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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 induces apoptosis of NSCLC cells.(A): A549 and H1975 cells were treated with Gefitinib (3 mM) or HHT at indicated concentrations for 24 h and stained with Hoechst 33258 assay. (B): A549 and H1975 cells were treated with HHT, lysed and the protein samples were analysed by western blot with indicated antibodies. (C): A549 and H1975 cells were treated with HHT at indicated concentration and the mitochondrial transmembrane potential (ΔΨ) was tested by confocal microscopy (Olympus Fluoview FV-1000, Tokyo, Japan). (D): Ca2+(i) was measured using Ca2+ indicator FLUO-4 (Invitrogen) by flow cytometry assay. (E): H1975 cells were treated with HHT for 24 h, lysed and analysed by western blot with indicated antibodies. (F): H1975 cells were pretreated with Z-VAD-FMK (20 mM) for 1 h and then treated with HHT at 2 mM for 24 h, and the inhibition rate was determined by MTT assay. The mean±SD of three independent experiments is shown. ***, P < 0.01. (G): A549 and H1975 cells were treated with HHT for 24 h, lysed and analysed by western blot with indicated antibodies. 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 2.
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f2: HHT induces apoptosis of NSCLC cells.(A): A549 and H1975 cells were treated with Gefitinib (3 mM) or HHT at indicated concentrations for 24 h and stained with Hoechst 33258 assay. (B): A549 and H1975 cells were treated with HHT, lysed and the protein samples were analysed by western blot with indicated antibodies. (C): A549 and H1975 cells were treated with HHT at indicated concentration and the mitochondrial transmembrane potential (ΔΨ) was tested by confocal microscopy (Olympus Fluoview FV-1000, Tokyo, Japan). (D): Ca2+(i) was measured using Ca2+ indicator FLUO-4 (Invitrogen) by flow cytometry assay. (E): H1975 cells were treated with HHT for 24 h, lysed and analysed by western blot with indicated antibodies. (F): H1975 cells were pretreated with Z-VAD-FMK (20 mM) for 1 h and then treated with HHT at 2 mM for 24 h, and the inhibition rate was determined by MTT assay. The mean±SD of three independent experiments is shown. ***, P < 0.01. (G): A549 and H1975 cells were treated with HHT for 24 h, lysed and analysed by western blot with indicated antibodies. 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 2.

Mentions: As indicated above, we tried to investigated the mechanism underlied the inhibition effect of HHT on Gefitinib-resistant NSCLC. By the optical light microscope, we found some dead A549 and H1975 cells floating in the medium treated with HHT. The cell death is reminiscent of the phenomena induced by apoptosis. Next, we tested the possibility of induction of apoptosis by HHT. Firstly, we investigated the nucleus morphological changes by Hoechst 33258 staining. As shown in Fig. 2A, we can find the nuclear condensation and fragmentation with HHT treatment which are typical changes in cell apoptosis. To identify the variation of apoptosis-related proteins, A549 and H1975 cells were treated with HHT at indicated concentration. By whole cell lysis extraction and western blot, HHT treatment resulted in a significant increase of cytochrome C release into cytoplasm and the decrease of the full length of Caspase 9, Caspase 3 and cleavage of poly(ADP-ribose) polymerase (PARP) in A549 and H1975 (Fig. 2B) cells in a dose-dependent manner. To further investigate the mitochondrial dysfunction in A549 and H1975 cells following HHT treatment, we measured mitochondrial transmembrane potential in situ. JC-1 accumulated in the mitochondria and formed red aggregates depending on the potential in healthy cells, and mitochondrial depolarization caused JC-1 release from mitochondria and exhibited green fluorescence. The untreated A549 and H1975 cells displayed strong red fluorescence, conversely the HHT-treated cells exhibited weak red fluorescence (Fig. 2C), suggesting the disruption of the mitochondrial transmembrane potential. Calcium (Ca2+) regulates many cellular functions and also participates in cell apoptosis. Mitochondrial Ca2+ uptake may lead to mitochondria swelling and in turn the release of mitochondrial apoptotic factors into the cytosol37. We examined HHT effects on intracellular calcium variation in H1975 using FLUO-4 (Invitrogen). We found there was a peak of intracellular Ca2+ levels at 4 h after HHT treatment and then decreased to basal level (Fig. 2D). Additionly, in lung, stromal interaction molecule 1 (STIM1)-Orai1 regulates store-operated Ca2+ entry (SOCE) in airway smooth muscle cells and its dysregulation correlates with pulmonary smooth muscle malignant cell proliferation. Orai1 overexpression causes the inflammatory response in cystic fibrosis and its knockdown inhibits endothelial cell migration and angiogenesis38. HHT had no effect on STIM1 and Orai1 during this period (Fig. 2E). To further identify the caspase dependent apoptosis induced by HHT, H1975 cells were pre-treated with a general caspase inhibitor Z-VAD-FMK (20 μM) for 1 h and then treated with HHT at 2 μM for 24 h. We found that HHT-induced apoptosis was significantly suppressed (Fig. 2F). These results indicated that HHT induced apoptosis through the mitochondrial pathway by activation of caspase cascade. Consequently, we examined the expression of several pro- and anti-apoptotic proteins and found that MCL1, a BCL2 family anti-apoptotic protein, and Survivin, other than BCL2 or BAX, decreased with HHT treatment in a concentration depend manner in A549 (Fig. 2G left panel) and H1975 cells (Fig. 2G right panel).


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 induces apoptosis of NSCLC cells.(A): A549 and H1975 cells were treated with Gefitinib (3 mM) or HHT at indicated concentrations for 24 h and stained with Hoechst 33258 assay. (B): A549 and H1975 cells were treated with HHT, lysed and the protein samples were analysed by western blot with indicated antibodies. (C): A549 and H1975 cells were treated with HHT at indicated concentration and the mitochondrial transmembrane potential (ΔΨ) was tested by confocal microscopy (Olympus Fluoview FV-1000, Tokyo, Japan). (D): Ca2+(i) was measured using Ca2+ indicator FLUO-4 (Invitrogen) by flow cytometry assay. (E): H1975 cells were treated with HHT for 24 h, lysed and analysed by western blot with indicated antibodies. (F): H1975 cells were pretreated with Z-VAD-FMK (20 mM) for 1 h and then treated with HHT at 2 mM for 24 h, and the inhibition rate was determined by MTT assay. The mean±SD of three independent experiments is shown. ***, P < 0.01. (G): A549 and H1975 cells were treated with HHT for 24 h, lysed and analysed by western blot with indicated antibodies. 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 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4499885&req=5

f2: HHT induces apoptosis of NSCLC cells.(A): A549 and H1975 cells were treated with Gefitinib (3 mM) or HHT at indicated concentrations for 24 h and stained with Hoechst 33258 assay. (B): A549 and H1975 cells were treated with HHT, lysed and the protein samples were analysed by western blot with indicated antibodies. (C): A549 and H1975 cells were treated with HHT at indicated concentration and the mitochondrial transmembrane potential (ΔΨ) was tested by confocal microscopy (Olympus Fluoview FV-1000, Tokyo, Japan). (D): Ca2+(i) was measured using Ca2+ indicator FLUO-4 (Invitrogen) by flow cytometry assay. (E): H1975 cells were treated with HHT for 24 h, lysed and analysed by western blot with indicated antibodies. (F): H1975 cells were pretreated with Z-VAD-FMK (20 mM) for 1 h and then treated with HHT at 2 mM for 24 h, and the inhibition rate was determined by MTT assay. The mean±SD of three independent experiments is shown. ***, P < 0.01. (G): A549 and H1975 cells were treated with HHT for 24 h, lysed and analysed by western blot with indicated antibodies. 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 2.
Mentions: As indicated above, we tried to investigated the mechanism underlied the inhibition effect of HHT on Gefitinib-resistant NSCLC. By the optical light microscope, we found some dead A549 and H1975 cells floating in the medium treated with HHT. The cell death is reminiscent of the phenomena induced by apoptosis. Next, we tested the possibility of induction of apoptosis by HHT. Firstly, we investigated the nucleus morphological changes by Hoechst 33258 staining. As shown in Fig. 2A, we can find the nuclear condensation and fragmentation with HHT treatment which are typical changes in cell apoptosis. To identify the variation of apoptosis-related proteins, A549 and H1975 cells were treated with HHT at indicated concentration. By whole cell lysis extraction and western blot, HHT treatment resulted in a significant increase of cytochrome C release into cytoplasm and the decrease of the full length of Caspase 9, Caspase 3 and cleavage of poly(ADP-ribose) polymerase (PARP) in A549 and H1975 (Fig. 2B) cells in a dose-dependent manner. To further investigate the mitochondrial dysfunction in A549 and H1975 cells following HHT treatment, we measured mitochondrial transmembrane potential in situ. JC-1 accumulated in the mitochondria and formed red aggregates depending on the potential in healthy cells, and mitochondrial depolarization caused JC-1 release from mitochondria and exhibited green fluorescence. The untreated A549 and H1975 cells displayed strong red fluorescence, conversely the HHT-treated cells exhibited weak red fluorescence (Fig. 2C), suggesting the disruption of the mitochondrial transmembrane potential. Calcium (Ca2+) regulates many cellular functions and also participates in cell apoptosis. Mitochondrial Ca2+ uptake may lead to mitochondria swelling and in turn the release of mitochondrial apoptotic factors into the cytosol37. We examined HHT effects on intracellular calcium variation in H1975 using FLUO-4 (Invitrogen). We found there was a peak of intracellular Ca2+ levels at 4 h after HHT treatment and then decreased to basal level (Fig. 2D). Additionly, in lung, stromal interaction molecule 1 (STIM1)-Orai1 regulates store-operated Ca2+ entry (SOCE) in airway smooth muscle cells and its dysregulation correlates with pulmonary smooth muscle malignant cell proliferation. Orai1 overexpression causes the inflammatory response in cystic fibrosis and its knockdown inhibits endothelial cell migration and angiogenesis38. HHT had no effect on STIM1 and Orai1 during this period (Fig. 2E). To further identify the caspase dependent apoptosis induced by HHT, H1975 cells were pre-treated with a general caspase inhibitor Z-VAD-FMK (20 μM) for 1 h and then treated with HHT at 2 μM for 24 h. We found that HHT-induced apoptosis was significantly suppressed (Fig. 2F). These results indicated that HHT induced apoptosis through the mitochondrial pathway by activation of caspase cascade. Consequently, we examined the expression of several pro- and anti-apoptotic proteins and found that MCL1, a BCL2 family anti-apoptotic protein, and Survivin, other than BCL2 or BAX, decreased with HHT treatment in a concentration depend manner in A549 (Fig. 2G left panel) and H1975 cells (Fig. 2G right panel).

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