Establishment and characterization of a new drug surviving cell line Am1010, derived directly from muscle metastases of a human lung adenocarcinoma patient with multi-drug-resistance to cisplatin, taxol, and gefitinib

Abstract

Aim: To Characterize a new human lung cancer cell line Am1010, derived from drug-surviving cells (DSCs).

Methods: The Am1010 cell line was established after 4 cycles of chemotherapy from an arm muscle metastatic tumor of a patient diagnosed with lung adenocarcinoma. The cell line has been remained in continuous culture for more than one year during this study.

Results: The Am1010 cell line demonstrated in vitro multi-drug-resistance to cisplatin, taxol, and gefitinib. The Am1010 cell doubling time without drug treatment was 42.395 h. The IC(50) value of cisplatin was 4.299 micromol/L and >10 micromol/L for the Am1010 and P0318 (a cell line derived from non-DSCs) cells, respectively. The IC(50) value of taxol was 0.067 micromol/L and >1 micromol/L for the Am1010 and P0318 cells, respectively. The IC(50) value of gefitinib was 15.233 micromol/L and >70 micromol/L for Am1010 and P0318 cells, respectively. 11 genes involved in the focal adhesion and cell adhesion pathways were found to be differentially expressed. The cells of Am1010 have a significantly larger chromosome number than most lung cancer cell lines.

Conclusion: This novel DSCs derived lung cancer cell line will be a valuable in vitro tool for the investigation of lung cancer drug resistance and metastasis.

Figure 1

The morphological characteristics of cultured Am1010 and P0318 cells. (A) Am1010 cells under phase contrast microscope. (a) A pleomorphic shape cell with prominent nuclei and rather scanty cytoplasm. (b) Cells with fusiform shape. (c) Spontaneous colonies of suspensing cells. (B) P0318 cells under phase contrast microscope. (C) Am1010 transplantation tumor in a nude mouse under light microscope. (D) The original tumor from the patient developed into Am1010 under light microscope. HE staining. ×200.

Figure 2

Ultrastructural morphology of Am1010 cells. (A) Smaller cells had higher electron density and smaller nuclei than the bigger cells. (B) Numerous mitochondria, endoplasmic reticulum and Golgi complexes observed in the bigger cells. (C) Lysosomes are frequently observed in smaller cells.

Figure 3

The chromosomes in one cell of Am1010. Chromosome number is 109 and the ploidy is hypertetraploidy in this cell. Mark chromosome is not found.

Figure 4

The figure shows effect of cisplatin and taxol on Am1010 and P0318 (cis=cisplatin; tax=taxol). The effect on both cell lines is dose-dependent and time-dependent. Both Am1010 and P0318 show resistance to cisplatin and taxol.

Figure 5

The expression profiling analysis. (A) The classification of the differentially expressed genes between DSCs cell line Am1010 and a non-DSCs lung adenocarcinoma cell line P0318 with MAS software. The top 10 was statistically significant (P<0.01). KEGG pathway are shown and the numbers indicate the numbers of the genes involved in this pathway. (B) The figure shows RT-PCR confirmation of the microarray data with 11 genes involved in cell adhesion and focal adhesion pathways. In the RT-PCR electrophoresis gel, A lane means Am1010 and C lane means control P0318 cell line. GAPDH and ACTB genes were used as normalization controls. In the microarray heatmap, it is shown the expressed ratio of Am1010 vs P0318, and column 1, 2 refer to the two slide hybridization ratio. (C) Color illustrations of different pathway and gene expression. Blue background color of pathway name indicates focal adhesion pathway and yellow does CAMs pathway. Red color of gene indicates higher expression in Am1010 than that in P0318 and green does lower.