Pt-DNA Binding Research Articles

Highly photoactive Ir(III)-Pt(IV) heterometallic conjugates for anticancer therapy.

For the first time, this study reported the photoactivatable activity of Ir(III)-Pt(IV) heterometallic conjugates, which were stable in the dark and activated to release oxaliplatin and Ir within 3 min of irradiation. The conjugates induced apoptosis and immunologic cell death through Pt-DNA binding and reactive oxygen species generation upon irradiation. This work developed photoactivatable heterometallic agents for anticancer therapy.

Abstract 4212: Natural compounds alone and in combination with platinum drugs found to show significant anti-tumour activity against ovarian cancer cell lines

Abstract Acquired drug resistance remains major problem in platinum based chemotherapy against ovarian cancer. It is believed that elevated expression of NF-kB and AKT/PKB is one of the prime reasons for acquired drug resistance. Since tumour active natural compounds can dampen NF-kB and AKT/PKB expression, it is thought that platinum drugs in combination with the natural compounds may produce synergism in killing tumor cells. The present study aimed to apply combinations of platinum drugs cisplatin, oxaliplatin, and carboplatin with natural compounds mycophenolic acid, honokiol, cholecalciferol and 6-shogaol to four ovarian cancer cell lines: A2780, A2780CisR, A2780ZD0473R and SKOV-3 towards overcoming drug resistance. IC50 values of single compounds were determined followed by administration of binary combinations. Studies on DNA damage, cellular platinum accumulation, and platinum-DNA binding levels were carried out. Proteomic studies will be done to explore the key proteins involve with drug resistance. Mycophenolic acid (IC50: 1.34-7.83 μM) from Penicillium brevicompactum and 6-shogaol (IC50: 6.79-10.20 μM) from Zingiber officinale showed significant activity against ovarian cancer cell lines. Ginger derived pure compound 6-shogaol showed prominent synergism in combination with platinums when administered as a bolus (0/0h) and with 0/4 h sequence. Honokiol from Magnolia virginia alone and in combination with cisplatin showed significant DNA damage in A2780 cell line whereas cholecalciferol did the same to A2780cisR cell line. Bolus combination of cisplatin and 6-shogoal produced increased cellular platinum accumulation and 3 times more Pt-DNA binding than cisplatin alone treated A2780 cells. Though honokiol and cisplatin (0/0 h) combination have half cellular platinum accumulation in compare to cisplatin alone treated A2780 cells but showed 9 times higher Pt-DNA binding. Cholecalciferol and 6-shogaol in combination with cisplatin also showed promising Pt-DNA in A2780CisR cell line. In conclusion, 6-shogaol, cholecalciferol and honokiol showed significant activity alone and in combination with cisplatin and oxaliplatin against ovarian cancer tumour models. Note: This abstract was not presented at the meeting. Citation Format: Md Sheikh Anwar, Jun Qing Yu, Philip Beale, Fazlul Huq. Natural compounds alone and in combination with platinum drugs found to show significant anti-tumour activity against ovarian cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4212. doi:10.1158/1538-7445.AM2017-4212

Abstract 3504: Synergism from combinations of monofunctional platinums with phytochemicals in human ovarian cancer cell lines

Abstract Aims To apply combinations of monofunctional platinums with selected phytochemicals in human ovarian tumor models towards overcoming platinum resistance. Recently research has been directed at combinations of platinum drugs with tumor active phytochemicals towards a means of overcoming platinum resistance in ovarian cancer. In this study, two designed monofunctional planaramineplatinum(II) complexes, tris(8-hydroxyquinoline)monochloroplatinum(II) chloride (LH3) and tris(benzimidazole)chloroplatinum(II) chloride (LH4), were investigated for activity against human ovarian A2780, cisplatin-resistant A2780 (A2780cisR) and ZD0473-resistant A2780 (A2780ZD0473R) cancer cell lines, alone and in combination with curcumin, genistein, querceitin, capsaicin and resveratrol. Methods LH3 and LH4 were synthesized starting with potassium tetracloroplatinate and phytochemicals were obtained from commercial sources. Cytotoxicity was determined using MTT reduction assay. Cellular levels of glutathione before and after treatment were also determined. Interaction of the designed compounds with pBR322 plasmid DNA and damage to DNA in A2780 and A2780cisR cell lines due to interaction with designed compounds alone and in combination with phytochemicals were also investigated. Proteomic studies involving 2D-gel electrophoresis and mass spectrometry were carried out to identify key proteins associated with drug resistance in A2780 and A2780cisR cell lines. Results and Discussion LH3 and LH4 were found to be much more active than cisplatin against the resistant tumour models and greatest synergism in activity was observed when combinations of LH3 and LH4 with the phytochemicals were administered as a bolus. For combinations of LH3 and LH4 with the phytochemicals, platinum accumulation and the level of Pt-DNA binding were greater in the resistant A2780cisR cell line than in the parental A2780 cell line. Proteomic studies confirmed significant changes in expression of a number of proteins in A2780cisR cell line compared to the A2780 parent cell line. Greater activity of LH3 and LH4 than cisplatin against the resistant ovarian cell lines indicates that monofunctional platinums can be novel drug candidates with different cytotoxicity profiles from those of Pt drugs currently in use. The steric properties conveyed by the heterocylic ligands play a significant role in modulating their cytotoxicities and binary combination with tumour active phytochemicals can serve to further enhance drug efficacy. Conclusion Monofunctional platinums containing bulky planaramine ligands in combination with tumor active phytochemicals have the potential of overcoming platinum resistance in ovarian cancer cell lines. Acknowledgment This research was partly supported by Biomedical Science Research Initiative Grant and Biomedical Science Cancer Research Donation Fund. Citation Format: Laila Arzuman, Fazlul Huq, Jun Qing Yu, Philip Beale. Synergism from combinations of monofunctional platinums with phytochemicals in human ovarian cancer cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3504. doi:10.1158/1538-7445.AM2015-3504

Impact of Individual PCNA-DNA Contacts on Clamp Loading and Function on DNA

Ring-shaped clamp proteins encircle DNA and affect the work of many proteins, notably processive replication by DNA polymerases. Crystal structures of clamps show several cationic residues inside the ring, and in a co-crystal of E. coli β clamp-DNA they directly contact the tilted duplex passing through. To investigate the role of these contacts in reactions involving circular clamps, we examined single arginine/lysine mutants of S. cerevisiae PCNA in RFC-catalyzed loading of the clamp onto primer-template DNA (ptDNA). Previous kinetic analysis has shown that ptDNA entry inside an ATP-activated RFC-PCNA complex accelerates clamp opening and ATP hydrolysis, which is followed by slow PCNA closure around DNA and product dissociation. Here we directly measured multiple steps in the reaction--PCNA opening, ptDNA binding, PCNA closure, phosphate release and complex dissociation to determine if mutation of PCNA residues R14, K20, R80, K146, R149 or K217 to alanine affects the reaction mechanism. Contrary to earlier steady state analysis of these mutants, pre-steady state data show that loss of single cationic residues can alter the rates of all DNA-linked steps in the reaction as well as movement of PCNA on DNA. These results explain an earlier finding that individual arginines and lysines inside human PCNA are essential for pol δ processivity. Mutations in the N-terminal domain have greater impact than in the C-terminal domain, indicating a positional bias in PCNA-DNA contacts that can influence its functions on DNA.

Impact of Individual Proliferating Cell Nuclear Antigen-DNA Contacts on Clamp Loading and Function on DNA

Ring-shaped clamp proteins encircle DNA and affect the work of many proteins, notably processive replication by DNA polymerases. Crystal structures of clamps show several cationic residues inside the ring, and in a co-crystal of Escherichia coli β clamp-DNA, they directly contact the tilted duplex passing through (Georgescu, R. E., Kim, S. S., Yurieva, O., Kuriyan, J., Kong, X. P., and O'Donnell, M. (2008) Structure of a sliding clamp on DNA. Cell 132, 43-54). To investigate the role of these contacts in reactions involving circular clamps, we examined single arginine/lysine mutants of Saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA) in replication factor C (RFC)-catalyzed loading of the clamp onto primer template DNA (ptDNA). Previous kinetic analysis has shown that ptDNA entry inside an ATP-activated RFC-PCNA complex accelerates clamp opening and ATP hydrolysis, which is followed by slow PCNA closure around DNA and product dissociation. Here we directly measured multiple steps in the reaction (PCNA opening, ptDNA binding, PCNA closure, phosphate release, and complex dissociation) to determine whether mutation of PCNA residues Arg-14, Lys-20, Arg-80, Lys-146, Arg-149, or Lys-217 to alanine affects the reaction mechanism. Contrary to earlier steady state analysis of these mutants (McNally, R., Bowman, G. D., Goedken, E. R., O'Donnell, M., and Kuriyan, J. (2010) Analysis of the role of PCNA-DNA contacts during clamp loading. BMC Struct. Biol. 10, 3), our pre-steady state data show that loss of single cationic residues can alter the rates of all DNA-linked steps in the reaction, as well as movement of PCNA on DNA. These results explain an earlier finding that individual arginines and lysines inside human PCNA are essential for polymerase δ processivity (Fukuda, K., Morioka, H., Imajou, S., Ikeda, S., Ohtsuka, E., and Tsurimoto, T. (1995) Structure-function relationship of the eukaryotic DNA replication factor, proliferating cell nuclear antigen. J. Biol. Chem. 270, 22527-22534). Mutations in the N-terminal domain have greater impact than in the C-terminal domain, indicating a positional bias in PCNA-DNA contacts that can influence its functions on DNA.

An Enthalpic “On/Off Switch” Linking DNA Binding and Nucleotide Incorporation Activity in Pol I DNA Polymerases

Primer-template DNA (pt-DNA) binding by DNA polymerases is the first step of the polymerization cycle. We have previously characterized the thermodynamics of pt-DNA binding with respect to temperature for the Klenow and Klentaq “large fragment domains” of DNA polymerase I, from Thermus aquaticus and Escherichia coli, respectively. DNA binding affinities for both polymerases are quite tight across wide temperature ranges, 5-70 °C for Klentaq and 5-37 °C for Klenow. Both polymerases show significant heat capacity changes upon binding, yielding curved ΔG versus temperature dependences. This results in large changes in ΔH and TΔS with temperature, including a sign change from positive to negative for both enthalpy and entropy as temperature increases. For both polymerases, DNA binding is enthalpy-driven near their respective physiological temperatures.Herein, nucleotide incorporation activity was measured with respect to temperature to examine how the thermodynamics of initial pt-DNA binding relates to the enzymatic activities of Klentaq and Klenow. It is found that both polymerases are enzymatically inactive until the temperature reaches the point where the enthalpy of binding becomes negative (favorable), despite the fact that they both bind DNA quite well at lower temperatures. The data suggest that, for both polymerases, a negative free energy of binding alone is insufficient to drive catalysis, and that a negative enthalpy of initial binding (ΔH) is required for nucleotide incorporation activity. This work is supported by the National Science Foundation.

A Central Swivel Point in the RFC Clamp Loader Controls PCNA Opening and Loading on DNA

Replication factor C (RFC) is a five-subunit complex that loads proliferating cell nuclear antigen (PCNA) clamps onto primer–template DNA (ptDNA) during replication. RFC subunits belong to the AAA+ superfamily, and their ATPase activity drives interactions between the clamp loader, the clamp, and the ptDNA, leading to topologically linked PCNA·ptDNA. We report the kinetics of transient events in Saccharomyces cerevisiae RFC-catalyzed PCNA loading, including ATP-induced RFC activation, PCNA opening, ptDNA binding, ATP hydrolysis, PCNA closing, and PCNA·ptDNA release. This detailed perspective enables assessment of individual RFC-A, RFC-B, RFC-C, RFC-D, and RFC-E subunit functions in the reaction mechanism. Functions have been ascribed to RFC subunits previously based on a steady-state analysis of ‘arginine-finger’ ATPase mutants; however, pre-steady-state analysis provides a different view. The central subunit RFC-C serves as a critical swivel point in the clamp loader. ATP binding to this subunit initiates RFC activation, and the clamp loader adopts a spiral conformation that stabilizes PCNA in a corresponding open spiral. The importance of RFC subunit response to ATP binding decreases as RFC-C>RFC-D>RFC-B, with RFC-A being unnecessary. RFC-C-dependent activation of RFC also enables ptDNA binding, leading to the formation of the RFC·ATP·PCNAopen·ptDNA complex. Subsequent ATP hydrolysis leads to complex dissociation, with RFC-D activity contributing the most to rapid ptDNA release. The pivotal role of the RFC-B/C/D subunit ATPase core in clamp loading is consistent with the similar central location of all three ATPase active subunits of the Escherichia coli clamp loader.

Increased understanding of platinum anticancer chemistry

The development of a few worldwide routinely used Pt(II) coordination compounds is described from a mechanistic point of view and related to the molecular aspects of Pt-DNA binding. Mechanistic knowledge developed from these studies is applied nowadays for the design and synthesis of new bifunctional and trifunctional compounds, aimed for use as improved anticancer drugs.

Studies on Two New Mixed Ligand Platinum Compounds with a Trans- Geometry

In this paper we report the synthesis and in vitro activity of two new mixed ligand platinum complexes: trans-[PtCl₂(thiazole)(imidazole) [JH3] and trans-[PtCl₂(thiazole)(3-hydroxypyridine) [JH4]. Although the compounds are less active than cisplatin against the parent ovarian cancer cell line A2780, they are more active than cisplatin against the resistant cell lines A2780(cisR) and A2780(ZD0473R), thus indicating that JH3 and JH4 have been better able to overcome mechanisms of resistance operating in A2780(cisR) and A2780(ZD0473R). When Pt-DNA binding levels at 24 h in A2780, A2780(cisR) and A2780(ZD0473R) cell lines are compared it is found that whereas for cisplatin the values in resistant cell lines are significantly lower than that in the parent cell line, for JH3 and JH4 Pt-DNA binding levels in the parent and resistant cell lines are comparable, thus providing an explanation for variations in activity of the compounds in the three cell lines.

Correlation Between DNA Binding Thermodynamics and Functional Behavior of Pol I DNA Polymerases

Primer-template DNA (pt-DNA) binding by DNA polymerases is the first step of the polymerization cycle. We have previously characterized the thermodynamics of pt-DNA binding with respect to temperature for the “large fragments” of DNA polymerase I, Klentaq and Klenow, from Thermus aquaticus and Escherichia coli, respectively. Results with both polymerases showed that DNA binding is enthalpy-driven near their respective physiological temperatures. Here, nucleotide incorporation activity was measured with respect to temperature to examine how the thermodynamics of initial pt-DNA binding relates to the enzymatic activities of Klentaq and Klenow. For both polymerases it is observed that a negative enthalpy of initial binding (ΔH) is required for nucleotide incorporation activity, and that a negative entropy of binding (TΔS) inhibits the catalytic activity. Nucleotide incorporation activity was also examined with respect to KCl concentration. As reported previously, thermodynamic linkage plots for pt-DNA binding with respect to KCl concentration (∂ln1/Kd versus∂ln[KCl]) exhibit negative slopes for both polymerases and indicate net ion releases of ∼3 and ∼5 ions upon pt-DNA binding by Klentaq and Klenow, respectively. Interestingly, linkage plots for the steady-state rate of incorporation activity with respect to KCl concentration (∂ln1/ssRateversus∂ln[KCl]) exhibit the same slopes as the linkage plots of pt-DNA binding. This result suggests that salt dependence of initial pt-DNA binding dictates the salt dependence of the overall incorporation activity. It is striking that for both salt and temperature dependences, the detailed thermodynamics of DNA binding so directly correlate with overall functional behavior.This work is supported by National Science Foundation.

Transfer RNA Bindings to Antitumor Estradiol-Platinum(II) Hybrid and Cisplatin

The anticancer platinum (Pt) drugs exert their antitumor activity by direct or indirect Pt-DNA binding. It has been shown that Pt drugs can induce major DNA damage and minor RNA damage during cancer treatment. A recent report showed that a new anticancer estradiol-Pt(II) hybrid molecule (CD-37) binds DNA bases indirectly, while being more effective than cis-diaminedichloroplatinum(II) (cisplatin) against several types of cancer. In this report, we examine the bindings of CD-37 and cisplatin drugs with transfer RNA (tRNA) in vitro and compare the results to those of the corresponding Pt-DNA complexes. Solutions containing various CD-37 or cisplatin concentrations were reacted with tRNA at physiological pH. Using Fourier transform infrared (FTIR), UV-visible, and circular dichroism spectroscopic methods, the drug binding mode, the binding constant, and RNA structural variations are determined for Pt-tRNA complexes in aqueous solution. Structural analysis showed direct binding of cisplatin drug to guanine and adenine N7 sites, while both direct and indirect interactions of CD-37 with tRNA bases and the backbone phosphate group were observed. The overall binding constants estimated were K(CD-37) = 2.77 (+/-0.90) x 10(4) M(1) and K(cisplatin) = 1.72 (+/-0.50) x 10(4) M(1). Major aggregation of tRNA occurs at high CD-37 concentrations, while RNA remains in the A-family structure.

Studies on activities, cell uptake and DNA binding of four trans-planaramineplatinum(II) complexes of the form: trans-PtL(NH 3)Cl 2, where L=2-hydroxypyridine, imidazole, 3-hydroxypyridine and imidazo(1,2-α)pyridine

Four trans-planaramineplatinum(II) complexes code named YH9, YH10, YH11 and YH12 each of the form trans-PtL(NH 3)Cl 2, where L=2-hydroxypyridine and 3-hydroxypyridine, imidazole, and imidazo(1,2-α)pyridine for YH9, YH10, YH11 and YH12, respectively, have been synthesized and the activity of the compounds against human cancer cell lines, cell uptake, DNA-binding and nature of interaction with pBR322 plasmid DNA have been studied. The compound having imidazo(1,2-α)pyridine ligand as one the carrier ligands in the trans-configuration is found to be significantly more active than cis-platin against ovarian A2780 cisR cancer cell line corresponding with higher Pt-DNA binding. All other compounds have resistance factors less than that for cis-platin in the A2780 and A2780 cisR cell lines. A greater prevention of BamH1 digestion with increasing concentration of the compounds indicates that as the compounds bind with nucleobases in DNA, the DNA conformation is changed sufficiently so as to prevent BamH1 digestion at the specific GG site. Gel electrophoresis results also indicate that as the compounds bind to DNA, unwinding of supercoiled form I DNA takes place to change it from the negatively supercoiled form I through relaxed circular form I to the positively supercoiled form I.

Enhancement of cisplatin and etoposide cytotoxicity after all-trans retinoic-acid-induced cellular differentiation of a murine embryonal carcinoma cell line.

The potential of a combination of differentiation induction and chemotherapy was analyzed. Treatment of the murine embryonal carcinoma (EC) cell line PCC4 in vitro with all-transretinoic acid (RA) was followed by exposure to cisplatin (CDDP) or etoposide (VP-16). The expression of EC-cell-specific markers decreased upon 96 hr exposure to 10(-9), 10(-8), 10(-7) and 10(-6) M RA, indicating a loss of embryonal phenotype of the cells, whereas expression of markers specific for cytokeratins and neurofilaments was increased after this treatment. These data suggest early somatic differentiation of PCC4 cells upon treatment with RA. Cellular growth rate of PCC4 cells was not affected by preincubation with RA at 10(-9) M for 96 hr, but was reduced at 10(-8) and 10(-7) M RA and inhibited at 10(-6) M RA. Culture of PCC4 cells in the presence of 10(-7) M RA for 96 hr led to accumulation in G1 of the cell cycle, whereas at 10(-8) M RA cell-cycle distribution was not affected. RA-treated and -untreated PCC4 cells were compared for CDDP and VP-16 sensitivity. Pre-treatment with 10(-9), 10(-8) and 10(-7) M RA increased CDDP sensitivity, resulting in a 1.9-, 2.7- and 2.6-fold decrease in the concentrations inhibiting survival by 50% (IC50s) respectively. Pre-treatment with 10(-8) and 10(-7) M RA increased VP-16 sensitivity 2.5- and 3.0-fold, respectively. Enhanced CDDP sensitivity at RA concentrations not affecting cell-cycle distribution was not attributable to changes in cellular platinum (Pt) accumulation, or to changes of Pt-DNA binding.(ABSTRACT TRUNCATED AT 250 WORDS)

A study of human small‐cell lung carcinoma (hsclc) cell lines with different sensitivities to detect relevant mechanisms of cisplatin (cddp) resistance

The cisplatin(CDDP)-resistant cell line GLC4-CDDP shows a variety of differences from the parent line GLC4. The aim of this study was to determine which of the observed changes correlated with the degree of resistance and was therefore relevant to the phenomenon of CDDP resistance. For these experiments we used cells of the sensitive hSCLC cell line GLC4 and the in vitro-acquired CDDP-resistant sublines GLC4-CDDP3 and GLC4-CDDP11, with a resistance factor (RF) of 3 and 11 respectively for CDDP and of 1.8 and 7.4 respectively for carboplatin. Carboplatin was used, in addition to CDDP in seeking relevant mechanisms. No consistency was found between the RF and the growth pattern or antigen expression, cellular volume, doubling time, cellular or nuclear platinum (Pt) content or the level of Pt-non-histone chromatin protein (NHCP) binding. A correlation was found between the RF and the level of glutathione (GSH), and a trend was found for the level of Pt-DNA binding, Pt-GG adduct content and the amount of interstrand cross-links (ISC). These changes might therefore be relevant for the development of resistance. These findings are compatible with a GSH-induced reduction of the amount of reactive Pt in the resistant cell, resulting in a lower net platination and toxic Pt-DNA adduct formation.

Characterization of a human small cell lung carcinoma cell line with acquired resistance to cis-diamminedichloroplatinum(II) in vitro : Hospers GAP, Mulder NH, De Jong B et al. Division of Medical Oncology Department of Internal Medicine, University Hospital, 9713 EZ Groningen. Cancer Res 1988;48:6803-7

A 6.4-fold cis-diamminedichloroplatinum(II) (CDDP) resistant human small cell lung carcinoma cell line (GLC4-CDDP) was developed to study acquired CDDP resistance in vitro. Compared to the sensitive cell line (GLC4), the GLC4-CDDP showed an increase in doubling time and a decrease in cloning efficiency, cellular size, double minutes per cell, cellular protein, and nuclear protein content. While a complete cross-resistance for tetraplatin and a partial cross-resistance for doxorubicin, melphalan, cadmium chloride, carboplatin, and cis-dichloro-trans-dihydroxo-cis-bis(isoprolylamine)platinum (IV) (resistance factor, respectively,4.0,5.8,2.1,1.5,2.9) was found, no cross-resistance for vincristine was found. In the GLC4-CDDP line in comparison to the GLC4 line, glutathione and total amount of sulfhydryl compounds was significantly increased, while glutathione S-transferase and glutathione reductase was the same. The platinum content in cells and nuclei was lower in the resistant line, but after correction for cellular protein or volume no difference was found. The amount of platinum bound to DNA was significantly lower in the GLC4-CDDP line. After a 1-h incubation with CDDP, the amount of Pt-GG adducts was the same and the amount of interstrand cross-links was reduced in the GLC4-CDDP line as compared to GLC4. In conclusion, in the GLC4-CDDP line the phenotype and genotype are changed and various mechanisms, such as decreased Pt-DNA binding, elevated glutathione, and reduced interstrand cross-links, play a role in the development of the CDDP resistance.