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Published ahead of print on December 20, 2007, doi:10.1165/rcmb.2007-0001OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 38, pp. 559-565, 2008
© 2008 American Thoracic Society
DOI: 10.1165/rcmb.2007-0001OC

HGF Increases Cisplatin Resistance via Down-Regulation of AIF in Lung Cancer Cells

Jin-Tang Chen*,1, Chih-Yang Huang*,2, Yung-Yen Chiang3, Wen-Heng Chen4, Shiow-Her Chiou2, Chih-Yi Chen5 and Kuan-Chih Chow4,5

1 Feng-Yuan Hospital, Feng-Yuan; 2 Graduate Institute of Veterinary Microbiology, and 4 Graduate Institute of Biomedical Sciences, National Chung Hsing University, Taichung; 3 Department of Dental Laboratory Technology, Central Taiwan University of Science and Technology, Taichung; and 5 Comprehensive Cancer Center, China Medical University Hospital, Taichung, Taiwan

Correspondence and requests for reprints should be addressed to Kuan-Chih Chow, PhD, Graduate Institute of Biomedical Sciences, National Chung Hsing University, 250 Kuo-Kuang Road, Taichung, 40227 Taiwan. E-mail: kcchow{at}dragon.nchu.edu.tw


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Our previous study had shown that advanced stages of lung adenocarcinomas (ADC) was frequently associated with overexpression of hepatocyte growth factor (HGF), which has multipotent and anti-apoptotic activities. In this study, we examined the effect of HGF on gene expression of apoptosis-inducing factor (AIF) and cisplatin sensitivity in lung ADC cells. Expression of AIF was determined by immunocytochemistry and confocal immunofluorescence microscopy. Our data show that addition of HGF suppressed AIF expression and increased cisplatin resistance. The effect could be through HGF receptor and its downstream effector, focal adhesion kinase (FAK). Interestingly, knockout of FAK gene increased AIF expression and drug sensitivity. Re-introduction of FAK gene, on the other hand, restored drug resistance. These results suggested that HGF might induce cisplatin resistance via c-Met to activate FAK and down-regulate AIF expression.

Key Words: apoptosis inducing factor • cisplatin resistance • hepatocyte growth factor • focal adhesion kinase • non–small cell lung cancer



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
In this study, we show that hepatocyte growth factor increases drug resistance and metastatic potential of lung cancer cells via focal adhesion kinase activation and apoptosis-inducing factor down-regulation.

 
Recently, by using suppression subtractive hybridization (SSH) and microarray techniques to examine the expression profiles of lung adenocarcinomas (ADC), we found that hepatocyte growth factor (HGF) was frequently overexpressed in the afflicted tissues, and HGF level was positively correlated with tumor stages and adverse prognosis (1). Because HGF is a potent pulmotrophic factor that mediates growth and migration of epithelial and cancer cells, our previous results suggest that HGF plays an essential role in tumor development, invasion, and disease progression of lung ADC (1). In vitro, HGF expression was frequently detected in both lung cancer and cultured alveolar type II (ATII) epithelial cells. Furthermore, HGF reduces adriamycin- and camptothecin-mediated cytotoxicity of Chinese hamster ovary cells (2), as well as suppresses N-methyl-D-aspartate (NMDA)-mediated cell death in cultured hippocampal neurons (3). Through receptor bindings, NMDA activates caspase-3 and nuclear translocation of apoptosis-inducing factor (AIF). Treatment of HGF, however, retards caspase-3 activation and AIF nuclear translocation, and hence decreases NMDA-related cytotoxicity (3).

AIF is a 67-kD protein that is normally located in intermembranous space of mitochondria (4). Upon challenge with drugs (e.g., adriamycin, camptothecin, and etoposide), AIF is released from mitochondria and translocated to the nucleus (5). After the entrance of AIF, nuclear events, such as DNA fragmentation, chromosome condensation, and formation of micronuclei, take place to initiate apoptosis. AIF was thus considered to be closely associated with drug-related cytotoxicity (6). Decrease of AIF level would reduce drug-mediated cell death, and increase of AIF level would then raise drug-related cytotoxicity. However, no report has studied the effect of HGF on AIF expression and how this can affect sensitivity of anticancer drugs. In this study, we investigated the effect of HGF on AIF expression and cisplatin sensitivity in lung ADC cells.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Lung Cancer Cell Lines
Seven lung cancer cell lines (H23, H226, H838, H1437, H2009, H2087, and A549) were used for evaluation of AIF expression in vitro (1). Cells were grown at 37°C in a monolayer in RPMI 1640 supplemented with 10% fetal calf serum, 100 I.U./ml penicillin, and 100 µg/ml streptomycin.

RNA Extraction and RT-PCR
Total RNA was isolated from lung ADC cells using an SNAP RNA column (Invitrogen, San Diego, CA). After measurement of RNA yield, cDNA was synthesized by AMV reverse transcriptase using oligo random primers. An aliquot of cDNA was then subjected to 35 cycles of PCR. The reaction mixture contained 1x Taq buffer (BRL, Betheda, MD), 1.5 mM MgCl2, 2 µM dNTP, 0.25 µM of respective 3' and 5' primers, 1 U of Taq DNA polymerase, and 2 µl of cDNA. PCR was performed according to standard procedures by denaturing cDNA template at 94°C for 30 seconds, hybridizing with primers at 52°C for 45 seconds, and elongating DNA synthesis at 72°C for 2 minutes. The primer sequences for AIF were 5'-GCCGAAATGTTCCGGTGTGGAGGCCTGGCGGC-3' (nts 37–68, AF100928) and 5'-GGCTTCAGTCTTCATGAATGTTGAATAGTTTGGCT-3' (nts 1888–1854); for mouse FAK, the sequences were 5'-GGATCAGTTACCTGACAGAC-3' (nts 836–855, AB030035) and 5'-CCTGGCTTCATCTATTCCAT-3' (nts 1278–1259).

The amplified products were analyzed in 1% agarose gel, and visualized by ethidium bromide staining. The AIF fragment was 1,852 base pairs (bp). The cDNA was inserted into plasmid pCRII, and DNA sequence was determined by an automatic DNA sequencer (ABI PRISM; Perkin-Elmer Applied Biosystems, Foster City, CA). After getting unsatisfactory results from several batches of commercially obtained antibodies, we decided to raise our own antibodies.

Preparation of Mouse Antibodies
DNA sequence corresponding to C-terminal amino acids 315 to 612 was amplified by primer sequences containing Eco RI (sense) and Not I (antisense) restriction sites, respectively. The primer sequences were 5'-CATGATGAATTCATGCTGGCCTGTGCTCTTGGC-3' (Eco RI site is underlined) and 5'-ATCGACGTGCGGCCGCTCAGTCTTCATGAATGTTGAATA-3' (Not I site is underlined).

The 900-bp cDNA of AIF was cloned into an expression vector pET-32b+ (pET32+-AIF; Promega KK, Tokyo, Japan). Bacterial colony containing the pET32+-AIF was selected, and induced by isopropyl-β-D-thiogalactopyranoside (IPTG) to mass-produce AIF. The recombinant protein was purified by a nickel-affinity column, and protein identity was determined by MALDI-TOF. Affinity-purified AIF was used to immunize BALB/c mice, and sensitivity of antiserum (OD405 > 0.3 at 1:6,000 dilution) was measured by enzyme-linked immunosorbent assay (ELISA). Specificity of antibodies was determined by showing distinct bands with molecular weight of 67 kD in the immunoblotting of lung cancer cell extract (4, 6). Monoclonal antibodies were produced by a hybridoma technique, and AIF-specific antibodies were screened by the above-mentioned methods.

Immunoprecipitation, Gel Electrophoresis, and Protein Analysis by MALDI-TOF
Total cell lysate was prepared by mixing 5 x 107 cells/100 µl phosphate-buffered saline with equal volume of 2 x NP-40 lysis buffer (40 mM Tris-HCl, pH 7.6, 2 mM EDTA, 300 mM NaCl, 2% NP-40, and 2 mM phenylmethylsulfonylfluoride [PMSF]). Protein G sepharose (Amersham Biosciences AB, Uppsala, Sweden) was pre-washed before mixing with 500 µg of total cell lysate. The reaction mixture was incubated at 4°C for 60 minutes, and then centrifuged at 800 x g for 1 minute. The supernatant was reacted with 5 µg of purified monoclonal antibodies, and 20 µl of fresh protein G sepharose at 4°C for 18 hours. The reaction mixture was centrifuged at 800 x g for 1 minute. After removal of the supernatant, the precipitate was washed with 1x PBS and dissolved in loading buffer (50 mM Tris, pH 6.8, 150 mM NaCl, 1 mM disodium EDTA, 1 mM PMSF, 10% glycerol, 5% β-mercaptoethanol, 0.01% bromophenol blue, and 1% SDS). Eletrophoresis was performed in two 10% polyacrylamide gels with 4.5% stacking. One gel was processed for immunoblotting (7), and the other gel was stained with Coomasie blue. Protein bands on the Coomasie-stained gel, which corresponded to the immunoblotting-positive bands, were extracted from the gel for identification by MALDI-TOF on a Voyager-DE pro biospectrometry workstation (Applied Biosystems, Milpitas, CA). Fragments of peptide fingerprints were matched with those on the SwissProt database by MS-fit (ProteinProspector 4.0.5.; The Regents of the University of California). After electrophoresis, proteins on the first gel were transferred to a nitrocellulose membrane for immunoblotting. The membrane was probed with specific antibodies. The signal was amplified by biotin-labeled goat anti-mouse IgG, and peroxidase-conjugated streptavidin. The protein was visualized by exposing the membrane to an X-Omat film (Eastman Kodak, Rochester, NY) with enhanced chemiluminescent reagent (NEN, Boston, MA).

Immunocytochemistry and Confocal Immunofluorescence Microscopy
Briefly, the cells were grown on the slide overnight, and fixed with cold methanol/acetone at 4°C for 10 minutes before staining. Immunologic staining was performed according to the immunoperoxidase method (8). For confocal immunofluorescence microscopy, peroxidase-conjugated second antibodies were replaced by those conjugated with fluorescence.

Cytotoxicity Assay
Cells were seeded at 1,000, 2,500, 5,000, and 10,000 cells/well 18 hours before drug challenge. The cells were then treated continuously with various concentrations of cisplatin (range, 1.6 µM to 1.0 mM) for 72 hours. The control group was only treated with drug diluent, PBS. After drug challenge, 10 µl of WST-1 (BioVision, Mountain View, CA) was added and continued incubation for 2 hours. Percent survival of cells was quantified by comparing the number of viable cells in the treatment group with that in the control group. All procedures were performed in triplicate. This assay measures both replicating and static cells.

Colony-Forming Assay
Cells were seeded at 100, 500, 2,000, and 10,000 cells/6-cm culture plate. The cells were treated with various concentrations of cisplatin for 12 hours, and then the drug was washed off with PBS. After drug challenge, the cells were incubated at 37°C for 10 days until cell colony (>= 50 cells/colony) appeared. Percent survival of cells was quantified by comparing the number of viable cell colonies in the treatment group with that in the control group. All procedures were performed in triplicate. This assay measures only replicating cells.

Enforced Expression of AIF
DNA sequences corresponding to the full-length AIF were amplified by primer sequences containing Eco RI (sense) and Not I (antisense) restriction sites, respectively. The primer sequences were 5'-TTGGAATTCATTATGTTCCGGTGTGAGCCTG-3' (nts 179–199, NM_004208, Eco RI site is underlined and the initiation codon is highlighted in bold) and 5'-CTAGAGCGGCCGCCAGTCTTCATGAATGTTGAA-3' (nts 2000–2019, NM_004208, Not I site is underlined and the stop codon is highlighted in bold). The 1,844-bp cDNA of the full-length AIF was cloned into a mammalian expression vector pcDNA3.1/myc-His (Invitrogen Taiwan, Taipei, Taiwan). Plasmid containing the full-length AIF gene was selected and amplified. The plasmid was introduced into cells by jetPEI, the polyethylenimine derivative reagent for DNA tranfection (Polyplus-Transfection Inc., New York, NY). For cytotoxicity assay, cells were harvested and seeded 24 hours after transfection. For immunoblotting, cells were harvested 48 hours after transfection.

Statistical Analysis
Statistical analysis was performed using GraphPad Prism5 statistical software (San Diego, CA). Statistical significance was set at P < 0.05.


   RESULTS
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 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Functional Characterization of Monoclonal Antibodies to AIF
Specificity of the monoclonal antibodies was determined by an immunoblotting analysis of the whole cell lysate. The unique 67-kD protein was precipitated by monoclonal antibodies and protein G sepharose (Figure 1A). The immunoprecipitated protein was characterized by MALDI-TOF. The peptide mass fingerprints of the 67-kD protein matched that of full-length AIF: UniProtKB/Swiss-Prot: O95831, Programmed cell death protein 8 (PDCD8), apoptosis-inducing factor. The matched peptides covered 49% (303/613 aa's) of the protein. Immunocytochemical staining showed that AIF was abundantly present in cytoplasm. The granularly subcellular structure suggested that AIF was predominantly present in mitochondria (Figure 1B). We used an uptake assay of MitoTracker green FM (Molecular Probes, Inc., Eugene, OR) and confocal fluorescence immunocytochemistry to confirm that AIF was localized in mitochondria (Figure 1C). Detection of nuclear AIF after staurosporine (STS) treatment further validated that our monoclonal antibodies recognized functional AIF (Figure 1D).


Figure 1
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  		Figure 1. Characterization of monoclonal antibodies to apoptosis-inducing factor (AIF). (A) A unique 67-kD protein, which was precipitated by monoclonal antibodies and protein G sepharose, was resolved by SDS-polyacrylamide gel electrophoresis and stained with Coomasie blue. (B) Immunocytochemical staining showed that AIF was abundantly present as distinct granules in the cytoplasm, which suggests that AIF can be located in mitochondria. (C) Confocal fluorescence immunocytochemistry of cells with (C1) AIF-specific monoclonal antibodies labeled with rhodamine. (C2) Cells fed with a mitochondria-specific dye, MitoTracker green FM. (C3) Nuclei were stained with fluorescent dye 4', 6-diamidino-2-phenylindole (DAPI). (C4) A merged image of C1, C2, and C3 confirms that AIF is located in mitochondria. (D) After treatment with 2 µM of staurosporine for 2 hours, detection of AIF protein in nuclei indicated that monoclonal antibodies specific to AIF recognized functional AIF. (D1) Confocal immunocytochemistry with AIF-specific monoclonal antibodies labeled with rhodamine. (D2) Cells fed with MitoTracker green FM. (D3) Nuclei stained with DAPI. (D4) A merged image of D1, D2, and D3.

 
Effect of HGF on AIF Expression and Cell Survival after Cisplatin Challenge in Lung Adenocarcinoma Cells
Six lung adenocarcinoma cells, H23, H838, A549, H1437, H2009, and H2087, examined by immunoblotting, expressed various levels of AIF. Expression of AIF was high in H23, A549, and H1437; intermediate in H2009; and low in H838 and H2087 (Figure 2A). The results were confirmed by RT-PCR. Specificity of the amplified cDNA fragments was determined by DNA sequencing. DNA sequence matched that of AIF (NM_004208.2) listed in the GenBank database (http://www.ncbi.nlm.nih.gov/). No mutation or polymorphism in AIF mRNA was found. Interestingly, cisplatin sensitivity of each cell line was proportional to the expression level of AIF (Figure 2B); that is, higher AIF level indicates higher drug sensitivity. In vitro, after treatment with HGF for 24 to 48 hours, AIF level decreased by 40 to 60% (Figure 2C). Although HGF treatment did not totally inhibit cisplatin-induced nuclear translocation of AIF (Figure 2D), HGF decreased cisplatin cytotoxicity of A549 cells (Figure 2E). Enforced AIF expression in H838 cells (Figure 3A), on the other hand, increased cisplatin sensitivity (Figure 3B). These results clearly indicated that HGF could indeed suppress AIF expression and that AIF level was proportional to cisplatin toxicity in ADC cells. Because FAK is a downstream effector of c-MET (HGFR), and activated c-MET could directly interact with FAK to initiate HGF-related cell effect, we therefore examined whether interference of FAK activity could affect AIF expression.


Figure 2
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  		Figure 2. AIF Expression and cisplatin sensitivity in cultured adenocarcinoma (ADC) cells. (A) Expression of AIF was detected in all six human lung ADC cell lines by immunoblotting. Expression of AIF was high in H23, A549, and H1437; intermediate in H2009; and low in H838 and H2087. (B) Sensitivity of ADC cells to cisplatin was measured by a colony-forming assay. Cytotoxicity to cisplatin was high in H23 (triangles), A549 (inverted triangles), and H1437 (squares), and low in H2087 (circles) and H838 (diamonds). (C) After treatment with HGF for 24 to 48 hours, AIF level in A549 cells decreased by 40 to 60%. (D) After incubation with HGF for 24 hours, A549 cells was treated with 2 µM STS for 2 hours, feeding with MitoTracker green FM for 15 minutes and then stained for confocal fluorescence immunocytochemical examination. Although HGF treatment decreased intracellular AIF level, it did not inhibit nuclear translocation of AIF (white arrows indicate AIF-positive nuclei). AIF: cells stained with AIF-specific monoclonal antibodies labeled with rhodamine (red). DAPI: fluorescent nuclear stain (blue). Merge: a merged image of the three fluorescences. (E) Pretreatment of A549 with various concentrations (0 [squares], 1.0 [solid triangles], and 2.0 µg/ml [open triangles]) of HGF for 24 hours before cisplatin challenge increased cell survival. F-test, P < 0.01.

 

Figure 3
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  		Figure 3. Effect of AIF expression level on cisplatin sensitivity of H838 cells. (A) Transfection with pcDNA3.1-AIF/myc-His carrying the full-length AIF increased the expression of the exogenous AIF/myc-His (indicated as AIF+) and endogenous AIF in H838 cells. (B) Transfection with pcDNA3.1-AIF/myc-His increased cisplatin sensitivity of H838 cells. Squares, H838; triangles, H838 transfected with pcDNA3.1/myc-His vector; inverted triangles, H838 transfected with pcDNA3.1-AIF/myc-His. F-test, P < 0.001.

 
Effect of FAK, a Downstream Effector of HGFR, on Cisplatin Resistance and AIF Expression in Lung Adenocarcinoma Cells and Murine Embryonic Fibroblasts
As shown in Figures 4A and 4B, AIF expression and cisplatin sensitivity markedly increased in CL1–5 cells after introduction of C-terminus–truncated FAK (FAK-N'), which is known to interfere with c-MET binding to wild-type FAK (9). Interestingly, murine embryonic fibroblasts (MEF) that carried FAK–/– genotype/phenotype had a higher level of AIF and was more sensitive to cisplatin than FAK+/+ cells (Figures 4C and 4D). Re-introduction of FAK gene into FAK–/– cells (FAK–/re) lowered AIF level and drug sensitivity. Loss of reintroduced FAK in FAK–/re cells, on the other hand, increased AIF level and drug sensitivity. In mutant MEF cells, which had inactive c-MET, enforced expression of FAK did not affect AIF expression or drug sensitivity (data not shown). These results indicate that functional c-MET, which can interact with HGF and transmit cell signaling, is required for FAK activation. These data also showed that the effect of HGF on AIF expression is in fact through c-MET and FAK activation.


Figure 4
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  		Figure 4. The effect of FAK on AIF expression and cisplatin sensitivity. (A) Introduction of C-terminus–truncated FAK (FAK-N'), an FAK competitor protein, increased AIF expression (by ~ 3.84-fold) in CL1–5 cells. (B) Interference with FAK activity by competitor FAK-N' markedly decreased cisplatin resistance (solid circles, wild-type CL1–5; open circles, FAK-N'-expressed CL1–5). (C) AIF level in FAK–/– mouse embryonic fibroblasts (MEF) was clearly higher than that in FAK+/+ MEF. Re-introduction of full-length FAK gene into FAK–/– MEF (FAK-/re MEF) decreased AIF level. Loss of re-introduced FAK in FAK-/re cells (FAK-/re, Day 15) increased AIF expression. (D) Relationship between FAK expression and cisplatin sensitivity in MEF. Solid circles, FAK+/+ MEF; triangles, FAK–/– MEF; open circles, FAK-/re MEF; diamonds, FAK-/re, day 15. F-test, P < 0.001.

 

   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
The results indicate that monoclonal antibodies generated in this study not only recognize the functional AIF, which is normally present in mitochondria (4), but also recognize apoptotic AIF in the nuclei after genotoxic challenge (2–6).

Several studies have shown that HGF-related cytoprotections are mediated by activation of Akt (10) and restoration of Bcl-2 expression (11). Bcl-2 proteins then inhibit caspase activation and caspase-related cell death. In addition, activated Akt phosphorylates Bax, which then inhibits oligomerization of Bax and reduces Bax-associated perforation of mitochondrial outer membrane (MOM) (12). Maintaining an intact MOM could contain the efflux of intermembranous molecules (e.g., cytochrome c and AIF) and prevent activation of caspases (3–5).

Gallego and coworkers showed that sensitivity of NSCLC U1810 cells to STS was proportional to AIF expression (13). Jankowski and colleagues demonstrated that, although both HGF and stromal-derived factor-1 (SDF-1) can increase metastatic potential of rhabdosarcoma cells, only HGF increases radiation resistance (14). Ishihara and associates found that HGF-associated decrease of NMDA-induced cell death is via inhibition of nuclear translocation of AIF in cultured hippocampal neurons (3). Although we did not observe that HGF inhibits nuclear translocation of AIF after treatment with genotoxic reagents, our results showed that correlation of AIF level with cell sensitivity to anticancer therapeutics was not only detected in lung cancer cells, but also in mouse embryonic fibroblasts. The difference between their results and our findings could be genotoxic agents used in the respective studies: Ishihara and coworkers used NMDA, of which the cytotoxic effect is via NMDA receptor and the mechanism is more or less like tumor necrosis factor–related apoptosis–inducing ligand (TRAIL)-mediated cell death (15); and we used STS and cisplatin, of which the effect is on kinase inhibition and DNA alkylation, a reaction that is associated with DNA repair.

As noted previously, Akt activation is frequently detected in HGF-related cytoprotections. It is thus reasonable to anticipate that HGF affects AIF expression via c-MET activity (1, 9). Moreover, since FAK is a downstream effector of HGF and c-MET, activated FAK could have an immediate effect on tumor cell growth and invasion (1, 9, 14). Our data suggested that decreased AIF level in ADC could be a result of cell growth–related acceleration of nuclear replication, during which mitochondria duplication might not be able to keep up with the pace. It is also possible that HGF, c-MET, and FAK pathway may somehow affect AIF expression via regulation at transcriptional or translational level.

It is worth noting that FERM (band four point one, Ezrin/Radxin/Moesin) region of FAK protein could directly interact with the N-terminal transactivation domain of p53 and activate MAPK, which is downstream in HGF/c-Met pathway and is closely associated with cell proliferation (16). Although the effect of p53 and MAPK activation on mitochondrial duplication and mitochondria-associated protein expression is yet to be examined (17), detection of overproduced glycolytic products (e.g., lactate) in peripheral blood of patients with cancer favors our expectation that there is an imbalance between mitochondrial and nuclear replication (18). Selective binding of p53 to FAK, moreover, may favor cell proliferation. Interestingly, ATM and ATR kinases, the two cell stress–responsive proteins that are essential for DNA repair (19), contain a stretch of focal adhesion targeting (FAT) and phosphoinositide 3-kinase (PI3K) motifs. Because p53 is a substrate of ATR and ATM kinases, it is possible that these enzymes may help elude the regulatory effect of p53 on cell cycle progression and AIF expression as well. In addition, AIF level is correlated with expression of a critical sensor protein of DNA damage, Nijmegen breakage syndrome 1 (Nbs1, nibrin), which is essential for mediating cell-cycle arrest and DNA-repair mechanism (20). These data considered with our current results clearly provide an explanation for how the activated FAK could affect AIF expression, and increase cytotoxicity of human melanoma cells to 5-fluorouracil, an antimetabolite that could introduce DNA damage during DNA replication (21). The impact of HGF on AIF expression remains to be determined if this is one of the pathophysiologic effects that influence drug resistance in tumor cells.

In conclusion, our data show that HGF decreases AIF expression and cisplatin sensitivity in lung ADC cells. Enforced AIF expression increased cisplatin sensitivity. In FAK–/– MEF and in FAK activity-inhibited lung cancer cells, AIF expression and cisplatin sensitivity increased. These results suggest that HGF/c-Met system plays an important role in increasing drug resistance of ADC cells through FAK activation to mediate down-regulation of AIF expression.


   Acknowledgments
 
The authors thank Dr. Po-Chao Chan for technical assistance in confocal immunofluorescence microscopy.


   Footnotes
 
* These authors contributed equally to this study. Back

This study was supported by the grants from the National Science Council, Taiwan (NSC94-2320-B-005-005 to K.-C.C.; and NSC95-2314-B-075A-015 to C.-Y.C.).

Originally Published in Press as DOI: 10.1165/rcmb.2007-0001OC on December 20, 2007

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form January 2, 2007

Accepted in final form October 22, 2007


   References
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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