DNA double-strand break repair

It has been indicated that DSB caused by topoisomerase poisons or oxidative free radicals could be repaired through both HR and NHEJ DNA repair pathways⁷⁰. We found that although sildenafil could not enhance the doxorubicin-induced DNA damage, combination of these two drugs significantly downregulated the levels of three crucial DSB repair proteins including p-DNA-PKcs (Thr2609) in NHEJ, and RPA32 and Rad51 in HR.

Ku70/Ku80 heterodimers are crucial DSB sensors to recognize and bind to the ends of DSB, which is required for directing DNA-PKcs to DSB and triggering its kinase activity for activation of NHEJ repair machinery⁷⁰. Our results demonstrated that doxorubicin combined with sildenafil not only lowered the level of p-DNA-PKcs (Thr2609), but also significantly decreased the DNA-end binding capacity of Ku80 without changing its protein level, confirming the decrease in NHEJ activity. Even though NHEJ is an error-prone DSB repair pathway compared to HR, it has been proved as a fast, efficient and indispensable way for repair of DSB induced by doxorubicin^{71, 72}. Therefore, the declined NHEJ activity may sensitize cancer cells to doxorubicin. For instance, one study has reported that KU-0060648, a potent dual inhibitor of DNA-PK and PI-3K, enhances etoposide and doxorubicin-induced in vitro and in vivo cytotoxicity in human breast and colon cancer cells⁷³. Hence, we suggested the impaired NHEJ activity caused by the drug combination may partly confer the improved efficacy of doxorubicin in PC-3 cells.

Besides the reduced NHEJ activity, we found that eight hours after the drug treatment, sildenafil downregulated doxorubicin-induced hyperphosphorylation of RPA32. RPA32 is a regulatory subunit of RPA, which is a heterotrimeric protein complex with two other subunits RPA70 and RPA14. RPA bind to single-strand DNA

during DNA replication or upon DNA damage to facilitate nascent strand synthesis, or activate cell-cycle checkpoint mediators and DNA repair machineries involved in HR or/and nucleotide excision repair. RPA undergoes both stress-dependent and -independent phosphorylation on the N-terminus of the RPA32 subunit. Under un-stressed cell cycle, RPA32 is phosphorylated by cyclin-cdk complexes at Ser-23 and Ser-29 during DNA replication and mitosis. Upon DNA damage, PIKKs hyperphosphorylate RPA at five or more additional sites out of possible nine sites, including Ser4, Ser8, Ser11, Ser12, Ser13, Thr21, Ser23, Ser29, and Ser33 on the N-terminus of RPA32^{74, 75}. The hyperphosphorylated form of RPA32 has been shown to have a significantly reduced mobility on SDS–PAGE and can be recognized by Western blot analysis^{76, 77}. It has been suggested that RPA32 hyperphosphorylation leads to a change in RPA conformation, which reduces the interaction between RPA and proteins involved in DNA replication but without changing the interaction with proteins involved in DNA repair. This indicates that RPA32 hyperphosphorylation promotes the DNA repair^{58, 75}. Therefore, the initial reduction in hyperphosphorylation of RPA32 caused by doxorubicin and sildenafil in HRPC cells may imply a decrease in HR-mediated repair of DSBs. The impairment of HR was further substantiated by a dramatic reduction of another protein, Rad51, which has been regarded playing a central role in HR pathway to accurately repair DSBs by catalyzing homology searching and strand exchange reactions. The data revealed that doxorubicin in combination with sildenafil apparently lowered the total protein expression and nuclear foci formation of Rad51 in HRPC cells, confirming the diminished HR-mediated DSB repair. This decrease in total and nuclear expression of Rad51 can be further discussed in three aspects including the down-regulation of Rad51 transcription, increased degradation and reduced nuclear localization of Rad51. According to the previous studies, the decreased

transcription of Rad51 in response to the specific stimulation in cancer cells has been associated with the regulation of the E2 factor (E2F) family of transcription factors.

Bindra and Glazer have demonstrated that the hypoxia-induced downregulation of Rad51 in MCF7 breast cancer cell line is mediated by the increased occupancy of transcriptional repressor E2F4/p130 complexes at the E2F site in RAD51 promoter⁷⁸. Besides, Bonavida and Yakovlev revealed that NO/RNS stimulation caused by inflammation-relevant concentrations (50–200μM) of NO/RNS donor, SNAP, would not directly cause DNA damage in MCF-10A and A549 cancer cells, but inhibit the transcription of DNA repair genes including Rad51 and BRCA1 by changing their promoter occupancy from complexes containing activator E2F1 to complexes containing repressor E2F4^{79, 80}. Similar mechanism is also found in the pan-histone deacetylase (HDAC) inhibitor-induced reduction of Rad51 transcription in both gastric and prostate cancer cells^{81, 82}. On the other hand, gefitinib, a tyrosine kinase inhibitor, has been reported to induce cytotoxicity in drug sensitive human non-small cell lung cancer cells through not only lowering the mRNA level of Rad51 but also enhancing the 26S proteasome-mediated degradation of Rad51⁸³. In the present study, the decreased level of Rad51 in nucleus in HRPC cells co-treated with doxorubicin and sildenafil might be a consequence of either the total reduction of cellular Rad51 or the decreased nuclear localization of Rad51. So far, two different mechanisms have been proposed to explain how Rad51 localizes in the nucleus. Since Rad51 does not have a nuclear localization signal (NLS), Rad51C, one of Rad51 paralogs, containing a functional C-terminal NLS has been shown to directly interact with Rad51 to assist its nuclear entry⁸⁴. After Rad51 enters the nucleus, CRM1/exportin 1 may bind to the nuclear export signal (NES) within Rad51 for nuclear export. Another mechanism indicates that the BRC4 domain of BRCA2 is able to mask the Rad51 NES, leading to nuclear retention of RAD51⁸⁵. Based

on these prior studies, the effect of doxorubicin or/and sildenafil on E2F transcription factors, Rad51 ubiquitination and Rad51C and BRCA2-mediated nuclear localization of Rad51 may be intriguing issues waited to be investigated.

4.4. Effect of PDE5 inhibitors or PDE5 knockdown on doxorubicin-induced cell death and DSB signaling and repair

Two other PDE5 inhibitors have been utilized to further identify the role of PDE5 in the sensitization mechanism. We found that inhibition of PDE5 activity using two other PDE5 inhibitors, vardenafil and tadalafil, could also potentiate the cell-killing effect of doxorubicin in both PC-3 and DU145 cells. We found that although sildenafil, vardenafil and tadalafil were all selective and potent PDE5 inhibitors approved by FDA, they exerted different abilities to increase doxorubicin-induced cell apoptosis (vardenafil sildenafil tadalafil), which closely correlated with their abilities to lower the protein expression of Rad51. This result strongly supported our proposed mechanism that impaired HR repair played a major part to contribute to the synergistic apoptosis. However, it has not been well identified whether the reduction of Rad51 level caused by the drug combination is an on-target or off-target effect of PDE5 inhibitors.

Therefore, we further knocked down PDE5 using siRNA to examine its synergy with doxorubicin and the effect on DNA damage response. The results revealed that knockdown of PDE5 could also sensitize PC-3 cells to doxorubicin with the evidence of an increased sub-G1 population using PI staining and flow cytometric analysis.

However, unexpected results of Western blot showed that knockdown of PDE5 followed by the treatment of doxorubicin for 48 hours reduced the level of cleaved caspase-3, and decreased the expression of DNA damage response and repair proteins including γ-H2A.X, p-Chk2 (Thr68), p-RPA32 and Rad51. Of note, although PARP-1

has been known as a substrate of caspase-3, an apoptotic marker, the level of cleaved PARP-1 was not decreased. It has been suggested that the caspase-independent PARP-1 cleavage may be related to other types of cell death but not apoptosis, such as cathepsin D/AIF (apoptosis-inducing factor)-mediated apoptosis-like programmed cell death or cathepsin B/D-mediated necrosis ^{86, 87}. However, it needs further investigation to know the complicated regulation for PARP-1. Based on these data, we concluded that knockdown of PDE5 changes the cell fate of PC-3 cells. For example, unlike the usage of PDE5 inhibitors, knockdown of PDE5 sensitize PC-3 cells to doxorubicin in a caspase-independent mechanism. Besides the possible changes of cell fate, we considered knockdown of PDE5 followed by the treatment of doxorubicin may resemble the sequential treatment with PDE5 inhibitor followed by doxorubicin, which could not completely mimic the co-treatment of these two drugs. Therefore, take these observations into consideration, PDE5 knockdown is not similar to the presence of PDE5 inhibitors and can not explain the relationship between DNA damage response proteins and the inhibition of PDE5 caused by the PDE5 inhibitors.

4.5. Effect of other topoisomerase inhibitors or/and sildenafil on cell apoptosis

Previous studies have indicated that PDE5 inhibitors could sensitize multidrug-resistant cancer cells to chemotherapy drugs by inhibiting drug efflux of the ATP-binding cassette transporters (ABC transporters) including ABCB1 (p-glycoprotein), ABCG2 and ABCC1025, 88, 89

. However, this transporter-relevant mechanism can only be used to explain the synergism observed in multidrug-resistant cancer cells overexpressing specific ABC transporters. The HRPC cell lines PC-3 and DU145 do not express p-glycoprotein, ABCG2 and ABCC10^90-94. Besides, p-glycoprotein and ABCG2 have been shown to pump both topoisomeraseⅠ and Ⅱ

inhibitors out of cells ⁹⁵. According to our results, we found that sildenafil could only sensitize PC-3 cells to topoisomeraseⅡ inhibitors including doxorubicin, etoposide and mitoxantrone, but not topoisomeraseⅠinhibitor camptothecin. The finding strongly supports that sildenafil may not improve the efficacy of doxorubicin in HRPC cells by interfering with drug efflux function of p-glycoprotein and ABCG2.

Chapter 5: Conclusion

Sildenafil does not increase doxorubicin-induced DNA double-strand breaks (DSBs) in hormone-refractory prostate cancer cells (HRPC), but it significantly impairs the doxorubicin-elicited DSB repair systems including homologous recombination (HR) and non-homologous end joining (NHEJ) pathways, thus leading to the synergistic increase in nucleosomal DNA fragments and enhanced activation of intrinsic and extrinsic cell apoptosis. Besides, both inhibition of PDE5 activity and knockdown of PDE5 can sensitize HRPC to doxorubicin, indicating PDE5 may partly contribute to the sensitization mechanism. However, the role of PDE5 in DSB repair still remains unclear (Fig. 19).

Figure 1. Effect of doxorubicin and/or sildenafil on cell cycle distribution in PC-3 cells.

PC-3 cells were treated with 0.1% DMSO, graded concentrations of doxorubicin (0.1, 0.3 or 1 μM) or/and sildenafil (5 or 10 μM) for 48 hours. The cells were fixed with 70%

ethanol and stained with propidium iodide to analyze the distribution of cell populations in cell cycle phases (sub-G1, G0/G1, S and G2/M phase) by FACScan flow cytometric analysis. The data are presented as meanSD of three independent experiments. **p <

0.01 and ***p < 0.001.

Figure 2. Effect of doxorubicin and/or sildenafil on cell cycle distribution in DU145 cells.

DU145 cells were treated with 0.1% DMSO, graded concentrations of doxorubicin (0.1, 0.3 or 1 μM) or/and sildenafil (5 or 10 μM) for 48 hours. The cells were fixed with 70%

ethanol and stained with propidium iodide to analyze the distribution of cell populations in cell cycle phases (sub-G1, G0/G1, Sand G2/M phase) by FACScan flow cytometric analysis. The data are presented as meanSD of three independent experiments. *p <

0.05 and **p < 0.01.

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B

Figure 3. Effect of sildenafil on doxorubicin-induced apoptosis in PC-3 cells.

PC-3 cells were treated with 0.1% DMSO, doxorubicin (1 μM) or/and sildenafil (10 μM) for indicated hours. Changes in cell morphology were observed by microscopic examination (A). Scale bar, 20 μm. Arrows, apoptotic cells. (B) Cell Death Detection ELISA^PLUS kit was employed to detect apoptotic cells through measuring the level of nucleosomal DNA fragments. The data are presented as meanSD of three independent experiments. **p < 0.01 and ***p < 0.001. (C) The expressions of apoptotic markers including caspases-3, -8 and-9 and PARP-1 were detected by Western blot.

C

A

B

Figure 4. Effect of doxorubicin and/or sildenafil on the expression of Bcl-2 family in PC-3 cells.

PC-3 cells were treated with 0.1% DMSO, doxorubicin (1 μM) or/and sildenafil (10 μM) for 8 or 24 hours. (A) The expression of several members in Bcl-2 family was detected by Western blot. (B) The relative protein levels were quantified using the Bio-Rad Quantity One software with α -tubulin as the internal control. The data are presented as meanSD of three to four independent experiments and normalized to the protein level of the control group (8 hours). *p < 0.05.

Figure 5. Effect of doxorubicin and/or sildenafil on ROS production in PC-3 cells.

PC-3 cells were pretreated with 0.1% DMSO (non-pretreatment group), NAC (1 mM) or trolox (0.3 mM) for 30 min, and then co-incubated with doxorubicin (1 μM) or/and sildenafil (10 μM) for 3 hours. Before the termination of incubation, cells were incubated with 10 μM DCFH-DA for 30 min to probe the intracellular ROS. ROS production (%) was detected using FACScan flow cytometry by measuring the percentage of DCF fluorescence-positive cells in total cells. The data are presented as meanSD of three independent experiments. *p < 0.05 and ***p < 0.001 vs. respective non-pretreatment group. ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 vs. control group without pre-treatment. ^††p < 0.01 vs. group of drug combination without pre-treatment.

Figure 6. Effect of ROS scavengers NAC and trolox on cell apoptosis induced by doxorubicin and sildenafil.

PC-3 cells were pretreated with 0.1% DMSO (non-pretreatment group), NAC (1 mM) or trolox (0.3 mM) for 30 min, and then co-incubated with doxorubicin (1 μM) or/and sildenafil (10 μM) for 48 h. After the indicated treatment, cells were fixed with 70%

ethanol and stained with propidium iodide to analyze the sub-G1 population (apoptotic cells) by FACScan flow cytometric analysis. The data are presented as meanSD of three independent experiments.*P < 0.05 and ***P < 0.001.

Figure 7. Effect of doxorubicin and/or sildenafil on the integrity of chromosomal DNA in PC-3 cells.

PC-3 cells treated with doxorubicin (1 μM) or/and sildenafil (10 μM) for 2 or 4 hours were examined for the integrity of chromosome DNA by measuring the amount of DNA single- and double-strand breaks using alkaline comet assay. The DNA integrity in individual cells was scored by the parameter of comet tail moment (Tail moment =

%DNAtail × Lengthtail) using TriTek CometScore^TM software, and at least 100 cells were randomly scored per sample for calculating the mean value of comet tail moment. The quantitative data are presented as the relative mean of comet tail moment (normalized to that of control group) ± SD of two (2 h) or three independent (4 h) experiments.

A

B

C

D

E

Figure 8. Effect of sildenafil on doxorubicin-induced DNA double-strand break (DSB) signaling and repair in PC-3 cells.

PC-3 cells were treated with 0.1% DMSO, doxorubicin (1 μM) or/and sildenafil (10 μM) for indicated hours. The expression of several proteins involved in DSB signaling (A) and repair pathways including homologous recombination (HR) (C), and non-homologous end joining (NHEJ) (F), was monitored by Western blot. (B, D, E and G) The relative protein levels were quantified using the Bio-Rad Quantity One software.

The data are presented as meanSD of two (for Rad51) or three independent experiments. *p < 0.05 and **p < 0.01.

F

G

Figure 9. Effect of sildenafil on doxorubicin-induced DNA double-strand break (DSB) signaling and repair in DU145 cells.

DU145 cells were treated with 0.1% DMSO, doxorubicin (1 μM) or/and sildenafil (10 μM) for indicated hours. The expression of several proteins involved in DSB signaling (A) and repair (B) was monitored by Western blot.

A

B

Figure 10. Effect of doxorubicin and/or sildenafil on DNA end-binding capacity, total and nuclear expression of Ku80 in PC-3 cells.

PC-3 cells treated with doxorubicin (1 μM) or/and sildenafil (10 μM) for 24 h were harvested, and whole cell lysates and nuclear fractions were then collected separately.

(A) The DNA end-binding activity of Ku80 contained in nuclear fractions was assayed using a Ku70/ Ku86 DNA Repair kit. The data are presented as meanSD of three independent experiments. *p < 0.05. (B) The expression of Ku80 in whole cell lysates and nuclear fractions was respectively detected by Western blot and quantified by Bio-Rad Quantity One software. Nucleolin was used as an internal control of nuclear proteins. The quantitative data are presented as meanSD of two or three independent experiments and normalized to the protein level of the control group.

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B

A

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B

Figure 11. Effect of doxorubicin and/or sildenafil on the nuclear foci formation and expression of Rad51 in PC-3 cells.

(A) PC-3 cells were treated with doxorubicin (1 μM) or/and sildenafil (10 μM) for 24 hours followed by 8 hours of cell recovery in drug-free medium, and then fixed with 4

% paraformaldehyde. The formation of nuclear Rad51 foci in PC-3 cells was determined by immunofluorescence staining. Images were captured using a Zeiss AxioImager A1 fluorescent microscope with a 63x oil immersion objective equipped with a CCD camera.Green: Rad51, FITC; Blue: Nuclei, DAPI. (B) The enlarged images of typical cells selected from (A) were shown. (C) The percentage of cells containing over five Rad51 foci in each condition was estimated with minimum of 100 cells counted. The data are presented as meanSD of three independent experiments. ***p <

0.001. (D) The expression of nuclear Rad51 in PC-3 cells treated with indicated drug for 24 h was evaluated by Western blot. Nucleolin was used as an internal control of nuclear proteins.

C

D

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Figure 12. Effect of other PDE5 inhibitors, vardenafil and tadalafil, on doxorubicin-induced cell apoptosis in PC-3cells.

PC-3 cells were treated with 0.1% DMSO, doxorubicin (1 μM) or/and other PDE5 inhibitors, vardenafil (5 or 10 μM) or tadalafil (20 or 40 μM), for indicated hours. (A) Cells were harvested and apoptotic cells were determined using Cell Death Detection ELISA^PLUS kit by measuring cellular level of nucleosomal DNA fragments. The data are presented as meanSD of three independent experiments. *p < 0.05, **p < 0.01 and

***p < 0.001 vs. control group. ^#p < 0.05 and ^###p < 0.001 vs. doxorubicin group. (B, C) The expression of apoptotic markers including caspases-3 and PARP-1 was detected by Western blot.

C

Figure 13. Effect of other PDE5 inhibitors, vardenafil and tadalafil, on doxorubicin-induced cell apoptosis in DU145 cells.

DU145 cells were treated with 0.1% DMSO, doxorubicin (1 μM) or/and other PDE5 inhibitors, vardenafil (5 or 10 μM, A) or tadalafil (20 or 40 μM, B), for 48 hours. The cells were fixed with 70% ethanol and stained with propidium iodide to determine the percentage of apoptotic cell population (sub-G1 population) using FACScan flow cytometric analysis. The data are presented as meanSD of three independent experiments.*p < 0.05, **p < 0.05 and ***p < 0.001.

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B

Figure 14. Effect of PDE5 inhibitors, vardenafil and tadalafil, on HR-mediated repair of DSB induced by doxorubicin in PC-3 cells.

PC-3 cells were treated with 0.1% DMSO, doxorubicin (1 μM) or/and other PDE5 inhibitors, 10 μM of vardenafil (A) or 40 μM of tadalafil (B), for indicated hours. The expression of HR-related proteins was monitored by Western blot.

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B

Figure 15. Correlation between Rad51 expression and the level of nucleosomal DNA fragments (apoptosis) in PC-3 cells treated with doxorubicin or in combination with different PDE5 inhibitors.

Data points are shown for 4 treatment groups in PC-3 cells, including doxorubicin (1μM) alone and its combination with three different PDE5 inhibitors (10 μM sildenafil, 10 Μm vardenafil, or 40 μM tadalafil). The x axis is the relative level of nucleosomal DNA fragments (apoptosis) detected by Cell Death Detection ELISA^PLUS kit between 4 treatment groups. The y axis is the relative protein expression of Rad51 monitored by Western blot between 4 treatment groups. Values of both x and y axis parameters are normalized to that in the doxorubicin alone group. The data are presented as meanSD of two or three independent experiments. The line is a linear regression fit, with R² = 0.93.

Figure 16. Effect of PDE5 knockdown on doxorubicin-induced cell death in PC-3cells.

PC-3 cells were transfected with 10 nmole control siRNA (siControl) or 25 nmole PDE5 siRNA (siPDE5) for 5 hours. After 5 hours of siRNA transfection followed by 48 hours of cell recovery in serum-containing medium, cells were treated with or without 1 μM doxorubicin for 24 or 48 hours. PC-3 cells were harvested to analyze cell cycle by PI staining and flow cytometry (A). (B) Quantitative bar-graph showed the relative ratio of sub-G1 population, normalized to that in siControl group without adding doxorubicin, between different treatment groups. The data are presented as meanSD of two independent experiments. *p < 0.05. (C) Western blot was used to exam the knockdown

PC-3 cells were transfected with 10 nmole control siRNA (siControl) or 25 nmole PDE5 siRNA (siPDE5) for 5 hours. After 5 hours of siRNA transfection followed by 48 hours of cell recovery in serum-containing medium, cells were treated with or without 1 μM doxorubicin for 24 or 48 hours. PC-3 cells were harvested to analyze cell cycle by PI staining and flow cytometry (A). (B) Quantitative bar-graph showed the relative ratio of sub-G1 population, normalized to that in siControl group without adding doxorubicin, between different treatment groups. The data are presented as meanSD of two independent experiments. *p < 0.05. (C) Western blot was used to exam the knockdown