## 3 Results
### Effect of Gefitinib and Caneritinib on PCNA, Caspase-3, pERK and pAKT  
To better understand the effect of gefitinib and caneritinib on signalling proteins that are commonly known with the activation of EGFR and HER-2, we investigated the immunostaining of PCNA, cleaved caspase-3, pERK and pAKT. As shown in Table 5A,B, of eight ascitic cell samples, gefitinib reduced the PCNA in two samples (patient numbers 3 and 20; \(p<0.05\)) in EGFR positive cells. Similarly, caneritinib also reduced PCNA in two samples (patient numbers 19 and 20; p \(<0.05\)). Two ascitic cell samples (patient numbers 10 and 20; \(p<0.05\)) increased cleaved caspase-3 protein expression after gefitinib treatment. Only cells from patient number 10 increased cleaved caspase-3 after exposure to caneritinib. The phosphorylation of ERK (Tyr 204) and AKT (Ser 473) was randomly and broadly affected. As shown in Table 5B, gefitinib reduced pERK in four ascitic cell samples (patient numbers 3, 4, 19, 20; \(p<0.05\)). Caneritinib reduced pERK in five samples (patient numbers 4, 6, 8, 19, 20; \(p<0.05\)). Gefitinib reduced pAKT in five cell samples (patient numbers 4, 6, 10, 19, 20; \(p<0.05\)). Caneritinib reduced pAKT in four cell samples (patient numbers 4, 8, 19, 20; \(p<0.05\)). \begin{table}
\begin{tabular}{c c c c c c} \hline \hline  & & \multicolumn{4}{c}{**(A) EGFR positive cells**} \\ \hline \multirow{3}{*}{**Patient ID**} & \multirow{3}{*}{**Control**} & **PCNA (\% Relative Expression to Control)** & \multicolumn{2}{c}{**Cleaved Caspase-3 (\% Relative Expression to Control)**} \\ \cline{3-6}  & & & **5 \(\upmu\)[PERSON]** & 5 \(\upmu\)[PERSON] & 5 \(\upmu\)[PERSON] & 5 \(\upmu\)[PERSON] \\ \hline
3 & 100 & \(81.8\pm 5.1\)* & 90.7 \(\pm\) 3.3 & 73.7 \(\pm\) 3.6 & 88.0 \(\pm\) 6.2 \\
4 & 100 & \(100\pm 7.2\) & 88.5 \(\pm\) 5.2 & 96.3 \(\pm\) 11.7 & 81.4 \(\pm\) 7.7 \\
6 & 100 & \(93.4\pm 4.1\) & 87.7 \(\pm\) 2.3 & 91.2 \(\pm\) 6.3 & 88.9 \(\pm\) 3.2 \\
8 & 100 & \(108.9\pm 6.2\) & 121.4 \(\pm\) 3.2 & 90.0 \(\pm\) 3.7 & 100 \(\pm\) 2.1 \\
10 & 100 & \(93.9\pm 3.12\) & 99.2 \(\pm\) 3.6 & 300 \(\pm\) 32* & 357.7 \(\pm\) 37.2* \\
17 & 100 & \(84.6\pm 3.1\) & 90.5 \(\pm\) 3.8 & 83.7 \(\pm\) 2.8 & 76.4 \(\pm\) 2.7 \\
19 & 100 & \(119.5\pm 3.8\) & 62.8 \(\pm\) 4.8* & 130.4 \(\pm\) 27 & 54.6 \(\pm\) 4.5 \\
20 & 100 & \(63.6\pm 3.0\)* & 56.7 \(\pm\) 4.3* & 165.7 \(\pm\) 11.9* & 128.9 \(\pm\) 14.2 \\ \hline \hline \end{tabular}
\end{table} 
Table 5: The protein level of PCNA, cleaved caspase-3, pERK (Tyr 204) and pAKT (Ser 473) in EGFR positive cells are affected by gefitinib (Gef) and caneritinib (Can). The changes of PCNA and cleaved caspase-3 in Gef and Can treated cells are not prominent (**A**). The decrease in pERK (Tyr 204) and pAKT (Ser 473) is random in some selected patient cells (**B**). The fluorescent intensity of control is normalised to 100% and the fluorescent intensity of treatment is calculated relatively to the control sample. Representative analysis was from a set of two independent experiments and each experiment had at least three duplicates. Mean \(\pm\) SE; \(n=2\) is presented and statistical significance was determined by a \(t\)-test (*, \(p<0.05\)). With the EGFR and HER-2 positive ascitic cells as shown in Table 6A,B, both gefitinib and canertinib showed a limited decrease in PCNA and an increase in cleaved caspase-3. Only one patient cell sample (patient number 9; \(p<0.05\)) showed reduced PCNA after gefitinib treatment. Both gefitinib and canertinib induced cleaved caspase-3 only in one patient cell sample (patient number 7; \(p<0.05\)). Table 6B shows the effect of gefitinib and canertinib on the phosphorylation of ERK (Tyr 204) and ATK (Ser 473). Canertinib showed a greater decrease in pERK (Tyr 204) in five ascitic cell samples (patient numbers 7, 9, 11, 13, 16; \(p<0.05\)) than gefitinib, which affected only one cell sample (patient number 16; \(p<0.05\)).