Two findings led to the use of PARP1 inhibitors as an adjuvant to cisplatin chemotherapy. First, it was found that cells with elevated activity of DNA repair enzymes are resistant to cisplatin while, cells with decreased DNA repair capacity, such as fibroblasts from patients with xeroderma pigmentosum are more sensitive to the drug, suggesting the importance of DNA repair inhibition . An early response of DNA repair signals involves activation of PARP1 on sensing DNA damage and binding to it via its DNA binding domain for initiating repair activities.
The need for high doses and long-term administration, limits the use of cisplatin in chemotherapy. Also, resistance to the drug due to change in cellular uptake, efflux of the drug, increased biotransformation, detoxification in liver is observed in many cases . PARP1 inhibitors have been effective as adjuvants in therapy of cancer but they can also be useful as preventive agents . They also operate in some inflammatory diseases that increase the chance of development of cancer . PARP1 activity is stimulated in cells by DNA breaks induced by cisplatin. PARP1 inhibitors like AG014699, Olaparib, Iniparib, Veliparib and MK4827 have been used in conjunction with cisplatin . ACPH, a potential anticancer agent with PARP1 inhibitory properties have been shown to potentiate the killing by cisplatin to make it effective at lower doses . We have seen earlier that 4NCO can bind to the NAD+ binding pocket of PARP1 enzyme to act as its inhibitor . 4NCO exhibited limited toxicity in normal cells, but was effective in inducing killing in cancer cell lines and was most found to be most effective in A375 melanoma cells as obtained from our earlier findings . Therefore, the effect of combined treatment of 4NCO with cisplatin was tested in A375 melanoma cells. The use of drug combinations to show that they are significantly better than single agents is of particular interest. When two or more drugs that have overtly similar effects or complement each other are given together, their effects are often greatly enhanced. Our earlier result showed that the IC50 value of cisplatin treatment was reduced by ~ 2 fold with ACPH in A375 cells . Olaparib, another PARP1 inhibitor decreased the IC50 value of cisplatin by ~ 5 fold in A375 cells . For combined treatment with cisplatin and 4NCO in A375 cells, the fold reduction IC50 was found to be ~ 12 fold at some concentrations chosen for the study, which indicated that 4NCO was much more productive in potentiating the sensitivity to cisplatin-induced killing. Melanoma cells with enhanced DNA repair activities often can grow in presence of cisplatin . They are found to exhibit unusual DNA repair activity; DNA polymerase zeta responsible for translesion synthesis is found to be enhanced in melanomas, which possibly accounts for the aggressiveness of such tumors . PARP1 inhibitors can be used effectively as adjuvants to DNA damaging anticancer drugs in tumors with altered DNA repair activities . Our finding showed 4NCO to be a very effective combination with cisplatin in A375 melanoma cell line.
When the combined effect of two agents is greater than that predicted by their individual potencies, the combination is said to be synergistic. A synergistic interaction allows the use of lower doses of the combination constituent, a situation that may reduce adverse reactions. Isobologram analysis was performed to assess whether the cytotoxic effect was synergistic in nature. This analysis provides a CI value, which is a quantitative measure of the degree of drug interaction between two or more agents. A CI < 1.0, = 1.0, and > 1.0 indicate synergism, additive effect and antagonism, respectively. From evaluation of the CI30, CI50 and CI70 values of the combination treatments with 4NCO and cisplatin revealed that in all cases the CI values were found to be < 1.0 (Table 2). This unveiled the efficaciousness of co-treatment of 4NCO with cisplatin, which acted in synergy.
Cisplatin is a platinum coordination compound that interacts with DNA to induce both intra- and inter-strand DNA crosslinks between purines to result in DNA damage. It has been used in the therapy of different types of cancer . Cisplatin is often used in the treatment of solid tumors . Cells at the core of solid tumors are often refractory to the therapeutic agents as they often contain hypoxic non-dividing cells. Such quiescent cells are proficient in DNA repair through potential lethal damage repair (PLDR). Density inhibited cells are arrested in G0/G1 phase . The DNA damaging action of cisplatin also occurs through metal induced free radicals that lead to cell killing through oxidative damage . In order to test for the efficacy of 4NCO in solid tumors, density inhibited plateau phase cells were used as model system. Due to the involvement of PARP1 in PLDR process, inhibitors of PARP1 are effective to potentiate killing in such cells. PARP1 inhibitors like 3AB, PD128763, NU1025, AG14361 have been shown to inhibit PLDR activity in Chinese hamster V79 and CHO cells . The acridine derivative ACPH which could act as a PARP1 inhibitor was also effective in sensitizing density inhibited cells to killing . Isobologram analysis of co-treatment of 4NCO and cisplatin in density inhibited A375 cells also revealed the synergistic action of the combination; the findings indicated its possible efficacy in treatment of solid tumors. Combination of cisplatin and 4NCO could reduce the IC50 value by ~ 5 fold in the density inhibited A375 cells.
Cisplatin induces apoptotic death in cells through mitochondria and TRAIL-mediated pathways . Suppression of apoptosis can lead to cisplatin resistance in cells . We found that there was a significant increase in apoptosis in 4NCO and cisplatin co-treated cells when non-toxic doses of the individual agents were used for the treatment (Fig. 3). PARP1 activation depletes the NAD+ content in cells as PARP1 utilizes NAD+ as its substrate for synthesis of PAR polymers. Cisplatin treatment resulted in a time-dependent depletion of intracellular NAD+ in cells that were completely inhibited when 4NCO was used for co-treatment with cisplatin. Increment of the level of PARP1 substrate - NAD+ with time indicated inhibition of PARP1 in A375 cells. This effect was much higher when compared to the established PARP1 inhibitor 3AB. 4NCO inhibited PARP1 at much lower doses than 3AB (Fig. 4). This conclusively demonstrated the PARP1 inhibitory action of 4NCO in the combined treatment.
In order to further confirm our findings we carried out molecular modeling studies with cisplatin-crosslinked DNA bound to PARP1 with 4NCO. The PDB structure of cisplatin-crosslinked DNA bound to PARP1 was unavailable, so we built a model for the same. It can be seen from the best modeled structure that PARP1 binds to the cisplatin adduct containing DNA through its DNA binding domain (spanning residues 1 to 353) as can be shown in Fig. 5(A). Figure 5 (B) depicts the changes induced in PARP1 on binding to the cisplatin-induced damaged DNA since different forms of DNA damage leads to different kinks and angular changes in the backbone of the DNA inducing conformational changes in a DNA binding protein to accommodate it accordingly. This structure was used as a receptor for blind docking of 4NCO. The best scored docked complex having the lowest binding free energy showed that 4NCO binds to the substrate binding site or the nicotinamide binding site, which are shown in Fig. 5(C) and (D). The binding free energy of the docked complex was found to be -31.29632 kcal/mol, thus, indicating this binding to be a thermodynamically favorable process. Our previous report showed that the binding free energy of the interaction between 4NCO and the free PARP1 protein was − 29.61673 kcal/mol . Therefore, the binding free energy values were comparable, which further confirmed the feasibility of the methods followed. Interactions with the amino acid residues His862, Gly863, Tyr889, Tyr896, Lys903, Ser904, Tyr907 and Glu988 are very important for binding of PARP1 to NAD+ . As shown earlier, 4NCO could interact with free PARP1 at all these residues and prevented the binding of NAD+ to PARP1 at the substrate binding site to act as its inhibitor . Our present findings show that the same residues are also present for the interaction of 4NCO with PARP1 even after the changes induced in its structure after binding cisplatin-crosslinked DNA. Therefore, we can conclude that 4NCO can also effectively inhibit PARP1 that has already bound to DNA with cisplatin-adduct. The binding of 4NCO at the NAD+ binding catalytic domain of PARP1 ensured that 4NCO could still inhibit repair at the cisplatin-adduct site even after repair activity was initiated. This highlighted the effectiveness of 4NCO and also corroborated the findings from our experimental observations.
Our current findings conclusively proved that 4NCO could inhibit PARP1 to act as an effective adjuvant with cisplatin in A375 melanoma cells. The combination therapy induced synergistic cytotoxicity in the melanoma cells at non-toxic doses of the individual agents, both in exponential growing and density inhibited state by inducing apoptotic death. The efficacy of 4NCO was also heightened as it can also inhibit PARP1 that has already bound to the damaged DNA. Our present findings could justify the effectiveness of 4NCO in A375 melanoma cells as a potential drug candidate acting as a chemotherapeutic adjuvant to cisplatin.