Medical Hypotheses 80 (2013) 841–846

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Medical Hypotheses

journal homepage: www.elsevier .com/locate /mehy

Targetting cancer with Ru(III/II)-phosphodiesterase inhibitor adducts: A novelapproach in the treatment of cancer

Raj Kumar Koiri ⇑, Aditi Mehrotra, Surendra Kumar TrigunBiochemistry and Molecular Biology Lab, Department of Zoology, Banaras Hindu University, Varanasi, Uttar Pradesh 221005, India

a r t i c l e i n f o a b s t r a c t

Article history:Received 3 November 2012Accepted 17 March 2013

0306-9877/$ - see front matter � 2013 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.mehy.2013.03.029

Abbreviations: cAMP, cyclic adenosine monophospmonophosphate; LDH, lactate dehydrogenase; PDE, cyterases; PDEi, cyclic nucleotide phosphodiesterases innitric oxide.⇑ Corresponding author. Tel.: +91 9453061011.

E-mail addresses: rajkumarfrombhu@yahoo.com (R(S.K. Trigun).

Lack of specificity and normal tissue toxicity are the two major limitations faced with most of the anti-cancer agents in current use. Due to effective biodistribution and multimodal cellular actions, duringrecent past, ruthenium complexes have drawn much attention as next generation anticancer agents. Thisis because metal center of ruthenium (Ru) effectively binds with the serum transferrin and due to higherconcentration of transferrin receptors on the tumor cells, much of the circulating Ru-transferrin com-plexes are delivered preferentially to the tumor site. This enables Ru-complexes to become tumor cellspecific and to execute their anticancer activities in a somewhat targeted manner. Also, there are evi-dences to suggest that inhibition of phosphodiesterases leads to increased cyclic guanosine monophos-phate (cGMP) level, which in turn can evoke cell cycle arrest and can induce apoptosis in the tumorcells. In addition, phosphodiesterase inhibition led increased cGMP level may act as a potent vasodilatorand thus, it is likely to enhance blood flow to the growing tumors in vivo, and thereby it can further facil-itate delivery of the drugs/compounds to the tumor site.

Therefore, it is hypothesized that tagging PDE inhibitors (PDEis) with Ru-complexes could be a relevantstrategy to deliver Ru-complexes-PDEi adduct preferentially to the tumor site. The Ru-complex taggedentry of PDEi is speculated to initially enable the tumor cells to become a preferential recipient of suchadducts followed by induction of antitumor activities shown by both, the Ru-complex & the PDEi, result-ing into enhanced antitumor activities with a possibility of minimum normal tissue toxicity due toadministration of such complexes.

� 2013 Elsevier Ltd. All rights reserved.

Introduction

Since the discovery of anticancer properties of cisplatin (cisdi-amminedichloro-platinum(II)) by Rosenberg in 1960s [1], the useof metal-based complexes invited special attention in the area ofchemotherapeutics [2]. This resulted in the development of plati-num-based anticancer agents which could respond against a widerange of tumors like, ovarian, testicular, bladder and lung cancers[3]. These complexes bind to N-7 of guanine and adenine basesof DNA and thereby, they produce intrastrand/interstrand DNAcross-links [4]. As a result, it disrupts DNA replication and geneticinformation flow in the tumor cells [5]. Moreover, platinum basedcomplexes were found to produce a number of serious side effectsmainly due to lack of specificity for the tumor cells and attacking

ll rights reserved.

hate; cGMP, cyclic guanosineclic nucleotide phosphodies-hibitors; Ru, ruthenium; NO,

.K. Koiri), sktrigun@sify.com

DNA which is now considered as an unselective target [6]. Addi-tionally, clinical utility of this drug is often limited due to the onsetof resistance acquired against these complexes by the tumor cells[7,8]. This could be the reason why out of over 3000 platinum com-pounds synthesized [9], only three; cisplatin, carboplatin and oxa-liplatin, came in routine clinical use [10–12].

Moreover, these limitations necessitated the development ofnon-platinum compounds as alternate anticancer drugs. The anti-cancer properties of the other transition metal drugs examined in-cluded complexes based on ruthenium, arsenic, gallium, titanium,copper, iron, rhodium, and tin [13–18]. However, till recent, onlyruthenium complexes could derive much attention as a relevantalternative to the platinum based drugs [19,20].

Anticancer Ru-complexes

NAMI-A, [imH][trans-RuIIICl4(dmso-S)(im)] (im = imidazole), is thefirst non platinum and first Ru complex that has successfully com-pleted phase I clinical trials as an anti-cancer agent and is currentlyin phase II clinical trials. Another Ru(III) drug, KP1019, indazolium[trans-tetrachlorobis(1H-indazole)-ruthenate(III)]2 have also success-

842 R.K. Koiri et al. / Medical Hypotheses 80 (2013) 841–846

fully completed phase I clinical trials [19,20]. Both these complexesexhibited low level general toxicity, but with strong antimetastaticactivities [20,21]. We have also described that a ruthenium(II) com-plex; Ru(II)-CNEB, could induce apoptosis in the DL (Dalton’s lym-phoma) cells in vivo via inhibiting lactate dehydrogenase, theenzyme which is known to be up regulated during tumor growth,and was found to show negligible toxicity to the normal tissues [22,23].

What is specific about ruthenium complexes as anticanceragents?

Ruthenium (Ru) based complexes exhibit a number of proper-ties that advocate them as a physiologically better acceptable com-pounds when administered to the tumor bearing subjects. Themost important one is that they exhibit multiple accessible oxida-tion states mimicking iron [20,24], which enables them to bindwith the circulating transferrin [25,26]. It has been reported thatas tumor cells need higher amount of iron, they express highamount of transferrin receptor [25,26]. This provides main mecha-nism for much of circulating Ru-complexes to preferentially accu-mulate in the tumor cells. Such a mechanism could be accountablefor minimum Ru-toxicity to the normal tissues when administeredto the tumor bearing animals [22]. Additionally, affinity of Ru-com-plexes for the serum albumin [27] could also attribute for theirminimum systemic toxicity and effective biodistribution.

Higher coordination number of ruthenium, compared to theplatinum, provides additional sites for fine-tuning the ligand ex-change and thereby, allowing modulation of therapeutic propertiesof these complexes [24]. Also, metal Ru-centers, being positivelycharged, can bind to the negatively charged biomolecules; like pro-teins and nucleic acids and thereby, they are likely to modulate di-verse cellular functions [21,23]. For example, protein bindingproperties of Ru-complexes have been shown to facilitate their dis-tribution and to modulate their pharmacokinetics & mechanism ofaction in a significant way [22,28].

Modulation of molecular targets by Ru-complexes

Following the pattern of cisplatin action, structural distortionsof DNA were initially speculated to be the main mechanism of anti-cancer activities of many ruthenium and other transition metalcomplexes [29]. Indeed some of them were shown to interact withDNA/G-quadruplex [30] and could damage DNA in the tumor cells[31]. Moreover, DNA is considered as unselective target. Recent ad-vances in bioanalytical techniques, with high sensitivity and selec-tivity, have revealed that other than DNA, metal-based drugs canundergo a wide range of biomolecular interactions and therefore,they have generated interest in proteins as relevant target formetallodrugs [32]. Obviously, one can speculate that formulationof ruthenium complexes targeted to proteins of critical cell func-tions would be a better strategy to ascertain therapeutic potentialof the ruthenium-complexes [28,33].

There are reports to suggest that some ruthenium complexescould modulate specific biochemical properties like, productionof ROS [34], inhibition of survival kinases [35] & redox reactionsin the cancer cells [36]. A Ru(III) complex was found to inhibitDNA replication and RNA synthesis in vivo [24]. NAMI-A could pro-duce cytotoxicity by interacting with DNA and by inhibiting typeIV collagenase activity as well [24].

Targeting non-DNA molecules: a better option for anticanceragents

Based on the recent advances in the area of cancer genomics, ascompared to the DNA-adduct formation guided design of

metallodrugs, it is now being argued to target non-nuclear mecha-nisms which are over activated in the cancer cells [37]. One of themost plausible cancer associated phenomena is the switching overto glycolytic phenotype acquired by most of the tumors to meettheir extra energy need [38]. As our understanding of molecularbiology and cell signaling becomes clearer, key molecular targetsof this pathway are being identified as relevant pharmacologicaltargets by many workers including evaluation of certain Ru(II)-complexes from our lab also [22,39].

Structural diversity makes Ru-complexes a better choice

The multimodal actions of Ru-complexes were attributed totheir structural diversity generated due to different ligands at-tached to the ruthenium metal center. As a result, many of themcould enhance their water solubility, stability, cellular specificityand could also overcome with the problem of drug resistance[28,33,40,41]. Some important ligands being tried have beenamines, imines, DMSO & polypyridyl arene compounds[24,25,41]. Amongst these, imidazole ligand containing NAMI-Asystem and water-soluble arene ruthenium complexes haveemerged as important molecules to design anticancer drugs withlow toxicity and high tumor specificity due to their greater affinityfor the cancer cells [25,28,41]. Taking together, it is evident thatcoupling of Ru-complexes with molecules of distinct cellular tar-gets could be one of the promising strategies for developing syner-gistic anticancer drugs [33,41].

Ru(II) vs. Ru(III)

As compared to Ru(III), Ru(II) complexes could be more effectivefor tumor cell toxicity [40]. This is because Ru(II) complexes havebeen found to bind more effectively with macromolecules andcan induce better cytotoxicity [37]. Indeed a number of Ru(II)-com-pounds with flavonoids based ligands have been reported to en-hance their affinity towards bio-molecules [42,43] and toprovoke better anticancer properties [22,43]. Nonetheless, in vivotumor cells run with relatively low electro-chemical potentialdue to the low oxygen content and low pH which can reduce Ru(III)to Ru(II) for their better cytotoxicity [37]. NMR studies have re-vealed that the Ru(III) centers in KP1019 [44] and NAMI-A [37] un-dergo reduction in the presence of reducing agents, like ascorbicacid and glutathione found in the cells [40]. Thus both, Ru(II) andRu(III), could be exploited to formulate effective anticancer agents.

Cyclic nucleotide phosphodiesterases (PDE) and the tumorgrowth

Modulation of cyclic nucleotide phosphodiesterases (PDEs) areone of the recent and evolving inclusions in the series of biochem-ical adaptations reported in the cancerous cells. PDEs catalyzehydrolytic cleavage of the 30-phosphodiester bond in the cyclicadenosine monophosphate (cAMP) and cyclic guanosine mono-phosphate (cGMP), converting them into AMP & GMP respectively[45]. cAMP & cGMP are termed as second messengers involved inmaintaining bioenergetic homeostasis by relaying signals fromreceptors on the cell surface to the target molecules inside the cell[46]. Importantly, deregulation of cGMP production and unusualactivation of cAMP-controlled genes have been described associ-ated with the tumor growth [47]. The former is linked to theover-expression of PDE isoforms in many tumors resulting into re-duced cGMP level [47]. During cancer progression, such changesare likely to interfere with drug delivery also.

Interestingly, it is now evident that these tumor associatedevents can be reversed using selective PDE inhibitors (PDEis)

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[47–50]. PDEi block PDE activity, resulting into passive accumula-tion of the intracellular cGMP (Fig. 1) which in many tumor cellshas been found to induce apoptosis and cell cycle arrest [47]. Fur-thermore, in some cases, PDEis have been demonstrated to downregulate expression of the multi-drug resistance protein, MRP5also [51]. This evolving mechanism is further highlighted by thefact that the mechanism of some anticancer drugs also involvesraising of cGMP level via NO mediated activation of guanylyl cy-clase [46]. Roles of other molecular partners to accomplish thisprocess have been summarized in Fig. 2. The intracellular targetsof cGMP signaling include cGMP dependent protein kinases(PKG) & cGMP gated ion channels. When activated, they initiateprotein phosphorylation cascades leading into dilation of the bloodvessels [52]. The signal is terminated by metabolic degradation ofcGMP by PDE [46] or by ATP dependent export of cGMP from thecell by MRP5 [51].

PDEis also up regulate the phosphoinositol-3-phosphate kinase(PI3K)/AKT pathway and increases VEGF level to facilitate angio-genesis in the tumors [53,54]. Employing this strategy, some PDEisare already in clinical use. One of the most common is Sildenafil(Viagra) which specifically inhibits cGMP-specific PDE type 5 andin doing so, it enhances the vasodilatory effects of cGMP in the cor-pus cavernosum, overcoming erectile dysfunciton. The same drugis also being investigated as a cardioprotective treatment for pa-tients with Duchenne muscular dystrophy [55]. Thus, it may be ar-gued that PDE inhibitors can potentially improve drug delivery dueto cGMP led increased vasodilation in the growing tumors.

Use of PDEi as anticancer agents

cGMP levels are significantly reduced in the tumor cells as com-pared with their normal counterparts [56]. Accordingly, increasedintracellular cGMP elicited by various guanylyl cyclase agonists[57], atrial natriuretic peptide [58], YC-1 [56], nitric oxide donors

Sol

c5’-cGMP

PDE inhibitor

X

cGK

(active)

Protein phosphor

Cellular respo

NONO donors

NO synth

Fig. 1. NO production via NOS/NO donors activate soluble guanylyl cyclase to form cGphosphodiester bond to form inactive 50-GMP. Inhibitors of PDE block this activity, resnucleotide-dependent protein kinases (cGK), leading into disturbance of constitutive ce

[59], and/or PDE5-selective inhibitors, for example, sildenafil andvardenafil have been reported to induce apoptosis and inhibit cellproliferation [47]. One of the PDE5 inhibitors has been reported toinhibit growth of the breast cancer cells by inhibiting cGMP hydro-lysis and activating PKG signaling to induce apoptosis in those can-cer cells [49]. Added to this, Zhu et al. [48] have demonstratedsuppression of PDE5 gene expression by antisense pZeoSV2/ASP5plasmid transfection resulting into a sustained increase in theintracellular cGMP led growth inhibition and apoptosis in the hu-man colon tumor HT29 cells. Recently, a phosphodiesterase 5inhibitor, vardenafil, has been shown to synergize the effect ofEGCG inducing cancer cell death [54].

Furthermore, PDE5 inhibitors have been reported to serve aschemo-sensitizers. The ABC transporters (such as ABCB1, ABCC4,ABCC5, ABCC10 and ABCG2), whose over expression is associatedwith multi-drug resistance (MDR) in cancer cells, were also foundto be the targets of PDE5 inhibitors and thereby enhancing the ac-tion of various antineoplastic drugs [51,60–62]. Importantly, mostof these PDE5 inhibitors have shown acceptable tolerability pro-files in the patients [62].

Presentation of the hypothesis

Review of the recent findings, as described in this article, sug-gests that both; Ru-complexes and PDEis, adopt specific mecha-nisms for their preferential accumulation in the tumor cells andthat both of them exhibit antitumor activities by interfering withtumor growth associated biochemical events. Additionally, Ru-me-tal center [Ru(II)/Ru(III)] can bind/accommodate a variety of li-gands and thereby, it provides greater versatility for designingpotent anticancer agents with high tumor specificity. On the otherhand, PDEis are known to enhance blood supply to the target tissueand thereby they can enhance relatively preferential drug targetingto the tumor site. Added to this, PDEis have recently been

uble guanylyl cyclase

GTPGMP

cGK

(inactive)

ylation

nse

Guanylyl cyclase receptor

Agonistase

MP from GTP. Phosphodiesterases degrade cGMP by hydrolytic cleavage of the 30-ulting into accumulation of cGMP, which in turn can affect the activities of cyclicllular homeostasis of the target cells.

RuPDE inhibitor

PKC ERK

iNOS / eNOS

NO

Guanylyl cyclase

GTPcGMP5’-cGMP

RuPDE inhibitor

X

ATP

MRP5

X

ATP ADP

PKG

Downstream effectors

CREB VEGFPI3K/AKT

Lowering Ca2+ Ca2+

VSM relaxation

vasodilation

Angiogenesis blood flow in hypoxic core Accumulation of Ru(III/II) in tumor cell

Regression of cancer Apoptosis

Fig. 2. Proposed hypothesis for the mechanism of action of Ru(III/II)-PDEis adduct as an anticancer agent. This adduct is likely to inhibit metabolic degradation of cGMP byPDE and thereby it can allow passive accumulation of cGMP. This in turn can activate cGMP dependent protein kinase (PKG) signaling in the tumor cells to enhancevasodilation and to evoke cell cycle arrest and apoptosis. The vasodilation function, once initiated by such adducts, is likely to allow more amount of adduct to be deliveredpreferentially at the tumor site. PDEis can also up regulate the phosphoinositol-3-phosphate kinase (PI3K)/AKT pathway and increases VEGF levels and thus, leading intoincreased angiogenesis in the hypoxic tumor mass. This will further facilitate efficient delivery of adduct at the hypoxic core allowing effective antitumor activities of both,the Ru-complex and the PDEis. BF – blood flow; cGMP – cyclic GMP; CREB – cAMP response element binding protein; ERK – extracellular regulated kinase; GC – guanylylcyclase; iNOS or eNOS – inducible or endothelial nitric oxide synthase; NO – nitric oxide; PI3K – phosphotidyl inositol-3 kinase; PKC – protein kinase C; PKG – protein kinaseG; Ru-PDEi – ruthenium phosphodiesterase inhibitor; VSM – vascular smooth muscle.

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described to exhibit anti-cancer activity at their own by modulat-ing an important cell signaling cascade associated with the tumorgrowth.

Therefore, it is reasonable to hypothesize a strategy involvingtagging of PDEis with the Ru-metal center to produce a Ru-PDEiadduct which is speculated to:

(1) Increase Ru-led preferential delivery of the adduct at tumorsite.

(2) Facilitate further PDEi mediated greater perfusion of thetumor cells and thereby preferential delivery of most ofthe successively administered Ru-PDEis adduct to thetumors in vivo.

(3) Provide double edge molecular mechanisms of Ru- & PDEisto execute more effectively their antitumor activities toinduce apoptosis in the tumor cells with a little chance toaffect normal tissues adversely.

(4) Overcome with the problem of drug resistance.

The Experimental plan hypothesized

The following experiments may be proposed to accomplish thehypothesis:

(A) Synthesis of Ru(III/II) complexes containing known PDEinhibitors (e.g., inhibitors of PDE5) as ligands, for example,sildenafil, tadalafil, zaprinast, vardenafil, DA-8159, cilosta-mide, milrinone, trequinsin, cilastazol, OPC-33540, etc.

(B) In vitro assay of Ru(III/II)-phosphodiesterase inhibitor com-plexes for their ability to inhibit the PDE5 activity and toincrease the level of cGMP in the cells in culture.

(C) In vivo assay of general cytotoxicity and delivery of adduct tothe tumor site.

(D) Assay of whether adduct is able to inhibit PDE5 leading intoincreased level of cGMP in the tumor cells in vivo.

(E) Studies on modulation of biochemical targets like, bioener-getic enzymes, NOS activity and antioxidant enzymes inthe tumor cells in vivo.

(F) Asssay of drug resistance proteins like BCRP, MDR1 & MRP1in the tumor cells from Ru-PDEi adduct treated cancerousanimals.

Summary & perspective

Modulation of tumor cell biochemistry by Ru-complexes result-ing into tumor apoptosis and PDEis mediated cell cycle arrest & tu-mor regression are the evolving mechanisms in cancer

R.K. Koiri et al. / Medical Hypotheses 80 (2013) 841–846 845

chemotherapy. The findings from the present hypothesis will leadto the identification of Ru-PDEis adducts effective against a num-ber of common cancers with little normal tissue toxicity. The find-ings on whether they are able to modulate multidrug resistanceproteins in the in vivo tumors will provide additional propertiesof such complexes. Taking together, the proposed hypothesis is anovel approach of combining antitumor properties of more thanone compounds leading into synthesis of a physiologically betteracceptable but more potent anticancer complexes, showing in-creased tumor specificity, decreased drug resistance and multiplecellular targets to ultimately induce apoptosis in the tumor cells.

Conflict of interest

The authors declare no conflict of interest with respect to thisarticle.

Acknowledgments

This work had a genesis from a Department of Biotechnology(DBT), Govt. of India project (No. BT/PR5910/BRB/10/406/2005)and results from a CSIR SRF to RKK. AM thanks ICMR, Govt. Indiafor awarding Senior Research Fellowship. The facilities provideddue to UGC-CAS and DST-FIST programmes to Department of Zool-ogy, BHU, are also acknowledged.

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