DOI: 10.1002/ijch.201100059

Light-Activated Pharmaceuticals: Mechanisms andDetectionDavid Kessel*[a] and John Reiners, Jr.[b]

1. Introduction

In the context of photodynamic therapy (PDT), singletoxygen (1O2) was identified as a major phototoxic ele-ment in the anti-tumor effects of hematoporphyrin deriv-ative (HPD) in 1976.[1] HPD was the first of the majorclinically useful photosensitizers. Since oxygen is requiredfor 1O2 formation, it was not surprising that hypoxic re-gions of tumors were insensitive to PDT.[2] As one of theeffects of PDT is vascular shutdown, adjustments in irra-diation protocols that lead to only periodic interruptionsof blood flow can promote efficacy.[3] It is generally con-sidered that 1O2 is the predominant factor in photokilling,although this may not be true for all photosensitizingagents. Foote proposed a distinction between �type I� and�type II� photochemistry.[4] In the former, a reaction be-tween the activated state of the photosensitizer and sub-strate or solvent yields radicals or radical ions. In thelatter, reaction occurs at oxygen to form either 1O2 orother reactive oxygen species (ROS).

Another view was expressed by Vid�czy, who proposedthat Foote�s definitions might be applicable only toPDT.[5] Rodgers provided yet another view, suggestingthat the distinction was actually between electron transferand energy transfer reactions.[6] In the case of the porphy-rin/phthalocyanine structures commonly used in PDT, O2

is the only biological molecule with a 1Dg state that canbehave as an energy acceptor. PDT effects are furthercomplicated by ROS downstream from 1O2, as summar-ized by Girotti.[7] These include the superoxide anion rad-ical (*O2

�), the OH radical (*OH), hydrogen peroxide(H2O2) and lipid peroxides. In his review, Girotti consid-ers *O2

� to be a Type I product. Crosstalk among the

species adds further complication, e.g., ascorbate can in-teract with 1O2 to produce H2O2.

[8] Moreover, reactive ni-trogen species (RNS) can be formed as a byproduct of in-teractions between nitric oxide and ROS.[9]

2. ROS and RNS Detection

Identification of the different ROS associated with photo-dynamic action is not a simple matter. An unambiguousmethod is electron spin resonance (ESR) spectroscopy,but this technique does not readily lend itself to studies inbiological cultures, and the necessary equipment is notfound in most laboratories. One common approach in-volves the use of fluorescent probes that are supposed tolight up when confronted with specific reactive species.Considerable specificity is often claimed for these probes,but they are generally not nearly as selective as adver-tised.

Abstract : Photodynamic therapy relies on the interaction be-tween light, oxygen and a photosensitizing agent. Its medi-cal significance relates to the ability of certain agents, usual-ly based on porphyrin or phthalocyanine structures, to local-ize somewhat selectively in neoplastic cells and their vascu-lature. Subsequent irradiation, preferably at a sufficientlyhigh wavelength to have a significant pathway through tis-sues, results in a photophysical reaction whereby the excited

state of the photosensitizing agent transfers energy to mo-lecular oxygen and results in the formation of reactiveoxygen species. Analogous reactive nitrogen species arealso formed. These contain both nitrogen and oxygenatoms. The net result is both direct tumor cell death anda shutdown of the tumor vasculature. Other processes mayalso occur that promote the anti-tumor response but theseare outside the scope of this review.

Keywords: apoptosis · autophagy · photodynamic therapy · reactive oxygen species · reactive nitrogen species

[a] D. KesselDepartment of PharmacologyWayne State University School of MedicineDetroit, MI 48201 (USA)phone: +001313 5771787e-mail: dhkessel@med.wayne.edu

[b] J. Reiners, Jr.Institute of Environmental Health SciencesWayne State UniversityDetroit, MI 48201 (USA)phone: +001313 5775594e-mail: ad5180@wayne.edu

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Review D. Kessel and J. J. Reiners, Jr.

Among the more common probes for ROS is reduceddichlorofluorescein (H2DCF). Oxidation to DCF yieldsa green fluorescence. This agent is often provided as thediacetate to promote cellular uptake, with the acetateslater cleaved by the action of intracellular esterases. Thespecificity of H2DCF is quite broad, with a spectrum thatincludes H2O2,

*OH, *O2� , ONOO� , OCl� and 1O2. Self-

oxidation is also observed in the presence of light.[10] Ourexperience with this and other probes is described later inthis review.

Dihydrorhodamine 123 (DHR) is often used as a probefor H2O2,

[11] but is also sensitive to *O2�[12] and the perox-

ynitrite anion (ONOO�).[13] Dihydroethidium (DHE) isoften claimed to be a selective fluorescent probe for *O2

�

formation,[14] but the fluorescence emission spectrummust be monitored to distinguish between the probe oxi-dation product produced by superoxide and that pro-duced by the OH radical. HPLC analysis is sometimesneeded to confirm the identity of the ROS.[15] Tang’sgroup described a naphthofluorescein derivative that flu-oresces in the presence of *O2

� .[16] Use of this probemight be compromised by the fact that naphthofluores-ceins are nonpolar and difficult to work with in aqueousenvironments. Moreover, fluorescence emission fromnaphthofluorescein is highly pH dependent with a pKa of~7.5. This will complicate fluorescence measurements, es-pecially if the probe accumulates in subcellular regions oflow pH. The sensitivity of Tang’s test for *OH also ap-pears suboptimal since it relies on production of *OH viathe Fenton reaction which is quite inefficient. Maeda’sapproach to superoxide detection resulted in a substantial-ly greater response to *O2

� than to *OH, using a probebased on a nitrobenzenesulfonyl ester structure.[17] Thisreagent also becomes fluorescent in the presence of thiol-containing compounds. Although this is noted in thereport, in a critical test only a 50 mM concentration of glu-tathione (GSH) was used, which is perhaps 1 % of the ex-pected intracellular GSH concentration.

Sites of photodamage can be assessed by fluorescentprobes specific for mitochondrial membrane potential(MitoTracker Orange), lysosomal integrity (LysoTrackerand LysoSensor), the endoplasmic reticulum (ER-Track-ers) or membrane integrity (propidium iodide or trime-

thylamino-diphenylhexatriene).[18] There is also a probesaid to report on lipid oxidation: C-11-BODIPY,[20] whichappears to provide qualitative rather than quantitativedata.

Of the probes we have examined, the most specific ap-pears to be aminophenoxyxanthene benzoic acid(APF),[21] a product developed for detection of *OH.[10]

APF can, however, also respond to 1O2.[21] A summary of

studies relating to probe specificity in a cell-free system isshown in Figure 1. Procedures generally followed the ex-perimental approach outlined by Setsukinai et al. ,[10]

except that Fe(NH4)SO4 was substituted for Fe(ClO4)2 inthe Fenton reaction. Horseradish peroxidase (10 mg mL�1)was present where specified. It is noteworthy that most ofthe probes were highly responsive to *OH and that addi-tion of horseradish peroxidase markedly promoted probeoxidation in most cases. Included in this survey was theprobe 4-amino-5-methylamino-2’,7’-difluorofluorescein(DAF). Although intended as a probe for *NO,[22] DAFcan also be oxidized by *OH. A probe for 1O2 has recent-ly appeared designated as �Singlet Oxygen Sensor Green�(SOSG). This is said to be cell impermeable, but recentreports have indicated that SOSG can be accumulated bycells and that it can yield 1O2 upon irradiation.[23]

In addition to the report by Girotti cited above,[7] thereare many reviews on the general topic of fluorescentprobes.[24] In the context of PDT, some additional factorsneed to be considered. The photosensitizers also fluo-resce, so care needs to be taken that the fluorescence ofthe photosensitizer is not confused with the fluorescenceof the probe.[25] Studies in cell culture or cell-free systemswill necessarily neglect important elements of PDT, suchas the effects of NO on vascular elements during PDT.[26]

A final caution is the occasional appearance of spontane-ous fluorescence that can mimic the effects of probe oxi-dation without actually involving ROS formation. An ex-ample is the fluorescence that is observed when the pro-apoptotic drug HA14-1 interacts with albumin in culturemedia.[27] The resulting fluorescence emission spectrum isan almost exact duplicate of that of DCF, and was inter-preted in one study as evidence that adding HA14-1 togrowth media initiated production of ROS.[28]

3. Effects of ROS Formed during PDT

PDT is intended to kill malignant cells ; this can take theform of either apoptotic or necrotic death.[29] The latteroften occurs when PDT conditions are sufficient to causemassive plasma membrane and organelle membranedamage, and destroy/inactivate the enzymatic processes(e.g., caspases) involved in the apoptotic program.[30] InPDT protocols the apoptotic program is commonly initi-ated by photosensitizers that accumulate in, and subse-quently cause photodamage to, organelles such as mito-chondria, lysosomes, and the endoplasmic reticulum

David Kessel has been involved instudies relating to photodynamic thera-py since 1976. He is the immediatePast President of the International Pho-todynamic Association and a Councilorand Historian for the American Societyfor Photobiology. His current researchdeals with modes of cell death afterPDT.

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(ER). In particular, photosensitizing agents that targetmitochondria and the ER upon illumination often photo-damage organelle-associated anti-apoptotic members ofthe Bcl-2 family (i.e., Bcl-2, Bcl-XL),[29,31] inhibit the activ-ity of the ER SERCA2 pump,[31,32] and activate Bax.[32a]

Such effects result in the release of ER calcium stores,Bax-mediated permeabilization of mitochondria, releaseof pro-apoptotic mitochondrial factors such as cyto-chrome c and apoptosis-inducing factor, and the subse-quent activation of procaspases that selectively cleavecertain proteins and results in the apoptotic phenotype.Photodamage to late endosomes/lysosomes can causetheir permeabilization and the release of lysosomal hy-drolases.[33] Depending upon the extent of their releasesuch hydrolases can cause either necrosis or apoptosis.With regards to the latter, apoptosis is mediated by lyso-somal cathepsin-mediated cleavage of the pro-apoptoticprotein Bid, and subsequent permeabilization of mito-chondria by the cleavage product tBid.[33b]

Macroautophagy (hereafter referred to as autophagy)is a process whereby the cytosol and entire organellesbecome encapsulated in a double-membrane cytosolicvesicle termed the autophagosome. Eventual fusion ofthe autophagosome with the lysosome forms an autolyso-some and results in the proteolytic breakdown and recy-cling of what was the autophagosome and its contents.[34]

Autophagy is induced by different types of stress, and ap-pears to play a major role in maintaining cellular homeo-stasis. While autophagy is generally considered to be a sur-

vival pathway, it can also be a death mode if the processbecomes excessive. Recent studies have documented theinduction of autophagy in a variety of cell types followinglight activation of the photosensitizers mTHPC, Pc 4, hy-pericin, NPe6, CPO and BPD.[31] The contribution of au-tophagy to the eventual fate of cells in PDT protocols isvariable. In some situations it appears to support cell sur-vival,[31,35] whereas in others it seems to contribute to celldeath.[31,36]

Although type II PDT reactions produce predominant-ly 1O2, a variety of secondary ROS species including*O2

� , H2O2, and *OH are also generated. Two importantquestions are whether the different ROS species havesimilar effects, and to what extent they contribute to thecytotoxicity observed in PDT protocols. Although anearly study by Weishaupt et al. did not characterize therole of putative secondary ROS species in HPD-sensi-tized cells, trapping studies with 1,3-diphenylisobenzofur-an clearly indicated that rapid removal of 1O2 markedlyattenuated HPD cytotoxicity.[37] Similarly, studies by Ko-chevar et al. demonstrated that the 1O2 generated by pho-toactivation of rose bengal could induce the apoptosis ofcultured cells, whereas radical species derived from rosebengal could not.[38] Interestingly, rose bengal radicals,but not 1O2, were very efficient at inducing lipid peroxida-tion. With regard to a role for *O2

� in PDT, treatmentwith 2-methoxyestradiol has been reported to increasethe efficacy of PDT in tumor-bearing mice, an effect at-tributed to its inhibition of superoxide dismutase

Figure 1. Fluorescence exhibited by five fluorescent probes (5 mM) upon treatment with different reactive species, as described by Setsuki-nai et al.[10] Horseradish peroxidase (10 mg mL�1) was present where specified. The numbers shown represent the mean fluorescence emis-sion intensity upon excitation at 490–510 nm. In four replicate determinations, the variation was less than �3 % of these values. Inset: thesame data plotted on a different scale so as to better illustrate the lower fluorescence intensities.

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Review D. Kessel and J. J. Reiners, Jr.

(SOD).[39] Inhibition of SOD activity would indeed be ex-pected to result in higher levels of *O2

� . However, Frido-vich has demonstrated that 2-methoxyestradiol does pro-mote *O2

� formation but not by a mechanism involvingSOD inhibition.[40] Price et al. have reported the genera-tion of H2O2 following the irradiation of cultures preload-ed with the photosensitizer BPD.[41] In these studies thecytotoxicity of BPD-PDT was enhanced through the in-hibition of catalase using 3-aminotriazole, which facili-tates the accumulation of H2O2. Conversely, the cytotox-icity of BPD-PDT was significantly reduced in cells inwhich peroxisomal levels of catalase had been augment-ed.[41] The mechanism by which PDT induces autophagyis not known. However, several non-PDT studies haveshown that exogenously added *O2

� or H2O2 induces au-tophagy in cultured cells.[42] Detailed mechanistic studiesby Chen have suggested that *O2

� is principally responsi-ble for the induction of autophagy,[42a] but in this report itwas assumed that reduced DCF is specific for hydrogenperoxide and DHE for the superoxide radical. As wasdemonstrated above, this is an oversimplification of theresponse pattern of these probes.

A new photosensitizer termed WST11 has been devel-oped[43] that has the unusual property of producingmainly *O2

� and *OH but not 1O2 upon irradiation.[43b] Ithas been reported that both *O2

� and H2O2 can lead tothe initiation of autophagy.[42] Formation of H2O2 from*O2

� can occur spontaneously and is also catalyzed bySOD. In preliminary studies, we observed that irradiationof WST11-treated murine hepatoma 1c1c7 cells elicitedan autophagic response. This effect can also occur whenthe photosensitizing agent is BPD,[35b] which is known toproduce significant levels of 1O2 upon irradiation.[44] How-ever, while autophagy appears to serve a cytoprotectivefunction when elicited by BPD,[35b] the same effect wasnot observed with WST11. These results will be reportedin detail elsewhere. Although WST11 is currently beingused to target the tumor vasculature,[43] the efficacy of anagent that does not produce significant levels of singletoxygen is a novel result that may suggest new pathwaysfor drug development.

4. Chemistry of Nitric Oxide and RNS

Nitric oxide (nitrogen monoxide, *NO) is a small, nonpo-lar, highly diffusible, and relatively stable free radical. Itis an important signaling molecule and influences a di-verse range of physiological processes including vasodila-tion, neurotransmission, angiogenesis, tumor metastasis,and antimicrobial and antitumor activities. It is generatedby a family of enzymes termed the nitric oxide synthases(NOS). This family includes two constitutively expressedand calcium/calmodulin-dependent isoforms that were in-itially identified in neuronal tissue (nNOS) and endothe-lial cells (eNOS). A third isoform is neither calcium-de-

pendent nor constitutively expressed, but can be inducedby a variety of stressors, and is therefore termed inducibleNOS (iNOS). Isoform expression and tissue activity/con-tent vary among cell types, and can be influenced by a va-riety of factors.

Although reasonably stable for a radical species, nitricoxide is not inert. In particular, it reacts with *O2

� at dif-fusion-controlled rates (~4–16 � 109 M�1 s�1) to formONOO� .[45] This rate is several-fold faster than that ofthe enzymatic dismutation of *O2

� by SOD.[46] Hence,*NO is a far better scavenger of *O2

� than SOD. Be-cause of its reactivity, many of the cytotoxic properties of*NO are often attributed to ONOO� . However, at phys-iological pH a considerable percentage of ONOO� is inequilibrium with peroxynitrous acid (ONOOH; pKa =6.8). Both species can participate in one- and two-elec-tron oxidation reactions with susceptible biomole-cules.[46,47] For example, CO2 readily reacts with ONOO�

to form carbonate (CO3*�) and nitrogen dioxide (*NO2)

radicals, both of which are potent, short-lived oxidants.[47]

In the case of ONOOH, this species can facilitate directtwo-electron oxidation of thiols to produce NO2

� and sul-fenic acid derivatives, which are subsequently convertedto disulfides (e.g., glutathione and cysteine disulfides).Alternatively, ONOOH can undergo homolytic fission togenerate *OH and *NO2 radicals, which play a role inthe initiation of lipid peroxidation. In addition, *NO2 canparticipate in the diffusion-controlled nitration of pro-teins, lipids and DNA.[47]

5. Generation of Nitric Oxide in PDT Protocols

Numerous investigators have used a variety of techniquesto document increased generation or accumulation ofRNS following illumination of cultured cells or tissuesthat had been preloaded with the photosensitizers Photo-frin,[48] 2-butylamino-2-demethoxyhypocrellin B,[49] amino-levulinic acid (ALA),[50] or Verteporfin.[51] The methodsused for assessing RNS generation in the above studiesincluded: 1) monitoring of the fluorescence generated fol-lowing *NO oxidation of 4,5-diaminofluorescein;[49,50a,b] 2)measurement of the *NO metabolites nitrite or nitra-te;[48,50c,51] 3) ESR spin-trapping of *NO;[49] and 4) immu-nological detection of protein-associated nitrotyrosines.[52]

In many of these studies, increased production of RNSwas preceded or accompanied by an increased cytosoliccalcium concentration, which would favor the activationof constitutive NOS isoforms, as well as modest increasesin constitutive NOS isoform content and/or dramatic in-creases in iNOS content.

Both in vitro and in vivo studies suggest that endoge-nously generated nitric oxide may play a protective rolein PDT. Specifically, in vivo suppression of NOS activitieswith the inhibitors N-nitro-l-arginine (l-NNA) or N-nitro-l-arginine methyl ester (l-NAME) markedly in-

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creased Photofrin-PDT-mediated cures of xenograftedtumor lines that constitutively expressed high levels of*NO (i.e., RIF-1 and SCCVII cells), but had only mar-ginal effects on xenografts consisting of tumors cellshaving low levels of *NO (i.e., EMTS and FsaRcells).[48,53] The enhancement of tumor cures by pretreat-ment or cotreatment with NOS inhibitors correlated withreduced xenograft blood flow and increased vascularleakage.[48] Similarly, Reeves et al. reported that pretreat-ment of RIF-1 or EMTS xenograft-bearing mice with l-NNA or l-NAME potentiated ALA-PDT-induceddamage to tumor and normal microvasculature, and alsoincreased macromolecular leakage in the RIF-1 tumors.[54]

Presumably, given the ability of *NO to easily diffuseacross membranes, elevated levels of tumor *NO mayoffset the effects of PDT on tumor vasculature by main-taining sufficient vasodilation to ensure adequate bloodsupply to the tumor. As for the protective effects of *NOon macromolecular leakage, permeabilization of mem-branes often occurs as a consequence of membrane lipidperoxidation. *NO is quite facile at interacting with otherradicals to generate a non�free radical adduct.[46] Ineffect, such radical-radical termination reactions wouldstop radical propagation reactions, such as those occur-ring during membrane lipid peroxidation. Indeed, Nizio-lek et al. have documented the ability of *NO to termi-nate lipid chain peroxidation induced by protoporphyrinIX–PDT in both model membranes and cultured cells.[55]

Although the above studies suggest that *NO may pro-tect tumor vasculature from damage in PDT protocols,additional studies indicate that *NO may also directlyprotect tumor cells. For example, knockdown of iNOS ex-pression with siRNA and inclusion of NOS inhibitors or*NO scavengers have been reported to enhance ALA-PDT-mediated killing of COH-BRI breast cancer cells.[56]

Conversely, supplementation of COH-BRI cultures withnonlethal concentrations of the *NO generator spermineNONOate suppressed ALA-PDT-induced necrosis.[55]

Similarly, Gomes et al. reported that incubation of humanlymphoblastoid cells with *NO donors ((Z)-1-[(2-amino-ethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-dio-late or S-nitroso-N-acetylpenicillamine) dramatically sup-pressed PDT-induced apoptosis when bisulfonated alumi-num phthalocyanine was used as a photosensitizer.[57]

Although there is a body of literature that indicates*NO is protective in PDT protocols, several reports sug-gest the opposite. The approaches employed in theselatter studies assessed the effects of either reducing or in-creasing cellular *NO content on PDT-induced toxicity.With regard to the former, Lu et al. demonstrated thataddition of an NOS inhibitor or *NO scavenger to MCF7cultures suppressed the cytotoxicity of the photosensitizer2-butylamino-2-demethoxyhypocrellin B in PDT proto-cols.[49] As for the latter approach, Yamamoto et al. re-ported that overexpression of iNOS in HEK293T cells(via transfection) markedly elevated basal *NO levels

and enhanced ALA-PDT-mediated cell killing.[50b] A simi-lar potentiation of ALA-PDT-mediated killing was ob-served in RAW264.7 cultures in which cytokines hadbeen used to induce iNOS prior to illumination.[50b] Inthis study, the inclusion of an *NO scavenger suppressedcell killing, thus confirming the contribution of *NO tothe observed cytotoxicity. Although the RNS responsiblefor the cytotoxicity observed in the above studies werenot identified, there is ample experimental precedent forassuming that it was not *NO, but rather peroxynitriteand radicals derived therefrom. Szabo et al. have pub-lished a comprehensive review on how peroxynitrite andits derivative radicals can contribute to the developmentof necrosis and apoptosis.[47]

6. Nitric Oxide and PDT-Induced Autophagy

We are currently unaware of any study that has investi-gated the relationship between PDT-induced increases in*NO and the induction of autophagy. However, the addi-tion of exogenous *NO donors to cultured osteoblasts,[58]

human mammary tumor cell lines,[59] and cardiomyo-cytes[60] resulted in the induction of autophagy. Althoughthe above studies used an exogenous source of *NO, en-dogenously generated *NO may also contribute to the in-duction of autophagy. Yuan et al. reported that pharma-cological inhibition of NOS suppressed LPS and tumornecrosis factor–mediated induction of autophagy in cardi-omyocytes.[60] Given these results and the ability of PDTto induce iNOS, it is conceivable that *NO may contrib-ute to the induction of autophagy in PDT protocols.

7. Summary and Outlook

Reactive oxygen and nitrogen species mediate the photo-toxic effects of photodynamic therapy. Depending on thelocalization of the photosensitizing agent, the cell pheno-type and the reactive species produced, the immediate ef-fects can include apoptosis, autophagy, necrosis or combi-nations thereof. Vascular shutdown can also occur whenin vivo systems are involved. The reactive species that aregenerated upon irradiation of photosensitizers can varyand the effects will depend on the localization site(s) ofthe agent. Apoptosis is a common response and has sever-al advantages, including that the PDT dose needed isoften small since the apoptotic process can be triggeredby a relatively low PDT dose. Autophagy can serve bothas a cytoprotective response and as a death mechanism,depending on several variables. It is not yet entirely clearwhich pathways to cell death are more effective. More-over, the identification of reactive species is not a simplematter, and the information provided by most fluorescentprobes is ambiguous. As new photosensitizing agents aredeveloped, an examination of their localization patterns

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and photochemistry may aid in the selection of optimalagents for tumor eradication.

Acknowledgements

We thank the NIH (CA R01-23378 and ES090302) forsupport of work carried out at Wayne State University.

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Received: June 2, 2011Accepted: March 24, 2012

Published online: July 12, 2012

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Review D. Kessel and J. J. Reiners, Jr.