Limits...
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 inhibits IL-6-induced STAT3 phosphorylation a dose- and time-dependent manner.(A): IL-6 production in MCF-10A, A549 and H1975 cells measured by ELISA. (B): Cells were starved and treated with different concentration IL-6. Protein samples were detected by western blot. (C–E): Cells were starved and pretreated with PBS or HHT for 4 h followed by IL-6 treatment. Protein samples were detected by western blot (C), the distribution variation of phosphorylated STAT3 (Y705) were examined by immunofluorescence (D) and nuclear (N) and cytoplasmic (C) isolation assay (E). (F): pSTAT3-TA-luc plasmids were transfected into H1975 cells followed by treatment with HHT for 4 h. Then H1975 cells were treated with IL-6 for another 20 h. Firefly luciferase activities were assayed. (G): Cells were starved and pretreated with HHT at different concentrations followed by IL-6 stimulation. Protein samples were examined by western blot. (H): Cells were starved and treated with 4 μM (in A549 cells) or 2 μM (in H1975 cells) for indicated time points (0 h–4 h). After HHT pretreatment, cells were treated with 5 ng/mL IL-6 for 30 min. Protein samples were detected by western blot. (I): Cells were starved and pretreated with 4 μM (in A549 cells) or 2 μM (in H1975 cells) HHT for 4 h. Discard the HHT-containing medium and add fresh medium without HHT. After indicated incubation times (0 h–24 h), cells were treated with 5 ng/mL IL-6 for 30 min. Protein samples were detected by western blot. (J): Cells were starved and then pretreated with 4 μM (in A549 cells) or 2 μM (in H1975 cells) for 4 h. Discard the HHT-containing medium and add fresh medium containing protein synthesis inhibitor CHX (10 μg/mL) without HHT. After 12 h, cells were stimulated with 5 ng/mL IL-6 for 30 min. Protein samples were detected by western blot. The blots shown are derived from multiple gels. Membrane was cut based on the molecular weight, probed with antibody of interest and band of interest is indicated with an arrow. All the full-length blots are presented in Supplementary Figure 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4499885&req=5

f4: HHT inhibits IL-6-induced STAT3 phosphorylation a dose- and time-dependent manner.(A): IL-6 production in MCF-10A, A549 and H1975 cells measured by ELISA. (B): Cells were starved and treated with different concentration IL-6. Protein samples were detected by western blot. (C–E): Cells were starved and pretreated with PBS or HHT for 4 h followed by IL-6 treatment. Protein samples were detected by western blot (C), the distribution variation of phosphorylated STAT3 (Y705) were examined by immunofluorescence (D) and nuclear (N) and cytoplasmic (C) isolation assay (E). (F): pSTAT3-TA-luc plasmids were transfected into H1975 cells followed by treatment with HHT for 4 h. Then H1975 cells were treated with IL-6 for another 20 h. Firefly luciferase activities were assayed. (G): Cells were starved and pretreated with HHT at different concentrations followed by IL-6 stimulation. Protein samples were examined by western blot. (H): Cells were starved and treated with 4 μM (in A549 cells) or 2 μM (in H1975 cells) for indicated time points (0 h–4 h). After HHT pretreatment, cells were treated with 5 ng/mL IL-6 for 30 min. Protein samples were detected by western blot. (I): Cells were starved and pretreated with 4 μM (in A549 cells) or 2 μM (in H1975 cells) HHT for 4 h. Discard the HHT-containing medium and add fresh medium without HHT. After indicated incubation times (0 h–24 h), cells were treated with 5 ng/mL IL-6 for 30 min. Protein samples were detected by western blot. (J): Cells were starved and then pretreated with 4 μM (in A549 cells) or 2 μM (in H1975 cells) for 4 h. Discard the HHT-containing medium and add fresh medium containing protein synthesis inhibitor CHX (10 μg/mL) without HHT. After 12 h, cells were stimulated with 5 ng/mL IL-6 for 30 min. Protein samples were detected by western blot. The blots shown are derived from multiple gels. Membrane was cut based on the molecular weight, probed with antibody of interest and band of interest is indicated with an arrow. All the full-length blots are presented in Supplementary Figure 3.

Mentions: Apart from EGFR, STAT3 can be activated via some inflammatory cytokines as well as growth factors. Previous studies have shown that EGFR mutation increased IL-6 expression and therefore activated STAT3 via IL-6/gp130/JAK1 signal pathway2544 and JAK1 was the critical JAK receptor kinase in IL-6/gp130/JAK/STAT3 signal pathway in lung cancer cells27. In vivo study also suggested IL-6 blockage inhibited STAT3 activation and therefore repressed H1650 xenografts cell growth27. We found that IL-6 expression in a very low level in non-transformed breast epithelial MCF-10A and wild type EGFR harboring A549 cells, but the IL-6 production (3723 pg/mL) was significantly elevated in H1975 cells harboring mutant EGFR (Fig. 4A), which was consistant with the previous results25. To examine if HHT can block IL-6-induced STAT3 activation, we firstly validated the IL-6 activation effect on STAT3 activation. A549 and H1975 cells were cultured in DMEM and RPMI-1640 medium for 12 h, and then were starved in serum-free mudium for another 12 h or more time. The cells were treated with IL-6 at different concentration for 30 min. As shown in Fig. 4B, IL-6 can activate STAT3 by phosphorylation in a dose-dependent manner, and we choose 5 ng/mL IL-6 for subsequent experiment. Next, with the same culture conditions, after starvation, A549 cells were pretreated with 2 μM or 4 μM HHT and H1975 cells were pretreated with 1 μM or 2 μM HHT for 4 h followed by 5 ng/mL of IL-6 stimulation for 30 min. Fig. 4C showed that IL-6 induced STAT3 phosphorylation but the induction was repressed by HHT-pretreated cells. Meanwhile, the total STAT3 expression level was not altered with IL-6 or HHT treatment. Previous studies have shown that STAT3 phosphorylation was associated with translocation between nuclear and cytoplasm42. To investigate whether HHT can block this IL-6-induced translocation, cells were pretreated with indicated concentration HHT for 4 h and then incubated the cells with 5 ng/mL IL-6 for 30 min. Cells were fixed with 4% paraformaldehyde diluted in 1× PBS and immunofluorescence-stained with anti-phosphorylated STAT3 (Y705) primary antibody and FITC-conjugated secondary antibody. The nucleuses were stained with 5 μg/mL DAPI. As shown in Fig. 4C, STAT3 was activated and translocated into nucleus with IL-6 stimulation, but there were weaker STAT3 activation and nucleus translocation with HHT pretreatment. To further confirm the HHT-induced inhibition of STAT3 translocation on IL-6 stimulation, A549 and H1975 cells were treated as above and the nuclear and cytoplasmic fractionations were isolated and analyzed by western blot. As shown in Fig. 4E, HHT pretreated cells showed relatively weaker phosphorylated STAT3 in the nucleus. Additionally, HHT also inhibited STAT3 transcription activity in H1975 cells, which further confirmed HHT inhibited the nuclear translocation of p-STAT3 (Fig. 4F). The above results suggested that HHT pretreatment could block IL-6-induced STAT3 phosphorylation and nuclear translocation.


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 inhibits IL-6-induced STAT3 phosphorylation a dose- and time-dependent manner.(A): IL-6 production in MCF-10A, A549 and H1975 cells measured by ELISA. (B): Cells were starved and treated with different concentration IL-6. Protein samples were detected by western blot. (C–E): Cells were starved and pretreated with PBS or HHT for 4 h followed by IL-6 treatment. Protein samples were detected by western blot (C), the distribution variation of phosphorylated STAT3 (Y705) were examined by immunofluorescence (D) and nuclear (N) and cytoplasmic (C) isolation assay (E). (F): pSTAT3-TA-luc plasmids were transfected into H1975 cells followed by treatment with HHT for 4 h. Then H1975 cells were treated with IL-6 for another 20 h. Firefly luciferase activities were assayed. (G): Cells were starved and pretreated with HHT at different concentrations followed by IL-6 stimulation. Protein samples were examined by western blot. (H): Cells were starved and treated with 4 μM (in A549 cells) or 2 μM (in H1975 cells) for indicated time points (0 h–4 h). After HHT pretreatment, cells were treated with 5 ng/mL IL-6 for 30 min. Protein samples were detected by western blot. (I): Cells were starved and pretreated with 4 μM (in A549 cells) or 2 μM (in H1975 cells) HHT for 4 h. Discard the HHT-containing medium and add fresh medium without HHT. After indicated incubation times (0 h–24 h), cells were treated with 5 ng/mL IL-6 for 30 min. Protein samples were detected by western blot. (J): Cells were starved and then pretreated with 4 μM (in A549 cells) or 2 μM (in H1975 cells) for 4 h. Discard the HHT-containing medium and add fresh medium containing protein synthesis inhibitor CHX (10 μg/mL) without HHT. After 12 h, cells were stimulated with 5 ng/mL IL-6 for 30 min. Protein samples were detected by western blot. The blots shown are derived from multiple gels. Membrane was cut based on the molecular weight, probed with antibody of interest and band of interest is indicated with an arrow. All the full-length blots are presented in Supplementary Figure 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: HHT inhibits IL-6-induced STAT3 phosphorylation a dose- and time-dependent manner.(A): IL-6 production in MCF-10A, A549 and H1975 cells measured by ELISA. (B): Cells were starved and treated with different concentration IL-6. Protein samples were detected by western blot. (C–E): Cells were starved and pretreated with PBS or HHT for 4 h followed by IL-6 treatment. Protein samples were detected by western blot (C), the distribution variation of phosphorylated STAT3 (Y705) were examined by immunofluorescence (D) and nuclear (N) and cytoplasmic (C) isolation assay (E). (F): pSTAT3-TA-luc plasmids were transfected into H1975 cells followed by treatment with HHT for 4 h. Then H1975 cells were treated with IL-6 for another 20 h. Firefly luciferase activities were assayed. (G): Cells were starved and pretreated with HHT at different concentrations followed by IL-6 stimulation. Protein samples were examined by western blot. (H): Cells were starved and treated with 4 μM (in A549 cells) or 2 μM (in H1975 cells) for indicated time points (0 h–4 h). After HHT pretreatment, cells were treated with 5 ng/mL IL-6 for 30 min. Protein samples were detected by western blot. (I): Cells were starved and pretreated with 4 μM (in A549 cells) or 2 μM (in H1975 cells) HHT for 4 h. Discard the HHT-containing medium and add fresh medium without HHT. After indicated incubation times (0 h–24 h), cells were treated with 5 ng/mL IL-6 for 30 min. Protein samples were detected by western blot. (J): Cells were starved and then pretreated with 4 μM (in A549 cells) or 2 μM (in H1975 cells) for 4 h. Discard the HHT-containing medium and add fresh medium containing protein synthesis inhibitor CHX (10 μg/mL) without HHT. After 12 h, cells were stimulated with 5 ng/mL IL-6 for 30 min. Protein samples were detected by western blot. The blots shown are derived from multiple gels. Membrane was cut based on the molecular weight, probed with antibody of interest and band of interest is indicated with an arrow. All the full-length blots are presented in Supplementary Figure 3.
Mentions: Apart from EGFR, STAT3 can be activated via some inflammatory cytokines as well as growth factors. Previous studies have shown that EGFR mutation increased IL-6 expression and therefore activated STAT3 via IL-6/gp130/JAK1 signal pathway2544 and JAK1 was the critical JAK receptor kinase in IL-6/gp130/JAK/STAT3 signal pathway in lung cancer cells27. In vivo study also suggested IL-6 blockage inhibited STAT3 activation and therefore repressed H1650 xenografts cell growth27. We found that IL-6 expression in a very low level in non-transformed breast epithelial MCF-10A and wild type EGFR harboring A549 cells, but the IL-6 production (3723 pg/mL) was significantly elevated in H1975 cells harboring mutant EGFR (Fig. 4A), which was consistant with the previous results25. To examine if HHT can block IL-6-induced STAT3 activation, we firstly validated the IL-6 activation effect on STAT3 activation. A549 and H1975 cells were cultured in DMEM and RPMI-1640 medium for 12 h, and then were starved in serum-free mudium for another 12 h or more time. The cells were treated with IL-6 at different concentration for 30 min. As shown in Fig. 4B, IL-6 can activate STAT3 by phosphorylation in a dose-dependent manner, and we choose 5 ng/mL IL-6 for subsequent experiment. Next, with the same culture conditions, after starvation, A549 cells were pretreated with 2 μM or 4 μM HHT and H1975 cells were pretreated with 1 μM or 2 μM HHT for 4 h followed by 5 ng/mL of IL-6 stimulation for 30 min. Fig. 4C showed that IL-6 induced STAT3 phosphorylation but the induction was repressed by HHT-pretreated cells. Meanwhile, the total STAT3 expression level was not altered with IL-6 or HHT treatment. Previous studies have shown that STAT3 phosphorylation was associated with translocation between nuclear and cytoplasm42. To investigate whether HHT can block this IL-6-induced translocation, cells were pretreated with indicated concentration HHT for 4 h and then incubated the cells with 5 ng/mL IL-6 for 30 min. Cells were fixed with 4% paraformaldehyde diluted in 1× PBS and immunofluorescence-stained with anti-phosphorylated STAT3 (Y705) primary antibody and FITC-conjugated secondary antibody. The nucleuses were stained with 5 μg/mL DAPI. As shown in Fig. 4C, STAT3 was activated and translocated into nucleus with IL-6 stimulation, but there were weaker STAT3 activation and nucleus translocation with HHT pretreatment. To further confirm the HHT-induced inhibition of STAT3 translocation on IL-6 stimulation, A549 and H1975 cells were treated as above and the nuclear and cytoplasmic fractionations were isolated and analyzed by western blot. As shown in Fig. 4E, HHT pretreated cells showed relatively weaker phosphorylated STAT3 in the nucleus. Additionally, HHT also inhibited STAT3 transcription activity in H1975 cells, which further confirmed HHT inhibited the nuclear translocation of p-STAT3 (Fig. 4F). The above results suggested that HHT pretreatment could block IL-6-induced STAT3 phosphorylation and nuclear translocation.

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