The reverse transcription inhibitor abacavir shows anticancer activity in prostate cancer cell lines

Abstract

Background: Transposable Elements (TEs) comprise nearly 45% of the entire genome and are part of sophisticated regulatory network systems that control developmental processes in normal and pathological conditions. The retroviral/retrotransposon gene machinery consists mainly of Long Interspersed Nuclear Elements (LINEs-1) and Human Endogenous Retroviruses (HERVs) that code for their own endogenous reverse transcriptase (RT). Interestingly, RT is typically expressed at high levels in cancer cells. Recent studies report that RT inhibition by non-nucleoside reverse transcriptase inhibitors (NNRTIs) induces growth arrest and cell differentiation in vitro and antagonizes growth of human tumors in animal model. In the present study we analyze the anticancer activity of Abacavir (ABC), a nucleoside reverse transcription inhibitor (NRTI), on PC3 and LNCaP prostate cancer cell lines.

Principal findings: ABC significantly reduces cell growth, migration and invasion processes, considerably slows S phase progression, induces senescence and cell death in prostate cancer cells. Consistent with these observations, microarray analysis on PC3 cells shows that ABC induces specific and dose-dependent changes in gene expression, involving multiple cellular pathways. Notably, by quantitative Real-Time PCR we found that LINE-1 ORF1 and ORF2 mRNA levels were significantly up-regulated by ABC treatment.

Conclusions: Our results demonstrate the potential of ABC as anticancer agent able to induce antiproliferative activity and trigger senescence in prostate cancer cells. Noteworthy, we show that ABC elicits up-regulation of LINE-1 expression, suggesting the involvement of these elements in the observed cellular modifications.

Figure 1. ABC induces a dose-dependent growth inhibition in prostate cancer cell lines.

(A) Endogenous RT activity was detected in PC3, LNCaP and WI-38 cells as described in material and methods; (+) positive control reaction with commercial RT. (B) Prostate cancer and normal human fibroblast WI-38 cells were treated with ABC at the concentration of 15 and 150 µM and cultured at the indicated time points. Data shown are representative of at least three independent experiments; bars, ± SD.

Figure 2. ABC affects cell cycle progression.

(A) Cell cycle distribution of PC3 and LNCaP cells exposed to 150 µM ABC. Cells were harvested at the indicated time points, incubated with propidium iodide and DNA content was analysed by flow cytometry. The percentage of cells in each phase is reported. (ND, not done). The data are representative of five independent experiments. (B) A representative experiment of cell cycle progression in PC3 treated cells. The arrows indicate cell accumulation at G2/M phase. (C) PC3 and LNCaP cells were synchronized in early S phase by aphidicolin and cell cycle distribution was analyzed in treated (150 µM ABC) and untreated cells.

Figure 3. ABC treatment induces senescence and cell death in prostate cancer cells.

(A and B) 5×10⁴ cells were seeded in 12-well plates with 150 µM ABC. After 0, 2, 4 and 6 days adherent and floating cells were harvested and resuspended in culture medium containing 0.2% trypan blue for the count of viable and death cells. (C and D) ß-galactosidase activity was evaluated in PC3 and LNCaP cells treated with 150 µM ABC and analyzed at different time points.

Figure 4. Treatment of PC3 cells with ABC induces distinct morphological changes.

(A) SEM image illustrate modification occurring in PC3 cells treated with 15 µM ABC at 48 h. (B) Morphological changes and morphometric analysis of the nuclei stained with Hoechst. Doses and time-points are indicated. (C) Immunofluorescence of nucleoli stained with anti-nucleus human serum.

Figure 5. ABC reduces migration and invasion potential of prostate cancer cells.

PC3 and LNCaP cells were seeded on the membrane in presence or absence of Matrigel and incubated with 15 and 150 µM ABC for 18 h. (A) representative image of migrating and invading PC3 cells stained with crystal violet. (B) Percentage of migration and invasion decrease respect to untreated cells analysed by Optilab software. (*) corresponds to p<0.001, calculated by the Mann-Whitney test.

Figure 6. PC3 microarray analysis results.

(A) Summarizing chart of the expressed and differentially expressed gene numbers resulting from microarray assay (four biological replicates for each condition). (B) Comparison of the top-ten biological functions at 15 and 150 µM ABC obtained by IPA analysis. On the y-axis the statistical significance, expressed as -log/p-value, is reported. Threshold p-value  = 0.05 (yellow line). The number of the genes involved in each function is reported on the top of each bar. (C) Venn diagram showing the fraction of common genes differentially expressed in PC3 cells at 15 and 150 µM ABC.

Figure 7. Impact of ABC treatment on LINE-1 mRNA expression.

Relative levels of LINE-1 mRNA transcripts (ORF1 and ORF2) in PC3 and LNCaP cells treated with 15 and 150 µM ABC at different time points. Data are expressed as mean ± SD. The (*) and (**) symbols denote a significant difference compared to untreated cells, with a p value <0.05 and <0.01 respectively.