Gleevec (STI-571) inhibits lung cancer cell growth (A549) and potentiates the cisplatin effect in vitro
© Zhang et al; licensee BioMed Central Ltd. 2003
Received: 10 December 2002
Accepted: 3 January 2003
Published: 3 January 2003
Gleevec (aka STI571, Imatinib) is a recently FDA approved anti-tumor drug for chronic myelogenous leukemia. Gleevec binds specifically to BCR-ABL tyrosine kinase and inhibit the tyrosine kinase activity. It cross-reacts with another two important membrane tyrosine kinase receptors, c-kit and PDGF receptors. We sought to investigate if Gleevec has a potential role in treatment of non-small cell lung cancer.
We have shown that Gleevec alone can inhibit the A549 lung cancer cell growth in dose-dependent manner, and the optimal concentration of Gleevec inhibition of A549 cell growth is at the range of 2–3 μM (IC50). We have also shown that A549 cells are resistant to cisplatin treatment (IC50 64 μM). Addition of Gleevec to the A549 cells treated with cisplatin resulted in a synergistic cell killing effect, suggesting that Gleevec can potentiate the effect of cisplatin on A549 cells. We also showed that the A549 lung cancer cells expresses the platelet derived growth factor receptor α, and the inhibitory effects of Gleevec on A549 cells is likely mediated through inhibition of PDGFR α phosphorylation. We further tested 33 lung cancer patients' tumor specimens to see the frequency of PDGFR-α expression by tissue micro-arrays and immunohistochemistry. We found that 16 of the 18 squamous carcinomas (89%), 11 of the 11 adenocarcinomas (100%), and 4 of the 4 small cell lung cancers (100%) expressed PDGFR-α.
These results suggest a potential role of Gleevec as adjuvant therapeutic agent for treatment of non-small cell lung cancer.
KeywordsLung cancer A549 cell Gleevec (STI571) PDGF receptor
Lung cancer is the leading killer of all cancer patients. It is generally classified as small cell carcinoma and non-small cell carcinoma. Treatment of lung cancer is less than optimal and the mean survival for advanced lung cancer patient is less than one year regardless what treatment regimen was used . A new approach other than conventional chemoradiation therapy is needed for prolonged survival of lung cancer. Emerging new treatment modalities are generally targeted to specific tyrosine kinases of the tumor cells through basically two independent approaches . One is to use highly specific monoclonal antibody to target the membrane receptors of growth factors important for tumor cell growth, and the antibody/antigen complex evokes host immune system to kill the tumor cells. This approach is exemplified by Her2/neu receptor in breast cancer patients and Herceptin . The second approach is to develop small organic molecules targeting the specific tyrosine kinases in the signaling pathway in the tumor cells that can easily gain access into the tumor cells. This approach is best exemplified by Gleevec and BCR-ABL fusion kinase in chronic myelogenous leukemia .
Gleevec (also known as STI571, Imatinib from Novartis Pharmaceutical Inc.), a recently FDA approved drug for chronic myelogenous leukemia, is an ATP analogue, and it competitively binds to and inhibits BCR-ABL tyrosine kinase, resulted from the chromosomal translocation t(9; 22). Gleevec has been shown to induce clinical, hematological and molecular remissions for CML patients . It cross-reacts with two other important growth factor receptors containing tyrosine kinase domains, c-kit and PDGF receptors, that play important roles in growth and proliferation of a variety of cell types . C-kit (also known as CD117) is frequently mutated in gastrointestinal stromal tumor (GIST), and the mutated c-kit shows higher tyrosine kinase activity. Gleevec has been demonstrated to inhibit the growth and proliferation of GIST, and induce complete or partial clinical remission in GIST patients [6, 7]. Recently Gleevec was showed to inhibit the growth of dermatofibrosarcma protuberans (DFSP) through inhibiting the PDGF receptor [8, 9].
As a part of effort to evaluate the newly emerging drugs for lung cancer, we studied the role of Gleevec on non-small cell lung cancer. We previously showed that non-small cell lung cancers express minimal (negligible) level of c-kit (Zhang, P., unpublished). Therefore it is unlikely that Gleevec exerts the effect, if any, through c-kit on non-small cell lung cancer. We reported here a study of inhibitory effect of Gleevec on lung cancer cells (A549 cells) in vitro. We showed that Gleevec alone can inhibit the growth and proliferation of A549 cells at the known therapeutic concentration for CML. Addition of Gleevec to A549 cells with cisplatin induced cell death synergistically, suggesting Gleevec can potentiate the cisplatin effect on A549 cells. We have demonstrated that A549 cells express PDGFR α, one of the known potential targets for Gleevec effect. The inhibitory effect of Gleevec on the A549 cells is likely mediated through inhibition of PDGF receptor α phosphorylation. We further tested 33 lung cancer patients' tumor specimens, and showed that most of the lung cancer tumor specimens expressed PDGFR-α. These results provided important in vitro data to support the notion that Gleevec can inhibit the A549 cell growth and proliferation, and may potentially offer a treatment option for lung cancer either alone or in conjunction with chemotherapy drug cisplatin.
Gleevec inhibited the A549 cell growth in a dose-dependent manner
Gleevec potentiated the cell-killing effect of cisplatin
Expression of PDGFR-α in the A549 lung cancer cells
Microarrays and immunohistochemical staining
Summary of PDFGR-α expression in lung cancer specimens
Small cell carcinoma
Gleevec is the first tyrosine kinase inhibitor that has been proven to be effective for clinical cancer patients, and its design is based purely on the inhibitory effect of the compound on the intracellular BCR-ABL tyrosine kinase. This represents a new direction in drug design targeting specific tyrosine kinases important for intracellular signal transduction pathways. There are many other small organic molecules targeting different tyrosine kinases in horizon. From the clinical standpoint, it will be of great importance to see this line of new molecules to synergize with conventional chemotherapy or sensitize the tumor cells to conventional chemoradiation therapy. Our current study showed that the small organic molecules such as Gleevec targeting the specific tyrosine kinases can not only inhibit the tumor cell growth alone, but also synergize with cisplatin in induction of tumor cell death.
Gleevec effects vs drug toxicity
We have shown in this study that Gleevec alone can inhibit the growth of A549 cells at the concentration of 2–3 μM (IC50). This is within the known therapeutic ranges for patients with CML, since the plasma levels of Gleevec inducing hematologic and cytogenetic response in patients with CML were reported to be in the range of 0.1–3.4 μg/ml (0.17–5.68 μM) after treatment with 25–600 mg /day . In our system, the level of Gleevec at the tested concentration was in the low micromolar ranges, and the inhibitory effect of Gleevec on A549 cells is likely to be genuine through binding to the receptor, not due to the toxic effects of the drug. This inhibitory effect can also be seen with the human kidney 293 cells with slight higher IC50. At the concentration of 4 μM or higher, the inhibitory effect of Gleevec on both A549 and 293 cells were found to be identical (Figure 2 and 3). Higher concentration than 6 μM in the cell culture condition is unlikely to be translated to clinical patients, since the adverse effects of the drug will become intolerable for the patients.
Potential target of Gleevec in lung cancers
Gleevec was designed to inhibit the BCR-ABL tyrosine kinase, and it cross-reacts with c-kit and PDGFR . In non-small cell lung cancer, BCR-ABL expression has never been reported, and it is unlikely that BCR-ABL fusion protein plays any role in lung cancer. There has been no report to link cellular ABL kinase mutation to any cancer patients except BCR-ABL in CML, although the detailed c-Abl function has not been extensively studied in lung cancer patients. CAbl is a tyrosine kinase that plays important roles in cell growth, differentiation and apoptosis. Although c-Abl mutations have not been reported in human cancers, c-Abl related protein, ARG (or Abl-2), was shown to be regulated in non-small cell lung cancers by hypermethylation (Dr. Steven Reynolds, National Institute of Occupational and Safety Health, Morgantown, WV, personal communication). ARG shares significant homology with c-Abl throughout the amino acid sequences. Gleevec has been shown to inhibit ARG tyrosine kinase (Dr. Jean Wang, Biology, University of California San Diego, personal communication), although the functional significance of ARG kinase in lung cancer is yet to be elucidated.
We have studied the expression of c-kit (CD117) in the non-small cell lung cancer patients, and we found that approximately 13% of the tumor specimens were weakly positive for c-kit. The level of expression is generally weak (1+). These results lead us to find an alternative target for Gleevec, because of the low expression level and low frequency of expression of c-kit in the lung cancer patients.
PDGFR expression in lung cancer
We have shown convincingly that lung cancer cell A549 expressed PDGFR-α both by immunostaining the cells and by Western blot analysis. PDGF receptors are widely expressed in a variety of tissues, and the levels of expression in normal tissues are minimal. Over-expression of PDGFR has been reported in a variety of human tumors including glioma and glioblastoma, pancreatic and colonic cancers, breast, bone and ovarian tumors . In normal cells, expression of PDGFR can be seen in the fibroblasts and the smooth muscle cells in the lung and airway . There is so far to our knowledge no report to link the expression of PDGFR to lung cancer. Since it has been reported that Gleevec can inhibit the function of PDGFR under the cell culture conditions, it is likely that Gleevec exerts its inhibitory effects on A549 lung cancer cells through inhibiting the PDGFR function.
Synergistic effect of Gleevec and cisplatin on A549 cells
We have shown that Gleevec can exert its effect synergistically with cisplatin. Cisplatin causes two types of DNA damage, DNA adducts and inter-stranded cross-linking, resulting in activation of apoptosis pathway in the target tumor cells [13, 14]. Our results suggest that inhibition of tyrosine kinase activity of the potential targets by Gleevec appear to potentiate the effect of cisplatin in DNA-damage induced apoptosis in A549 cells. Although the underlying mechanism of such synergism is unclear, it is of great interest to combine the two drugs in clinical lung cancer patients to see if the synergy of the two drugs exists under the physiological conditions. It has been widely thought, but never been proven that the specific tyrosine kinase inhibitors such as Gleevec can be used as adjuvant therapy in combination with conventional chemotherapy, because the limited clinical trial data suggests only a marginal benefits using this kind of drug to treat the cancer alone (John Rogers, WVU Cancer center, ECOG trial member, personal communication). Current study results suggests that Gleevec can potentiate the cisplatin effect on A549 lung cancer cells, and these findings provide important in vitro data for further testing the possibility of using Gleevec as adjuvant therapy for clinical lung cancer patients.
Cell culture and drug testing
Lung cancer cell line A549 cells were cultured in DMEM with 5% fetal calf serum as described. The cells are seeded in 96-well plate at the cell density of 2–4 × 104 per well as indicated, and the tumor cells were cultured in the medium containing serum. Cisplatin was purchased from Sigma Chemicals (St. Louis, MO), and dissolved in water at the concentration of 32 mM. The Gleevec (Novartis Pharmaceuticals. Inc., NJ) was purchased from the outpatient pharmacy at West Virginia University and dissolved in water at the concentration of 1 mM. The experimental procedure for the cisplatin and Gleevec treatment was the following: The A549 cells and human 293 cells were plated into the 96 well plates at the cell density indicated and the cells were allowed to attach overnight. The attached cells in the plates were washed once with PBS and replaced with fresh medium containing various concentrations of drugs indicated. The MTT assays were performed after 48 hours of continuous incubation with medium containing the drugs. The values shown in the figures are mean readings from five wells in each experiments and representative of three independent experiments.
We have performed a series of MTT assays to determine the effects of the anti-tumor drug STI571 on the number of A540 lung cancer cells and human 293 kidney cells. The assay is based upon the cleavage of the yellow tetrazolium salt MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] to purple formazan crystals by metabolically active cells . The tested concentration of Gleevec ranged from 0.5 to 8 μM. The tumor cells were seeded into 96-well culture plate, and maintained for culture for 24 hours before the first compound was added to the medium. The cultured cells were incubated in a medium containing 5% serum and treated with Gleevec for 48 hours. After treatment, 10 μl of MTT labeling reagent were added to each well and plates were incubated at 37°C for 4 h. Following MTT incubation, the cultures were solublized and the spectrophotometric absorbance of the samples was detected by using a microtiter plate reader. The wavelength to measure absorbance of formazan product is 570 nm, with a reference wavelength of 750 nm.
Immunofluorescent staining of the cultured cells
The A549 cells were cultured as described on the coverslips, and fixed with 3.7% paraformaldehyde for 10 minutes at room temperature. The fixed cells were permeablized with 3% Triton X-100 in PBS and directly used for immunofluorescent and immunocytochemical stainings. The primary anti-PDGFR-α antibody was purchased from Santa Cruz Biotechnologies Inc (Santa Cruz, CA). The secondary antibody for immunofluorescent labeling was from Molecular Probe Inc. (Portland, OR).
Lung tumor tissue microarrays and immunohistochemical staining
The high-density lung cancer tissue microarray slides were made in Clinomics Bisciences Inc. MA, and was used for immunohistochemical staining for PDGFR-a. Briefly, the tissue microarray slides were sectioned at 5 micron in thickness and heated to 65°C for 10 minutes to de-parafinize the tissues. The slides were washed three times in xylene, then dehydrated and rehydrated in 100%, 95% and 70% ethanol. The tissue sections were finally washed with PBS once before going through the antigen retrieval process. The antigen retrieval was performed using citrate buffer (pH 7.4) at 90°C for 30 minutes. The immunohistochemical staining was performed using Ventana Bench Mark II automated staining device following the manufacturer's instruction (Ventana Medical Inc. Tucson, AZ). The primary PDGFR-α antibody was from Santa Cruz Biotechnologies Inc (Santa Cruz, CA). The tumor section slides and the immunostaining patterns were reviewed by two independent practicing surgical pathologists, and the signal intensities were scored at 1+, 2+ and 3+. The staining characteristics of all tumors were summarized in Table I.
The A549 cells were maintained in the culture condition as described above. Western blotting analyses of PDGF receptors were performed using a previously described method . Briefly, the tumor cells were treated with Gleevec at the various concentrations for 6 hours. The whole cell extracts were prepared and used for Western blot with anti-PDGF receptor α using RIPA buffer containing SDS. The whole cell extracts were separated on the 7.5% SDS PAGE and transferred onto nitrocellulose membrane by electroblotting. The primary antibody was incubated in the 5% non-fat milk with the proteins on the membrane overnight at 4°C, and the protein of interest was visualized by the enhanced chemiluminescent method (ECL). The anti-PDGF receptor α antibody was used at 1:500 (Santa Cruz Biotechnologies Inc. CA).
PZ designed, organized the whole study and analyzed all the data. WG performed a majority of the experiments in the study. ST provided micro-array slides for lung cancers, and discussed extensively regarding the design and execution of the study. BSD reviewed the patient's tumor specimens. All authors read and approved the final manuscript.
This study was support in part by Sara Crile Allen & James F. Allen endowment fund for lung cancer research. We like to thank Ms. Patrician Turner for her superb technical assistance in immunohistochemical staining of lung cancer tumor specimens.
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