JAK2 inhibitor TG101348 overcomes erlotinib-resistance in non-small cell lung carcinoma cells with mutated EGF receptor

Abstract

Non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations are responsive to EGFR-tyrosine kinase inhibitor (EGFR-TKI). However, NSCLC patients with secondary somatic EGFR mutations are resistant to EGFR-TKI treatment. In this study, we investigated the effect of TG101348 (a JAK2 inhibitor) on the tumor growth of erlotinib-resistant NSCLC cells. Cell proliferation, apoptosis, gene expression and tumor growth were evaluated by diphenyltetrazolium bromide (MTT) assay, flow cytometry, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining, Western Blot and a xenograft mouse model, respectively. Results showed that erlotinib had a stronger impact on the induction of apoptosis in erlotinib-sensitive PC-9 cells but had a weaker effect on erlotinib-resistant H1975 and H1650 cells than TG101348. TG101348 significantly enhanced the cytotoxicity of erlotinib to erlotinib-resistant NSCLC cells, stimulated erlotinib-induced apoptosis and downregulated the expressions of EGFR, p-EGFR, p-STAT3, Bcl-xL and survivin in erlotinib-resistant NSCLC cells. Moreover, the combined treatment of TG101348 and erlotinib induced apoptosis, inhibited the activation of p-EGFR and p-STAT3, and inhibited tumor growth of erlotinib-resistant NSCLC cells in vivo. Our results indicate that TG101348 is a potential adjuvant for NSCLC patients during erlotinib treatment.

Keywords: TG101348; cancer therapy; drug resistance; erlotinib; non-small cell lung cancer.

Figure 1. TG101348 induces apoptosis and inhibits JAK2/STAT3 signaling in NSCLC cells with EGFR-mutation

(A-D) TG101348 induces apoptosis of NSCLC cells. H1975 cells (A, C) and H1650 cells (B, D) were treated with erlotinib or TG101348 at the indicated doses (A, B) and time (C, D) for 48 hours. Cell apoptosis was determined by TUNEL. The data are means ± SD of three independent experiments. (E-F) TG101348 inhibits JAK2/STAT3 signaling in NSCLC cells. H1975 cells (E) and H1650 cells (F) were treated with TG101348 at the indicated doses for 24 hours. Protein expression was determined by Western Blot.

Figure 2. TG101348 sensitizes NSCLC cells to the cytotoxicity of erlotinib

(A-B) Erlotinib and TG101348 inhibit cell proliferation. PC-9, H1975 and H1650 cells were treated with erlotinib (A) and TG101348 (B) at the indicated doses for 72 hours. (C-D) TG101348 sensitizes the cytotoxicity of erlotinib to NSCLC cells. H1975 cells (C) and H1650 cells (D) were treated with erlotinib at the indicated doses alone or combined with TG101348 (1 μM) for 48 hours. Cell proliferation was determined by MTT. The data are means ± SD of three independent experiments. (*p < 0.05, compared to erlotinib treatment alone).

Figure 3. TG101348 inhibits colony formation of NSCLC cells

(A) Colony forming ability of H1975 in soft agar. H1975 cells were plated in 0.3% agar with 0.25 μM TG101348 and cultured for 2 weeks. Representative photos were taken at 2 weeks after the cell seeding. (B-C) Quantification analysis of clone number. H1975 cells (B) and H1650 cells (C) were plated in 0.3% agar for 2 weeks. Clone numbers were counted under microscope. Data represents the pooled means ± SD of 3 independent experiments, each consisting of triplicate samples for a total n = 9 for each treatment. *p < 0.01 compared to single treatment. #p < 0.01 compared to DMSO control.

Figure 4. TG101348 enhances erlotinib-induced apoptosis in NSCLC cells

(A) erlotinib induced apoptosis in NSCLC cells. PC-9, H1975 and H1650 cells were treated with erlotinib (2.5 μM) for 72 hours. Apoptosis was determined by TUNEL. *p < 0.01, compared to PC-9 cells. (B-C) TG101348 enhances erlotinib-induced apoptosis in erlotinib-resistant NSCLC cells. H1975 cells (B) and H1650 cells (D) were treated with TG101348 (1 μM), erlotinib (2.5 μM), or a combination of TG101348 (1 μM) and erlotinib (2.5 μM) for 48 hours. Apoptosis was determined by TUNEL. The data presented are means ± SD of three independent experiments. ^#p < 0.01, compared to DMSO treatment; *p < 0.01, compared to TG101348 or erlotinib treatment alone. (D) TG101348 regulates expressions of EGFR and apoptosis-related proteins. H1975 cells were treated with TG101348 (1 μM), erlotinib (2.5 μM) or their combination for 48 hours. Protein expressions were determined by Western Blot.

Figure 5. TG101348 potentiates the anti-tumor effect of erlotinib in vivo

(A-B) TG101348 enhances the inhibition of tumor growth of erlotinib in vivo. H1975 cells (A) and H1650 cells (B) were injected s.c. into the flanks of athymic nude mice. The tumor-bearing mice received TG101348, erlotinib or their combination treatment for 5 days. Tumor volume was measured weakly for 6 week after the treatment. The tumor volume presented is means ± SD of five mice. (C-D) Tumor weight was weighed at the last day of the experiment above. Tumor weights presented are means ± SD of five mice. *p < 0.01, compared to TG101348 or erlotinib treatment alone; #p < 0.01, compared to DMSO treatment.

Figure 6. TG101348 enhances erlotinib-induced apoptosis and inhibition of gene expression in vivo

(A) Tumor sections from the mice indicated in Figure 5 were subjected to cleavage caspase 3 staining (as an indicator of apoptosis), p-EGFR and p-STAT3 staining. Representative images are shown (Original magnification × 400). (B-D) Quantification of apoptotic index and gene expression was described in “Materials and Methods.” Data presented are means ± SD of five mice. ^#p < 0.01, compared to DMSO group; ^*p < 0.01, compared to single treatment DMSO.

Figure 7. The combination of TG101348 and erlotinib prolonged the survival of mice

(A) Tumor-bearing mice survival culture. H1975 cells were injected s.c. into the flanks of athymic nude mice. When tumor size reached 100 mm³, tumor-bearing athymic nude mice were treated with TG101348, erlotinib or their combination for 5 days. Mice were observed until the time point of death of all mice treated with the combination of TG101348 and erlotinib. (B) Histology analysis of main organs (heart, liver, kidney and lung) of mice treated indicated reagents 5 weeks after treatment (Original magnification × 200) are shown.