β1-integrin modulates cellular phenotype by mediating signals from the extracellular matrix (ECM). Although overexpression of integrin molecules in hepatocellular carcinoma (HCC) has been reported, the role of overexpressed β1-integrin in the disease process of HCC is not fully understood. The authors investigated the effects of β1-integrin on apoptosis in hepatoma cells.
Human hepatoma cell lines HepG2, Huh7, and HLE were stably transfected with full-length β1-integrin. Cells underwent apoptosis induced by chemotherapeutic reagents, including cis-platinum (II)-diammine dichloride, etoposide, and docetaxel. Cell survival and intracellular signaling pathways dependent on β1-integrin-mediated apoptosis effects were analyzed by treating cells with PD98059 (ERK inhibitor), SB203580 (p38MAP kinase inhibitor), wortmannin (phosphatidyl inositol-3-kinase inhibitor), and herbimycin A (tyrosine kinase inhibitor).
All three hepatoma cell lines overexpressing β1-integrin were protected from apoptosis induced by chemotherapeutic reagents, whereas parental or mock transfected cells were not. Treatment with PD98059 or SB203580 abolished the protective effect on apoptosis in cells overexpressing β1-integrin. Neither herbimycin nor wortmannin blocked the protective effects of β1-integrin overexpression.
Integrins are cell surface adhesion receptors composed of α- and β-subunits, which mediate cell-extracellular matrix (ECM) and cell-cell interactions.1 β1-integrin transduces biochemical signals from the extracellular environment, especially with respect to the growth, differentiation, invasive, and metastatic aspects of malignant cells.2, 3 Altered expression of integrins has been reported to be involved in both tumor suppression and tumor progression.4–6 We and others have described changes in integrin expression in hepatocellular carcinoma (HCC), including overexpression of β1-integrin and a role for β1-integrin in the progression of HCC.7–10
Chemotherapeutic drugs are cytotoxic by inducing apoptosis.11, 12 Patients with HCC have been treated with many reagents, including cis-platinum(II)-diamine dichloride (cisplatin), etoposide, and docetaxel, all of which induce apoptosis in tumor cells.13–15 However, these regimens have failed to show significant efficacy in HCC patients, suggesting resistance to chemotherapy in HCC. Multidrug resistance proteins, which are abundantly expressed in the liver, have been reported to contribute to chemotherapy resistance in HCC.16Bcl-2 and p53 mutations promote resistance to apoptosis by chemotherapy.17, 18 Recently, apoptosis of tumor cells by various stimuli has been blocked by β1-integrin-mediated cell adhesion to ECM,19, 20 suggesting that β1-integrin-mediated signals may protect cancer cells against chemotherapeutic drug-induced apoptosis. The role of β1-integrin overexpression in HCC cells in chemotherapy-induced apoptosis has not been studied. In the current report, we investigated the effects of several chemotherapeutic reagents on HCC cells; we showed resistance to chemotherapy-induced apoptosis in β1-integrin overexpressing-HCC cells and determined the responsible signal transduction pathway.
MATERIALS AND METHODS
Cisplatin and etoposide were purchased from Sigma (St. Louis, MO), and docetaxel was purchased from Rhone-Poulenc Rorer (Antony, France). PD98059 was purchased from Promega (Madison, WI), and SB203580, wortmannin, and herbimycin A were purchased from Sigma.
The human hepatoma cell lines HepG2, Huh7, and HLE were obtained from the Japanese Cancer Research Resources Bank (Osaka, Japan). Cells were cultured and maintained in Dulbecco's modified Eagle's medium (GIBCO-BRL, Gaithersburg, MD) containing 10% fetal calf serum (GIBCO-BRL).
A full-length complementary DNA (cDNA) coding human β1A-integrin cloned in pBJ-1 vector was given by Dr. Y. Takada (Scripps Research Institute, La Jolla, CA).21 The full-length β1A-integrin lacking a stop codon was constructed by polymerase chain reaction (PCR) amplification introducing BamHI and XhoI sites, and cloned into pT7Blue vector (Novagen, Madison, WI) to generate pT7GFβ1A. The BamHI-XhoI fragment of β1A-integrin cDNA was subcloned into the Bam HI-XhoI site of pEGFP-N1 vector (Clonetech, Palo Alto, CA) to generate pEGFP-N1/GFβ1A to express a β1A-integrin-GFP fusion gene under the control of the immediate early promoter of human cytomegalovirus.
Stable Transformation of HCC Cells
A full-length cDNA coding the human β1A-integrin cloned into pEGFP-N1 (pEGFP-N1/GFβ1A) or pEGFP-N1 alone was introduced into HepG2, Huh7, or HLE cells using lipofectamine (GIBCO-BRL) according to the manufacturer's instructions. Transfected HCC cells were treated with 500 μg/mL of G418 for two weeks and selected. Individual clones of HCC cells transduced with human β1-integrin were analyzed for transgene expression, and clones overexpressing β1-integrin were subjected to treatment with chemotherapeutic drugs.
RNA Isolation and Semiquantitative Reverse Transcription (RT)-PCR
Total RNA was extracted from cultured HCC cells using ISOGEN (Nippon Gene, Tokyo, Japan) according to the manufacturer's instructions. The concentration of RNA was determined spectrophotometrically, and the integrity of all samples was confirmed by visualizing 28S and 18S ribosomal RNA bands under ultraviolet (UV) light after gel electrophoresis. Semi-quantitative RT-PCR was performed as described previously.22 Briefly, 1 μg of total RNA was reverse transcribed with reverse transcriptase (Takara, Kyoto, Japan) using random primers. Subsequently, each RT reaction mixture was subjected to PCR amplification using Taq Gold polymerase (Perkin-Elmer, Branchburg, NJ) with cycle numbers varying from 15 to 40. Cycles consisted of heat denaturation (94° C for 1 minute), annealing (55° C for 1 minute), and extension (72° C for 2 minutes). The PCR products were size-fractionated on a 2% agarose gel, and visualized under UV light. Sequences of oligonucleotide primers used for RT-PCR to determine expression of the target gene are listed, preceded by accession number for Gene Bank or references, and followed by expected transcript sizes: GFP (U55762): sense 5′-GCAAGCTGACCCTGAAGTTCATC-3′, antisense 5′-GGATCTTGAAATTCACCTTGATGC-3′, 384 bp; β1A-integrin:23 sense 5′-AGAATCCAGAGTGTCCCACTGG-3′, antisense 5′-TTCCCTCATACTTCGGATTGA-3′,238 bp; Glyceraldehyde 3-phosphate dehydrogenase24 sense: 5′-ACGCATTTGGTCGTATTGGG-3′, antisense: 5′-TGATTTTGGAGGGATCTCGC-3′, 231 bp.
For the detection of β1A-integrin-green fluorescent protein (GFP) fusion mRNA, the sense primer of β1A-integrin and the antisense primer of GFP were used to generate a 785 bp RT-PCR product.
β1-integrin expression was determined by Western blotting. Cultured HCC cells were lysed with extraction buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 5 mM ethylene diamine tetraacetic acid (EDTA) (pH 8.0), 1 mM PMSF, 10 μg/mL trypsin inhibitor, and 50 mM iodoacetamide. After 30 minutes at 4° C, debris was eliminated by centrifugation at 15,000 rpm for 20 minutes; and the supernatant was collected. After measurement of protein concentration with a protein assay kit (Bio-Rad, Hercules, CA), 40 μg of protein was mixed with NuPAE sample buffer (Novex, San Diego, CA), separated by SDS polyocrylamide gel electrophoresis, transferred to a polyvinylidene difluoride membrane (Bio-Rad), and blocked with 0.1% Tween and 5% skim milk overnight. The immunoblots were incubated with anti β1-integrin mouse monoclonal antibody (Santa Cruz, CA) in phosphate buffered saline (PBS) with 1% bovine serum albumin for 1 hour. Rabbit polyclonal anti-human β-actin antibody (Biomedical Technologies, Stoughton, MA) was used as a control. The membranes were washed three times with 0.1% Tween 20 in PBS and stained with horseradish peroxidase-conjugated secondary antibody. All immunoblots were detected by the ECL system (Amersham, Buckinghamshire, England) according to the manufacturer's instructions.
Cell Proliferation Assay
The sensitivity of HCC cell lines to chemotherapeutic drugs was determined by the WST-1 cell proliferation assay kit (Takara) as previously described.25 Cells were seeded in 24 well culture plates at a density of 104 cells/well, incubated at 37° C for 24 hours, and incubated with chemotherapeutic reagents at the indicated concentration for the next 48 hours. The cells were then incubated with WST-1 reagents and absorbance of formazan products at 450 nm was measured with a CS-9300PC microplate reader (Shimadzu, Tokyo, Japan).
Detection of Apoptosis
For analysis of DNA laddering characteristic of apoptotic cell death, DNA was isolated from cells treated with chemotherapeutic drugs at a dose of 50% growth inhibition (IC50). Cells were resuspended in lysis buffer containing 10 mM EDTA, 10 mM Tris (pH 8.0), and 0.5% Triton X-100 at 4° C for 10 minutes and centrifuged at 16,000 rpm for 20 minutes. The supernatant was treated with RNase A at 37° C for 1 hour and proteinase K at 50° C for 30 minutes, then precipitated with isopropanol. The DNA was resuspended and electrophoresed in a 2% agarose gel at 50 V for three hours. DNA was visualized by ethidium bromide staining and photographed under UV light. Morphologic changes in the nuclear chromatin of HCC cells undergoing apoptosis were detected by staining with Hoechst 33342 (Wako, Osaka, Japan).26 Briefly, cells were plated on 96 well plates (2 × 105 cells/well), treated with chemotherapeutic drugs at the IC50 concentration for 24 hours; stained with Hoechst 33342, and then observed under fluorescence microscopy. One thousand cells were observed in five randomly chosen areas. Cells with condensed or fragmented nuclei were considered apoptotic. The data are expressed as the percentage of apoptotic cells.
Differences were determined using the Student t test, and P < 0.05 was considered significant. All experiments were performed in triplicate or more. Data are shown as the mean ± standard deviation.
Generation of β1-integrin Overexpressing Cells
HepG2, Huh7, and HLE cells were transfected with a full-length β1A-integrin fused with GFP (pEGFP-N1/GFβ1A) or pEGFP-N1 vector alone. After transfection and G418 selection for two weeks, 10 of each growing clone were picked and screened to confirm transgene expression by RT-PCR or Western blot. Results from parental cells, mock transfected cells, and two representative clones of each native HCC cell line are shown in Figure 1A and B. Green fluorescent protein mRNA expression was found in mock and β1-integrin transfected cells, and β1-integrin and GFP-fused mRNA expression was detected in β1-integrin transfected cells but not in mock cells. Increased β1-integrin mRNA was also found in β1-integrin transfected HCC cells compared to parental or mock transfected cells. Western blots showed increased levels of β1-integrin protein in β1-integrin transfected cells (Fig. 1B).
Green fluorescent protein expression was detected by fluorescence microscopy (Fig. 1C). Green fluorescent protein was diffusely located in the nucleus and cytoplasm of Huh7 mock cells, whereas it was present on the membrane, in the cytoplasm, and in the region forming the adhesion macula of Huh7 cells transfected with β1-integrin-GFP. Similar results were obtained in HepG2 and HLE cells (data not shown). The cells overexpressing β1-integrin were used for further experiments.
Overexpression of β1-integrin Protects HCC Cells from Chemotherapy Induced Growth Inhibition
To investigate the sensitivity of HCC cells to chemotherapeutic reagents, native cells were treated with cisplatin, etoposide, or docetaxel, and cell proliferation was determined after 48 hours. As shown in Figure 2A, growth of HCC cells was inhibited by chemotherapeutic drugs in a dose-dependent manner, although sensitivity differed among cells. Growth inhibition 50% (IC50) was observed with 1 μg/mL of cisplatin, 50 μg/mL of etoposide, and 100 ng/mL of docetaxel in HepG2; with 1 μg/mL of cisplatin, etoposide, and docetaxel in Huh7; and with 2 μg/mL of cisplatin, 10 μg/mL of etoposide, and 10 ng/mL of docetaxel in HLE cells. The respective concentrations corresponding to IC50 were used for the following experiments.
To investigate whether HCC cells overexpressing β1-integrin exhibit had altered sensitivity to chemotherapeutic reagents, the proliferation of transfected HCC cells was examined after cisplatin, etoposide, and docetaxel administration. As shown in Figure 2B, all HCC cells overexpressing β1-integrin showed significantly decreased cytotoxic effects of these three chemotherapeutic drugs as compared to parental or mock-transfected cells. To eliminate the possibility that resistance to chemotherapy was due to an increased proliferation rate, the proliferative ability of these transfected cells was examined. No significant differences in proliferation were observed between parental cells, mock-transfected cells, or β1-integrin transfected cells (data not shown). Therefore, overexpression of β1-integrin protects HCC cells from the growth inhibitory effects of cisplatin, etoposide, and docetaxel.
Protection of HCC Cells Overexpressing β1-integrin from Apoptosis Induced by Chemotherapeutic Reagents
Chemotherapeutic drugs are known to induce apoptosis of hepatoma cells.11 To determine the induction of apoptosis in HCC cells treated with cisplatin, etoposide, or docetaxel, electrophoretic analysis of DNA fragments was performed. We first investigated DNA ladder formation after treatment with chemotherapeutic reagents in parental, mock-transfected, and β1-integrin overexpressing HCC cells (Fig. 3). There was an increase in DNA-laddering in parental and mock transfected Huh7 cells, indicating apoptosis induced by cisplatin, etoposide, and docetaxel. In contrast, a substantial decrease in DNA fragmentation was observed in Huh7 cells overexpressing β1-integrin after treatment with chemotherapeutic drugs.
For quantification of apoptotic HCC cells induced by chemotherapeutic reagents, Hoechst 3342 staining was performed. After treatment, staining revealed that the number of cells showing small, condensed, and fragmented nuclei (indicative of apoptosis) was increased in native Huh7 cells (Fig. 4A). The ratio of apoptotic cells was calculated in parental, mock-transfected, and β1-integrin transfected HCC cells treated with cisplatin, etoposide, or docetaxel. As shown in Figure 4B, Huh7 cells overexpressing β1-integrin were substantially protected from apoptosis induced by chemotherapeutic drugs as compared to parental or mock-transfected Huh7 cells. Two representative clones of β1-integrin transfectants showed 48% and 55%, 67% and 75%, and 38% and 47% reductions in apoptotic cells compared to mock-transfected cells treated with 1 μg/mL of cisplatin, etoposide, and docetaxel, respectively. Similarly, the β1-integrin transfectants of HepG2 and HLE cells also showed substantial reductions in apoptosis induced by cisplatin, etoposide, and docetaxel (data not shown). Overexpression of β1-integrin in HCC cells increases their resistance to apoptosis induced by chemotherapeutic drugs.
Contribution of a MAP Kinase Pathway in β1-integrin Mediated Resistance to Apoptosis
To understand the mechanisms of β1-integrin mediated-resistance to apoptosis, signal transduction pathways involved in the process were investigated. Specific inhibitors of ERK MAP kinase (PD98059), p38 MAP kinase (SB203580); phosphatidyl inositol–3 (PI3) kinase (wortmannin), and tyrosine kinase (herbimycin A) were used. When HepG2 cells transfected with β1-integrin were pretreated with PD98059 or SB203580, the protective effects of β1-integrin on cell viability after chemotherapeutic drugs were markedly inhibited (Fig. 5A). Similar results were obtained in β1-integrin transfected Huh7 and HLE cells treated with these inhibitors (data not shown). The effects of these inhibitors on apoptosis were also determined in HepG2 cells transfected with β1-integrin. As shown in Figure 5B, pretreatment of HepG2 cells with PD90059 or SB203580 abolished the β1-integrin overexpression mediated protection from apoptosis induced by cisplatin, etoposide, and docetaxel; wortmannin and herbimycin did not enhance apoptosis. Furthermore, a substantial increase in nucleosomal DNA laddering was observed when HepG2 cells transfected with β1-integrin were pretreated with PD90059 or SB203580, but not with wortmannin or herbimycin (Fig. 5C).
Although administration of various chemotherapeutic drugs has been used for the treatment of HCC, few treatment protocols show response rates > 20%.27 One reason for resistance to chemotherapy in HCC is explained by the expression of the multidrug resistance gene (MDR, P-glycoprotein) and MRP.16, 28–30 These membrane proteins are expressed on HCC cells and promote efflux of intracellular chemotherapeutic drugs, thereby reducing the sensitivity of tumor cells to chemotherapy-induced apoptosis.
However, MDR protein is not the sole mechanism for resistance of cancer cells to apoptosis induced by chemotherapy. Factors such as p53, Bcl-2, and β1-integrin have been shown to be involved in the apoptotic process induced by chemotherapeutic drugs.17, 18, 31, 32 β1-integrin mediates cell-ECM or cell-cell interactions, and can transmit multiple signals.1 β1-integrin is essential for normal development,33 and disruption of this cell-matrix interaction causes apoptosis, showing β1-integrin as a survival factor.34 Several mechanisms have been proposed to account for β1-integrin-mediated suppression of apoptosis, including upregulation of Bcl-2 expression, suppression of interleukin 1β converting enzyme expression, and activation of MAP and PI-3 kinase.35–38 Overexpression of integrin molecules including β1-integrin in HCC has been shown,7–10 and several in vitro studies have shown that levels of constitutive activity of β1-integrins are correlated with invasive and metastatic abilities of HCC cells.39, 40 Alternatively, a knockout of α6β1-integrin expression in HepG2 cells showed a reduced ability to adhere and migrate on laminin matrices and to invade Matrigel, as well as significantly lower growth rates.41 These observations suggest the involvement of β1-integrin in the disease progression of HCC through invasion and metastasis.
Recently, the role of β1-integrin in chemotherapeutic drug-induced apoptosis has been examined. A study in murine tumor derived endothelial cells showed that antibody activation of β1-integrin prevented cells from etoposide induced apoptosis.31 In another study using human small cell lung carcinoma cells, Sethi et al.32 showed that ECM protected cancer cells from cisplatin and etoposide and that blocking antibody against β1-integrin abrogated this ECM-mediated protection from apoptosis. We have now investigated the effects of β1-overexpression in HCC cells on apoptosis induced by chemotherapy.
Overexpression of β1-integrin protected HCC cells from apoptosis induced by cisplatin, etoposide, and docetaxel. We also treated HCC cells overexpressing β1-integrin with several inhibitors of signal transduction pathways. As summarized in Figure 6 in bold lines, ERK and p38 MAP kinase pathways are involved in the blockade of apoptosis induced by cisplatin, etoposide, and docetaxel in β1-overexpressing HCC cells, but PI-3 kinase and nonreceptor protein tyrosine kinases are not involved. Although MAP kinase, PI-3 kinase, and protein tyrosine kinases assist hepatocytes and HCC cells to resist activation-induced apoptosis,42, 43, the pathway involved in protection from apoptosis is dependent on the specific apoptosis-stimulation signal or factor which blocks apoptosis, such as epidermal growth factor, tumor necrosis factor-α (TNF-α), or hepatocyte growth factor.42, 44 Figure 6 shows relationships of signaling pathways which interact with specific apoptotic stimuli. The current results suggest that MAP kinase is a key signaling pathway for protection from apoptosis in β1-integrin overexpressing HCC cells treated with chemotherapeutic reagents. Insights into the interactions of β1-integrin mediated signals will provide a basis for the development of better therapies to enhance apoptosis.
The incidence of HCC development is accelerated by the presence of hepatic fibrosis in patients infected with the hepatitis C virus.45 Animal studies show that HCC develops more frequently in fibrotic livers than in livers without fibrosis.46 However, the mechanisms by which the development of HCC is enhanced in fibrotic livers have not been clarified. Immunohistochemic studies show that β1-integrin was increased in fibrotic livers, along with α integrin subunits.7 Serum β1-integrin was increased in patients with liver fibrosis, and this may be a useful marker for hepatic fibrosis.47 Our recent data confirmed increased expression of β1-integrin mRNA in fibrotic livers, as well as a further increase of β1-integrin mRNA in HCC tissue.10
Our current results suggest that increased β1-integrin expression (which mediates signals from the ECM) might promote hepatocarcinogenesis in fibrotic livers by protecting hepatocytes from apoptosis caused by TGF-β, TNF-α, or the Fas/Fas ligand system, all known to cause hepatocyte apoptosis.48 Increased β1-integrin expression may promote progression of HCC by increasing the resistance of HCC cells to therapeutic reagents. Further investigation is required to clarify the role of β1-integrin in the regulation of apoptosis during hepatocarcinogenesis in the fibrotic liver.
In conclusion, the current data strongly suggest that β1-integrin overexpression protects HCC cells from chemotherapeutic drug-induced apoptosis via ERK and p38 MAP kinase pathways. Specific strategies directed at β1-integrin-mediated survival signals may improve the therapeutic response in HCC.
The authors thank Dr. Y. Takada (Scripps Research Institute, La Jolla, CA) for providing β1A-integrin expression vector.