Nimotuzumab enhances radiation sensitivity of NSCLC H292 cells in vitro by blocking epidermal growth factor receptor nuclear translocation and inhibiting radiation-induced DNA damage repair

Figures & data

Figure 1 Illustration of nimotuzumab viability influence in H292 and H1975 cells.

Notes: MTT assay revealed that nimotuzumab alone increased H292 viability suppression with time and dose escalation (A and C), but barely influenced H1975 viability (B and D). The suppression ability in H292 cells was still feeble and it was merely 42.5% (calculated separately, not shown in the figure) at maximum dose 1,000 μg/mL.
Abbreviation: MTT, methylthiazolyldiphenyl-tetrazolium bromide.

Figure 2 Cell clonogenic survival apoptosis and γ-H2AX formation.

Notes: H292 and H1975 distinct dose-survival curve was illustrated by Figure (A and B) (clonogenic survival assay n=3). Cell apoptosis distribution was shown by (C and D). The total apoptosis percentage was marked at the upper space of the right two quarterns (n=3). There was no statistical significance, but it seemed that H292 cells exhibited the trend of higher apoptotic percentage compared with H1975 cells (P=0.08 and P=0.90 for H292 and H1975 cells, respectively). γ-H2AX formed in both the cytoplasm and the nucleus. Protein formation was more significant in H292 cells (E and G) compared with H1975 cells (F and H). Figure (I) illustrated the brief profile of protein expression (n=3). Each bar represents the mean ± SD and *indicates significant difference (P<0.05).
Abbreviations: h-R3, nimotuzumab group; RT, irradiation group.

Table 1 Radiation parameters fitted to classic multitarget single-hit model

Parameters	H292		H1975
	Control	Combination	Control	Combination
D0	1.396	1.070	2.039	1.866
Dq	2.053	1.747	1.063	0.922
N	5.581	5.358	1.421	1.347
SF2	0.782	0.592	0.487	0.342
SER	1.304		1.092

Notes: Control group: cells receiving 6 Gy radiation; combination group: cells pretreated with 100 μg/mL nimotuzumab for 24 hours before receiving 6 Gy radiation; SER = D0 control group/D0 combination group.

Abbreviations: D0, mean lethal dose; Dq, quasi-threshold dose; N, extrapolation number; SF2, surviving fraction at 2 Gy; SER, sensitivity enhancement ratio.

Table 2 Cell cycle distribution in H292 cells ($\overline{x}$ ± s)

	G0/G1 (%)	G2/M (%)	S phase (%)
Control	77.77±1.20	9.14±0.58	13.10±1.59
h-R3	82.80±2.95	5.84±1.09	11.36±3.91
RT	63.62±2.83*	24.74±4.49	11.64±1.67
h-R3+RT	84.42±3.28*	7.79±7.30	7.79±4.09

Notes: h-R3: cells pretreated with nimotuzumab for 24 hours; RT: cells receiving 6 Gy radiation; h-R3+RT: cells pretreated with nimotuzumab and receiving 6 Gy radiation. Data was in the form of Mean ± SD.

* Indicates P<0.05.

Abbreviations: h-R3, nimotuzumab group; RT, irradiation group.

Table 3 Cell cycle distribution in H1975 cells ($\overline{x}$ ± s)

	G0/G1 (%)	G2/M (%)	S phase (%)
Control	89.60±3.31	1.92±2.04	8.48±1.26
h-R3	90.45±1.42	2.93±1.15	6.62±2.45
RT	73.00±5.95	17.55±7.47	9.45±1.66
h-R3+RT	78.29±3.07	13.03±2.87	8.68±0.20

Notes: h-R3: cells pretreated with nimotuzumab for 24 hours; RT: cells receiving 6 Gy radiation; h-R3+RT: cells pretreated with nimotuzumab and receiving 6 Gy radiation. Data was in the form of Mean ± SD.

Abbreviations: h-R3, nimotuzumab group; RT, irradiation group.

Figure 3 Illustration of EGFR/p-EGFR expression in both cytoplasm and nucleus.

Notes: Cytoplasmic p-EGFR expression was decreased in both cell lines when comparing the RT group with the RT+h-R3 group. The decrease was more significant in H292 cells, but the decrease did not reach statistical significance (B and C). Nuclear EGFR and p-EGFR was significantly decreased in H292 cells (D and F, P<0.05), and the decrease in H1975 cells was not statistically significant (E and G). (A) illustrates the brief profile of protein expression (n=3). Each bar represents the mean ± SD and *indicates significant difference (P<0.05).
Abbreviations: EGFR, epidermal growth factor receptor; p-EGFR, phospho-epidermal growth factor receptor; h-R3, nimotuzumab group; RT, irradiation group.

Figure 4 DNA damage repair after irradiation with or without nimotuzumab.

Notes: The p-DNA-PK expression was related to the DNA damage repair activity. Nimotuzumab suppressed H292 cytoplasmic p-DNA-PK expression and scarcely influenced p-DNA-PK expression in H1975 cells (B and C). Nuclear p-DNA-PK in H292 cells was suppressed (P<0.05), while it almost remained the same in H1975 cells (D and E). (A) illustrates the brief profile of protein expression (n=3). Each bar represents the mean ± SD and *indicates significant difference (P<0.05).
Abbreviations: p-DNA-PK, phosphorylated DNA-PK; h-R3, nimotuzumab group; RT, irradiation group.