The radiation response of androgen-refractory prostate cancer cell line C4-2 derived from androgen-sensitive cell line LNCaP

Abstract

Radiation therapy is a relatively effective therapeutic method for localized prostate cancer (PCa) patients. However, radioresistance occurs in nearly 30% of patients treated with potentially curative doses. Therapeutic synergy between radiotherapy and androgen ablation treatment provides a promising strategy for improving the clinical outcome. Accordingly, the androgen deprivation-induced signaling pathway may also mediate radiosensitivity in PCa cells. The C4-2 cell line was derived from the androgen-sensitive LNCaP parent line under androgen-depleted condition and had acquired androgen-refractory characteristics. In our study, the response to radiation was evaluated in both LNCaP and C4-2. Results showed that C4-2 cells were more likely to survive from irradiation and appeared more aggressive in their resistance to radiation treatment compared with LNCaP, as measured by clonogenic assays and cell viability and cell cycle analyses. Gene expression analyses revealed that a set of genes involved in cell cycle arrest and DNA repair were differentially regulated in LNCaP and C4-2 in response to radiation, which was also consistent with the radiation-resistant property observed in C4-2 cells. These results strongly suggested that the radiation-resistant property may develop with progression of PCa to androgen-independent status. Not only can the LNCaP and C4-2 PCa progression model be applied for investigating androgen-refractory progression, but it can also be used to explore the development of radiation resistance in PCa.

Figure 1

Growth curve of LNCaP and C4-2 cells after irradiation. 5 000 LNCaP (A) and 4000 C4-2 (B) cells were irradiated with 0, 2.5, 5 and 10 Gy and the cell viability was determined by MTT assay at 0, 6, 12, 24, 48 and 72 h post-radiation. Each point is an average of three experiments. Each data point is presented as mean ± SD.

Figure 2

Colony formation of LNCaP (A) and C4-2 (B) cells after irradiation. Cells were seeded into six-well plates at 2 000–8 000 cells per well and were then irradiated with 5 Gy. Cell survival was determined by colony formation assay.

Figure 3

Soft agar assay. A total of 2 000–8 000 cells were suspended in 2 mL of 0.34% low-melting-point agarose and irradiated with 5-Gy. After 20 days of anchorage-independent growth, colonies with at least 15 cells were counted. Each data point is presented as mean ± SD of triplicate independent experiments.

Figure 4

G₂ checkpoint activation and G₂/M arrest induced by irradiation treatment in LNCaP and C4–2. LNCaP and C4-2 were exposed to 5-Gy radiation. Cell cycle distribution was detected by flow cytometry at 6, 12, 24, 48 and 72 h post-treatment.

Figure 5

Real-time PCR analysis of gene expression associated with DNA repair and cell cycle arrest in LNCaP and C4–2. (A): P53, p21, ATR, CHK1 and CHK2 mRNA expression were examined using real-time PCR assays in LNCaP and C4–2. (B)–(G): Gene expression changes induced by 5- or 10-Gy radiation in LNCaP and C4-2 were examined in p53, p21, ATR, MRE11, CHK1 and CHK2 at 24h post-radiation. Data shown are mean ± SD of triplicate independent measurements. The relative amounts of mRNA of target genes to β-actin were calculated using the equation 2^{−(C(t)target−C(t)β−actin)}.

Figure 6

Immunoblotting analysis of gene expression changes in response to radiation in LNCaP and C4–2. p53, p21 and Chk2 protein expression changes in response to radiation were evaluated in LNCaP and C4-2 at 24h post-radiation using Western blot assays, with β-actin used as normalizing control.