A12B4C3, an inhibitor of polynucleotide kinase/phosphatase enhances radio-sensitivity in PC-3 cells exposed to carbon ion beam

Pallavi Srivastava¹, Asitikantha Sarma², Chandra Mohini Chaturvedi¹

Introduction

World wide prostate cancer prevalence is highest among American men of Caucasian and African origin [1]. In USA, prostate cancer incidence is 19% and it is the second most common cause of death due to cancer in men after lung and bronchus cancer. Prostate cancer (PCa) alone accounting for almost one in five new diagnoses [2]. Cancer is rising as a major killer in developing countries like India[3].Prostate is the second most leading site of cancer among males, in big Indian cities like Delhi, Kolkata, Pune and Thiruvananthapuram and third leading site of cancer in Bangalore and Mumbai. It is among the top ten leading sites of cancers in the rest of the population based cancer registries (PBCRs) of India. PCa incidence rate are constantly and rapidly increasing in all the PBRCs. The cancer projection data reveals that the number of cases will become doubled by 2020 [4]. Major obstacle in the therapy of cancer is recurrence after radiotherapy and it is due to inherent damage repair property of the cell. This provides a strong rationale for exploration of potential inhibitors which can suppress the DNA damage repair pathway of cancerous cells and thus kinase/phosphatase) plays vital roles in mammalian base excision repair (BER), single strand break (SSB) repair, double strand break (DSB) repair and non-homologous end joining (NHEJ) making it an attractive therapeutic target [5].

In recent years, level of interest has increased to inhibit DNA-repair pathways as an approach to potentiate the radio-therapeutic cancer treatments. Inhibitors of several enzymes involved in the repair of DNA strand breaks works as radiomodulator and are currently at various stages of the drug development process [6-7]. Therefore, targeting signaling networks involved in DNA break repair is a promising approach for enhancing cellular 12 High LET (Linear Energy Transfer) Carbon ion radiation has many potential advantages over the low LET radiation such as gamma radiation or X-rays. Heavy 12 ion beam or Carbon ion radiation as typical high LET radiation creates cluster damage to DNA and produces high Relative Biological Effectiveness (RBE), utilizing Spread Out Bragg Peak (SOBP) and resulting in an increased therapeutic ratio [8]. A major reason for the physical selectivity of the inverted dose profile is a sharp and lateral scattering can be neglected for heavy particles such as carbon ions. This permits dose escalation within the tumor which might result in a better tumor control [9]. Radiation induces DNA breaks with incompatible termini i.e. 3´-phosphate and 5´-hydroxyl moieties which must be processed for SSBs & DSBs repair pathway to complete repair. These lesions create a barrier for DNA polymerase and ligase to replace missing bases and seal the breaks because these enzymes have a strict requirement for the presence of a 3´-OH & in addition DNA ligases requires a 5´-PO group. It is now clear that the major enzyme 4 responsible for phosphorylation of 5´-OH termini and dephosphorylation of 3´-PO termini in human cells is 4 polynucleotide kinase/phosphatase (hPNKP) [10]. hPNKP or PNKP contains both 5´kinase and 3´- phosphatase activities required for restoration of 3´- hydroxyls and 5´-phosphates needed to seal the broken DNA.

Till now most of the focus for radiomodulator has centered on the inhibitors of poly [ADP-ribose] polymerase (PARP) and protein kinase (PK). It has been suggested that the terminal base pairs of double-stranded substrates near the 3´-phosphate are destabilized by PNKP to allow substrate access to the active site [5]. It has also been suggested that inhibiting the PNKP may sensitize cancer cells to chemotherapy and radiotherapy [11-12]. Allinson et al suggested PNKP as a potential target in the treatment of cancer [12]. Inhibition of PNKP represents a DNA repair targeted therapy, which sensitizes cancer cells to DNA damage and enhances anticancer potential. Obviously, new inhibitory compounds need to be identified and optimized for clinical use.

The hPNKP inhibitor, A12B4C3 is reported to inhibit the mammalian PNKP activity. It disrupts the secondary structure of PNKP and acts as a noncompetitive inhibitor that allosterically regulates the phosphatase activity of human PNKP [13]. A12B4C3 is 2-(1-hydroxyundecyl)-1- (4-nitrophenylamino)-6-phenyl-6,7a-dihydro-1Hpyrrolo [3,4-b]pyridine-5,7(2H,4aH)-dione, a polysubstituted piperidine (Figure1) [14-15]. A12B4C3 has ability to inhibit phosphatase activity of PNKP in vitro and it was evidenced that it radiosensitizes the wild type A-549 cells to ionizing radiation but not A-549 PNKP knockdown cells suggesting PNKP is its only target. It enhanced the radiosensitivity in human A549 lung carcinoma and MDA-MB-231 breast adenocarcinoma cells at a very low non-toxic dose 1 μmol/L [14]. Therefore we have selected A12B4C3 to inhibit the PNKP activity. A12B4C3 treatment was shown to radio-sensitizes human A549 cells, MDA-MB-231cells and human myeloid leukemia cells with γ-radiation but has not been studied in a radio resistant cells with C-ion radiation [16-17].

In our previous study we have reported that after 24 hr of 12 C ion exposure, A12B4C3 (an inhibitor of PNKP prostate cancer cells [18]. This data intrigue us to check the effect of A12B4C3 after 48 hr of carbon ion exposure to compare and assures the post radiation effect in PC-3 cells. Hence, present study was designed to monitor the regrowth/recurrence in PC-3 cells after 48 hr of carbon ion radiation in combination with PNKPi (PNKP

amino]-6-phenyl-1H-pyrrolo[3,4-b]pyridine-5,7(2H,6H)- dione].

Experimental

Materials and methods

Cell culture

Prostate cancer PC-3 cells were purchased from our National repository NCCS (National Centre for Cell Science, Pune, India) and maintained in nutrient mixture F-12 Ham, K medium (Himedia, India) supplemented with 10% fetal bovine serum (Himedia, India) in presence of antibiotic solution (Himedia, India) at 5% CO in a 2 humidified atmosphere at 37°C. 70-80% confluent cultured cells were used for experiment.

Treatment

5 0.5×10 cells/plate were treated with the PNKP inhibitor A12B4C3 (Sigma Aldrich, USA) 2-4 hr. before irradiation. A12B4C3 (PNKPi) stock solution was made in dimethyl sulfoxide (DMSO) and dilution was prepared in culture medium. We have used very low concentration of PNKPi 10μM based on our previous study [18].

Carbon ion beam/radiation

15 UD Pelletron accelerator at Inter University Acclerator Centre (IUAC), New Delhi, India, was utilized for irradiation of cells. Cells were cultured and maintained in Radiation Biology Laboratory at IUAC, New Delhi. The irradiation of cells was done using heavy ion irradiation facility ASPIRE software, where the dosimetry is done 12 using silicon surface barrier detectors [19-20]. C beam with 85MeV (equivalent to 7.08 MeV/nucleon) energy from the accelerator was used. The energy of the beam on the cell surface was 62 MeV (equivalent 5.16 MeV/nucleon) with entrance LET 287 kev/μm as calculated by SRIM software [20-21]. The beam flux was 5 maintained at about 2 x 10 particles/sq.cm/sec. 2Gy and 4Gy radiation dose was used for the present study. The dose in Gy was calculated using the following standard relation, where the cell is taken to be water equivalent.

-9 Dose (Gy) = 1.6 x 10 x LET (keV/um) x Fluence (particles/sq. cm) [22].

MTT assay

Cell viability was evaluated by MTT assay according to Plumb et al. (1989) [23]. MTT tetrazolium salt was dissolved in PBS (pH 7.4) at 5 mg/ml. After 48hr incubation period, 30 μl MTT solution was added to each well, mixed thoroughly and all plates were again placed in the incubator. After 4 hr incubation, 100 μl DMSO was added to each well and mixed thoroughly in order to dissolve the formed blue crystals of formazan. All plates were placed on shaker for 5 min. The absorbance was TM measured at a wavelength of 490 nm using VICTOR X5 multilabel Plate Reader (PerkinElmer).

Viability of cells was calculated by using the following formula:

% Viability = OD of PNKPi treated cells + OD of radiation treated cells /OD of control cells (without PNKPi) X 100.

Apoptotic body formation

Apoptotic body formation was observed under R flurorescence microscope after staining with Prolong Gold Antifade reagent. % of apoptotic bodies were counted (approx. 150 cells at each dose) after 48 hr of C- ion irradiation.

Cell cycle distribution by FACS

Propidium Iodide (PI) staining was used to analyze the phases of cell cycle. After irradiation, cells were incubated for 48hr and then collected in medium by scraping, rinsed with PBS, centrifuged, put in 70 % 0 precooled ethanol, fixed at 4 C overnight. After fixation, cells were washed thrice with cold PBS and then stained with staining solution (10μg/ml Propidium Iodide, 100μg/ml of DNase-free RNase and 0.1% v/v triton-100) 0 and incubated at 37 C for 15 min. The phases of cell TM cycle were detected on the BD Accuri Flow Cytometer (B.D. Biosciences, USA) and data analyzed by BD Accuri C6 software.

Statistical analysis

Data were averaged from at least three biological repeats and presented as mean ± standard deviation wherever required. Different samples means were compared by one-way analysis of variance (ANOVA) followed by Newman–Keuls multiple comparison tests. P<0.05 was considered significant. All statistical analysis was done using Prism ver. 5 (GraphPad Software Inc., USA).

Results and discussion

Effects on cell morphology and viability when carbon ion radiation was combined with PNKPi

Our previous study suggested that at 24 hr time point, PNKPi enhanced the radiosensitivity in PC-3 cells after exposure of carbon ion radiation [18]. Present study reveals that after 48 hr of carbon ion radiation appearance of cells changed when combined with PNKPi (Figure 2A). Cell viability data showed that viability of cells decreased to 71.32±0.02%, 61.48±0.12%, 47.38±0.04% and 35.36±0.02% in 2Gy, 2Gy+PNKPi, 4Gy and 4Gy+PNKPi group as compared to control (100%) and 62.77±0.02%, 75.14±0.03% in the 2Gy+PNKPi and 4Gy+PNKPi groups as compared to cells that were irradiated only i.e. 2Gy and 4Gy respectively (Figure 2B).

Figure 2. (A) Morphological changes of PC-3 cells with and without PNKPi (A12B4C3) treatment after 48 hr of

12

2Gy and 4 Gy C beam (B)Cell viability (%) decreased

12

after 48 hr of radiation when C beam is combined with PNKPi. ***p<0.001, significance of difference from

###

control and p<0.001, significance of difference from only irradiated group respectively.

Combined treatment of carbon ion radiation and PNKPi stimulate cell death through apoptotic body formation

Many reports suggested that carbon ion radiation induced cell death through apoptosis and independent of p53 status [24]. Since PC-3 cell line is null for p53 so, it is undergoing p53-independent apoptosis. After 48 hr of irradiation apoptotic body formation was significantly higher (2.61±1.02, 1.72±0.03 fold higher) in cells which were treated with PNKPi in combination with C-ion irradiation as compare to only irridiated cells i.e. 2Gy and 4Gy. We found 2.83±0.72, 6.00±0.57, 9.5±0.98, 16.55±2.02 fold increase in number of apoptotic bodies in 2Gy, 2Gy+PNKPi, 4Gy and 4Gy+PNKPi groups as

after 48 hr of radiation.***p<0.001, significance of

## ### difference from control and p<0.01, p<0.001,

significance of difference compare to the only irradiated group.

Carbon ion radiation induces significant S phase arrest after 48 hr when combined with PNKPi

Cell cycle progression is controlled by many checkpoints at different phases; in which G1/S check point is one of the most critical for control of cell proliferation. Failure of this check point can lead to abnormal growth or apoptosis. Prostate cancer cells (PC-3) showed, a significant cell cycle arrest (1.43±0.02, 1.23±0.01 fold change) at S phase after 48 hr of carbon ion radiation in the presence of PNKPi as compare to only irradiated cells i.e. 2Gy and 4 Gy. We observed 1.21±0.04, 1.73±0.08, 2.13±0.13, 2.62±0.13 fold enhancement at S phase arrest in 2 Gy, 2Gy+PNKPi, 4Gy, 4Gy+PNKPi groups as compared to control cells (Figure 4).

80

significance of difference compared to control and

#

p<0.05 in comparison with the only irradiated group.

Conclusion

In view of the fact that recurrence is major obstacle in the treatment of cancer, targeting DNA repair process could be an attractive approach for cancer therapy. Present experimental study confirmed that, PNKP inhibition by A12B4C3 may be a suitable approach to reduce the recurrence after exposure of carbon ion radiation in prostate cancer cell PC-3. It is also suggested that 10μM, a low concentration of A12B4C3 is effective after 48 hr of carbon ion radiationin 2 Gy-4Gy range. However, further understanding of the molecular mechanism behind the radio-sensitization of PNKPi will strengthens the use of PNKP inhibitor A12B4C3 as a possible radiomodulator to reduce the recurrence of cancerous cells following radiotherapy.

Acknowledgment

We are highly thankful to Inter University Accelerator Centre, New Delhi for financial support grant UFR-56310 and National Institute of Immunology, New Delhi for providing their FACS facility. Council of Scientific & Industrial Research (CSIR), India, is highly acknowledged for senior research fellowship to Pallavi Srivastava.

References

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