doi: 10.1111/j.1349-7006.2009.01226.x Cancer Sci | September 2009 | vol. 100 | no. 9 | 1581–1584© 2009 Japanese Cancer Association

Blackwell Publishing AsiaReview Article

Target-selective degradation of cancer-related proteins by novel photosensitizers for molecular-targeted photodynamic therapyKazunobu Toshima,1 Shuho Tanimoto, Kana Tsumura, Kazuo Umezawa and Daisuke Takahashi

Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku Yokohama 223-8522, Japan

(Received April 25, 2009/Revised May 12, 2009/Accepted May 13, 2009/Online publication June 11, 2009)

Proteins are key players in many biological events including cancers.The development of novel photosensitizers for the selectivedegradation of cancer-related proteins has attracted much attentionin the fields of photodynamic therapy for various cancers. In thisreview article, several novel photosensitizers, which selectivelydegrade cancer-related proteins under photoirradiation and mildconditions without any further additives, are introduced. This novelclass of photosensitizers promises bright prospects for findingmolecular-targeted drugs for cancer photodynamic therapy in thenear future. (Cancer Sci 2009; 100: 1581–1584)

Photodynamic therapy (PDT) is used to treat diseasescharacterized by neoplastic growth, including various

cancers.(1,2) Cancer cell death is induced by the photoexcitationof a photosensitizer, generally through the production of singletoxygen. In the absence of light, the photosensitizer is benign, sothe systemic toxicity is relatively low and treatment may berepeated without acquired resistance (Fig. 1). Molecular-targetedphotosensitizers should allow greater advantages than areachievable with conventional photosensitizers as a consequenceof preventing the delocalization of photosensitizers that lead toserious side effects such as light irritation. Proteins are keyplayers in many biological events including cancers. Thedevelopment of new photosensitizers for the selective degradationof only cancer-related proteins is of considerable importance incancer PDT. Thus, the possibility of developing a photosensitizerthat can selectively degrade target proteins upon irradiation by aspecific wavelength of light under mild conditions and withoutany additives (such as metals or reducing agents) has attractedmuch attention. However, there have not been any reports onphotosensitizers that are used for the selective degradation of atarget protein. In this review article, several novel photosensitizersthat selectively degrade only cancer-related proteins underphotoirradiation and mild conditions are introduced. To the bestof our knowledge, this is the first demonstration of the target-selective degradation of proteins by light switching.

2-Phenylquinoline as a novel photosensitizer for protein degradation

Certain 2-phenylquinoline derivatives were found to be efficientagents for DNA photodegradation in our previous study.(3) Basedon these findings, we expected that if a 2-phenylquinolinederivative could be made to produce a radical or a reactiveoxygen species (ROS) by photoexcitation, this could be used forthe degradation not only of DNA, but also of protein molecules.To investigate this hypothesis, we selected 2-phenylquinolineitself as a protein photodegrading agent, and estrogen receptor

(ER)-α as the target protein (Fig. 2). It has been previouslyreported that the 2-phenylquinoline scaffold has some similaritiesto estrogen in terms of its affinity with ER.(4) Furthermore,modulation of the ER-α function is an important factor in avariety of diseases including breast cancer.(5) Therefore, we firstexamined the photoinduced protein-degrading activity of 2-phenylquinoline against ER-α using a long-wavelength UVlamp (365 nm, 100 W) for photoirradiation. The UV light is notharmful to the human body, and we can observe the light withouta pair of glasses for protection. The progress of the photo-degradation reaction was monitored by SDS-PAGE.(6) Theresults showed that 2-phenylquinoline is capable of degrading aprotein, ER-α, upon irradiation by long-wavelength UV lightand without further additives, although its ability is not particularlyhigh. Because degradation of ER-α by 2-phenylquinoline didnot take place in the absence of light, it was confirmed that UVlight functioned as a trigger to initiate protein degradation by 2-phenylquinoline. The pattern obtained for ER-α degradation by2-phenylquinoline from the SDS-PAGE analysis containedfaded and smeared bands, and no aggregation of the protein wasobserved at the top of the gels. Furthermore, no peakscorresponding to degraded peptide fragments were detected byMALDI-TOF MS analysis. These results suggest that thedegradation reaction occurred non-site-specifically and ER-αwas degraded into peptide fragments that were too small toallow for the SDS-PAGE and MALDI-TOF MS analyses.Therefore, it was concluded that the degradation of ER-α by 2-phenylquinoline took place in a random fashion.(7,8)

2-Phenylquinoline–steroid hormone hybrid for ER-a-selective photodegradation

In order to improve the ER-α-degrading ability and selectivityof 2-phenylquinoline, we designed a hybrid molecule consistingof 2-phenylquinoline and estradiol (Fig. 2). Estradiol has a verystrong and selective affinity for ER-α. It was suggested that the2-phenylquinoline–estradiol hybrid had a good affinity withER-α based on computational molecular modeling study usingMolecular Operating Environment software (version 2007.09;Chemical Computing Group, Montreal, QC, Canada). The 2-phenylquinoline–estradiol hybrid was synthesized by an effectivechemical synthetic method using commercially available materials.It was then confirmed that the binding ability of the 2-phenylquinoline–estradiol hybrid against ER-α was comparablewith an agonist, tamoxifen, by a fluorescence polarization method

1To whom correspondence should be addressed. E-mail: toshima@applc.keio.ac.jp

1582 doi: 10.1111/j.1349-7006.2009.01226.x© 2009 Japanese Cancer Association

that measured the capacity of the 2-phenylquinoline–estradiol hybridto displace a high-affinity fluorescent ligand, fluorescentnon-steroid estrogen, from ER-α using the Beacon 2000 Fluor-escence Polarization Instrument (Invitrogen Corp., Carlsbad, CA,USA) with a 490 nm excitation filter and 535 nm emission filter.(9)

With the designed 2-phenylquinoline–estradiol hybrid in hand,we next examined its application for the target-selective photo-degradation of proteins. The photoinduced degradation of threetypes of protein (ER-α, BSA, and hen egg lysozyme [Lyso])was carried out using the 2-phenylquinoline–estradiol hybrid,and the reaction progress was monitored by SDS-PAGE. Theresults showed that when the hybrid was exposed to ER-αduring photoirradiation, significant degradation took place. Thedegradation ability of the 2-phenylquinoline–estradiol hybridwas found to be much greater than that of 2-phenylquinoline.This result was in sharp contrast to those obtained using theother proteins (BSA and Lyso), which showed no degradationor even slight degradation during photoirradiation with the 2-phenylquinoline–estradiol hybrid. Furthermore, it was noteworthythat when ER-α and other proteins were both present in thereaction mixture, only ER-α was degraded by the hybrid. Theseresults clearly indicate that the 2-phenylquinoline–estradiol hybrid

causes selective degradation only of the target protein, ER-α,upon photoirradiation, without any additives and under neutralconditions (Fig. 2).

The ER-α-degrading activity of the 2-phenylquinoline–estradiolhybrid was found to decrease in the presence of other estradiolderivatives that also show strong affinity for ER-α. This resultagain indicates that the strong and selective photodegradingability of the 2-phenylquinoline–estradiol hybrid against ER-αdepends on the high recognition ability of the estradiol moietyfor ER-α. In addition, the activity of the hybrid decreased in thepresence of the O2•

– and H2O2 scavengers Tiron and potassiumiodide (KI). Furthermore, photoirradiation of the hybrid in thepresence of the spin trap 5,5-dimethyl-1-pyrroline-N-oxide(DMPO) gave products with electron spin resonance (ESR)spectrum characteristics of the DMPO–superoxide anion spinadduct DMPO/•OOH and the DMPO–hydroxyl radical spin adductDMPO/•OH, which result from the reaction of DMPO with O2•

–

and •OH respectively.(10,11) Therefore, degradation of the ER-αprotein must be due to ROS produced by the photoexcitation of2-phenylquinoline and O2. The life time of ROS is generally veryshort; therefore, the target selectivity is generated by the locationof the photosensitizer 2-phenylquinoline–estradiol hybrid.

Fig. 1. Principle of photodynamic therapy. ROS, reactive oxygen species.

Fig. 2. Target-selective degradation of estrogenreceptor (ER)-α by a 2-phenylquinoline–steroidhormone hybrid (2) under long-wavelength ofUV photoirradiation, and a complex of the 2-phenylquinoline–steroid hormone hybrid withER-α supported by molecular modeling study.

Toshima et al. Cancer Sci | September 2009 | vol. 100 | no. 9 | 1583© 2009 Japanese Cancer Association

In the present study, we have developed a novel photosensi-tizer for the selective degradation of target and cancer-relatedprotein by photoirradiation using a 2-phenylquinoline–steroidhormone hybrid under neutral conditions.(12) Based on these results,the general strategy, that hybridization of a photosensitizer forproteins with no specificity and a targeted-protein recognitionmolecule make it possible to selectively degrade only targetproteins including cancer-related ones, has been demonstratedfor the first time.

Porphyrin derivative for ER-a-selective photodegradation

Certain porphyrin derivatives are well known to be efficientagents for DNA photodegradation, and have been used for PDTagainst cancer.(13–19) However, there are no reports of methodsusing a light-activated porphyrin for the degradation of proteins.If a porphyrin derivative could be made to produce a radical ora ROS by photoexcitation, we hypothesized that this could beused for the degradation of not only DNA, but also of proteinmolecules. One of the methods for finding the desired novelmolecules is random screening from known and availablecompounds. Therefore, to investigate this hypothesis, we selected10 commercially available porphyrin derivatives as candidatesfor protein photodegrading agents, and BSA as the protein forthe preliminary experiments.

First, we examined the photoinduced protein-degrading activityof the selected porphyrin derivatives against BSA using a long-wavelength UV lamp (365 nm, 100 W) for the photoirradiation.The progress of the photodegradation reaction was monitored bySDS-PAGE. Among them, four porphyrin derivatives were foundto degrade BSA in a dose-dependent manner (3–6 in Fig. 3).These results show, for the first time, that some porphyrin deriv-atives are capable of degrading a protein, BSA, upon irradiationby long-wavelength UV light and without further additives.Because the degradation of BSA by these porphyrin derivativesdid not take place in the absence of light, UV light triggers theinitiation of protein degradation by the porphyrin derivatives. Inaddition, because the SDS gels contained both faded andsmeared bands, the degradation of BSA by the porphyrin deriv-atives apparently occurs in a random fashion.

In order to examine whether visible (VIS) light can initiatethe porphyrin-mediated photodegradation of proteins, we firstmeasured the UV/VIS spectra of the porphyrin derivatives (3–6in Fig. 3). It was found that each porphyrin derivative showedabsorption in both the UV and VIS regions. These resultsprompted us to examine the photodegrading activity of the por-phyrin derivatives under VIS irradiation. It was found thatwhether VIS (diffuse sunlight, 75 W xenon lamp) or UV was used,

the photodegrading abilities of the porphyrin derivatives 3–6were quite similar.

Porphyrin derivative 3 has some similarity to estrogens suchas estradiol, which has a high affinity for ER due to its intraphe-nolic structure and hydrophobicity.(20,21) Therefore, we expectedthat porphyrin derivative 3 might selectively degrade the tran-scription factor ER-α. Indeed, the distance between thehydroxyl group of the A-ring and the 17β-hydroxyl group of theD-ring in estradiol is similar to that of the two phenolichydroxyl groups in porphyrin derivative 3 (Fig. 3). We thenexamined the application of porphyrin derivative 3 for the target-selective photodegradation of proteins. The photoinduced degra-dation of three proteins (ER-α, BSA, and Lyso) was carried outusing porphyrin derivative 3 against each protein under VISirradiation, and the reaction was monitored by SDS-PAGE. Itwas found that when porphyrin derivative 3 was exposed toER-α under photoirradiation, significant degradation took place.The degradation of ER-α by porphyrin derivative 3 was muchgreater (approximately 1000 times) than the degradation ofBSA. The use of catalytic amounts of porphyrin derivative 3was sufficient to effectively degrade ER-α. This result is insharp contrast to the other proteins (BSA and Lyso), whichshowed no degradation under the same conditions. Furthermore,it is noteworthy that when ER-α and other proteins were bothpresent in the reaction mixture, only ER-α was degraded by por-phyrin derivative 3. These results clearly indicate that porphyrinderivative 3 causes selective degradation of only the target pro-tein, ER-α, upon VIS photoirradiation, in the absence of anyadditives and under neutral conditions (Fig. 4).

ER-α degradation by porphyrin derivative 3 was found to sig-nificantly decrease in the presence of 17α-ethynylestradiol,(22)

which also shows a strong affinity for ER-α. Furthermore, por-phyrin derivatives 4 and 6, which have a bulky substituent ortwo hydroxyl groups at the phenolic moiety, respectively, didnot photodegrade ER-α with high efficiency. These results indi-cate that the strong and selective photodegradation ability ofporphyrin derivative 3 toward ER-α depends on the high degreeof recognition of ER-α by porphyrin derivative 3. A scavengerassay was conducted in order to investigate the mechanismunderlying this photodegradation reaction. The photodegradingactivity of porphyrin derivative 3 significantly decreased in thepresence of the HO•, H2O2, and the 1O2 scavengers DMSO, KI,and histidine. Furthermore, the photoirradiation of porphyrinderivative 3 in the presence of the spin trap DMPO and 2,2,6,6-tetramethylpiperidin-4-one (4-oxo-TEMP) gave products withESR spectrum characteristics of the DMPO–hydroxyl radicalspin adduct DMPO/•OH and the 4-oxo-TEMP-N-oxidized spinproduct 4-oxo-TEMPO, which result from the reaction of DMPO

Fig. 3. (a) Protein-photodegrading porphyrin derivatives discovered by random screening. (b) Structural similarity of porphyrin derivative 3 andestradiol.

1584 doi: 10.1111/j.1349-7006.2009.01226.x© 2009 Japanese Cancer Association

with •OH and 4-oxo-TEMP with 1O2 respectively. Therefore, thedegradation of ER-α must be due to ROS produced by thephotoexcitation of porphyrin and O2.

In present study, we have developed a new method for selec-tively degrading a target protein, ER-α, by not only UV photoir-radiation, but also by VIS photoirradiation using a porphyrinderivative.(23) Because VIS is more benign than UV light towardthe human body, and the transmission efficacy of VIS to biotis-sues is higher than that of UV light, this molecular-targetedphotosensitizer should find alternative applications in molecular-targeted PDT.

Summary

In conventional PDT, photosensitizers show no specificityagainst target biomacromolecules (specific DNA, proteins, oroligosaccharides). As the result, light irritation caused by aphotosensitizer located on an undesired part of the human bodyis a serious issue, and a patient treated with PDT may have to

stay in the dark for a long time. Thus, PDT is now not verypopular for cancer therapy. To overcome this problem, thedevelopment of molecular-targeted photosensitizers is indispens-able, which makes it possible to kill only cancer cells in completetime- and space-control manners without any side effects usinga molecular-targeted photosensitizer with a specific wavelengthof light. From these points of view, although the result presentedhere is preliminary, this is the first demonstration of molecular-targeted photosensitizers and should open a new and innovativearea of molecular-targeted PDT for various cancers in the nextgeneration.(24-26)

Acknowledgments

This research was supported in part by the 21st Century COE Program‘Keio Life-Conjugated Chemistry’, High-Tech Research Center Projectfor Private Universities: Matching Fund Subsidy, 2006–2011, andScientific Research (B) (no. 20310140) from the Ministry of Education,Culture, Sports, Science, and Technology of Japan (MEXT).

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Fig. 4. Target-selective degradation of estrogen receptor (ER)-α by porphyrin derivative 3 under visible (VIS) photoirradiation.