Erica J. Peterson

DNA methyltransferase 1, cytosine methylation, and cytosine hydroxymethylation in mammalian mitochondria.

Mitochondrial DNA (mtDNA) has been reported to contain 5-methylcytosine (5mC) at CpG dinucleotides, as in the nuclear genome, but neither the mechanism generating mtDNA methylation nor its functional significance is known. We now report the presence of 5-hydroxymethylcytosine (5hmC) as well as 5mC in mammalian mtDNA, suggesting that previous studies underestimated the level of cytosine modification in this genome. DNA methyltransferase 1 (DNMT1) translocates to the mitochondria, driven by a mitochondrial targeting sequence located immediately upstream of the commonly accepted translational start site. This targeting sequence is conserved across mammals, and the encoded peptide directs a heterologous protein to the mitochondria. DNMT1 is the only member of the three known catalytically active DNA methyltransferases targeted to the mitochondrion. Mitochondrial DNMT1 (mtDNMT1) binds to mtDNA, proving the presence of mtDNMT1 in the mitochondrial matrix. mtDNMT1 expression is up-regulated by NRF1 and PGC1α, transcription factors that activate expression of nuclear-encoded mitochondrial genes in response to hypoxia, and by loss of p53, a tumor suppressor known to regulate mitochondrial metabolism. Altered mtDNMT1 expression asymmetrically affects expression of transcripts from the heavy and light strands of mtDNA. Hence, mtDNMT1 appears to be responsible for mtDNA cytosine methylation, from which 5hmC is presumed to be derived, and its expression is controlled by factors that regulate mitochondrial function.

Luminescent Ruthenium Complexes for Theranostic Applications

The water-soluble and visible luminescent complexes cis-[Ru(L-L)2(L)2](2+) where L-L = 2,2-bipyridine and 1,10-phenanthroline and L= imidazole, 1-methylimidazole, and histamine have been synthesized and characterized by spectroscopic techniques. Spectroscopic (circular dichroism, saturation transfer difference NMR, and diffusion ordered spectroscopy NMR) and isothermal titration calorimetry studies indicate binding of cis-[Ru(phen)2(ImH)2](2+) and human serum albumin occurs via noncovalent interactions with K(b) = 9.8 × 10(4) mol(-1) L, ΔH = -11.5 ± 0.1 kcal mol(-1), and TΔS = -4.46 ± 0.3 kcal mol(-1). High uptake of the complex into HCT116 cells was detected by luminescent confocal microscopy. Cytotoxicity of cis-[Ru(phen)2(ImH)2](2+) against proliferation of HCT116p53(+/+) and HCT116p53(-/-) shows IC50 values of 0.1 and 0.7 μmol L(-1). Flow cytometry and western blot indicate RuphenImH mediates cell cycle arrest in the G1 phase in both cells and is more prominent in p53(+/+). The complex activates proapoptotic PARP in p53(-/-), but not in p53(+/+). A cytostatic mechanism based on quantification of the number of cells during the time period of incubation is suggested.

The Bioinorganic Chemistry of Apoptosis: Potential Inhibitory Zinc Binding Sites in Caspase-3†

Zn(2+) inhibits the action of several of the caspases and thus may act as a regulator of apoptosis. Reversal of this inhibition is one possible approach for the development of apoptosis-based therapies. Few studies describe the molecular details of the Zn(2+)-caspase interaction, the understanding of which is essential for the success of any therapeutic strategies. Enzyme kinetics and biophysical studies have shown that the inhibition is of mixed type with prominent (ca. 60 % of inhibition) uncompetitive characteristics and an IC50 of 0.8 μM under the conditions used. Fluorescence-based techniques confirmed that, during inhibition in the sub-micromolar range, substrate binding remains unaffected. A new zinc binding site composed of the catalytic histidine and a nearby methionine residue, rather than the catalytic histidine and cysteine dyad, is proposed based on the experimental observations. DFT models were used to demonstrate that the proposed site could be the preferred inhibitory zinc binding site.

Heparan Sulfate Proteoglycan-Mediated Entry Pathway for Charged Tri-Platinum Compounds: Differential Cellular Accumulation Mechanisms for Platinum

We examined the mechanism of accumulation of charged polynuclear platinum complexes (PPCs) based on analogy of polyarginine interactions with the cell surface heparan sulfate proteoglycan (HSPG) family of protein-linked glycosoaminoglycan polysaccharides (GAGs). GAGS such as heparan sulfate (HS) and chondroitin sulfate (CS) mediate the cellular entry of many charged molecules. Fluorescence microscopy and flow cytometry showed that PPCs, but not the neutral cisplatin or oxaliplatin, blocked the cellular entry of TAMRA-R(9) (a nonarginine peptide, R(9)) coupled to the TAMRA fluorescent label 5-(and 6-)carboxytetramethylrhodamine) in Chinese hamster ovary (CHO), human colon carcinoma (HCT116), and osteosarcoma (SAOS-2) cells. Furthermore, detection of platinum accumulation in wt CHO, mutant CHO-pgsD-677 (lacking HS), and CHO-pgsA (lacking HS/CS) cells confirms that HSPG-mediated interactions are an important mechanism for PPC internalization but not so for uncharged cisplatin and oxaliplatin. Endocytosis inhibitor studies show that macropinocytosis, a mechanism of cell entry for heparan sulfate GAGs and arginine-rich peptides, is important in the cellular accumulation of noncovalent TriplatinNC and, to a lesser degree, the covalently binding BBR3464. Clathrin-mediated endocytosis, however, was not involved in either case. Overall, the results suggest a new proteoglycan-mediated mechanism for cellular accumulation of PPCs not shared by cisplatin or oxaliplatin. The results have significant implications for the rational design of platinum antitumor drugs with distinct biological profiles in comparison to those of the clinically used agents as well as expanding the chemotypes for HS proteoglycan-dependent receptors.

Nucleolar targeting by platinum: p53-independent apoptosis follows rRNA inhibition, cell-cycle arrest, and DNA compaction.

TriplatinNC is a highly positively charged, substitution-inert derivative of the phase II clinical anticancer drug, BBR3464. Such substitution-inert complexes form a distinct subset of polynuclear platinum complexes (PPCs) interacting with DNA and other biomolecules through noncovalent interactions. Rapid cellular entry is facilitated via interaction with cell surface glycosoaminoglycans and is a mechanism unique to PPCs. Nanoscale secondary ion mass spectrometry (nanoSIMS) showed rapid distribution within cytoplasmic and nucleolar compartments, but not the nucleus. In this article, the downstream effects of nucleolar localization are described. In human colon carcinoma cells, HCT116, the production rate of 47S rRNA precursor transcripts was dramatically reduced as an early event after drug treatment. Transcriptional inhibition of rRNA was followed by a robust G1 arrest, and activation of apoptotic proteins caspase-8, -9, and -3 and PARP-1 in a p53-independent manner. Using cell synchronization and flow cytometry, it was determined that cells treated while in G1 arrest immediately, but cells treated in S or G2 successfully complete mitosis. Twenty-four hours after treatment, the majority of cells finally arrest in G1, but nearly one-third contained highly compacted DNA; a distinct biological feature that cannot be associated with mitosis, senescence, or apoptosis. This unique effect mirrored the efficient condensation of tRNA and DNA in cell-free systems. The combination of DNA compaction and apoptosis by TriplatinNC treatment conferred striking activity in platinum-resistant and/or p53 mutant or null cell lines. Taken together, our results support that the biological activity of TriplatinNC reflects reduced metabolic deactivation (substitution-inert compound not reactive to sulfur nucleophiles), high cellular accumulation, and novel consequences of high-affinity noncovalent DNA binding, producing a new profile and a further shift in the structure–activity paradigms for antitumor complexes.

Ligand modulation of a dinuclear platinum compound leads to mechanistic differences in cell cycle progression and arrest

Despite similar structures and DNA binding profiles, two recently synthesized dinuclear platinum compounds are shown to elicit highly divergent effects on cell cycle progression. In colorectal HCT116 cells, BBR3610 shows a classical G2/M arrest with initial accumulation in S phase, but the derivative compound BBR3610-DACH, formed by introduction of the 1,2-diaminocyclohexane (DACH) as carrier ligand, results in severe G1/S as well as G2/M phase arrest, with nearly complete S phase depletion. The origin of this unique effect was studied. Cellular interstrand crosslinking as assayed by comet analysis was similar for both compounds, confirming previous in vitro results obtained on plasmid DNA. Immunoblotting revealed a stabilization of p53 and concomitant transient increases in p21 and p27 proteins after treatment with BBR3610-DACH. Cell viability assays and cytometric analysis of p53 and p21 null cells indicated that BBR3610-DACH-induced cell cycle arrest was p21-dependent and partially p53-dependent. However, an increase in the levels of cyclin E was observed with steady state levels of CDK2 and Cdc25A, suggesting that the G1 block occurs downstream of CDK/cyclin complex formation. The G2/M block was corroborated with decreased levels of cyclin A and cyclin B1. Surprisingly, BBR3610-DACH-induced G1 block was independent of ATM and ATR. Finally, both compounds induced apoptosis, with BBR3610-DACH showing a robust PARP-1 cleavage that was not associated with caspase-3/7 cleavage. In summary, BBR3610-DACH is a DNA binding platinum agent with unique inhibitory effects on cell cycle progression that could be further developed as a chemotherapeutic agent complementary to cisplatin and oxaliplatin.

A new approach to glycan targeting: enzyme inhibition by oligosaccharide metalloshielding

Metalloglycomics - the effects of defined coordination compounds on oligosaccharides and their structure and function - opens new areas for bioinorganic chemistry and expands its systematic study to the third major class of biomolecules after DNA/RNA and proteins.

Comparison of Metal–Ammine Compounds Binding to DNA and Heparin. Glycans as Ligands in Bioinorganic Chemistry

We present spectroscopic and biophysical approaches to examine the affinity of metal-ammine coordination complexes for heparin as a model for heparan sulfate (HS). Similar to nucleic acids, the highly anionic nature of heparin means it is associated in vivo with physiologically relevant cations, and this work extends their bioinorganic chemistry to substitution-inert metal-ammine compounds (M). Both indirect and direct assays were developed. M compounds are competitive inhibitors of methylene blue (MB)-heparin binding, and the change in the absorbance of the dye in the presence or absence of heparin can be used as an indirect reporter of M-heparin affinity. A second indirect assay uses the change in fluorescence of TAMRA-R9, a nonaarginine linked to a fluorescent TAMRA moiety, as a reporter for M-heparin binding. Direct assays are surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). The Kd values for TriplatinNC-heparin varied to some extent depending on the technique from 33.1 ± 2 nM (ITC) to 66.4 ± 1.3 nM (MB absorbance assay) and 340 ± 30 nM (SPR). The differences are explained by the nature of the technique and the use of heparin of differing molecular weight. Indirect probes using the displacement of ethidium bromide from DNA or, separately, fluorescently labeled oligonucleotide (DNA-Fl) can measure the relative affinities of heparin and DNA for M compounds. These assays showed essentially equivalent affinity of TriplatinNC for heparin and DNA. The generality of these methods was confirmed with a series of mononuclear cobalt, ruthenium, and platinum compounds with significantly lower affinity because of their smaller overall positive charge but in the order [Co(NH3)6]3+ > [Ru(NH3)6]3+ > [Pt(NH3)4]2+. The results on heparin can be extrapolated to glycosoaminoglycans such as HS, emphasizing the relevance of glycan interactions in understanding the biological properties of coordination compounds and the utility of the metalloglycomics concept for extending bioinorganic chemistry to this class of important biomolecules.

Abstract 5117: Triplatin preferably suppress lung metastasis of breast cancer, and peritoneal carcinomatosis of colon and pancreatic cancer

Background: Since metastatic spread is often directly associated with the poor outcome, an effective treatment for metastatic lesion is expected to improve overall survival. This is particularly the case in peritoneal carcinomatosis of pancreatic cancer where there is no effective treatment. Recent clinical trials demonstrated that Cisplatin is effective in certain metastatic breast cancer, however with severe side effects. Polynuclear platinum analog, Triplatin, was developed to overcome the severe toxicity. Methods: Murine cancer cell lines, 4T1-luc2 and E0771 (breast), Panc02-luc (pancreas) and CT26-luc (colon) were used. Cell proliferation was quantified by CCK8 assay. Syngeneic orthotopic implantation of 4T1-luc2 cells was used for metastatic breast cancer model. For mastectomy model, primary tumors were surgically removed 8 days after inoculation. Syngeneic intraperitoneal injection of CT26-luc cells and Panc02-luc cells were used for peritoneal carcinomatosis models of colon and pancreatic cancer respectively. Triplatin (0.3 mg/kg) or vehicle was administered intraperitoneally every four days three times and tumor burden was quantified by bioluminescence imaging. Results: Triplatin suppressed cell growth of both breast cancer and pancreatic cancer in a dose dependent manner in vitro (IC50 of 4T1-luc2, E0771 and Panc02-luc cells were 0.08, 0.51 and 0.07 μM, respectively). Triplatin did not suppress the growth of 4T1-luc2 primary breast tumor, however, ex vivo results showed that lung metastases were significantly reduced to 14% of the control (p = 0.034) in orthotopic model. This result was reproduced in post-mastectomy “adjuvant therapy” model where Triplatin treatment was began after primary implanted tumor was removed, which suppressed lung metastases down to 1.9% of control by Day21 (p = 0.038), and significantly prolonged survival (p = 0.007). This result led us to use Triplatin in CT26-luc colon cancer carcinomatosis model, where Triplatin suppressed total tumor burden to 1.9% of control by Day13 (p = 0.029) and significantly prolonged survival (p = 0.001). Finally, Triplatin reduced total tumor burden of Panc02-luc pancreatic cancer peritoneal carcinomatosis model to 35% of the control by Day 9 (p = 0.337) and the survival was significantly prolonged (p = 0.025). Conclusion: Triplatin demonstrated significant suppression lung metastatic tumor of 4T1-luc2 breast cancer, and peritoneal carcinomatosis of CT26-luc colon cancer and Panc02-luc pancreatic cancer. Newer platinum compounds with less toxicity and favorable pharmacokinetics warrant further investigation for advanced metastatic cancer. Citation Format: Eriko Katsuta, Erica J. Peterson, Samantha J. Katner, Nicholas P. Farrell, Kazuaki Takabe. Triplatin preferably suppress lung metastasis of breast cancer, and peritoneal carcinomatosis of colon and pancreatic cancer [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 5117. doi:10.1158/1538-7445.AM2017-5117

Abstract 3064: Newer platinum agent, Triplatin NC, is as effective as Triplatin for metastatic breast cancer and pancreatic cancer carcinomatosis

Introduction: Since metastatic cancer is the most advanced disease and is associated with the worst outcome, an effective treatment for this Stage is expected to directly improve overall survival. Recent clinical trials demonstrated that Cisplatin is effective in certain metastatic breast cancer, however with severe side effects. Polynuclear platinum analog, Triplatin, was developed to overcome the severe toxicity, however, pharmacokinetics limited its clinical efficacy in Phase II clinical trials. Recent development of Triplatin NC resolved the PK issue, and it is expected to be more clinically useful if it is as effective as Triplatin with less cytotoxic properties. Methods: HUVEC cells were used for tube formation assays. After orthotopic implantation of Murine breast cancer 4T1-luc2 cells, female Balb/C mice were randomized on Day 1 to Day 1, 5, 9 i.p. administration of Triplatin (0.3mg/kg), Triplatin NC (25mg/kg) or Saline and mice were sacrificed on Day 20 when lung metastasis was evaluated by ex vivo imaging. Pancreatic cancer carcinomatosis, generated by ip injection of 1 million Panc02-luc cells into C57/Blk6 mice. Animals were treated every 4 days beginning on day 1 by i.p. injection with either Triplatin (0.3mg/kg), Triplatin NC (25mg/kg) or Vehicle. Results: IC50 of 4T1-luc cells at 72h were 9μM and 8μM for Triplatin and Triplatin NC, respectively. 10μM of Triplatin or Triplatin NC demonstrated significant suppression of tube formation compared from no treatment (p = 0.005 and p = 0.002, respectively). Additionally, 0.1μM of Triplatin NC demonstrated significant suppression of tube formation as well (p = 0.003). In 4T1-luc2 implanted model, bioluminescent imaging on Day 7 demonstrated reduced tumor growth in Triplatin and Triplatin NC to 54% and 57% of the control, respectively. By Day 20, Triplatin and Triplatin NC suppressed the tumor size to 20% and 43% of the control, respectively. Triplatin and Triplatin NC significantly reduced the amount of lung metastasis to 14% and 21% of the control, respectively. In pancreatic cancer peritoneal carcinomatosis model, Triplatin and Triplatin NC reduced total tumor burden to 35% and 39% of the control on Day 9, respectively. Mouse survival was significantly enhanced by Triplatin treatment group in comparison with the control (p Conclusion: Both Triplatin and Triplatin NC effectively suppressed 4T1-luc2 breast cancer cell growth, and angiogenesis in vitro. Both agents demonstrated similar growth suppression not only of the primary tumor, but also in lung metastasis and panc02-luc carcinomatosis. New platinum compounds with less toxicity and favorable pharmacokinetics warrant further investigation for advanced metastatic cancer. Citation Format: Eriko Katsuta, Stephanie C. DeMasi, Samantha J. Katner, Hiroaki Aoki, Erica J. Peterson, Nicholas P. Farrell, Kazuaki Takabe. Newer platinum agent, Triplatin NC, is as effective as Triplatin for metastatic breast cancer and pancreatic cancer carcinomatosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3064.