Formaldehyde-releasing Prodrugs Research Articles

An evaluation of the interaction of pixantrone with formaldehyde-releasing drugs in cancer cells.

Pixantrone is a synthetic aza-anthracenedione currently used in the treatment of non-Hodgkin's lymphoma. The drug is firmly established as a poison of the nuclear enzyme topoisomerase II, however, pixantrone can also generate covalent drug-DNA adducts following activation by formaldehyde. While pixantrone-DNA adducts form proficiently in vitro, little evidence is presently at hand to indicate their existence within cells. The molecular nature of these lesions within cancer cells exposed to pixantrone and formaldehyde-releasing prodrugs was characterized along with the cellular responses to their formation. In vitro crosslinking assays, [14C] scintillation counting analyses and alkaline comet assays were applied to characterize pixantrone-DNA adducts. Flow cytometry, cell growth inhibition and clonogenic assays were used to measure cancer cell kill and survival. Pixantrone-DNA adducts were not detectable in MCF-7 breast cancer cells exposed to [14C] pixantrone (10-40µM) alone, however the addition of the formaldehyde-releasing prodrug AN9 yielded readily measurable levels of the lesion at ~ 1 adduct per 10kb of genomic DNA. Co-administration with AN9 completely reversed topoisomerase II-associated DNA damage induction by pixantrone yet potentiated cell kill by the drug, suggesting that pixantrone-DNA adducts may promote a topoisomerase II-independent mechanism of cell death. Pixantrone-DNA adduct-forming treatments generally conferred mild synergism in multiple cell lines in various cell death and clonogenic assays, while pixantrone analogues either incapable or relatively defective in forming DNA adducts demonstrated antagonism when combined with AN9. The features unique to pixantrone-DNA adducts may be leveraged to enhance cancer cell kill and may be used to guide the design of pixantrone analogues that generate adducts with more favorable anticancer properties.

Abstract 992: A switch in mechanism of action attenuates doxorubicin-mediated cardiotoxicity

Abstract Chemotherapy remains as the mainstay systemic therapy for triple negative breast cancer patients since targeted therapies are not suitable for this breast cancer subtype. Among the chemotherapies for triple negative breast cancer, doxorubicin is known to be effective but its use is hampered by a risk of developing doxorubicin-mediated cardiomyopathy. Doxorubicin causes cardiotoxicity primarily through topoisomerase-IIβ poisoning, resulting in cardiomyocyte apoptosis. Notably, cardioprotection has been reported after combination treatment of doxorubicin with an acyloxyalkyl ester, AN-7 in an in vivo model. As an acyloxyalkyl ester, AN-7 also releases formaldehyde upon esterase hydrolysis. Therefore, the aim of this study was to elucidate the mechanism of cardioprotection conferred by the co-treatment of doxorubicin with formaldehyde-releasing prodrugs in BALB/c mice bearing mammary 4T1.2 tumours. Both experimental and clinically available formaldehyde-releasing prodrugs were investigated in this triple negative breast cancer model. Cardiac damage was induced with a single dose of doxorubicin at 16 mg/kg in mammary tumour-bearing mice. Combined treatment of doxorubicin and formaldehyde-releasing prodrugs significantly reduced γH2AX foci (a marker for topoisomerase-IIβ poisoning) and protected the heart from doxorubicin-induced myocardial apoptosis. To investigate if the combination treatments were cardioprotective due to alteration in doxorubicin biodistribution, the initial response to the first dose of doxorubicin was investigated by treating tumour-bearing mice with 4 mg/kg 14C-doxorubicin. Addition of formaldehyde-releasing prodrugs enhanced formation of formaldehyde-mediated doxorubicin-DNA adducts in the hearts, independent of doxorubicin biodistribution in the heart, blood and tumours. This doxorubicin-DNA adduct forming treatment was associated with a novel cardiac transcriptomic profile as compared to doxorubicin single agent treatment, as assessed by RNA-sequencing. These combination treatments also maintained doxorubicin antitumour efficacy with respect to inducing dsDNA breaks, promoting apoptosis and inhibiting mammary tumour proliferation. The switch in doxorubicin action from cardiac topoisomerase-IIβ poisoning to doxorubicin-DNA adduct formation protects the heart from doxorubicin-mediated cardiac damage. Hence, the combination treatment of doxorubicin and formaldehyde-releasing prodrugs may be a promising cardioprotective therapy that does not compromise doxorubicin activity against mammary tumours. Citation Format: Alison Cheong, Sean McGrath, Tina Robinson, Ruqaya Maliki, Alex Spurling, Ada Rephaeli, Abraham Nudelman, Don R. Phillips, Belinda S. Parker, Salvatore Pepe, Suzanne M. Cutts. A switch in mechanism of action attenuates doxorubicin-mediated cardiotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 992.

A switch in mechanism of action prevents doxorubicin-mediated cardiac damage

Cancer patients treated with doxorubicin are at risk of congestive heart failure due to doxorubicin-mediated cardiotoxicity via topoisomerase IIβ poisoning. Acute cardiac muscle damage occurs in response to the very first dose of doxorubicin, however, cardioprotection has been reported after co-treatment of doxorubicin with acyloxyalkyl ester prodrugs. The aim of this study was to examine the role played by various forms of acute cardiac damage mediated by doxorubicin and determine a mechanism for the cardioprotective effect of formaldehyde-releasing prodrug AN-9 (pivaloyloxymethyl butyrate).Doxorubicin-induced cardiac damage in BALB/c mice bearing mammary tumours was established with a single dose of doxorubicin (4 or 16 mg/kg) administered alone or in combination with AN-9 (100 mg/kg). AN-9 protected the heart from doxorubicin-induced myocardial apoptosis and also significantly reduced dsDNA breaks, independent from the level of doxorubicin biodistribution to the heart. Covalent incorporation of [14C]doxorubicin into DNA showed that the combination treatment yielded significantly higher levels of formaldehyde-mediated doxorubicin-DNA adducts compared to doxorubicin alone, yet this form of damage was associated with cardioprotection from apoptosis. The cardiac transcriptomic analysis indicates that the combination treatment initiates inflammatory response signalling pathways.Doxorubicin and AN-9 combination treatments were cardioprotective, yet preserved doxorubicin-mediated anti-tumour proliferation and apoptosis in mammary tumours. This was associated with a switch in doxorubicin action from cardiac topoisomerase IIβ poisoning to covalent-DNA adduct formation. Co-administration of doxorubicin and formaldehyde-releasing prodrugs, such as AN-9, may be a promising cardioprotective therapy while maintaining doxorubicin activity in primary mammary tumours.

Potential Therapeutic Advantages of Doxorubicin when Activated by Formaldehyde to Function as a DNA Adduct-Forming Agent.

Doxorubicin has been in use as a key anticancer drug for forty years, either as a single agent or in combination chemotherapy. It functions primarily by interfering with topoisomerase II activity but in the presence of formaldehyde, it forms adducts with DNA, mainly with the exocyclic amine of guanine at GpC sites and these adducts are more cytotoxic than topoisomerase II induced damage. High levels of adducts form spontaneously from the endogenous level of formaldehyde in tumour cells (1,300 adducts per cell after a 4 hr treatment with doxorubicin), but substantially higher levels form with the addition of exogenous sources of formaldehyde, such as formaldehyde releasing prodrugs. The enhanced cytotoxicity of adducts has been confirmed in mouse models, with adduct-forming conditions resulting in much improved inhibition of tumour growth, as well as cardioprotection. Doxorubicin cardiotoxicity has been attributed to topoisomerase II poisoning, and the cardioprotection is consistent with a mechanism switch from topoisomerase II poisoning to covalent adduct formation. Although the adducts have a half-life of less than one day, a population remains as essentially permanent lesions. The capacity of doxorubicin to form adducts offers a range of potential advantages over the conventional use of doxorubicin (as a topoisomerase II poison), including: enhanced cell kill; tumour-selective activation, hence tumour-selective cell kill; decreased cardiotoxicity; decreased resistance to prolonged doxorubicin treatment. There is therefore enormous potential to improve clinical responses to doxorubicin by using conditions which favour the formation of doxorubicin-DNA adducts.

Abstract C279: Novel anthracenedione derivatives demonstrating several potential modes of actions.

Abstract DNA has been regarded as the primary target of numerous non-specific cytotoxic anticancer agents such as the nitrogen mustards and anthracycline antibiotics. The anthracyclines, most notably doxorubicin, proved to be highly potent and were thus incorporated into numerous chemotherapeutic regimens. However, their dose dependent redox activity that presumably contributes to their broad spectrum versatility is also associated with potentially lethal cardiotoxic adverse effects and thus, inspired the development of synthetic analogues including the anthracenediones. Like the anthracylines, their structural frame constitutes the DNA intercalating anthraquinone chromophore with functional amino groups stabilizing its insertion via hydrogen bonds and Van der Waal interactions. This interaction poisons the topoisomerase II cleavable complex which induces widespread DNA strand breaks culminating in the induction of apoptosis. We have shown that the availability of the methyl donating molecule, formaldehyde, generates adduct formation via a covalent aminal linkage between the exocyclic N2 amino group of guanine residue at specific CpG dinucleotides and an amino group on the anthracenediones side chains. The formaldehyde mediated DNA adduct formation, achievable via simultaneous formaldehyde-releasing prodrug administration, increases cancer cell toxicity and potentially endows general cardioprotective activity. Such cardioprotection has been associated with formaldehyde-mediated doxorubicin-DNA adducts We have synthesised a new series of anthracenediones that vary in their potential to induce covalent DNA adducts. We now show that the new anthracenediones including pixantrone (unlike the anthracyclines) also exhibits a preference for forming adducts with single stranded DNA and RNA, and these have similar characteristics to adducts that are formed with DNA. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C279. Citation Format: Paul P. Pumuye, Jelena E. Medan, Brad Sleebs, Keith Watson, Benny J. Evison, Donald R. Phillips, Suzanne M. Cutts. Novel anthracenedione derivatives demonstrating several potential modes of actions. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C279.

Abstract A29: Bcl-2 overexpression provides short-term resistance to doxorubicin-DNA adducts but does not protect against longer-term treatments in HL-60 cells.

Abstract The anthracycline anticancer agent doxorubicin functions primarily as a topoisomerase II inhibitor but forms more cytotoxic DNA adducts in the presence of formaldehyde. For this reason, the efficacy of doxorubicin can be greatly enhanced with the co-administration of formaldehyde-releasing prodrugs such as AN-9. The combination of doxorubicin with AN-9 results in DNA adduct formation and synergistic apoptosis in HL-60 leukemic cells after 6 hr. Under the same conditions HL-60 cells overexpressing the anti-apoptotic protein Bcl-2 (HL-60/Bcl-2 cells) are completely resistant to doxorubicin/AN-9 treatments. This resistance can be overcome with the use of the small molecule Bcl-2/Bcl-xL/BCL-W inhibitor ABT-737. While it appears that Bcl-2 overexpression confers resistance and thus may limit the therapeutic potential of doxorubicin-DNA adduct forming treatments, this is shown to be only a short-term effect. The use of a DNA adduct forming doxorubicin-formaldehyde conjugate, doxazolidine, produced similar results to the doxorubicin/AN-9 combination with HL-60/Bcl-2 cells being resistant to the drug after 4 hr treatment. This resistance was maintained up to 12 hr treatment, however, after 18 hr the HL-60/Bcl-2 cells started to display classical hallmarks of apoptosis including DNA fragmentation, and chromatin condensation which increased over time (18–36 hr). These results suggest that the Bcl-2 protection is only short-lived and that over time the resistance is overcome and the cells die via classical apoptosis. The exact mechanisms involved in overcoming this resistance in HL-60/Bcl-2 cells are currently being investigated and may involve changes in expression of Bcl-2 family proteins, or Bcl-2 interactions with pro-apoptotic proteins which tip the balance in favour of apoptosis. These findings imply that the damage caused by these DNA adduct forming compounds may be sufficient to overcome Bcl-2 mediated resistance observed in tumors in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A29.

Multifunctional 5-aminolevulinic acid prodrugs activating diverse cell-death pathways

Herein we describe a series of multifunctional 5-aminolevulinic-acid (ALA) prodrugs for photodynamic dependent and independent cancer therapy (PDT). We studied the cell-death mechanisms in glioblastoma U251 cells treated with four ALA-prodrugs: (1) AlaAcBu, that releases ALA, acetaldehyde, and butyric acid; (2) AlaFaBu, that releases ALA, formaldehyde, and butyric acid; (3) AlaFaPi, that releases ALA, formaldehyde and pivalic acid (4) AlaAcPi that releases ALA, acetaldehyde and pivalic acid. We examined the light-activated and dark cell-death mechanisms of the active metabolites released from the prodrugs by unspecific cellular hydrolases. The active moieties accelerated biosynthesis of protoporphyrin IX (PpIX) due to upregulated porphobilinogen deaminase (PBGD) activity. AlaAcBu was found to be the superior prodrug for PDT due to its ability to induce the highest PpIX synthesis. Photo-irradiation of AlaAcBu-treated cells led to dissipation of the mitochondrial membrane potential and reduction in the mitochondria metabolic activities; apoptosis and necrosis. Electron microscopy analyses of these cells revealed mitochondrial and endoplasmic reticulum swelling, membrane blebbing, apoptotic bodies and necrotic cell rupture. The formaldehyde-releasing prodrugs AlaFaBu and AlaFaPi induced low PDT efficacy, moreover sequestering the formaldehyde with semicarbazide resulted in high PpIX synthesis, suggesting that formaldehyde inhibited its synthesis. ALA and AlaAcBu phototherapy resulted in a dramatic accumulation of ubiquitinated proteins due to reduced proteasome activity and expression. In conclusion, the PDT potency of the prodrugs was in the order: AlaAcBu, AlaAcPi > AlaFaBu ≥ ALA > AlaFaPi, and the superiority of AlaAcBu stems from lower molar concentrations of AlaAcBu and lower light intensity needed to activate cell death following PDT.

Abstract 3507: Facilitating pixantrone-induced DNA damage and subsequent cell kill through formaldehyde activation and improvements in drug design

Abstract Pixantrone is a novel aza-anthracenedione originally developed to improve the therapeutic profile of mitoxantrone, a DNA-interactive agent currently used in the clinical management of a range of haematological malignancies and solid tumours. Like mitoxantrone, pixantrone interacts with DNA via intercalation and stimulates topoisomerase II-mediated DNA cleavage, presumably by stabilising the cleavable complex. Despite the drug's ability to stimulate DNA cleavage via topoisomerase II impairment, this form of DNA damage does not directly correlate with drug cytotoxicity, suggesting that pixantrone may be operating by a distinct, currently undefined mechanism of cell kill. A novel form of pixantrone-DNA interaction has recently emerged in which the drug can be efficiently activated by formaldehyde to yield covalent drug-DNA adducts. In cellular systems, pixantrone exhibits a mild synergistic relationship with the formaldehyde-releasing prodrug AN-9, indicating a favourable role for formaldehyde in mediating pixantrone-induced cell kill. A variety of methods including in vitro transcription and mass spectrometry have established that formaldehyde-activated pixantrone is a monofunctional DNA alkylator that binds selectively to CpG and CpA dinucleotides via the exocyclic N2 amino group of guanine within DNA. Crucially, formaldehyde provides the methylene bridge that covalently links DNA with a single drug side-chain. A major functional limitation of the monoadduct is its poor intrinsic stability (t1/2 = 75 min). It was subsequently rationalised that an increase in drug side-chain length may permit the drug to completely bridge the two strands of duplex DNA, much like an interstrand crosslink. Accordingly, new anthracenediones were synthesised that incorporated symmetrical side chains of the nature -NH-(CH2)n-NH2 (where n = 2—5). An in vitro crosslinking assay demonstrated that side-chain extension generally conferred a remarkable increase in the temporal stability of formaldehyde-activated anthracenedione-DNA adducts. A clear and direct relationship existed between side-chain length and drug-DNA adduct half-life with MX5 (n = 5) exhibiting exceptional stability (t1/2 > 2 days). Significantly, the enhanced drug-DNA adduct stability was reflected in breast adenocarcinoma MCF-7 cells where derivatives with longer side-chains were synergistic in combination with the formaldehyde-releasing prodrug AN-9. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3507.

ABT-737 overcomes Bcl-2 mediated resistance to doxorubicin–DNA adducts

Doxorubicin is an anthracycline anticancer agent that functions primarily by inhibiting topoisomerase II, but also forms covalent DNA adducts depending on the cellular availability of formaldehyde. The combination of formaldehyde-releasing prodrugs (such as AN-9) with doxorubicin has been shown to result in synergistic doxorubicin–DNA adduct formation and synergistic apoptosis in HL-60 leukemic cells, offering the potential for lower concentrations of doxorubicin to be used clinically in order to minimize side-effects. However, the overexpression of Bcl-2 confers resistance to doxorubicin/AN-9 DNA adduct forming treatments, thus limiting the therapeutic potential of this drug combination. The small molecule inhibitor, ABT-737, which binds to and inhibits Bcl-2, Bcl-xL and Bcl-w, was used in combination with doxorubicin/AN-9 treatments to overcome resistance to doxorubicin–DNA adducts in Bcl-2 overexpressing HL-60 cells (HL-60/Bcl-2). The combination treatment of doxorubicin and AN-9 (and all single agent controls) failed to induce an apoptotic response in HL-60/Bcl-2 cells, however, the addition of low nanomolar (sub-lethal) concentrations of ABT-737 was able to greatly increase apoptosis levels. Various control compounds were used to demonstrate that the mechanism of cell kill in response to the ‘triple treatment’ (doxorubicin, AN-9 and ABT-737) is dependent on DNA adduct formation. Therefore, the ability of ABT-737 to inhibit Bcl-2 renders previously resistant HL-60 cancer cells highly sensitive to doxorubicin–DNA adducts, leading to a classical apoptotic response. In conclusion, the data obtained provides promising evidence that the anticancer activity of doxorubicin–DNA adducts can be substantially enhanced in Bcl-2 overexpressing cancers with the use of the small molecule Bcl-2 inhibitor, ABT-737.

Development of Pluronic Micelle-Encapsulated Doxorubicin and Formaldehyde-Releasing Prodrugs for Localized Anticancer Chemotherapy

The chemotherapeutic agent doxorubicin forms drug-DNA adducts that are enhanced by formaldehyde-releasing prodrugs such as AN-9. One of the major limitations of doxorubicin is dose-limiting cardiotoxicity; therefore, the use of a targeting strategy that enables drug delivery and release at tumor sites is of great interest. The major aim of this study was to use the Pluronic-ultrasound delivery system to encapsulate doxorubicin and formaldehyde-releasing prodrugs within Pluronic micelles, and then use ultrasound to trigger controlled drug release from micelles. Pluronic micelles themselves were not stable upon dilution and required the use of a stabilizing agent DSPE-PEG2000 to form stable "mixed micelles." Following the separation of free doxorubicin, approximately 60% of doxorubicin remained encapsulated within mixed micelles with a retention half-life of approximately 12 h. The formaldehyde-releasing prodrugs, however, were not retained within mixed micelles, but could potentially be administered separately to doxorubicin-loaded micelles to achieve tumor-localized formation of doxorubicin-DNA adducts. The use of low-frequency, high-power ultrasound (20 kHz, 100 W/cm2) released 7-10% of doxorubicin from mixed micelles. Collectively, these results indicate that the Pluronic-ultrasound system could be used to deliver and release doxorubicin with the potential of forming cytotoxic DNA adducts at tumor sites with coadministrated formaldehyde-releasing prodrugs.

Formaldehyde-releasing prodrugs specifically affect cancer cells by depletion of intracellular glutathione and augmentation of reactive oxygen species

Histone deacetylase inhibitory prodrugs that are metabolized to carboxylic acid(s) and aldehyde(s) possess antineoplastic properties. Formaldehyde-releasing prodrugs were shown to be the most potent. The objective of this study was to gain understanding on the mode of action of these prodrugs in cancer cells. HL-60 and MCF-7 cells in the presence of N-acetylcysteine or glutathione were protected from death induced by formaldehyde-releasing prodrugs but not from death caused by the homologous acetaldehyde-releasing ones. Cell death induced by the former was accompanied by depletion of intracellular glutathione and increased reactive oxygen species that were attenuated by N-acetylcysteine. At fourfold higher concentration, acetaldehyde-releasing prodrugs increased reactive oxygen species that were further augmented by N-acetylcysteine. In HL-60 cells, formaldehyde-releasing prodrugs dissipated the mitochondrial membrane potential and glutathione or N-acetylcysteine restored it. Although acetaldehyde-releasing prodrugs dissipated mitochondrial membrane potential, it occurred at 20-fold greater concentration and was unaffected by the antioxidants. Formaldehyde-releasing prodrugs abrogated c-myc protein expression and elevated c-Jun and H2AX phosphorylation, N-acetylcysteine partially reversed these changes. Herein, we show that formaldehyde-releasing prodrugs diminish the level of glutathione most likely by forming S-formylglutathione adducts resulting in increase of reactive oxygen species followed by signaling events that lead to cancer cells death.

Activation of clinically used anthracyclines by the formaldehyde-releasing prodrug pivaloyloxymethyl butyrate

The anthracycline group of compounds is extensively used in current cancer chemotherapy regimens and is classified as topoisomerase II inhibitor. However, previous work has shown that doxorubicin can be activated to form DNA adducts in the presence of formaldehyde-releasing prodrugs and that this leads to apoptosis independently of topoisomerase II-mediated damage. To determine which anthracyclines would be useful in combination with formaldehyde-releasing prodrugs, a series of clinically relevant anthracyclines (doxorubicin, daunorubicin, idarubicin, and epirubicin) were examined for their capacity to form DNA adducts in MCF7 and MCF7/Dx (P-glycoprotein overexpressing) cells in the presence of the formaldehyde-releasing drug pivaloyloxymethyl butyrate (AN-9). All anthracyclines, with the exception of epirubicin, efficiently yielded adducts in both sensitive and resistant cell lines, and levels of adducts were similar in mitochondrial and nuclear genomes. Idarubicin was the most active compound in both sensitive and resistant cell lines, whereas adducts formed by doxorubicin and daunorubicin were consistently lower in the resistant compared with sensitive cells. The adducts formed by doxorubicin, daunorubicin, and idarubicin showed the same DNA sequence specificity in sensitive and resistant cells as assessed by lambda-exonuclease-based sequencing of alpha-satellite DNA extracted from drug-treated cells. Growth inhibition assays were used to show that doxorubicin, daunorubicin, and idarubicin were all synergistic in combination with AN-9, whereas the combination of epirubicin with AN-9 was additive. Although apoptosis assays indicated a greater than additive effect for epirubicin/AN-9 combinations, this effect was much more pronounced for doxorubicin/AN-9 combinations.

Doxorubicin-DNA Adducts Induce a Non-Topoisomerase II–Mediated Form of Cell Death

Doxorubicin (Adriamycin) is one of the most commonly used chemotherapeutic drugs and exhibits a wide spectrum of activity against solid tumors, lymphomas, and leukemias. Doxorubicin is classified as a topoisomerase II poison, although other mechanisms of action have been characterized. Here, we show that doxorubicin-DNA adducts (formed by the coadministration of doxorubicin with non-toxic doses of formaldehyde-releasing prodrugs) induce a more cytotoxic response in HL-60 cells than doxorubicin as a single agent. Doxorubicin-DNA adducts seem to be independent of classic topoisomerase II-mediated cellular responses (as observed by employing topoisomerase II catalytic inhibitors and HL-60/MX2 cells). Apoptosis induced by doxorubicin-DNA adducts initiates a caspase cascade that can be blocked by overexpressed Bcl-2, suggesting that adducts induce a classic mode of apoptosis. A reduction in the level of topoisomerase II-mediated double-strand-breaks was also observed with increasing levels of doxorubicin-DNA adducts and increased levels of apoptosis, further confirming that adducts exhibit a separate mechanism of action compared with the classic topoisomerase II poison mode of cell death by doxorubicin alone. Collectively, these results indicate that the presence of formaldehyde transfers doxorubicin from topoisomerase II-mediated cellular damage to the formation of doxorubicin-DNA adducts, and that these adducts are more cytotoxic than topoisomerase II-mediated lesions. These results also show that doxorubicin can induce apoptosis by a non-topoisomerase II-dependent mechanism, and this provides exciting new prospects for enhancing the clinical use of this agent and for the development of new derivatives and new tumor-targeted therapies.

Formaldehyde-Releasing Prodrugs in Combination With Adriamycin Can Overcome Cellular Drug Resistance

The anticancer drug Adriamycin is widely used in cancer chemotherapy and is classified as a topoisomerase II inhibitor. However, in the presence of formaldehyde, Adriamycin also forms high levels of DNA adducts. In this study, a new series of butyric acid and formaldehyde-releasing drugs related to AN9 (pivaloyloxymethyl butyrate) was assessed for their ability to facilitate Adriamycin-DNA adduct formation in Adriamycin-sensitive and -resistant cell lines (HL60 and HL60/MX2; MES-SA and MES-SA/Dx5). Drugs that released two molar equivalents of formaldehyde per mole of prodrug were superior in their ability to enhance adduct formation compared to those that released one molar equivalent. Adduct formation (as assessed by binding of radiolabeled Adriamycin to genomic DNA) was always lower in the resistant cell lines compared to the sensitive cell lines. However, in growth inhibition experiments, prodrug combinations were able to overcome Adriamycin resistance to varying degrees, and the combination of Adriamycin with selected prodrugs that release two moles of formaldehyde totally overcame resistance in HL60/MX2 cells. These HL60-derived cells express altered levels of topoisomerase II and also express a mutant form of the enzyme. Combinations of Adriamycin with selected prodrugs that release one or two moles of formaldehyde partially overcame P-glycoprotein-mediated resistance in MES-SA/Dx5 cells. Formaldehyde-releasing prodrugs (as single agents) overcame both forms of resistance in the two resistant cell lines, demonstrating that they were not substrates of these resistance mechanisms. Collectively, these results suggest that changing the mechanism via which Adriamycin exerts its anticancer effect by dramatically increasing adduct levels (requiring coadministration of formaldehyde-releasing prodrugs) may be a useful means of cancer treatment, as well as for overcoming Adriamycin-induced resistance.

The Role of Intracellularly Released Formaldehyde and Butyric Acid in the Anticancer Activity of Acyloxyalkyl Esters

Previous studies described a family of anticancer histone deacetylase inhibitor prodrugs of formula Me(CH(2))(2)COOCH(R)OR(1), which upon intracellular hydrolysis release acids and aldehydes. This study examines the mechanisms by which the prodrugs affect tumor cells and the contribution of the released aldehyde (formaldehyde or acetaldehyde) and acids to their anticancer activity. Type I prodrugs release 2 equiv of a carboxylic acid and 1 equiv of an aldehyde, and of Type II release 2 equiv of acids and 2 equiv of an aldehyde. SAR studied inhibition of proliferation, induction of differentiation and apoptosis, histone acetylation, and gene expression. Formaldehyde, measured intracellularly, was the dominant factor affecting proliferation and cell death. Among the released acids, butyric acid elicited the greatest antiproliferative activity, but the nature of the acid had minor impact on proliferation. In HL-60 cells, formaldehyde-releasing prodrugs significantly increased apoptosis. The prodrugs affected to a similar extent the wild-type HL-60 and MES-SA cell lines and their multidrug-resistant HL-60/MX2 and MES-Dx5 subclones. In a cell-free histone deacetylase (HDAC) inhibition-assay only butyric acid inhibited HDAC activity. The butyric acid and formaldehyde induced cell differentiation and increased p53 and p21 levels, suggesting that both affect cancer cells, the acid by inhibiting HDAC and the aldehyde by an as yet unknown mechanism.

Formation of mitoxantrone adducts in human tumor cells: potentiation by AN-9 and DNA methylation.

The ability of mitoxantrone to form DNA adducts was investigated in a series of human tumor cell lines consisting of human cervical cancer (HeLa), human breast cancer (MCF-7), and human neuroblastoma (IMR-32) cells. The mitoxantrone-resistant human promyelocytic leukemia cell line HL60/MX2 was also compared to the parental cell line HL60 in terms of adduct formation in cellular DNA, RNA, and protein. DNA adduct formation detected using [14C]mitoxantrone as a single agent occurred at very low levels but addition of the formaldehyde-releasing prodrug AN-9 (pivaloyloxymethyl butyrate) increased adduct formation considerably in all cell lines tested. Adduct formation increased when increasing ratios of AN-9 were used, and were observed at maximal levels when AN-9 addition was 4 h after the addition of mitoxantrone. However, low levels of adducts were observed when AN-9 addition was 16 h prior to mitoxantrone. The ability of [14C]mitoxantrone to form adducts with DNA, RNA, and protein was assessed in HL60 cells, and DNA was found to be the major substrate for adduct formation. RNA was also shown to be a good substrate while protein adduct levels were consistently very low. In mitoxantrone-resistant HL60/MX2 cells, DNA adduct levels were approximately fourfold lower. To establish the influence of DNA methylation on the ability of mitoxantrone to form adducts in cells, decitabine was used to reduce DNA methylation levels in cells prior to mitoxantrone treatment. This was clearly shown to influence adduct formation, with increasing decitabine levels leading to a decrease in the level of adducts observed in both IMR-32 and MCF-7 cell lines. Collectively, these results suggest that two major factors that influence the extent of mitoxantrone adduct formation in cells are the availability of formaldehyde and the extent of genomic DNA methylation.

Prodrugs of butyric acid. Novel derivatives possessing increased aqueous solubility and potential for treating cancer and blood diseases

The synthesis and biological activities of acidic, basic and neutral types of butyric acid (BA) prodrugs possessing increased aqueous solubility are described. The compounds are butyroyloxyalkyl derivatives of carboxylic acids, which possess functionalities suitable for aqueous solubilization. The anticancer activity of the prodrugs in vitro was evaluated by examining their effect on the growth of human colon, breast and pancreatic carcinoma cell lines, and their solubility in aqueous media was determined. The most promising compounds, with respect to activity and solubility, were found to be the butyroyloxymethyl esters of glutaric 2a and nicotinic acids 4a and phosphoric acid as its diethyl ester 10a, which displayed IC 50 values of 100 μM or lower. These prodrugs are expected to release formaldehyde upon metabolic hydrolysis. The corresponding butyroyloxyethyl esters ( 2b, 4b and 10b) that release acetaldehyde upon metabolism were significantly less potent. A similar correlation was observed for growth inhibition of the human prostate carcinoma cell lines PC-3 and LnCap and for induction of differentiation and apoptosis in the human myeloid leukemia cell line HL-60. The higher biological activity of the formaldehyde-releasing prodrugs 2a and 10a was further confirmed when induction of hemoglobin (Hb) synthesis in the human erythroleukemic cell line K562 was measured. Moreover, a therapeutic index (IC 50/ED 50) of ca. 5 was observed. The acute i.p. toxicity LD 50 in mice for 2a, 2b, 10a and 10b was similar and in the range of 400–600 mg kg –1. The results obtained support the potential use of the butyric acid prodrugs for the treatment of neoplastic diseases and β-globin disorders.