Enhanced cytotoxicity from deoxyguanosine-enriched T-oligo in prostate cancer cells

Abstract

Prostate cancer represents approximately 10 percent of all cancer cases in men and accounts for more than a quarter of all cancer types. Advances in understanding the molecular mechanisms of prostate cancer progression, however, have not translated well to the clinic. Patients with metastatic and hormone-refractory disease have only palliative options for treatment, as chemotherapy seldom produces durable or complete responses, highlighting the need for novel therapeutic approaches. T-oligo, a single-stranded deoxyribonucleic acid with partial sequence homology to human telomeric DNA, has elicited cytostatic and/or cytotoxic effects in multiple cancer cell types. In contrast, normal primary cells of varying tissue types are resistant to cytotoxic actions of T-oligo, underscoring its potential utility as a novel targeted cancer therapeutic. Mechanistically, T-oligo is hypothesized to interfere with normal telomeric structure and form G-quadruplex structures, thereby inducing genomic stress in addition to aberrant upregulation of DNA damageresponse pathways. Here, we present data demonstrating the enhanced effectiveness of a deoxyguanosine-enriched sequence of T-oligo, termed (GGTT)4, which elicits robust cytotoxic effects in prostate cancer cells at lower concentrations than the most recent T-oligo sequence (5'-pGGT TAG GTG TAG GTT T 3') described to date and used for comparison in this study, while exerting no cytotoxic actions on nontransformed human prostate epithelial cells. Additionally, we provide evidence supporting the T-oligo induced activation of cJun N-terminal kinase (JNK) signaling in prostate cancer cells consistent with G-quadruplex formation, thereby significantly advancing the understanding of the T-oligo mechanism of action.

FIG. 1.

Effects of T-oligo on cell cycle profiles in human prostate cancer cells. DU145 (A), PC-3 (B), and LNCaP (C) prostate cancer cell lines were exposed to T-oligo or C-oligo (each at 40 μM) or diluent for 48 or 96 hours. DNA content was analyzed by propidium iodide staining and flow cytometry.

FIG. 2.

Effects of T-oligo on cell cycle profiles of human prostate epithelial cells. The pZ-HPV-7 immortalized human prostate epithelial cell line was exposed to either diluent, T-oligo, or C-oligo (each at 40 μM) for 48 or 96 hours. Cells were analyzed by flow cytometry for DNA content profiles and for hypodiploid cell populations (bottom panels). In this panel, the population identified as M1 represents the hypodiploid fraction (<2N DNA content).

FIG. 3.

T-oligo-induced apoptosis of human prostate cancer cell lines. DU145 prostate cancer cells were treated for 96 hours with either diluent or T-oligo (40 μM). Cells were harvested and stained with annexin 5-fluorescein (FITC) and propidium iodide, and analyzed by flow cytometry for apoptosis and DNA content.

FIG. 4.

Duration of exposure to T‐oligo required to generate cytotoxic effects. (A) Top panel: DU145 cells were continuously exposed to T‐oligo (T), C‐oligo (C), or diluent (DW), and harvested at the indicated time points and viable cells were enumerated. Bottom panel: DU145 cells were exposed to T‐oligo (T), C‐oligo (C), or diluent (DW) for 72 hours, then medium was washed out and replaced with growth medium free of oligonucleotide and culture was continued. Cells were harvested at the indicated time points, including 24 and 48 hours after washout and viable cells were enumerated. Error bars represent standard error of the mean (SEM); p values were determined using Student's t‐test (*p<0.05 for 24‐hour timepoint; ^†p<0.05 for 48‐hour timepoint). (B) Cell cycle profiles following washout of oligonucleotides. DU145 prostate cancer cells were exposed continuously for 96 hours to either diluent, T‐oligo, or C‐oligo (each at 40 μM) (continuous treatment). In parallel, a different cohort of DU145 were exposed in an identical fashion for 72 hours, followed by washout of the oligonucleotides and continuation of culture for 24 hours (washout post 24‐hour treatment). Cells were harvested, stained with propidium iodide, and analyzed by flow cytometry.

FIG. 4.

Duration of exposure to T‐oligo required to generate cytotoxic effects. (A) Top panel: DU145 cells were continuously exposed to T‐oligo (T), C‐oligo (C), or diluent (DW), and harvested at the indicated time points and viable cells were enumerated. Bottom panel: DU145 cells were exposed to T‐oligo (T), C‐oligo (C), or diluent (DW) for 72 hours, then medium was washed out and replaced with growth medium free of oligonucleotide and culture was continued. Cells were harvested at the indicated time points, including 24 and 48 hours after washout and viable cells were enumerated. Error bars represent standard error of the mean (SEM); p values were determined using Student's t‐test (*p<0.05 for 24‐hour timepoint; ^†p<0.05 for 48‐hour timepoint). (B) Cell cycle profiles following washout of oligonucleotides. DU145 prostate cancer cells were exposed continuously for 96 hours to either diluent, T‐oligo, or C‐oligo (each at 40 μM) (continuous treatment). In parallel, a different cohort of DU145 were exposed in an identical fashion for 72 hours, followed by washout of the oligonucleotides and continuation of culture for 24 hours (washout post 24‐hour treatment). Cells were harvested, stained with propidium iodide, and analyzed by flow cytometry.

FIG. 5.

Comparison of effects of T‐oligo and (GGTT)₄ on prostate cancer cell proliferation and cell cycle profiles. (A) DU145 cells were exposed to either diluent (DW), (GGTT)₄ at 20 μM, T‐oligo at 40 μM (T), or C‐oligo at 40 μM (C). Viable cells were enumerated after 72 and 96 hours of exposure. Error bars represent SEM; p values were determined by Student's t‐test (*p<0.005). (B) DU145 cells were exposed to either diluent (DW), (GGTT)₄ at 20 μM, or T‐oligo at 20 μM (T) for 24 hours. The medium on all cultures was then changed and replaced with fresh growth medium. Viable cells were enumerated 96 hours later. Error bars represent SEM. Cell numbers in the cultures exposed to T‐oligo compared to those exposed to (GGTT)₄ were significantly different at day 5 (*p<0.02; determined by Student's t‐test). (C) DU145, LN4, LNCaP, or PC‐3 cells were exposed to either diluent (DW), (GGTT)₄ at 20 μM, T‐oligo at 40 μM (T), or C‐oligo at 40 μM (C). Viable cells were enumerated 96 hours later. Error bars represent SEM. Values for the (GGTT)₄‐ or T‐oligo‐treated cultures compared to the vehicle‐ or C‐oligo‐treated cultures were significant for the DU145, LN4, and PC3 cell lines ( p<0.01; determined by Student's t‐test). (D) Cells were exposed to either diluent, (GGTT)₄ at 20 μM, T‐oligo at 40 μM, or C‐oligo at 40 μM. Cells were collected at 72 and 96 hours, stained with propidium iodide, and analyzed by flow cytometry.

FIG. 5.

Comparison of effects of T‐oligo and (GGTT)₄ on prostate cancer cell proliferation and cell cycle profiles. (A) DU145 cells were exposed to either diluent (DW), (GGTT)₄ at 20 μM, T‐oligo at 40 μM (T), or C‐oligo at 40 μM (C). Viable cells were enumerated after 72 and 96 hours of exposure. Error bars represent SEM; p values were determined by Student's t‐test (*p<0.005). (B) DU145 cells were exposed to either diluent (DW), (GGTT)₄ at 20 μM, or T‐oligo at 20 μM (T) for 24 hours. The medium on all cultures was then changed and replaced with fresh growth medium. Viable cells were enumerated 96 hours later. Error bars represent SEM. Cell numbers in the cultures exposed to T‐oligo compared to those exposed to (GGTT)₄ were significantly different at day 5 (*p<0.02; determined by Student's t‐test). (C) DU145, LN4, LNCaP, or PC‐3 cells were exposed to either diluent (DW), (GGTT)₄ at 20 μM, T‐oligo at 40 μM (T), or C‐oligo at 40 μM (C). Viable cells were enumerated 96 hours later. Error bars represent SEM. Values for the (GGTT)₄‐ or T‐oligo‐treated cultures compared to the vehicle‐ or C‐oligo‐treated cultures were significant for the DU145, LN4, and PC3 cell lines ( p<0.01; determined by Student's t‐test). (D) Cells were exposed to either diluent, (GGTT)₄ at 20 μM, T‐oligo at 40 μM, or C‐oligo at 40 μM. Cells were collected at 72 and 96 hours, stained with propidium iodide, and analyzed by flow cytometry.

FIG. 6.

Effect of (GGTT)₄ exposure on cJun N-terminal kinase (JNK) signaling. (A) Top panel: DU145 cells were exposed to either diluent, (GGTT)₄, or T-oligo at the concentrations indicated for 12 hours, then harvested for isolation of proteins. Lysates were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies against total JNK and phospho-JNK (p-JNK). Bottom panel: normalized quantification of p-JNK band intensity from top panel of Figure 6A. (B) Top panel: DU145 cells were exposed to either (GGTT)₄ at 20 μM or to JNK inhibitor SP600125 at 10 μM, or both simultaneously for 12 hours, then harvested for isolation of proteins. Lysates were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies against p-cJun or total cJun. Bottom panel: normalized quantification of p-JNK band intensity from top panel of Fig. 6B. (C) DU145 cells were cultured in the presence of diluent (control), (GGTT)₄ at 20 μM, or JNK inhibitor SP600125 at 10 μM, or to both (GGTT)₄ and SP600125 simultaneously [(GGTT)₄+SP]. Viable cells were enumerated 96 hours later. Error bars represent SEM. Differences between cultures exposed to (GGTT)₄ versus (GGTT)₄ plus SP600125 were significant (*p<0.01; determined by Student's t-test).