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Homoharringtonine induces apoptosis and inhibits STAT3 via IL-6/JAK1/STAT3 signal pathway in Gefitinib-resistant lung cancer cells.

Cao W, Liu Y, Zhang R, Zhang B, Wang T, Zhu X, Mei L, Chen H, Zhang H, Ming P, Huang L - Sci Rep (2015)

Bottom Line: Unfortunately, treatment with Gefitinib for a period of time will result in drug resistance and cause treatment failure in clinic.NCI-H1975 cells with EGFR T790M mutation are more sensitive to HHT treatment compared with that of A549 cells with wild type EGFR.HHT reversiblely inhibited IL-6-induced STAT3 Tyrosine 705 phosphorylation and reduced anti-apoptotic proteins expression.

View Article: PubMed Central - PubMed

Affiliation: 1] School of Life Sciences, Tsinghua University, Beijing, 100084, China [2] The Shenzhen Key Laboratory of Gene and Antibody Therapy, State Key Laboratory of Health Science and Technology (prep), Center for Biotechnology &Biomedicine and Division of Life &Health Sciences, Graduate School at Shenzhen, Tsinghua University, Shenzhen, Guangdong, 518055, China.

ABSTRACT
Tyrosine kinase inhibitors (TKIs) are mostly used in non-small cell lung cancer (NSCLC) treatment. Unfortunately, treatment with Gefitinib for a period of time will result in drug resistance and cause treatment failure in clinic. Therefore, exploring novel compounds to overcome this resistance is urgently required. Here we investigated the antitumor effect of homoharringtonine (HHT), a natural compound extracted from Cephalotaxus harringtonia, on Gefitinib-resistant NSCLC cell lines in vitro and in vivo. NCI-H1975 cells with EGFR T790M mutation are more sensitive to HHT treatment compared with that of A549 cells with wild type EGFR. HHT inhibited cells growth, cell viability and colony formation, as well as induced cell apoptosis through mitochondria pathway. Furthermore, we explored the mechanism of HHT inhibition on NSCLC cells. Higher level of interleukin-6 (IL-6) existed in lung cancer patients and mutant EGFR and TGFβ signal requires the upregulation of IL-6 through the gp130/JAK pathway to overactive STAT3, an oncogenic protein which has been considered as a potential target for cancer therapy. HHT reversiblely inhibited IL-6-induced STAT3 Tyrosine 705 phosphorylation and reduced anti-apoptotic proteins expression. Gefitinib-resistant NSCLC xenograft tests also confirmed the antitumor effect of HHT in vivo. Consequently, HHT has the potential in Gefitinib-resistant NSCLC treatment.

No MeSH data available.


Related in: MedlinePlus

HHT inhibitory effects on NSCLC cells.(A): Chemical structure of HHT. (B–E): The inhibitory effects of HHT on A549 (B and C) and H1975 (D and E) cells evaluated by MTT assay. (F and G): Cell viability inhibition effect of HHT on A549 and H1975 cells analyzed by trypan blue exclusion assay. (H and I): The soft-agar colony formation assays of A549 and H1975 cells treated with HHT at indicated concentration. (J): A549 and H1975 cells were treated with HHT or Gefitinib for 24 h, lysed and the protein samples were analyzed by western blot with indicated antibodies. All the full-length blots are presented in Supplementary Figure 1.
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f1: HHT inhibitory effects on NSCLC cells.(A): Chemical structure of HHT. (B–E): The inhibitory effects of HHT on A549 (B and C) and H1975 (D and E) cells evaluated by MTT assay. (F and G): Cell viability inhibition effect of HHT on A549 and H1975 cells analyzed by trypan blue exclusion assay. (H and I): The soft-agar colony formation assays of A549 and H1975 cells treated with HHT at indicated concentration. (J): A549 and H1975 cells were treated with HHT or Gefitinib for 24 h, lysed and the protein samples were analyzed by western blot with indicated antibodies. All the full-length blots are presented in Supplementary Figure 1.

Mentions: In this study, we firstly investigated the cytotoxicity of HHT on human NSCLC cell lines, A549 (wild type EGFR) and NCI-H1975 (H1975, mutant EGFR with L858R and T790M), using Gefitinib as a control. By 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, we found that HHT had moderate cytotoxicity to A549 with an IC50 of 3.7 μM and H1975 cells were more sensitive to HHT with an IC50 of 0.7 μM . We also found that HHT inhibited the cell proliferation and growth of A549 cells (Fig. 1B,C) and H1975 cells (Fig. 1D,E) in a time- and dose-dependent manner through MTT assay. By trypan blue exclusion assay, we found that HHT rapidly reduced viable A549 (Fig. 1F) and H1975 cells (Fig. 1G) in a dose- and time-dependent manner. We investigated HHT’s effect on cell colony formation activity, and the results showed that HHT significantly inhibited the clonogenic ability of A549 (Fig. 1H) and H1975 cells(Fig. 1I). These results suggested that HHT inhibited the anchorage-dependent (cell proliferation) and anchorage-independent (colony formation) growth of NSCLC cells.


Homoharringtonine induces apoptosis and inhibits STAT3 via IL-6/JAK1/STAT3 signal pathway in Gefitinib-resistant lung cancer cells.

Cao W, Liu Y, Zhang R, Zhang B, Wang T, Zhu X, Mei L, Chen H, Zhang H, Ming P, Huang L - Sci Rep (2015)

HHT inhibitory effects on NSCLC cells.(A): Chemical structure of HHT. (B–E): The inhibitory effects of HHT on A549 (B and C) and H1975 (D and E) cells evaluated by MTT assay. (F and G): Cell viability inhibition effect of HHT on A549 and H1975 cells analyzed by trypan blue exclusion assay. (H and I): The soft-agar colony formation assays of A549 and H1975 cells treated with HHT at indicated concentration. (J): A549 and H1975 cells were treated with HHT or Gefitinib for 24 h, lysed and the protein samples were analyzed by western blot with indicated antibodies. All the full-length blots are presented in Supplementary Figure 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4499885&req=5

f1: HHT inhibitory effects on NSCLC cells.(A): Chemical structure of HHT. (B–E): The inhibitory effects of HHT on A549 (B and C) and H1975 (D and E) cells evaluated by MTT assay. (F and G): Cell viability inhibition effect of HHT on A549 and H1975 cells analyzed by trypan blue exclusion assay. (H and I): The soft-agar colony formation assays of A549 and H1975 cells treated with HHT at indicated concentration. (J): A549 and H1975 cells were treated with HHT or Gefitinib for 24 h, lysed and the protein samples were analyzed by western blot with indicated antibodies. All the full-length blots are presented in Supplementary Figure 1.
Mentions: In this study, we firstly investigated the cytotoxicity of HHT on human NSCLC cell lines, A549 (wild type EGFR) and NCI-H1975 (H1975, mutant EGFR with L858R and T790M), using Gefitinib as a control. By 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, we found that HHT had moderate cytotoxicity to A549 with an IC50 of 3.7 μM and H1975 cells were more sensitive to HHT with an IC50 of 0.7 μM . We also found that HHT inhibited the cell proliferation and growth of A549 cells (Fig. 1B,C) and H1975 cells (Fig. 1D,E) in a time- and dose-dependent manner through MTT assay. By trypan blue exclusion assay, we found that HHT rapidly reduced viable A549 (Fig. 1F) and H1975 cells (Fig. 1G) in a dose- and time-dependent manner. We investigated HHT’s effect on cell colony formation activity, and the results showed that HHT significantly inhibited the clonogenic ability of A549 (Fig. 1H) and H1975 cells(Fig. 1I). These results suggested that HHT inhibited the anchorage-dependent (cell proliferation) and anchorage-independent (colony formation) growth of NSCLC cells.

Bottom Line: Unfortunately, treatment with Gefitinib for a period of time will result in drug resistance and cause treatment failure in clinic.NCI-H1975 cells with EGFR T790M mutation are more sensitive to HHT treatment compared with that of A549 cells with wild type EGFR.HHT reversiblely inhibited IL-6-induced STAT3 Tyrosine 705 phosphorylation and reduced anti-apoptotic proteins expression.

View Article: PubMed Central - PubMed

Affiliation: 1] School of Life Sciences, Tsinghua University, Beijing, 100084, China [2] The Shenzhen Key Laboratory of Gene and Antibody Therapy, State Key Laboratory of Health Science and Technology (prep), Center for Biotechnology &Biomedicine and Division of Life &Health Sciences, Graduate School at Shenzhen, Tsinghua University, Shenzhen, Guangdong, 518055, China.

ABSTRACT
Tyrosine kinase inhibitors (TKIs) are mostly used in non-small cell lung cancer (NSCLC) treatment. Unfortunately, treatment with Gefitinib for a period of time will result in drug resistance and cause treatment failure in clinic. Therefore, exploring novel compounds to overcome this resistance is urgently required. Here we investigated the antitumor effect of homoharringtonine (HHT), a natural compound extracted from Cephalotaxus harringtonia, on Gefitinib-resistant NSCLC cell lines in vitro and in vivo. NCI-H1975 cells with EGFR T790M mutation are more sensitive to HHT treatment compared with that of A549 cells with wild type EGFR. HHT inhibited cells growth, cell viability and colony formation, as well as induced cell apoptosis through mitochondria pathway. Furthermore, we explored the mechanism of HHT inhibition on NSCLC cells. Higher level of interleukin-6 (IL-6) existed in lung cancer patients and mutant EGFR and TGFβ signal requires the upregulation of IL-6 through the gp130/JAK pathway to overactive STAT3, an oncogenic protein which has been considered as a potential target for cancer therapy. HHT reversiblely inhibited IL-6-induced STAT3 Tyrosine 705 phosphorylation and reduced anti-apoptotic proteins expression. Gefitinib-resistant NSCLC xenograft tests also confirmed the antitumor effect of HHT in vivo. Consequently, HHT has the potential in Gefitinib-resistant NSCLC treatment.

No MeSH data available.


Related in: MedlinePlus