - ). Moreover, the a

57、noiisassay showedthat 549/DDP cells formed larger cellular aggregates than 549 cells, which mainly werefound as individual cells and smaller cellular aggregates (Fig. 2C). Flow cytometry analysis of these suspended cells demonstrated that 549 cells had a higher proportion of anoiis than 549/DDP cells (Fig. 2D). Furthermore, the expression of pluripotent marers (CD44, OCT-4 and Nanog) was higher in 549/DDP cells than in 549 cells (Fig. 2E). Collectiv