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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 supresses the phosphorylation of STAT3.(A and B): A549 and H1975 cells treated with HHT, and the STAT3 phosphorylation and its target genes (A) and the upstream key efftors (B) were examined by western blot with indicated antibodies. (C): With pan-JAK inhibitor P6 (1 μM) and HHT (1 μM) treatment for 12 h, A549 and H1975 cell extracts were conducted western blot with indicated antibodies. (D): H1975 cells were treated with P6 and HHT together or alone and conducted MTT assay. (E): H1975 cells transfected with STAT3C or empty vector were treated with HHT and the inhibition rate was determined by MTT assay (P < 0.01). 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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f3: HHT supresses the phosphorylation of STAT3.(A and B): A549 and H1975 cells treated with HHT, and the STAT3 phosphorylation and its target genes (A) and the upstream key efftors (B) were examined by western blot with indicated antibodies. (C): With pan-JAK inhibitor P6 (1 μM) and HHT (1 μM) treatment for 12 h, A549 and H1975 cell extracts were conducted western blot with indicated antibodies. (D): H1975 cells were treated with P6 and HHT together or alone and conducted MTT assay. (E): H1975 cells transfected with STAT3C or empty vector were treated with HHT and the inhibition rate was determined by MTT assay (P < 0.01). 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: Previous studies have shown that the Mcl1 and Survivin promoters contain a common potential transcription factor STAT3 binding site39. Additionally, STAT3 constitutive activation promotes tumorigenesis partly through upregulation of certain antiapoptotic proteins expression including Bcl-xL, Bcl2, Survivin, and Mcl1, and has been discovered in a variety of hematological tumors and solid tumors including 22%~65% NSCLC. This aberrant constitutive activation plays malignant roles in lung cancer cell proliferation and associated with resistance to chemotherapy4041. STAT3 is activated by a variety of cytokines and growth factors. In clinical studies, STAT3 or pSTAT3 is associated with poor prognosis40. We tested the expression of STAT3 and its phosphorylation states in A549 and H1975 cells treated with HHT, and showed that the STAT3 phosphorylation at Tyr705 (p-STAT3 Y705), which is related STAT3 dimerization, nucleocytoplasmic shuttling, and DNA binding42, was decreased, but not the phosphorylation at Ser727 (Fig. 3A). Pim-1 combined with c-Myc participates in cell transformation and protects cells from apoptosis. Additionally, Pim-1 and c-Myc are STAT3 target genes upon gp130 stimulation43. We also found that Pim-1 and c-Myc expression decreased upon HHT treatment in both A549 and H1975 cells (Fig. 3A). Previous studies have shown that IL-6/JAK, PI3K/AKT and RAS/MAPK/ERK signal pathways are three major upstream STATs activators, so we tested the JAK1, PDK1, AKT and ERK active status. As shown in Fig. 3B, JAK1 phosphorylation (Tyr1022/1023) was significantly decreased but the PDK1, AKT and ERK phosphorylation was not changed after HHT treatment. Additionally, Yao et al. demonstrated that IL-6 upregulation mediated by TGF-β signal pathway conferred NSCLC cell drug resistance in an EGFR independent manner and promoted NSCLC cell proliferation9. As shown in Fig. 3B, the total protein level and phosphorylation of Smad2 and Smad3 were not inhibited by HHT treatment. These results suggested that HHT inhibited the STAT3 aberrant activation through IL-6/JAK/STAT3 pathway without interference with TGFβ signal. Furthermore, we investigated the inhibition effect of combination of pan-JAK inhibitor Pyridone 6 (P6, 1 μM) and HHT (1 μM) treatment for 12 h. Extracts from A549 and H1975 were analyzed for phospho- and total STAT3, Caspase 3 and PARP by western blot. As shown in Fig. 3C, the phosphorylated STAT3 was further inhibited by P6 and HHT combination with no variation of total STAT3. The Caspase 3 was also activated by the combination. Additionally, the cell inhibition rates were also augmented by the combination assayed by MTT assay in H1975 cells (Fig. 3D). To further investigate the essential role of STAT3 inhibition in HHT-induced cell death, H1975 cells transfected with EF.STAT3C.Ubc.GFP were treated with 2 μM HHT for 24 h and the cell inhibition rate was determined by MTT assay. As shown in Fig. 3E, the overexpression of constitutively active mutant of STAT3 (STAT3C) exhibited resistance to HHT treatment and the inhibition rate significantly decreased compared with control group.


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 supresses the phosphorylation of STAT3.(A and B): A549 and H1975 cells treated with HHT, and the STAT3 phosphorylation and its target genes (A) and the upstream key efftors (B) were examined by western blot with indicated antibodies. (C): With pan-JAK inhibitor P6 (1 μM) and HHT (1 μM) treatment for 12 h, A549 and H1975 cell extracts were conducted western blot with indicated antibodies. (D): H1975 cells were treated with P6 and HHT together or alone and conducted MTT assay. (E): H1975 cells transfected with STAT3C or empty vector were treated with HHT and the inhibition rate was determined by MTT assay (P < 0.01). 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

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

f3: HHT supresses the phosphorylation of STAT3.(A and B): A549 and H1975 cells treated with HHT, and the STAT3 phosphorylation and its target genes (A) and the upstream key efftors (B) were examined by western blot with indicated antibodies. (C): With pan-JAK inhibitor P6 (1 μM) and HHT (1 μM) treatment for 12 h, A549 and H1975 cell extracts were conducted western blot with indicated antibodies. (D): H1975 cells were treated with P6 and HHT together or alone and conducted MTT assay. (E): H1975 cells transfected with STAT3C or empty vector were treated with HHT and the inhibition rate was determined by MTT assay (P < 0.01). 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: Previous studies have shown that the Mcl1 and Survivin promoters contain a common potential transcription factor STAT3 binding site39. Additionally, STAT3 constitutive activation promotes tumorigenesis partly through upregulation of certain antiapoptotic proteins expression including Bcl-xL, Bcl2, Survivin, and Mcl1, and has been discovered in a variety of hematological tumors and solid tumors including 22%~65% NSCLC. This aberrant constitutive activation plays malignant roles in lung cancer cell proliferation and associated with resistance to chemotherapy4041. STAT3 is activated by a variety of cytokines and growth factors. In clinical studies, STAT3 or pSTAT3 is associated with poor prognosis40. We tested the expression of STAT3 and its phosphorylation states in A549 and H1975 cells treated with HHT, and showed that the STAT3 phosphorylation at Tyr705 (p-STAT3 Y705), which is related STAT3 dimerization, nucleocytoplasmic shuttling, and DNA binding42, was decreased, but not the phosphorylation at Ser727 (Fig. 3A). Pim-1 combined with c-Myc participates in cell transformation and protects cells from apoptosis. Additionally, Pim-1 and c-Myc are STAT3 target genes upon gp130 stimulation43. We also found that Pim-1 and c-Myc expression decreased upon HHT treatment in both A549 and H1975 cells (Fig. 3A). Previous studies have shown that IL-6/JAK, PI3K/AKT and RAS/MAPK/ERK signal pathways are three major upstream STATs activators, so we tested the JAK1, PDK1, AKT and ERK active status. As shown in Fig. 3B, JAK1 phosphorylation (Tyr1022/1023) was significantly decreased but the PDK1, AKT and ERK phosphorylation was not changed after HHT treatment. Additionally, Yao et al. demonstrated that IL-6 upregulation mediated by TGF-β signal pathway conferred NSCLC cell drug resistance in an EGFR independent manner and promoted NSCLC cell proliferation9. As shown in Fig. 3B, the total protein level and phosphorylation of Smad2 and Smad3 were not inhibited by HHT treatment. These results suggested that HHT inhibited the STAT3 aberrant activation through IL-6/JAK/STAT3 pathway without interference with TGFβ signal. Furthermore, we investigated the inhibition effect of combination of pan-JAK inhibitor Pyridone 6 (P6, 1 μM) and HHT (1 μM) treatment for 12 h. Extracts from A549 and H1975 were analyzed for phospho- and total STAT3, Caspase 3 and PARP by western blot. As shown in Fig. 3C, the phosphorylated STAT3 was further inhibited by P6 and HHT combination with no variation of total STAT3. The Caspase 3 was also activated by the combination. Additionally, the cell inhibition rates were also augmented by the combination assayed by MTT assay in H1975 cells (Fig. 3D). To further investigate the essential role of STAT3 inhibition in HHT-induced cell death, H1975 cells transfected with EF.STAT3C.Ubc.GFP were treated with 2 μM HHT for 24 h and the cell inhibition rate was determined by MTT assay. As shown in Fig. 3E, the overexpression of constitutively active mutant of STAT3 (STAT3C) exhibited resistance to HHT treatment and the inhibition rate significantly decreased compared with control group.

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