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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

In vivo therapeutic efficiency of HHT on mice xenograft bearing human Gefitinib-resistant H1975 cells.(A): Murine models were treated with Gefitinib or HHT and the tumor volumes were calculated every two days. (B): Images of xenograft tumors obtained from mice with different treatment after 3 weeks. (C) HHT treatment did not affect the murine model body weight. (D): Phosphorylated STAT3(Y705), MCL1 expression level, phosphorylated AKT and ERK of tumor sample lysates were analyzed by western blot with indicated antibodies. Vehicle (Line 1–3), Gefitinib (Line 4–6) and HHT (Line 7–9). 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 5. (E): Phosphorylated STAT3(Y705) expression was examined by tumor immunofluorescence staining.
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f7: In vivo therapeutic efficiency of HHT on mice xenograft bearing human Gefitinib-resistant H1975 cells.(A): Murine models were treated with Gefitinib or HHT and the tumor volumes were calculated every two days. (B): Images of xenograft tumors obtained from mice with different treatment after 3 weeks. (C) HHT treatment did not affect the murine model body weight. (D): Phosphorylated STAT3(Y705), MCL1 expression level, phosphorylated AKT and ERK of tumor sample lysates were analyzed by western blot with indicated antibodies. Vehicle (Line 1–3), Gefitinib (Line 4–6) and HHT (Line 7–9). 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 5. (E): Phosphorylated STAT3(Y705) expression was examined by tumor immunofluorescence staining.

Mentions: To access the anti-tumor effect on NSCLC in vivo, we subcutaneously inoculated 2.5 × 106 H1975 cells in 100 μL serum-free RPMI-1640 medium into the right flank of nude mice to generate xenografted murine models. When the tumors grow to a measurable size, each group (10 mice for each random allocation) were administrated with vehicle control, Gefitinib (30 mg/kg) and HHT (10 mg/kg) 5 times per week for 3 weeks. Tumor bearing mice were humanely killed when their tumors reached 2 cm indiameter or when paralysis or major compromise in their quality of life occurred. To our expections, we found that HHT efficiently repressed tumor growth compared to vehicle control or Gefitinib (P < 0.05) (Fig. 7A,B). Additionally, HHT treatment did not reduce the mice body weight, which suggested that HHT had no apparent side effect (Fig. 7C). All the mice were euthanized, the tumors were isolated and imaged and the tumor sample cells were harvested to extract protein for determination if HHT inhibited STAT3 phosphorylation via western blot. As seen in Fig. 7D, the level of STAT3 phosphorylation and MCL1 from HHT treatment group was significantly decreased compared to vehicle control or Gefitinib treatment. Meanwhile, consistant with the results in the above, AKT1/2/3 and ERK1/2 phosphorylation was not inhibited with HHT treatment.To further examine the STAT3 phosphorylation in the xenograft tumor samples with different treatments, the tumor samples were frozen and cutted into 10 μm sections for fluorescent immunohistochemistry. Figure 7E showed that HHT treatment inhibited STAT3 phosphorylation compared to vehicle control or Gefitinib treatment. These results suggested that HHT had immense potential for Gefitinib-resistant NSCLC therapy.


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)

In vivo therapeutic efficiency of HHT on mice xenograft bearing human Gefitinib-resistant H1975 cells.(A): Murine models were treated with Gefitinib or HHT and the tumor volumes were calculated every two days. (B): Images of xenograft tumors obtained from mice with different treatment after 3 weeks. (C) HHT treatment did not affect the murine model body weight. (D): Phosphorylated STAT3(Y705), MCL1 expression level, phosphorylated AKT and ERK of tumor sample lysates were analyzed by western blot with indicated antibodies. Vehicle (Line 1–3), Gefitinib (Line 4–6) and HHT (Line 7–9). 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 5. (E): Phosphorylated STAT3(Y705) expression was examined by tumor immunofluorescence staining.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: In vivo therapeutic efficiency of HHT on mice xenograft bearing human Gefitinib-resistant H1975 cells.(A): Murine models were treated with Gefitinib or HHT and the tumor volumes were calculated every two days. (B): Images of xenograft tumors obtained from mice with different treatment after 3 weeks. (C) HHT treatment did not affect the murine model body weight. (D): Phosphorylated STAT3(Y705), MCL1 expression level, phosphorylated AKT and ERK of tumor sample lysates were analyzed by western blot with indicated antibodies. Vehicle (Line 1–3), Gefitinib (Line 4–6) and HHT (Line 7–9). 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 5. (E): Phosphorylated STAT3(Y705) expression was examined by tumor immunofluorescence staining.
Mentions: To access the anti-tumor effect on NSCLC in vivo, we subcutaneously inoculated 2.5 × 106 H1975 cells in 100 μL serum-free RPMI-1640 medium into the right flank of nude mice to generate xenografted murine models. When the tumors grow to a measurable size, each group (10 mice for each random allocation) were administrated with vehicle control, Gefitinib (30 mg/kg) and HHT (10 mg/kg) 5 times per week for 3 weeks. Tumor bearing mice were humanely killed when their tumors reached 2 cm indiameter or when paralysis or major compromise in their quality of life occurred. To our expections, we found that HHT efficiently repressed tumor growth compared to vehicle control or Gefitinib (P < 0.05) (Fig. 7A,B). Additionally, HHT treatment did not reduce the mice body weight, which suggested that HHT had no apparent side effect (Fig. 7C). All the mice were euthanized, the tumors were isolated and imaged and the tumor sample cells were harvested to extract protein for determination if HHT inhibited STAT3 phosphorylation via western blot. As seen in Fig. 7D, the level of STAT3 phosphorylation and MCL1 from HHT treatment group was significantly decreased compared to vehicle control or Gefitinib treatment. Meanwhile, consistant with the results in the above, AKT1/2/3 and ERK1/2 phosphorylation was not inhibited with HHT treatment.To further examine the STAT3 phosphorylation in the xenograft tumor samples with different treatments, the tumor samples were frozen and cutted into 10 μm sections for fluorescent immunohistochemistry. Figure 7E showed that HHT treatment inhibited STAT3 phosphorylation compared to vehicle control or Gefitinib treatment. These results suggested that HHT had immense potential for Gefitinib-resistant NSCLC therapy.

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