| Objective:To investigate the radio sensitivity enhancenment of Nimotuzumab at different administration times on human lung cancer A549 or Calu-6 lines cell lines, observe the differences of experiment results in two cell lines and explore the underlying reasons.Methods:Human lung adenocarcinoma A549 or Calu-6 cell lines were cultured in vitro. EGFR protein expression was measured by Western blot analysis. The subjects were divided into 6 groups:control group (O group), Nimotuzumab group (N group), radiation group (R group), Nimotuzumab administration before irradiation group (N→R group), Nimotuzumab administration upon irradiation group (NR group), Nimotuzumab administration after irradiation group (R→N group). The antiproliferative activity of Nimotuzumab was determined by MTT analysis, and the 20% and 50% inhibitory concentrations (IC20 and IC50) of two cell lines were caculated, respectively. The IC20 were needed for the next subjects. Cell-survival rates were evaluated by colony-forming assays.Cell-apoptosis and cell-cycle distribution were investigated using flow cytometry; meanwhile, the expression of phosphorylated DNA-PKcs, Bcl-2, Bax, P21, Akt and pAkt after irradiation and/or Nimotuzumab were determined by Western blot analysis.Results:1. Two human lung adenocarcinoma cell lines expressed differential EGFR levels. The A549 cell lines exhibited higher EGFR protein expression level than Calu-6 cell lines.2. The effect of the dose and time dependent growth inhibition was observed by the MTT analysis in the two cell lines. The IC20 and IC50 of A549 were (1.15±0.18)μg/ml and (24.78±1.16)μg/ml in 24 hours, (0.84±0.05)μg/ml and (11.08±0.30)μg/ml in 48 hours, and (0.72±0.35)μg/ml and (8.99±0.12)μg/ml in 72 hours for Nimotuzumab, respectively. The IC2o and IC50 of Calu-6 were (2.45±0.32)μg/ml and (51.15±1.58)μg/ml in 24 hours, (2.31±0.21)μg/ml and (48.49±0.59)μg/ml in 48 hours, and (1.06±0.08)μg/ml and (36.41±0.85)μg/ml in 72 hours for Nimotuzumab, respectively.3. Clonogenic radiation survival assays showed that the Dq,Do,N(cell-survival curve extrapolation number) (N→R) group was lower than those in (NR) group or (R→N) group both in the two cell lines. The SER of (N→R) group, NR group and (R→N) group was 2.02,1.50 and 1.26 in A549 cell lines, respectively. And the SER of (N-R) group, NR group and (R→N) group was 1.88,1.09 and 0.97 in Calu-6 cell lines, respectively. The enhanced SER in Nimotuzumab treated cells indicated that 24h before irradiation was found to be the optimal administration time for the enhancement of radiosensitivity.4. The Flow cytometry showed that Nimotuzumab increase G1/G0 phase arrest rates, but not in Calu-6 cell lines. Nimotuzumab administration before irradiation could obviously increase G2/M phase arrest and enhance radiation-induced apoptosis both in the two cell lines.5. In the two cell lines, Western blot showed that Bax protein was increased and reached a peak in groups where Nimotuzumab was administrated before irradiation. On the contrary, Bcl-2 protein was decreased and showed lowest value in the same groups. As an important factor of the G2/M cell cycle checkpoint, P21 protein showed a maximum value in (N→R) group. phosphorylated DNA-PKcs protein root activity was low in (O) group, while increasing in (R) group, and then decreasing when irradiation was combined with Nimotuzumab especially in (N→R) group. The Calu-6 cell lines exhibited higher Akt/or pAkt protein expression level than A549 cell lines. In the two cell lines, the expression of Akt protein had no difference in any group. Nimotuzumab could inhibit the expression of pAkt in A549 cell lines and radiation can enhance the expression of pAkt in two cell lines. The expression of pAkt was lowest in (N→R) group both in A549 and Calu-6 cell lines.Conclusions: The best radiosensitizing effect was obtained when Nimotuzumab delivered before irradiation both in A549 and Calu-6 cell lines. Nimotuzumab differently enhanced radiosensitivity effects in the two cell lines, probably via EGFR/Akt/or pAkt expression. However, detailed mechanism remains to be investigated. |
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