Limits...
NVP-BEZ235 overcomes gefitinib-acquired resistance by down-regulating PI3K/AKT/mTOR phosphorylation.

Sun Z, Li Q, Zhang S, Chen J, Huang L, Ren J, Chang Y, Liang Y, Wu G - Onco Targets Ther (2015)

Bottom Line: We show that NVP-BEZ235 effectively inhibited the growth of H1975 cells in vivo as well as in vitro.Similarly, H1975 cell migration was reduced by NVP-BEZ235.Taken together, we suggest that NVP-BEZ235 inhibits gefitinib-resistant tumor growth by downregulating PI3K/AKT/mTOR phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Oncology department, Xiangyang central Hospital, Xiangyang, Hubei, People's Republic of China.

ABSTRACT

Background: Patients harboring activating mutations in epidermal growth factor receptors (EGFR) are particularly sensitive to EGFR tyrosine kinase inhibitors (TKIs). However, most patients develop an acquired resistance after a period of about 10 months. This study focuses on the therapeutic effect of NVP-BEZ235, a dual inhibitor of phosphatidylinositol-3-kinase/mammalian target of rapamycin (PI3K/mTOR), in gefitinib-resistant non-small cell lung cancer.

Methods: H1975 cell line was validated as a gefitinib-resistant cell model by the nucleotide-sequence analysis. We used the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to detect the growth of H1975 cell line in vitro. H1975 cells' migration was detected by the migration assay. Xenograft models were used to investigate the growth of gefitinib-resistant non-small cell lung cancer in vivo. Western blot and immunohistochemical analysis were used to investigate the level of PI3K/protein kinase B(AKT)/mTOR signaling pathway proteins.

Results: We show that NVP-BEZ235 effectively inhibited the growth of H1975 cells in vivo as well as in vitro. Similarly, H1975 cell migration was reduced by NVP-BEZ235. Further experiments revealed that NVP-BEZ235 attenuated the phosphorylation of PI3K/AKT/mTOR signaling pathway proteins.

Conclusion: Taken together, we suggest that NVP-BEZ235 inhibits gefitinib-resistant tumor growth by downregulating PI3K/AKT/mTOR phosphorylation.

No MeSH data available.


Related in: MedlinePlus

H1975 cell line was resistant to gefitinib and inhibited by BEZ235.Notes: (A) H1975 cells were treated with different concentrations of BEZ235 as well as with combinations with gefitinib. After 3 days, viable cell numbers were estimated by MTT assay. Mean values of three independent measurements (± SD) are shown. (B) We extracted H1975 cells’ DNA, and then polymerase chain reaction was used to amplify the exons 18–21 of EGFR; the products were directly sequenced in both sense and antisense directions by Invitrogen. *indicates the the site of base change in the mutation.Abbreviations: B+G, NVP-BEZ235 combination with gefitinib; BEZ, NVP-BEZ235; EGFR, epidermal growth factor receptor; GEF, gefitinib; L858R, leucine-to-arginine substitution at codon 858; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; SD, standard deviation; T790M, threonine-to-methionine substitution at codon 790.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4321659&req=5

f1-ott-8-269: H1975 cell line was resistant to gefitinib and inhibited by BEZ235.Notes: (A) H1975 cells were treated with different concentrations of BEZ235 as well as with combinations with gefitinib. After 3 days, viable cell numbers were estimated by MTT assay. Mean values of three independent measurements (± SD) are shown. (B) We extracted H1975 cells’ DNA, and then polymerase chain reaction was used to amplify the exons 18–21 of EGFR; the products were directly sequenced in both sense and antisense directions by Invitrogen. *indicates the the site of base change in the mutation.Abbreviations: B+G, NVP-BEZ235 combination with gefitinib; BEZ, NVP-BEZ235; EGFR, epidermal growth factor receptor; GEF, gefitinib; L858R, leucine-to-arginine substitution at codon 858; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; SD, standard deviation; T790M, threonine-to-methionine substitution at codon 790.

Mentions: To understand whether the H1975 cell line is an ideal model of gefitinib resistance, we assessed the cellular proliferation by MTT assay, and amplification of the EGFR exons 18–21 in the H1975 cell line. After incubation with gefitinib for 72 hours, as shown in Figure 1A, H1975 cell growth was not inhibited until the concentration of gefitinib was over 3 μmol/L (P<0.05). The IC50 was 22.61 μmol/L, which was consistent with the result of a previous report.8 It has been reported previously that the H1975 cell line carries L858R-T790M EGFR mutation and is resistant to gefitinib and erlotinib.6,19 To confirm the secondary T790M mutation in the H1975 cell line, polymerase chain reaction was performed to amplify the exons 18–21 of EGFR. The nucleotide sequencing exhibited that there existed a C-to-T point mutation in exon 20, which was due to the threonine-to-methionine substitution at codon 790 (T790M). Another T-to-G point mutation in exon 18, which led to arginine being substituted by leucine at codon 858 (L858R) was also observed (Figure 1B).


NVP-BEZ235 overcomes gefitinib-acquired resistance by down-regulating PI3K/AKT/mTOR phosphorylation.

Sun Z, Li Q, Zhang S, Chen J, Huang L, Ren J, Chang Y, Liang Y, Wu G - Onco Targets Ther (2015)

H1975 cell line was resistant to gefitinib and inhibited by BEZ235.Notes: (A) H1975 cells were treated with different concentrations of BEZ235 as well as with combinations with gefitinib. After 3 days, viable cell numbers were estimated by MTT assay. Mean values of three independent measurements (± SD) are shown. (B) We extracted H1975 cells’ DNA, and then polymerase chain reaction was used to amplify the exons 18–21 of EGFR; the products were directly sequenced in both sense and antisense directions by Invitrogen. *indicates the the site of base change in the mutation.Abbreviations: B+G, NVP-BEZ235 combination with gefitinib; BEZ, NVP-BEZ235; EGFR, epidermal growth factor receptor; GEF, gefitinib; L858R, leucine-to-arginine substitution at codon 858; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; SD, standard deviation; T790M, threonine-to-methionine substitution at codon 790.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4321659&req=5

f1-ott-8-269: H1975 cell line was resistant to gefitinib and inhibited by BEZ235.Notes: (A) H1975 cells were treated with different concentrations of BEZ235 as well as with combinations with gefitinib. After 3 days, viable cell numbers were estimated by MTT assay. Mean values of three independent measurements (± SD) are shown. (B) We extracted H1975 cells’ DNA, and then polymerase chain reaction was used to amplify the exons 18–21 of EGFR; the products were directly sequenced in both sense and antisense directions by Invitrogen. *indicates the the site of base change in the mutation.Abbreviations: B+G, NVP-BEZ235 combination with gefitinib; BEZ, NVP-BEZ235; EGFR, epidermal growth factor receptor; GEF, gefitinib; L858R, leucine-to-arginine substitution at codon 858; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; SD, standard deviation; T790M, threonine-to-methionine substitution at codon 790.
Mentions: To understand whether the H1975 cell line is an ideal model of gefitinib resistance, we assessed the cellular proliferation by MTT assay, and amplification of the EGFR exons 18–21 in the H1975 cell line. After incubation with gefitinib for 72 hours, as shown in Figure 1A, H1975 cell growth was not inhibited until the concentration of gefitinib was over 3 μmol/L (P<0.05). The IC50 was 22.61 μmol/L, which was consistent with the result of a previous report.8 It has been reported previously that the H1975 cell line carries L858R-T790M EGFR mutation and is resistant to gefitinib and erlotinib.6,19 To confirm the secondary T790M mutation in the H1975 cell line, polymerase chain reaction was performed to amplify the exons 18–21 of EGFR. The nucleotide sequencing exhibited that there existed a C-to-T point mutation in exon 20, which was due to the threonine-to-methionine substitution at codon 790 (T790M). Another T-to-G point mutation in exon 18, which led to arginine being substituted by leucine at codon 858 (L858R) was also observed (Figure 1B).

Bottom Line: We show that NVP-BEZ235 effectively inhibited the growth of H1975 cells in vivo as well as in vitro.Similarly, H1975 cell migration was reduced by NVP-BEZ235.Taken together, we suggest that NVP-BEZ235 inhibits gefitinib-resistant tumor growth by downregulating PI3K/AKT/mTOR phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Oncology department, Xiangyang central Hospital, Xiangyang, Hubei, People's Republic of China.

ABSTRACT

Background: Patients harboring activating mutations in epidermal growth factor receptors (EGFR) are particularly sensitive to EGFR tyrosine kinase inhibitors (TKIs). However, most patients develop an acquired resistance after a period of about 10 months. This study focuses on the therapeutic effect of NVP-BEZ235, a dual inhibitor of phosphatidylinositol-3-kinase/mammalian target of rapamycin (PI3K/mTOR), in gefitinib-resistant non-small cell lung cancer.

Methods: H1975 cell line was validated as a gefitinib-resistant cell model by the nucleotide-sequence analysis. We used the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to detect the growth of H1975 cell line in vitro. H1975 cells' migration was detected by the migration assay. Xenograft models were used to investigate the growth of gefitinib-resistant non-small cell lung cancer in vivo. Western blot and immunohistochemical analysis were used to investigate the level of PI3K/protein kinase B(AKT)/mTOR signaling pathway proteins.

Results: We show that NVP-BEZ235 effectively inhibited the growth of H1975 cells in vivo as well as in vitro. Similarly, H1975 cell migration was reduced by NVP-BEZ235. Further experiments revealed that NVP-BEZ235 attenuated the phosphorylation of PI3K/AKT/mTOR signaling pathway proteins.

Conclusion: Taken together, we suggest that NVP-BEZ235 inhibits gefitinib-resistant tumor growth by downregulating PI3K/AKT/mTOR phosphorylation.

No MeSH data available.


Related in: MedlinePlus