The Reverse Effect Of X-ray Irradiation On Acquired Gefitinib Resistance In Non-small Cell Lung Cancer Cell Line NCI-H1975in Vitro

Objective: Lung cancer is the most commonly diagnosed malignancy with thehighest morbidity and mortality currently. Non-small cell lung cancer (NSCLC)accounts for80%~85%. For a long time, the main treatment for NSCLC has beensurgical resection combined with chemoradiotherapy. But the application of traditionalchemotherapeutics, which were mostly DNA synthesis inhibitors, was vastly restricteddue to their poor specificity and severe side effects on patients. In recent years,epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), representedby gefitinib, has demonstrated dramatic clinical benefits in the treatment of NSCLC.Some retrospective studies have previously shown that patients with NSCLC harboringsomatic activating mutations in exon18,19,21of EGFR, which were discoveredfrequently in adenocarcinoma of Asians, women, and nonsmokers, were usuallysensitive to TKI. Nevertheless, most of NSCLC patients being sensitive initially to TKIwould develop acquired TKI resistance after continuous drug administration. Therefore,it is imperative to solve the problem of acquired TKI resistance clinically. As reported,the mechanisms of acquired TKI resistance are mainly as follows: the T790M mutationin exon20of EGFR; the encoding gene mutations as KRAS in EGFR downstream ofthe signal transduction pathway, the abnormal amplification of other oncogenes as MET,and epithelial-mesenchymal transition (EMT). Since most of these mechanismsmentioned were related to DNAchanges, for example, EGFR T790M mutation has been regarded as the specific reason for acquired TKI resistance. It is well-known that onemechanism of anti-cancer from radiotherapy is to damage DNA of cancer cells. Itshould be thought that the acquired TKI resistance would be probably reversed bychanging DNAof cancer cells.In this research, we chose a human NSCLC cell line NCI-H1975(harboring boththe L858R mutation in exon21and the T790M mutation in exon20of EGFR) as theparent cell line to establish an acquired gefitinib-resistant cell line NCI-H1975/GR. Andthen NCI-H1975/GR was irradiated with X-ray to develop the X-ray irradiated cell lineNCI-1975/GR/XR. By comparing the sensitive state to gefitinib of the three cell lines,we aimed to investigate the possibility that X-ray irradiation would reverse the acquiredgefitinib resistance in NSCLC cell line NCI-H1975in vitro, to explore the mechanismand hoped to provide some help for more effective using of gefitinib in theindividualized treatment of NSCLC.Methods:1. Establishment of cell lines1.1Mutation status of EGFR, KRAS and BRAF gene of NCI-H1975cell line wasdetected by HRMA;1.2The acquired gefitinib-resistant cell line named as NCI-H1975/GR wasestablished by exposing parental cell line NCI-H1975to gefitinib in increasingconcentrations gradually over a year.1.3The NCI-H1975/GR cells were irradiated by an X-ray generator at a dose of6Gy. Irradiation at the same dose was repeated after the cells resumed normalproliferation status as that of the cell unirradiated. After4times irradiated, the X-rayirradiation cell line was estabilished, named as NCI-H1975/GR/XR.2. The growth and resistant condition to gefitinib of three cell lines were detectedThe viable cell numbers were calculated and the inhibition curves of three celllines to gefitinib were depicted from Trypan blue assay; IC50and gefitinib resistanceindex were analyzed with MTT; The cellular morphologies and growth conditions ofthree cell lines were directly observed under inverted microscope; The growth curves were portrayed from cytometry, and the cell population doubling times of three celllines were figured out;The changes of cell cycle distributions and apoptosis of three celllines treated with and without gefitinib were detected by flow cytometry.3. The protein expressions of E-cadherin and vimentin from three cell lines weredetected by western-blot analysis.4. Mutation status of EGFR, KRAS and BRAF gene in NCI-H1975/GR/XR andNCI-H1975/GR cells was performed by HRMA.Results:1. The detection results of HRMA displayed that there existed the L858R mutationin exon21and the T790M mutation in exon20of EGFR in NCI-H1975cell line.2. The growth and resistant condition to gefitinib of three cell lines2.1The results of Trypan blue showed that compared with NCI-H1975/GR, theviable cell numbers of NC-H1975/GR/XR treated with gefitinib did not significantlydecrease on the1stday (P>0.05), but they decreased significantly from the2ndday to the4thday on dose-dependence (P<0.05). There was a negative correlation of viable cellnumbers of cell line NCI-H1975/GR/XR to gefitinib concentration in the same effecttime.2.2The IC50calculated from MTT assay of NCI-H1975/GR/XR, NCI-H1975/GRand NCI-H1975were respectively6.40±0.17μmol/L,12.42±0.09μmol/L and6.15±0.22μmol/L. The resistance index of NCI-H1975/GR/XR cell line returned to1.04from2.02of NCI-H1975/GR.2.3There were obvious morphological differences in three cell lines. The resistantcells NCI-H1975/GR became spindle from the parent cells NCI-H1975being longspindle, and the cells became smaller; however, part of X-ray irradiation cellsNCI-H1975/GR/XR returned back to long spindle.2.4The population doubling time of three cell lines were46.68±2.68h,47.73±3.50h and38.70±5.92h respectively; the time of NCI-1975/GR/XR was1.05h（P>0.05）shorter than that of NCI-H1975/GR.2.5After being treated with gefitinib for24h, the cell proportions of phase G0/G1 in three cell lines obviously increased (P<0.05); the extent of the cell proportion ofphase G0/G1increased in NCI-H1975/GR/XR was more obvious than that inNCI-H1975/GR (P<0.05).2.6There was no visible difference (P>0.05) in the apoptosis cell proportionsbetween NCI-H1975/GR/XR and NCI-H1975/GR treated with gefitinib at lowconcentrations of20and40μmol/L, but at the high concentration of80μmol/L, theapoptosis cell proportion of NCI-H1975/GR/XR was significantly higher than that ofNCI-H1975/GR (P<0.05).3. The results of western-blot showed that the expression of E-cadherin was lower,while the expression of vimentin was higher in NCI-H1975/GR than those inNCI-H1975. The expression of E-cadherin was higher (P<0.05) and the expression ofvimentin was (P<0.05) lower in NCI-H1975/GR/XR than those in NCI-H1975/GR.4. New EGFR, KRAS and BRAF mutations were not found in NCI-H1975/GR/XRand NCI-H1975/GR compared with those of NCI-H1975cells by HRMA.Conclusion:1. X-ray irradiation could reverse acquired gefitinib resistance in non-small celllung cancer cell line NCI-H1975in vitro.2. The mechanism of X-ray reversing acquired gefitinib resistance may be relatedwith “mesenchymal-epithelial transformation”.