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Inhibition of non-small cell lung cancer (NSCLC) growth by a novel small molecular inhibitor of EGFR.

Li J, Deng H, Hu M, Fang Y, Vaughn A, Cai X, Xu L, Wan W, Li Z, Chen S, Yang X, Wu S, Xiao J - Oncotarget (2015)

Bottom Line: WB-308 decreased NSCLC cell proliferation and colony formation, by causing G2/M arrest and apoptosis.Furthermore, WB-308 inhibited the engraft tumor growths in two animal models in vivo (lung orthotopic transplantation model and patient-derived engraft mouse model).WB-308 was less cytotoxic than Gefitinib.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopedic Oncology, Changzheng Hospital, The Second Military Medical University, Shanghai 200003, China.

ABSTRACT
The epidermal growth factor receptor (EGFR) is a therapeutic target (oncotarget) in NSCLC. Using in vitro EGFR kinase activity system, we identified a novel small molecule, WB-308, as an inhibitor of EGFR. WB-308 decreased NSCLC cell proliferation and colony formation, by causing G2/M arrest and apoptosis. Furthermore, WB-308 inhibited the engraft tumor growths in two animal models in vivo (lung orthotopic transplantation model and patient-derived engraft mouse model). WB-308 impaired the phosphorylation of EGFR, AKT, and ERK1/2 protein. WB-308 was less cytotoxic than Gefitinib. Our study suggests that WB-308 is a novel EGFR-TKI and may be considered to substitute for Gefitinib in clinical therapy for NSCLC.

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WB-308 exerts the same molecular mechanism with Gefitinib in EGFR mutated NSCLC cells(A) Establishment of the EGFR-WT, EGFR-L858R and the vehicle over-expressing stable lines. The 3 stable cell lysates were subjected to Western blotting analysis with antibodies against p-EGFR (Tyr1068) and EGFR. Anti-β-actin antibody (1:10 000 dilution) was used as a loading control. (B) WB-308 increases NSCLC cell's sensitivity EGFR mutation. H1299 (EGFR-WT) and H1299 (EGFR-L858R) stable cell lines were exposed to indicated concentrations of WB-308 for 4 hours and cell lysates were subjected to Western blotting analysis with antibodies against p-EGFR (Tyr1068) and EGFR. (C) WB-308 suppresses NSCLC cell's proliferation in EGFR mutation. The cell viability assay stained by SRB was performed as described in Materials and Methods. Columns, mean; bars, SE (n = 3; t-test, P < 0.05).
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Figure 2: WB-308 exerts the same molecular mechanism with Gefitinib in EGFR mutated NSCLC cells(A) Establishment of the EGFR-WT, EGFR-L858R and the vehicle over-expressing stable lines. The 3 stable cell lysates were subjected to Western blotting analysis with antibodies against p-EGFR (Tyr1068) and EGFR. Anti-β-actin antibody (1:10 000 dilution) was used as a loading control. (B) WB-308 increases NSCLC cell's sensitivity EGFR mutation. H1299 (EGFR-WT) and H1299 (EGFR-L858R) stable cell lines were exposed to indicated concentrations of WB-308 for 4 hours and cell lysates were subjected to Western blotting analysis with antibodies against p-EGFR (Tyr1068) and EGFR. (C) WB-308 suppresses NSCLC cell's proliferation in EGFR mutation. The cell viability assay stained by SRB was performed as described in Materials and Methods. Columns, mean; bars, SE (n = 3; t-test, P < 0.05).

Mentions: It has been previously reported that EGFR-TKIs drug sensitivity-related mutations commonly occur at exons 18–21, 49% of which are exon 19 del (Del E746–A750) mutations, and 45% of which contain the L858R mutation at exon 21. When these two types of EGFR mutations are present, NSCLC cell's sensitivity to clinical chemotherapy drugs, including Gefitinib, increases [24]. To confirm that WB-308 operates via the same mechanism as Gefitinib in response to EGFR mutations, we employed a de novo system to test WB-308′s working model in wild type NSCLC cell line H1299 in order to exclude artificial results and background. Considering the mutant efficiency and experiment simplicity, we chose the L858R mutation to confirm our result because to construct a point mutant is much easier that to construct a deletion mutant. We established stable cell lines that could stably express WT EGFR and L858R-mutated EGFR by transfecting with plasmid constructs in NCI-H1299 cells, which is an EGFR low-expressed cell line. The empty vector plasmid was employed as negative control in this experiment. Western blotting analysis demonstrated that these 3 stable cell lines, namely H1299 (vector), H1299 (EGFR–WT), and H1299 (EGFR–L858R), were successfully established (Figure 2A). Next we treated these cells with WB-308 and Gefitinib, respectively. Within our expectations, WB-308 showed the same effects as Gefitinib, further emphasizing the same effective model, and inhibited the phosphorylation of EGFR–L858R more than that of EGFR-WT, in a dose dependent manner. At a concentration of 0.5 μM, WB-308 decreased the level of p-EGFR of EGFR-L858R stable cells even more severely than that in EGFR-WT stable cells at the concentration of 5 μM, which indicated that the NSCLC cell's sensitivity to WB-308 increased after EGFR mutation occurred (Figure 2B).


Inhibition of non-small cell lung cancer (NSCLC) growth by a novel small molecular inhibitor of EGFR.

Li J, Deng H, Hu M, Fang Y, Vaughn A, Cai X, Xu L, Wan W, Li Z, Chen S, Yang X, Wu S, Xiao J - Oncotarget (2015)

WB-308 exerts the same molecular mechanism with Gefitinib in EGFR mutated NSCLC cells(A) Establishment of the EGFR-WT, EGFR-L858R and the vehicle over-expressing stable lines. The 3 stable cell lysates were subjected to Western blotting analysis with antibodies against p-EGFR (Tyr1068) and EGFR. Anti-β-actin antibody (1:10 000 dilution) was used as a loading control. (B) WB-308 increases NSCLC cell's sensitivity EGFR mutation. H1299 (EGFR-WT) and H1299 (EGFR-L858R) stable cell lines were exposed to indicated concentrations of WB-308 for 4 hours and cell lysates were subjected to Western blotting analysis with antibodies against p-EGFR (Tyr1068) and EGFR. (C) WB-308 suppresses NSCLC cell's proliferation in EGFR mutation. The cell viability assay stained by SRB was performed as described in Materials and Methods. Columns, mean; bars, SE (n = 3; t-test, P < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4466647&req=5

Figure 2: WB-308 exerts the same molecular mechanism with Gefitinib in EGFR mutated NSCLC cells(A) Establishment of the EGFR-WT, EGFR-L858R and the vehicle over-expressing stable lines. The 3 stable cell lysates were subjected to Western blotting analysis with antibodies against p-EGFR (Tyr1068) and EGFR. Anti-β-actin antibody (1:10 000 dilution) was used as a loading control. (B) WB-308 increases NSCLC cell's sensitivity EGFR mutation. H1299 (EGFR-WT) and H1299 (EGFR-L858R) stable cell lines were exposed to indicated concentrations of WB-308 for 4 hours and cell lysates were subjected to Western blotting analysis with antibodies against p-EGFR (Tyr1068) and EGFR. (C) WB-308 suppresses NSCLC cell's proliferation in EGFR mutation. The cell viability assay stained by SRB was performed as described in Materials and Methods. Columns, mean; bars, SE (n = 3; t-test, P < 0.05).
Mentions: It has been previously reported that EGFR-TKIs drug sensitivity-related mutations commonly occur at exons 18–21, 49% of which are exon 19 del (Del E746–A750) mutations, and 45% of which contain the L858R mutation at exon 21. When these two types of EGFR mutations are present, NSCLC cell's sensitivity to clinical chemotherapy drugs, including Gefitinib, increases [24]. To confirm that WB-308 operates via the same mechanism as Gefitinib in response to EGFR mutations, we employed a de novo system to test WB-308′s working model in wild type NSCLC cell line H1299 in order to exclude artificial results and background. Considering the mutant efficiency and experiment simplicity, we chose the L858R mutation to confirm our result because to construct a point mutant is much easier that to construct a deletion mutant. We established stable cell lines that could stably express WT EGFR and L858R-mutated EGFR by transfecting with plasmid constructs in NCI-H1299 cells, which is an EGFR low-expressed cell line. The empty vector plasmid was employed as negative control in this experiment. Western blotting analysis demonstrated that these 3 stable cell lines, namely H1299 (vector), H1299 (EGFR–WT), and H1299 (EGFR–L858R), were successfully established (Figure 2A). Next we treated these cells with WB-308 and Gefitinib, respectively. Within our expectations, WB-308 showed the same effects as Gefitinib, further emphasizing the same effective model, and inhibited the phosphorylation of EGFR–L858R more than that of EGFR-WT, in a dose dependent manner. At a concentration of 0.5 μM, WB-308 decreased the level of p-EGFR of EGFR-L858R stable cells even more severely than that in EGFR-WT stable cells at the concentration of 5 μM, which indicated that the NSCLC cell's sensitivity to WB-308 increased after EGFR mutation occurred (Figure 2B).

Bottom Line: WB-308 decreased NSCLC cell proliferation and colony formation, by causing G2/M arrest and apoptosis.Furthermore, WB-308 inhibited the engraft tumor growths in two animal models in vivo (lung orthotopic transplantation model and patient-derived engraft mouse model).WB-308 was less cytotoxic than Gefitinib.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopedic Oncology, Changzheng Hospital, The Second Military Medical University, Shanghai 200003, China.

ABSTRACT
The epidermal growth factor receptor (EGFR) is a therapeutic target (oncotarget) in NSCLC. Using in vitro EGFR kinase activity system, we identified a novel small molecule, WB-308, as an inhibitor of EGFR. WB-308 decreased NSCLC cell proliferation and colony formation, by causing G2/M arrest and apoptosis. Furthermore, WB-308 inhibited the engraft tumor growths in two animal models in vivo (lung orthotopic transplantation model and patient-derived engraft mouse model). WB-308 impaired the phosphorylation of EGFR, AKT, and ERK1/2 protein. WB-308 was less cytotoxic than Gefitinib. Our study suggests that WB-308 is a novel EGFR-TKI and may be considered to substitute for Gefitinib in clinical therapy for NSCLC.

Show MeSH
Related in: MedlinePlus