Metuzumab enhanced chemosensitivity and apoptosis in non-small cell lung carcinoma

Abstract

Targeted therapeutics is used as an alternative treatment of non-small cell lung cancer (NSCLC); however, treatment effect is far from being satisfactory, and therefore identification of new targets is needed. We have previously shown that metuzumab inhibit tumor growth in vivo. The present study was performed to investigate the anti-tumor efficacy of metuzumab combined with gemcitabine and cisplatin (GP), paclitaxel and cisplatin (TP) or navelbine and cisplatin (NP) regimens in multiple NSCLC cell lines. Our results demonstrate that, in comparison to single agent metuzumab or GP treated cells, metuzumab combined with GP display inhibitory effects on tumor growth. Furthermore, we found that metuzumab elevated the sensitivity of cell lines to gemcitabine, which was identified by MTT assay. Flow cytometric analysis showed that metuzumab combined with gemcitabine (GEM) treatment led to an obvious G1 arrest and an elevated apoptosis in A549, NCI-H460 and NCI-H520 cells. Western blot analysis also demonstrated a significantly reduced level of cyclin D1, Bcl-2, and an obviously increase level of Bax and full-length caspase-3 in A549, NCI-H460 and NCI-H520 cells treated with metuzumab/gemcitabine combination in comparison with single agent treated cells. In addition, metuzumab/gemcitabine treated A549, NCI-H460 and NCI-H520 cells also demonstrated a significantly increase in deoxycytidine kinase (dCK) protein level compared with single agent metuzumab or gemcitabine treated cells. Xenograft models also demonstrated that this metuzumab/gemcitabine combination led to upregulation of dCK. Taken together, the mechanisms of metuzumab combined with GP repress tumor growth were that the combined treatment significantly inhibited the tumor cell proliferation, apoptosis and cell cycle in vitro and in vivo and at least partially by induction of dCK expression. Our results suggested that metuzumab could significantly enhance chemosensitivity of human NSCLC cells to gemcitabine. Metuzumab/gemcitabine combination treatment may be a potentially useful therapeutic regimen for NSCLC patients.

Keywords: CD147; chemosensitivity; gemcitabine; metuzumab; monoclonal antibody; non-small-cell lung cancer; targeted therapy.

Figure 1.

The antitumor efficacy of metuzumab combined with GP, TP or NP in vitro and in vivo. (A) Tumor growth inhibition with metuzumab alone or combined with GP, TP or NP in Balb/c nude mice bearing A549 (left), NCI-H460 (middle) and NCI-H520 (right) human lung cancer cell line xenografts. Data are presented as tumor volume (mm³) in the means ± SD (n = 10). (B) Analysis of cell-growth rate by modified MTT assay. (C) Representative images of metuzumab distribution in nude mice bearing A549, NCI-H460 and NCI-H520 cell xenograft. Each experiment was repeated for at least 3 times.

Figure 2.

Metuzumab combined with GP represses cell proliferation and induces apoptosis in vivo. (A) Western blot analysis on the level of CD147 expression in A549, NCI-H460 or NCI-H520 cells. β-actin was used as the internal control. The experiments were performed triplicate. Gray-scale value was analyzed by Image J software for 3 times. The numbers indicate relative protein expression normalized to β-actin. (B) FACS analysis was used to examine the level of CD147 expression in A549, NCI-H460 or NCI-H520 cells. (C) Representative images of immunohistochemical staining for Ki-67, TUNEL, Bax and Bcl-2 in nude mice bearing A549 cell xenograft received metuzumab, GP or metuzumab combined with GP treatment. Scale bar in Ki-67, Bax and Bcl-2 represents 50 μm, scale bar in TUNEL represents 20μm. Histogram represents the integrated optical density (IOD) of Ki-67, Bax and Bcl-2 expression and percentage of TUNEL positive nuclei from the studied group. The bars represent each sample performed in triplicate, and the error bars indicate mean ± SD. **p < 0.01. ***p < 0.001.

Figure 3.

Combined effect of metuzumab and gemcitabine on cell cycle in A549, NCI-H460 and NCI-H520 cells. (A) Representative cell cycle profiles obtained by FACS analysis from propidium iodide stained A549, NCI-H460 or NCI-H520 cells treated with human IgG1, metuzumab, gemcitabine or metuzumab combined with gemcitabine. X-axis values correspond to DNA content; Y-axis values correspond to the number of events detected. Graph of the percentage of cells within each phase of the cell cycle in A549 (B), NCI-H460 (C) or NCI-H520 (D) cells obtained by FACS analysis. And data are presented as mean ± SD. (E) Western blot analysis on the expression levels of PCNA, cyclin D1 in A549, NCI-H460 or NCI-H520 cells treated with metuzumab or cHAb18, gemcitabine, and metuzumab or cHAb18 combined with gemcitabine for 24 hour. β-actin was used as the internal control. Gray-scale value was analyzed the same as above. The experiments were performed triplicate. The error bars indicate mean ± SD.

Figure 4.

The effects of metuzumab combined with gemcitabine on apoptosis in A549, NCI-H460 and NCI-H520 cells. (A) Representative apoptosis profiles obtained by FACS analysis from Annexin V and propidium iodide stained in A549, NCI-H460 or NCI-H520 cells treated with human IgG1, metuzumab, gemcitabine or metuzumab combined with gemcitabine. Graphic presentation of data obtained from cell apoptosis analysis of A549 (B), NCI-H460 (C) or NCI-H520 (D) cells. Values are expressed as means (Q2+ Q3) ± SD of at least 3 independent experiments. (E) Western blot analysis on the expression levels of Bax, Bcl-2 and full length caspase-3 in A549, NCI-H460 or NCI-H520 cells treated with metuzumab or cHAb18, gemcitabine and metuzumab or cHAb18 combined with gemcitabine for 24 hour. β-actin was used as the internal control. Gray-scale value was analyzed the same as above. The numbers indicate relative protein expression normalized to β-actin. The bars represent each sample performed in triplicate, and the error bars represent mean ± SD. ***p < 0.001.

Figure 5.

Metuzumab induces dCK expression in vitro and in vivo. (A) Western blot analysis on the level of dCK expression in A549, NCI-H460 or NCI-H520 cells treated with human IgG1, metuzumab or cHAb18 for 24 hour. β-actin was used as the internal control. Gray-scale value was analyzed the same as above. The numbers indicate relative protein expression normalized to β-actin. (B) Immunohistochemical staining for dCK expression in nude mice bearing A549 cell xenograft received saline or metuzumab treatment. Scale bar represents 50 μm. Histogram represents the integrated optical density (IOD) of dCK expression. The bars represent each sample performed in triplicate, and the error bars indicate mean ± SD. **p < 0.01. ***p < 0.001.