Poly(ADP-ribose) polymerase (PARP) inhibitors enhance the effect of DNA alkylating agents on
Poly(ADP-ribose) polymerase (PARP) enzyme inhibitors are emerging as a valuable new drug class in the treatment of cancer. PARP-1 is the founding member of a family of 18 PARP members that have been identified thus far. PARP-1 functions as a key molecule in the repair of DNA single-strand breaks (SSBs) via the base excision DNA repair (BER) pathway (
Repurposing existing drugs is another strategy for drug development in which safety pharmacology studies have been already done, which reduces the time and cost of approving the compounds for clinical use (
The MDA-MB-436, MDA-MB-231 and MCF7 human breast cancer cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). MDA-MB-436 cells harbor 5396+1G > A (spliced donor site of exon 20), a BRCA1 mutation. MCF-7 and MDA-MB-231 cells express wild-type BRCA1 while MDA-MB-231 cells are hemizygous for
PARP activity
The MCF-7, MDA-MB-436 and MDA-MB-231 cells were grown in chamber slides (Sigma-Aldrich) at a 70% of confluence. Immunostaining for poly (ADP-ribose) (PAR) was performed on cells fixed in ice-cold 4% paraformaldehyde in PBS for 10 min. The cells were fixed 1 h after treatment with NAM at concentrations of 0.5, 1, 5 and 10 mM and exposed to H2O2 (1 mmol/l, 10 min, 37°C). Controls were treated with medium alone. The primary antibody used was anti-pADPR (H10, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Poly ADP-ribosylation immunostaining was developed following NAM treatment. The secondary antibody used was the Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (Molecular Probes, Life Technologies, Carlsbad, CA, USA). Nuclear counterstaining with DAPI was performed after removal of excess secondary antibody. Immunostaining was visualized with a Carl Zeiss confocal multiphotonic laser microscope 780 NLO (Hamburg, Germany).
Cell viability was assessed by crystal violet staining. Semi-confluent culture flasks were trypsinized and 2.5×104 cells were seeded in 12-well plates. After 24 h, the cells were exposed to NAM (5, 10, 20, 40, 60, 80 and 100 mM) and cisplatin (20, 40, 60 and 80 μM) at the indicated concentrations. After 72 h, the cells were rinsed with PBS, fixed in 2% formaldehyde for 5 min and stained with 1% crystal violet. Relative cell viability was obtained by scanning with an ELISA plate reader at 540 nm.
Synergism or additivity was determined by calculating the combination index (CI) using the equation: CIx = (D1/Dx1) + (D2/Dx2) + a(D1)(D2)/(Dx1)(Dx2), where CIx is the CI value for x% effect, Dx1 and Dx2 are the doses of agents 1 and 2 required to exert x% effect alone, and D1 and D2 are the doses of agents 1 and 2 that elicit the same x% effect in combination with the other agent, respectively. The factor indicates the type of interaction: a =0 for mutually exclusive drugs (similar mechanisms of action), and a =1 for mutually non-exclusive drugs (independent modes of action), with the equation being resolved for a =1. A CI of 1 indicates additivity, a CI of <1 synergism and a CI of >1 antagonism.
Cells were seeded in 12-well plates Nunc (Thermo Scientific™ Nunclon, Waltham, MA, USA) at 15×103 cells/well into 0.5 ml of Optimem (Applied Biosystems Life Technologies, Carlsbad, CA, USA). After 24 h, the cells were transfected with lipofectamine and PARP1 siRNA (cat no. 4390824; Ambrion, Minneapolis, MN, USA) or RNAiMAX containing siRNA Scramble (cat no. 4390844; Ambrion). Cisplatin was added for 24 h and the cells were cultured at 37°C in humidified atmosphere containing 5% CO2. After 72 h, the medium was aspirated and the cells were processed for western blotting and cell viability.
Ionizing radiation (IR) of cells was performed at room temperature in culture medium, using a Theratron Phoenix (60Co) irradiator (Best Theratronics Ltd., Ottawa, Ontario, Canada) with an average energy of 1.25 MeV in a field of 20×20 cm2, to a distance isocenter of 80 cm. Cell lines were irradiated at a range of 0.5–6 Gy in the presence of different concentrations of NAM (0.5–20 mM). Control cells were treated with medium only.
Exponentially proliferating cells were plated into a 25-cm2 cell culture flask and incubated for 48 h to allow cells to reach their optimum proliferation rate. NAM (0.5, 1, 5, 10 and 20 mM) was added to the dishes and incubated for 72 h and the cells were irradiated. Control cells were not treated. Cells were collected and cultured in drug-free medium in 60 mm Petri dishes for up to 21 days, depending on the proliferation rate of the individual cell line. Colonies were fixed in methanol and acetic acid (3:1 v/v), stained with crystal violet and counted with a stereoscopic microscopy [Leica Microsystems, (Schweiz) AG, Heerbrugg, Switzerland]. Data are expressed as the percentage of colonies in NAM-treated cultures compared with control cultures. Lethal concentration 50 (LC50) was calculated for each cell line in each independent experiment. Each assay was performed in triplicate for each concentration. Plating efficiencies and the surviving fractions were calculated.
Data are expressed as the mean ± SD values. For statistical analysis the ANOVA and Bonferroni post-tests were used to mediate GraphPad Prism 5. P<0.05 was considered to indicate a statistically significant difference.
To examine whether NAM can inhibit PARP activity
To confirm that NAM inhibits poly-ADP-ribosylation, we induced DNA damage with H2O2 (10 mmol/l for 10 min) leading to poly-ADP ribosylation in the three cell lines after treatment with 0.5, 0.75, 1, 5 and 10 mM of NAM. We performed a series of immunofluorescent stainings using anti-pADPR.
To determine whether PARP inhibition with NAM sensitizes cell to cisplatin-induced death, we treated MDA-MB-436, MDA-MB-231 and MCF-7 cell lines with cisplatin (0–100 μM) and NAM (5, 10, 20, 40, 60, 80 and 100 mM) and determined the IC50 value.
We also analyzed the effect of co-treatment of NAM and cisplatin using a lower dose of cisplatin (IC20) and varying doses of NAM (5, 10, 20 and 40 mM). The results demonstrated that a combination of NAM and cisplatin significantly decreased the viability of MDA-MB-436 and MCF-7 cells as compared to NAM alone (P<0.001,
To determine to what extent the downregulation of PARP1 inhibited cell growth, MDA-MB-436 were subjected to siRNA against PARP1. The results showed that at 72 h there was complete knockdown of mRNA of PARP1 and cell growth was inhibited by 10%. When cisplatin was added this inhibition increased as compared to cisplatin alone or PARP1 inhbition. These differences were statistically significant. No growth effects were observed for scramble siRNA with or without cisplatin (
The effect of NAM in combination with ionizing radiation (IR) through clonogenic assay was analyzed to determine the colony-forming ability from a cell after inducing DNA damage. Survival curves were performed to determine the median lethal doses (LD50), which were defined as the absorbed dose of ionizing radiation (Gy) required to induce 50% cell death. The results demonstrated a different sensitivity to IR in cell lines. MDA-MB-436 cells were more sensitive to IR than MDA-MB-231 and MCF-7 cells (data not shown). Subsequently, we analyzed the effect of NAM combined with IR. A significant decrease in cell survival levels in MDA-MB-436 and MDA-MB-231 cells exposed to the combination compared with NAM only was observed (P<0.001,
The results of this study show that NAM inhibits the growth of breast cancer cell lines in a dose-dependent manner. In addition, depletion of PARP1 mRNA reduces cell viability and NAM increases the cytotoxic effects of cisplatin and induces radiosensitization to various degrees in MDA-MB-436, MDA-MB-231 and MCF-7 breast cancer cell lines.
A number of PARP inhibitors are being developed in the clinic as single agents and/or in combination with other drugs as potential enhancers of DNA-damaging cytotoxic agents, such as alkylating agents or radiation therapy. The chemistry of most of these agents is that of reversible NAD+ mimetics, although they have different bioavailability and molar equivalence for PARP enzyme inhibition (
Despite the long-established activity of NAM as a PARP1 inhibitor this drug has not been extensively evaluated as anticancer agent. However, it has largely been used in the clinic at pharmacological doses over many years with a low incidence of side effects and toxicity for diverse conditions including dermatological, metabolic and psychiatric disorders (
It has been reported that PARP inhibition may potentiate the effects of antineoplastic DNA-damaging agents such as temozolomide, cyclophosphamide and platinum in BRCA1-deficient cells (
Inhibition of PARP activity reduced the single-strand breaks (SSBs) repair range and increased sensitivity to ionizing radiation and antineoplastic agents. As such, PARP inhibition exerts radiosensitization by facilitating the conversion of an unrepaired SSB to double-strand breaks (DSBs) during the S phase of the cell cycle (
The results of the siRNA demonstrate that depletion of PARP1 has only a modest effect on reducing cell viability. However, it is known that NAM exerts a number of biological actions including inhibition of SIRT1 (silent mating-type information regulation 2, homolog 1), which is a NAD+-dependent deacetylase that regulates the processes of stress response and cell survival (
Our findings and those of other studies on the antitumor effects of NAM in a number of cancer models suggest that this drug that can be clinically tested as a repositioned cancer drug. A major drawback for its potential application is that the antitumor effects of NAM require drug concentrations in millimolar ranges (>10 mM) when used as single agent. However, when used for radiosensitization or in combination with cytotoxic drugs, the effects are seen at lower molar concentrations. Pharmacokinetic studies in cancer patients receiving oral high-dose NAM show that at doses of 6 g daily, peak plasma concentrations can be as high as >200 μg/ml, which corresponds to molar concentrations >2 mM. No clinical significant toxicity other than easily controlled nausea and vomiting were observed (
The authors would like to thank Dr Patricia García López for valuable comments concerning this study as well as M.C. Roberto Lazzarini for technical assistance. This study was supported by CONACYT Mexico grant (203457).
PARP inhibition by nicotinamide. (A) Nicotinamide inhibits
Endogenous PARP inhibition of ADP-ribosylation in cell nuclei. Nicotinamide inhibits the nuclear formation of polymers (ADP-ribosylation, pADP, green) in the presence of DNA damage induced by H2O2. Nuclei were counterstained with DAPI.
Growth inhibition by nicotinamide and cisplatin. MDA-MB-436, MDA-MB-231 and MCF-7 cell lines were treated with cisplatin (0–100 μM) and NAM (5, 10, 20, 40, 60, 80 and 100 mM) to determine their IC50 and IC20 values, respectively. (A–C) IC50 values for NAM were 30.09, 20.09 and 20.01 mM. (D–F) The corresponding IC20 for cisplatin was 0.5, 5 and 4 μM.
Chemosensitization by nicotinamide. Cells were treated with different doses of NAM for 72 h and cisplatin by 24 h. Average and SD from at least three experiments are shown. *Statistically significant with respect to NAM vs NAM+CIS (p<0.001). **Statistically significant with regard to CIS vs NAM (p<0.001).
Growth inhibition by knockdown of PARP1 by siRNA. MDA-MB-436 cells were subjected to PARP1 knockdown by siRNA for 24 h and then treated with cisplatin for 24 h. PARP1 protein expression and cell viability were assessed 24 h later. Scramble siRNA was used as a negative control. *Statistically significant differences in cell growth inhibition as compared to the control (p<0.01).
Radiosensitization by nicotinamide. (A) MDA-MB-436, (B) MDA-MB-231 and (C) MCF-7 cells were exposed to different concentrations of NAM for 72 h and IR. The IR dose varied depending on the cell line. Averages and SD from at least three experiments are shown. *Statistically significant as compared to NAM vs. NAM+IR (p<0.001); **Statistically significant as compared to IR vs NAM+IR (P<0.001).
Combination index of nicotinamide and cisplatin.
Cell line | Doses (mM) | ||||||
---|---|---|---|---|---|---|---|
Drugs in combination | Drugs alone | Control growth | Combination index (Cix) | Interaction | |||
Nicotinamide (D1) | Cisplatin (D2) | Nicotinamide (Dx1) | Cisplatin (Dx2) | (x%) | (ICx) | ||
MDA-MB-436 | 5 | 0.0005 | 11.1 | 0.0018 | 76 | 0.73 | Synergistic |
10 | 0.0005 | 18.65 | 0.0041 | 64 | 0.66 | Synergistic | |
20 | 0.0005 | 39.14 | 0.11 | 45 | 0.52 | Synergistic | |
30 | 0.0005 | 121 | 0.12 | 31 | 0.25 | Synergistic | |
40 | 0.0005 | 122 | 0.13 | 19 | 0.33 | Synergistic | |
MDA-MB-231 | 5 | 0.005 | 7.33 | 0.0082 | 82 | 1.29 | |
10 | 0.005 | 10.03 | 0.0133 | 75 | 1.37 | ||
20 | 0.005 | 20.27 | 0.0345 | 52 | 1.13 | ||
30 | 0.005 | 28.35 | 0.0516 | 39 | 1.16 | ||
40 | 0.005 | 35.9 | 30.03 | 30 | 1.28 | ||
MCF-7 | 5 | 0.004 | 14.34 | 0.0055 | 64 | 1.08 | |
10 | 0.004 | 19.73 | 0.0082 | 54 | 0.99 | ||
20 | 0.004 | 34.28 | 0.0166 | 34 | 0.82 | Synergistic | |
30 | 0.004 | 51 | 0.15 | 20 | 0.62 | Synergistic | |
40 | 0.004 | 66 | 0.2 | 12 | 0.63 | Synergistic |