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Oncol Rep
2013 Mar 01;293:1094-100. doi: 10.3892/or.2012.2198.
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Effect of PF-02341066 and radiation on non-small cell lung cancer cells.
Tumati V
,
Kumar S
,
Yu L
,
Chen B
,
Choy H
,
Saha D
.
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Recently, a fusion protein of echinoderm microtubule associated protein like-4 (EML4) and anaplastic lymphoma kinase (ALK) has been found in non-small cell lung cancer (NSCLC) patients. In addition, endogenous expression of phosphorylated c-Met was found to be increased in many invasive NSCLC cases. PF-02341066 (crizotinib) is a novel dual c-Met and EML4-ALK inhibitor, and preclinical studies have shown that treatment with ALK inhibitors leads to drastic tumor regression in xenograft models. A phase I trial of PF-02341066 yielded a 53% response rate and a disease control rate of 79%. We evaluated crizotinib as a potential radiation-sensitizing agent in multiple established NSCLC cell lines with varying expression levels of c-Met and EML4-ALK. The combined effect of ionizing radiation (IR) and PF-02341066 was determined by the surviving cell fraction, cell cycle distribution, apoptosis, DNA double-strand break repair in 5 NSCLC cell lines (A549, H460, H3122, H2228 and H1993) and in in vivo xenograft studies. Treatment of NSCLC cells with either PF-02341066 alone or PF-02341066 + IR did not significantly alter cellular radiosensitivity, DNA repair kinetics and cell cycle distribution; no significant enhancement of tumor growth delay was noted in response to the combined treatment of PF-02341066 + IR. EML4-ALK and c-Met inhibition leads to activation of parallel pathways that converge on Akt signaling which abrogates any radiation-sensitizing effect. Although PF-02341066 is an effective therapy able to suppress tumor growth in tumors that exhibit positivity for either EML4-ALK or c-Met, it did not affect the intrinsic radiation response of tumor cell lines. In the present study, we demonstrated that PF-02341066 did not enhance radiation sensitivity in a panel of NSCLC cell lines.
Figure 1. Effect of PF-02341066 on NSCLC cell lines. (A) Concentration of PF-02341066 necessary to inhibit clonogenic growth by 50% was determined by clonogenic surviving assay. (B) H2228 cells were treated with PF-02341066 for 4 h after which the drug was removed from the culturing medium. Samples were collected at the times noted. (C-E) H3122, H460 and A549 cells were treated with HGF (50 ng/ml) in the presence or absence of PF-02341066, and samples were collected as indicated for western blot analysis.
Figure 2. Effect of radiation and PF-02341066 on NSCLC cells. Cells were grown to 70% confluence prior to treatment with PF-02341066 for 2 h and then cells were irradiated with increasing doses of radiation (2, 4, 6 and 8 Gy). Cells were then washed, trypsinized and plated for colony formation assay. (A) H460 cells (100 nM); (B) A549 cells (100 nM); (C) H2228 cells (13 nM); (D) H3122 cells (21 nM) and (E) H1993 cells (3.8 nM).
Figure 3. Effect of radiation and PF-02341066 on NSCLC xenografts. (A and B) One million cells in 100 μl volume with Matrigel (1:1) were injected subcutaneously into female nude mice. Mice were divided into the following treatment groups: control, PF-02341066 only (50 mg/kg), IR (2 Gy × 5) and IR + PF 02341066. Treatments were initiated when the tumor diameter was 3–5 mm. (C) H460 cells, which are non-responsive to PF-02341066 treatment, were injected into female nude mice without Matrigel and the tumor-bearing animals were divided into 4 groups and treated as described above.
Figure 4. Effect of PF-02341066 on radiation-induced DNA-double-strand break repair. Cells were treated with varying concentrations of PF-02341066 as indicated, irradiated with 2 Gy, and fixed in paraformaldehyde at the designated time points for H2AX staining. (A) A549; (B) H1933; (C) H460; (D) H3122 and (E) H2228 cells.
Figure 5. Effect of PF-02341066 and radiation on the cell cycle distribution. Cells were plated and treated as previously described. The radiation groups received 2 Gy radiation while the drug treatment groups were treated at the cell line-specific EC50 values. (A) All cell lines show typical G2/M arrest associated with ionizing radiation, and addition of PF-02341066 failed to increase the proportion of cells that were arrested. H1993 and H2228 cells showed strong G1 arrest following treatment of PF-02341066 only. (B) Representative flow cytometric data from H1993 cells. Cells that were treated with 3.8 nM PF progressively underwent G1 arrest with the greatest effect noted at 24 h.
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