cancers

Article Photodynamic Synergistic Effect of Pheophorbide a and Doxorubicin in Combined Treatment against Tumoral Cells

Rubén Ruiz-González 1, Paula Milán 2, Roger Bresolí-Obach 1, Juan Carlos Stockert 2, Angeles Villanueva 2, Magdalena Cañete 2,* and Santi Nonell 1,*

1 Institut Químic de SarriÒ, Universitat Ramon Llull, Via Augusta 390, 08017 Barcelona, Spain; [email protected] (R.R.-G.); [email protected] (R.B.-O.) 2 Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, Darwin 2, 28049 Cantoblanco-Madrid, Spain; [email protected] (P.M.); [email protected] (J.C.S.); [email protected] (A.V.) * Correspondence: [email protected] (M.C.); [email protected] (S.N.); Tel.: +34-914-976-256 (M.C.); +34-932-672-028 (S.N.)

Academic Editor: Michael R. Hamblin Received: 8 November 2016; Accepted: 11 February 2017; Published: 17 February 2017

Abstract: A combination of therapies to treat cancer malignancies is at the forefront of research with the aim to reduce drug doses (ultimately side effects) and diminish the possibility of resistance emergence given the multitarget strategy. With this goal in mind, in the present study, we report the combination between the chemotherapeutic drug doxorubicin (DOXO) and the photosensitizing agent pheophorbide a (PhA) to inactivate HeLa cells. Photophysical studies revealed that DOXO can quench the excited states of PhA, detracting from its photosensitizing ability. DOXO can itself photosensitize the production of singlet oxygen; however, this is largely suppressed when bound to DNA. Photodynamic treatments of cells incubated with DOXO and PhA led to different outcomes depending on the concentrations and administration protocols, ranging from antagonistic to synergic for the same concentrations. Taken together, the results indicate that an appropriate combination of DOXO with PhA and red light may produce improved cytotoxicity with a smaller dose of the chemotherapeutic drug, as a result of the different subcellular localization, targets and mode of action of the two agents.

Keywords: photodynamic therapy; doxorubicin; pheophorbide a; synergic treatment; HeLa cells

1. Introduction Chemotherapy is the most common strategy applied to treat cancer, with up to 90 types of chemotherapy drugs already approved [1]. It is less invasive than surgery or radiotherapy, so the overall negative impact on the patient is smaller. This type of therapy is very efficient in killing tumor cells and preventing metastasis. Despite these facts, resistance to chemotherapy is the major pitfall that exists in this therapeutic approach. Resistance may be inherent, i.e., from the beginning of treatment, or acquired, appearing after a partial response to the drug. Some of the mechanisms by which resistance to chemotherapy develops include exclusion of the drug from the tumor cells, failure in the activation of the pro-drug to its active form, increased detoxification, altered drug targeting, increased tumor cell repair after injury or escape of the apoptotic response [2,3]. These processes can be caused by the expression of efflux proteins that decrease intracellular levels of the drug (i.e., P-glycoprotein, MRP1), induction of genes encoding anti-apoptotic proteins (i.e., Bcl-2, Bcl-XL), increased DNA repair or the regulation of the expression of biotransformation enzymes [4].

Cancers 2017, 9, 18; doi:10.3390/cancers9020018 www.mdpi.com/journal/cancers Cancers 2017, 9, 18 2 of 18

On the other hand, photodynamic therapy (PDT) is a type of non-invasive treatment that involves the use of light-active agents named photosensitizers (PSs), which produce reactive oxygen species (ROS) upon illumination with light of a specific wavelength, causing unspecific cell damage and ultimately killing the cancer cells [5,6]. Among its advantages, PDT enables treating tumors that cannot be removed by surgery, can be applied to vulnerable patients provided its minimal side effects and may help overcome cancer drug resistance [7,8]. Of special interest, and unlike chemotherapeutic drugs, the PS is regenerated in most cases after ROS are produced, which enables it to participate in several therapeutic cycles. The removal of the tumor via PDT depends on the type of PS, its concentration and location, the irradiation wavelength and light dose, the oxygenation levels and the type of ROS generated [6]. From a mechanistic point of view, the action of the initially-formed ROS is localized in the vicinity of the site where the PS has accumulated. Oxidation of biomolecules, such as lipids, amino acids and proteins, can cause irreversible damage to organelles and cell destruction [9]. Cell death can occur via apoptosis, necrosis or autophagy, the final mechanistic outcome being dictated by intracellular PS accumulation [10–12]. Most PSs are capable of accumulating in tumor tissue; however, they can also be targeted to the microvessels of the neovasculature that nourishes it (vascular PDT) causing additionally direct and indirect effect to the tumor microenvironment [13–15]. Moreover, their photodynamic action also helps to activate the immune system, which can further stimulate tumor suppression (immuno PDT) [16–19]. Provided that PDT-mediated immune reactions are likely involved in final tumor eradication, combinations of PDT with immunotherapy have been explored, including cytokine therapy, microbial adjuvants or antibodies (photoimmunotherapy (PIT)) [20]. Recent approaches in anticancer therapies include the application of combined chemotherapeutic drugs, delivered together in order to reduce the individual toxicity of each drug (and ultimately side effects) and decrease the likelihood of generating resistance [21,22]. The combination of different anticancer therapies is also being actively explored [23,24] as different cell signaling pathways are simultaneously activated. Hence, tumor cells are destroyed in a more efficient manner by the additive or synergistic effect of both treatments, allowing a reduction in the dose of the most toxic therapeutic agent [23,24]. The combination of a chemotherapeutic drug with a photosensitizing agent is an increasingly growing area of study in vitro, in vivo or even in clinical trials [25]. Several studies have been addressed to evaluate proposed combinations in cell culture models. One of the first ones was conducted by Peterson et al. and evaluated the interaction between doxorubicin and meso-chlorin e6 monoethylene diamine (Mce6) against human epithelial ovarian carcinoma, showing additive or synergic effects depending on the dose at which each of the therapeutic agents was administered [26]. Datta et al. [27] studied the photodynamic effect of 5-aminolevulinic acid (ALA) in combination with mitomycin C on J82 bladder cell lines, either regular or mitomycin resistant. In this scenario, results on cell viability indicated a higher sensitivity to PDT for the mitomycin-sensitive cell line. Moreover, the combination treatment resulted in an enhanced effect when the chemotherapeutic agent was given first both for parental and mitomycin-sensitive cells [27]. Another study was the application of cisplatin and porfimer sodium (HpD) to L5178Y lymphoma cells where the authors described a marked synergistic effect and increased apoptosis death [28]. In the same line, low doses of cisplatin or gemcitabine in combination with HpD-based or indocyanine-green-based PDT achieved an additive or synergic effect in lung and breast cancer cells, respectively [29,30]. These positive results were complemented by mechanistic evaluation of the treatment responses. Combining sodium talaporfin with oxaliplatin, cis-diaminodichloroplatinum or gemcitabine to inactivate NOZ gallbladder carcinoma cells, there appears in all cases a synergistic result in the combined treatments with respect to the inactivation produced by each of the drugs separately [31]. These treatments not only significantly increase cell inactivation, but also reduce the concentrations of PS and, more importantly, the antineoplastic agent as described in HpD and cisplatin treatments in mouse cancer cells [32]. The use of ALA in combination treatments with doxorubicin or vincristine in murine leukemic cells does not always produce an additive effect, but avoids the multiresistance Cancers 2017, 9, 18 3 of 18

Cancers 2017, 9, 18 3 of 18 of cells to treatments [33]. However, in some combinations of both therapies, there may be produced synergic,not a synergic, but antagonistic but antagonistic effect, as effect, described as described for erlotinib for or erlotinib cetuximab or when cetuximab applied when in combination applied in withcombination PDT using with PDTmeso-tetraphenylporphine using meso-tetraphenylporphine [34]. With [34 ].the With aim the aimto achieve to achieve better better selectivity, selectivity, approaches combining combining chemotherapy chemotherapy with with PIT PIT have have also also been been conducted. conducted. Combination Combination effects effects have beenhave beenassessed assessed in human in human ovarian ovarian cancer cancer cell cell line liness for for the the addition addition of of chemotherapeutic agents (cisplatin or 2,5-bis(5-hydroxymethyl-2-thienyl)furan2,5-bis(5-hydroxymethyl-2-thienyl)furan (SOS (SOS thiophene) thiophene) and and chlorin chlorin PSs linked to antibody fragments against different carcinoma antigens with promising results [[35,36].35,36]. In the same line, Rizvi et al.al. achieved the reductionreduction ofof chemotherapeuticchemotherapeutic cycles when including EGFR-targeted Mce6 PITPIT for for an an ovarian ovarian carcinoma carcinoma murine model [37]. [37]. In the present present study, we have analyzed the effect on the cell survival survival of of a a photosensitizing photosensitizing compound (pheophorbide a (PhA)) in combination with the traditional antineoplastic agent agent doxorubicin doxorubicin (DOXO) (DOXO) (Figure 11).). PhAPhA isis aa photosensitive photosensitive chlorophyllchlorophyll metabolitemetabolite withwith immunostimulationimmunostimulation activityactivity [[38],38], which atat adequateadequate concentrations concentrations causes causes the the apoptosis apoptosis of tumor of tumor cells [cells39]. Despite[39]. Despite its negligible its negligible toxicity toxicityto healthy to cellshealthy in the cells dark, in the exposure dark, toexposure light elicits to light apoptosis, elicits apoptosis, cell cycle arrest cell cycle at sub-G1, arrest abolition at sub-G1, of abolitionantiapoptotic of antiapoptotic protein Bcl-2, protein cytochrome-C Bcl-2, releasecytochrome-C to the cytosol release and to activationthe cytosol of procaspasesand activation 3 and of procaspases9 [40]. PhA has 3 and been 9 shown[40]. PhA to inducehas been apoptosis shown into Jurkatinduce leukemia apoptosis cells in [Jurkat41], human leukemia hepatocellular cells [41], humancarcinoma hepatocellular [42] or uterine carcinoma carcinosarcomas [42] or uterine [43] in photodynamiccarcinosarcomas treatments. [43] in photodynamic On the chemotherapeutic treatments. Onside, the DOXO chemotherapeutic is a nonselective side, anthracycline DOXO is antibiotic a nonsel classective I whose anthracycline mechanism antibiotic of action isclass the inhibitionI whose mechanismof enzymes responsibleof action is forthe DNA inhibition replication. of enzymes This drug responsible intercalates for inDNA the DNAreplication. double This helix drug and intercalatesinterferes with in the topoisomerases DNA double I andhelix II, and thereby interferes preventing with topoisomerases the binding of the I and DNA II, strandsthereby andpreventing leading theto cell binding death of [44 the]. DOXO DNA strands is often usedand leading in the treatment to cell death of solid [44]. tumors; DOXO however,is often used it has in significant the treatment side ofeffects, solid suchtumors; as cardiomyopathies however, it has significant [45]. side effects, such as cardiomyopathies [45].

Figure 1. Structures of the active compounds studied in this work.

2. Results A potential problem arising from the combination of a PS and a chemotherapeutic drug is the interaction between the the two, two, which which could could result result in in the the quenching quenching of of the the excited excited states states of of the the PS, PS, thereby detracting from its photodynamic activity. This was assessed by a series of photophysical experiments described below.

2.1. Photophysical Studies

2.1.1. Absorption and Fluorescence As a first first approach, we measured the absorption and fluorescence fluorescence spectra of solutions of DOXO and PhA alone in two different solvents, namely et ethanolhanol (EtOH) and water, taken as simple models of the the cell cell membrane membrane and and the the cytoplasm, cytoplasm, respective respectivelyly (Figure (Figure 2).2 ).In InEtOH, EtOH, both both the theabsorption absorption and fluorescenceand fluorescence spectra spectra showshow narrow narrow structured structured bands, bands, and the and fluorescence the fluorescence excitation excitation spectra spectra match thematch corresponding the corresponding absorption absorption spectra (Figure spectra 2a,b). (Figure These2a,b). observations These observations indicate that indicate both compounds that both existcompounds as monomers. exist as monomers.In contrast, In the contrast, absorption the absorption spectra in spectra water in (Figure water (Figure 2c) show2c) show broader broader and non-structured bands, red-shifted in the case of DOXO and blue-shifted for PhA (Table 1). Notwithstanding, the fluorescence emission (Figure 2d) and excitation (Figure 2c, inset) spectra are very similar to those recorded in EtOH, although much weaker in intensity, particularly for PhA.

Cancers 2017, 9, 18 4 of 18 and non-structured bands, red-shifted in the case of DOXO and blue-shifted for PhA (Table1).

Notwithstanding,Cancers 2017, 9, 18 the fluorescence emission (Figure2d) and excitation (Figure2c, inset) spectra4 of 18 areCancers very 2017 similar, 9, 18 to those recorded in EtOH, although much weaker in intensity, particularly for4 PhA. of 18 TheseThese findingsfindings indicateindicate thatthat both compounds exist as a mixture of fluorescentfluorescent monomers and essentially non-fluorescentnon-fluorescentThese findings indicate aggregates that inboth aqueous aqueous compounds solutions solutions exist (J-type (J-type as a mixture in in the the case of case fluorescent of of DOXO DOXO monomersand and H-type H-type and PhA). PhA). essentially non-fluorescent aggregates in aqueous solutions (J-type in the case of DOXO and H-type PhA).

Figure 2. Absorption (a,c) and fluorescence (b,d) spectra of DOXO (green) and PhA (red) in EtOH (a,b) Figure 2. Absorption (a,c) and fluorescence (b,d) spectra of DOXO (green) and PhA (red) in EtOH (a,b) andFigure water 2. Absorption (c,d). DOXO (a, cwas) and excited fluorescence at 455 (nmb,d )and spectra PhA of at DOXO 610 nm. (green) Insets: and (a ,PhAc) excitation (red) in EtOHspectra (a ,ofb) and water (c,d). DOXO was excited at 455 nm and PhA at 610 nm. Insets: (a,c) excitation spectra of the and water (c,d). DOXO was excited at 455 nm and PhA at 610 nm. Insets: (a,c) excitation spectra of fluorescencethe fluorescence at 705 at nm705 fornm PhAfor PhA and and 610 nm610 fornmDOXO. for DOXO. the fluorescence at 705 nm for PhA and 610 nm for DOXO. Figure 3 shows the fluorescence decays for DOXO (green traces) and PhA (red traces). In EtOH, DOXOFigureFigure decays3 3 shows shows with the athe biexponential fluorescence fluorescence decaysfunction decays for for with DOXO DOXO time (green (greenconstants traces) traces) of and1.4 and ns PhA PhA (93% (red (red weight) traces). traces). and In In EtOH, EtOH,4.3 ns DOXODOXO decaysdecays withwith aa biexponentialbiexponential functionfunction withwith timetime constantsconstants ofof 1.41.4 nsns (93%(93% weight)weight) andand 4.34.3 nsns (7% weight). In D2O, however, only one lifetime (4.0 ns) was needed to adequately fit the decay. On (7%the(7% other weight). weight). hand, In In D DPhA2O,2O, however, showshowever, a onlymonoexponential only one one lifetime lifetime (4.0 decay(4.0 ns) ns) wasin was EtOH needed needed with to adequatelytothe adequately lifetime fit of thefit 5.9 the decay. ns, decay. whereas On theOn otherthe other hand, hand, PhA PhA shows shows a monoexponential a monoexponential decay decay in EtOH in EtOH with with the lifetimethe lifetime of 5.9 ofns, 5.9whereas ns, whereas the the weak emission in D2O showed two components with lifetimes of 4.8 ns (61% weight) and 2.6 ns weak(31%the weak weight). emission emission in D in2O D showed2O showed two two components components with with lifetimes lifetime ofs of 4.8 4.8 ns ns (61% (61% weight) weight) and and 2.62.6 nsns (31%(31% weight).weight).

Figure 3. Time-resolved fluorescence (TRF) decays for DOXO (green) and PhA (red) in EtOH (a) and Figure 3. Time-resolved fluorescence (TRF) decays for DOXO (green) and PhA (red) in EtOH (a) and FigureD2O (b 3.). GreyTime-resolved traces represent fluorescence the instrument (TRF) decays respon forse DOXO function. (green) DOXO and was PhA excited (red) in at EtOH 504 nm (a) andand D2O (b). Grey traces represent the instrument response function. DOXO was excited at 504 nm and DPhA2O( atb ).654 Grey nm. traces Observation represent wavelengths the instrument were response660 nm and function. 710 nm, DOXO respectively. was excited at 504 nm and PhAPhA atat 654654 nm.nm. ObservationObservation wavelengthswavelengths werewere 660660 nmnm andand 710710 nm,nm, respectively.respectively.

Cancers 2017, 9, 18 5 of 18

Cancers 2017, 9, 18 5 of 18 Table 1. Optical and photochemical properties of the compounds studied in this work. Table 1. Optical and photochemical properties of the compounds studied in this work. a b c Compound Solvent λAbs/nm λFluo/nm τF1/ns τF2/ns Φ∆ Compound Solvent λAbs/nm a λFluo/nm b τF1/ns c τF2/ns ΦΔ d,e EtOH 497 582 1.4 (0.93) 4.3 (0.07) d,e 0.03 DOXO EtOH 497 582 1.4 (0.93) 4.3 (0.07) 0.03 d,e DOXOD 2O 489 590 4.0 - 0.01 D2O 489 590 4.0 - 0.01 d,e EtOH 668 674 5.9 - 0.61 d PhA EtOH 668 674 5.9 - 0.61 d d PhA D2O 682 666 4.8 (0.61) 2.6 (0.39) 0.04 D2O 682 666 4.8 (0.61) 2.6 (0.39) 0.04 d a Maximum of intensity for DOXO and maximum of the lowest energy absorption band for PhA; b maxima of the a Maximum of intensity forc DOXO and maximum of the lowest energy dabsorption bande for PhA; most intense fluorescence band; values in brackets are the fractional amplitudes; λexc = 532 nm; λexc = 473 nm. b maxima of the most intense fluorescence band; c values in brackets are the fractional amplitudes; d λexc = 532 nm; e λexc = 473 nm. In separate experiments, we recorded the absorption and fluorescence spectra and the fluorescence decay ofIn aqueous separate and experiments, ethanolic solutions we recorded of PhA the in absorption the presence and of 1fluorescence mM DOXO spectra (Figure and4). Inthe both fluorescence decay of aqueous and ethanolic solutions of PhA in the presence of 1 mM DOXO solvents, the absorption bands of PhA broadened and decreased their amplitude (Figure4a,b). On the (Figure 4). In both solvents, the absorption bands of PhA broadened and decreased their amplitude other hand, the shape of the fluorescence spectra and the decay lifetime were unaffected by the (Figure 4a,b). On the other hand, the shape of the fluorescence spectra and the decay lifetime were additionunaffected of DOXO, by the butaddition the fluorescenceof DOXO, but intensitythe fluorescence decreased intensity by approximatelydecreased by approximately 40% in EtOH 40%and almostin EtOH completely and almost in water completely (Figure in4c–f). water These (Figure observations 4c–f). Theseindicate observations that theindicate PhA that and the DOXO PhA and form a ground-stateDOXO form complex a ground-state that causes complex static that quenching causes static of the quenching PhA singlet of the excited PhA singlet state. excited state.

Figure 4. Absorption spectra (a,b), fluorescence spectra (c,d) and fluorescence decay (e,f) of PhA Figure 4. Absorption spectra (a,b), fluorescence spectra (c,d) and fluorescence decay (e,f) of PhA 25 µM 25 μM alone (red solid lines) and in the presence of 1 mM DOXO (dashed violet lines) in EtOH (left alone (red solid lines) and in the presence of 1 mM DOXO (dashed violet lines) in EtOH (left panels) and panels) and H2O (right panels) solutions. For TRF measurements, λexc = 654 nm and λobs = 750 nm. H O (right panels) solutions. For TRF measurements, λ = 654 nm and λ = 750 nm. Grey dashed 2 Grey dashed traces correspond to the instrument responseexc function. obs traces correspond to the instrument response function. 2.1.2. Singlet Oxygen Photosensitization by Doxorubicin 2.1.2. Singlet Oxygen Photosensitization by Doxorubicin The strong fluorescence of DOXO suggests that it might generate ROS upon exposure to light. ItsThe capacity strong to fluorescence photosensitize of 1 DOXOO2 was suggestsassessed in that D2O it and might EtOH generate by time-resolved ROS upon detection exposure of tothe light. 1 1 1 Its capacitynear-infrared to photosensitize O2 phosphorescenceO2 was at 1275 assessed nm. The inD lumi2Onescence and EtOH observed by time-resolved is assigned to detection O2 because of the 1 1 1 near-infraredthe transientO 2is phosphorescencecompletely quenched at 1275 in presence nm. The of luminescencesodium azide (NaN observed3, a well-known is assigned O to2 quencher;O2 because 1 the transient is completely quenched in presence of sodium azide (NaN3, a well-known O2 quencher; Cancers 2017, 9, 18 6 of 18

Cancers 2017, 9, 18 6 of 18 1 25 mM) [46]. As shown in Figure5, DOXO does indeed generate 1 O2 with quantum yield Φ∆ = 0.03 in Cancers25 mM) 2017 [46]., 9, 18As shown in Figure 5, DOXO does indeed generate O2 with quantum yield ΦΔ = 0.036 of in18 EtOHEtOH and Φand∆ =Φ 0.01∆ = 0.01 in Din2 DO,2O, irrespective irrespective ofof thethe excitationexcitation wavelength. wavelength. The The decrease decrease of Φ ofΔ inΦ ∆Din2O Dis2 O is consistent25consistent mM) with [46]. with the As the occurrenceshown occurrence in Figure of of aggregation 5,aggregation DOXO does in in thisindeedthis solvent. generate 1O2 with quantum yield ΦΔ = 0.03 in EtOH and Φ∆ = 0.01 in D2O, irrespective of the excitation wavelength. The decrease of ΦΔ in D2O is consistent with the occurrence of aggregation in this solvent.

Figure 5. Singlet oxygen phosphorescence kinetics at 1275 nm in EtOH (a,c) and D2O (b,d) solutions Figure 5. Singlet oxygen phosphorescence kinetics at 1275 nm in EtOH (a,c) and D O(b,d) solutions of DOXO (green), PhA (red) and reference photosensitizers (PSs) (brown). The signals2 completely of DOXO (green), PhA (red) and reference photosensitizers1 (PSs) (brown). The signals completely Figuredisappeared 5. Singlet in the oxygen presence phosphorescence of 25 mM NaN ki3, neticsa typical at 1275O2 quencher nm in EtOH (grey). (a, cRose) and Bengal D2O (b (RB),d) solutions in EtOH disappeared in the presence of 25 mM NaN , a typical 1O quencher (grey). Rose Bengal (RB) in EtOH ofand DOXO flavin (green),mononucleotide PhA (red) (FMN) and reference in D2O3 were photosen usedsitizers as 2references. (PSs) (brown). The excitation The signals wavelength completely was and flavin mononucleotide (FMN) in D O were used as references. The excitation wavelength was 532 disappeared532 nm for (a in,b )the and presence 473 nm offor 25 (c mM,d).2 NaN3, a typical 1O2 quencher (grey). Rose Bengal (RB) in EtOH a b c d nm forand ( ,flavin) and mononucleotide 473 nm for ( ,(FMN)). in D2O were used as references. The excitation wavelength was 2.1.3.532 Interaction nm for (a ,betweenb) and 473 Doxorubicin nm for (c,d). and DNA 2.1.3. InteractionDOXO is between well known Doxorubicin to localize and in DNA the nucleus where it binds to DNA. Spectral and 2.1.3. Interaction between Doxorubicin and DNA DOXOphotophysical is well consequences known to localize of DNA in the binding nucleus are where shown it in binds Figure to DNA.6: (i) the Spectral absorption and photophysicalspectrum consequencesappearsDOXO more of is DNAstructured well bindingknown (Figure to are localize6a); shown (ii) thein in fluorethe Figure nucleusscence6: (i) iswhere thestrongly absorption it bindsquenched to spectrum DNA. (Figure Spectral 6b); appears (iii) andthe more 1 photophysicalamount of O2 producedconsequences is strongly of DNA reduced binding (Figure are sh 6c);own and in Figure(iv) the 6: triplet (i) the lifetime absorption is extended, spectrum as 1 structured (Figure6a); (ii) the fluorescence is strongly quenched (Figure6b); (iii) the amount of O2 appearsdemonstrated more structuredby the slower (Figure rise 6a); of (ii) the the 1 Ofluore2 phosphorescencescence is strongly signal. quenched On the (Figure other 6b); hand, (iii) the produced is strongly reduced (Figure6c); and (iv) the triplet lifetime is extended, as demonstrated by amountfluorescence of 1O lifetime2 produced does is not strongly change reduced (Figure 6d).(Figure These 6c); observations and (iv) the are triplet consistent lifetime with is extended, strong static as the slower rise of the 1O phosphorescence signal. On the other hand, the fluorescence lifetime does demonstratedquenching of the by DOXO the2 slower excited rise states of andthe shielding1O2 phosphorescence from oxygen uponsignal. binding On the to DNA.other hand, the not changefluorescence (Figure lifetime6d).These does not observations change (Figure are 6d). consistent These observations with strong are static consistent quenching with strong of the static DOXO excitedquenching states and of the shielding DOXO excited from oxygen states and upon shielding binding from to oxygen DNA. upon binding to DNA.

Figure 6. Spectroscopic and photophysical properties of DOXO in D2O solutions in the absence (green traces) and presence (blue traces) of 60 μg/mL DNA: absorption spectra (a); fluorescence emission

Figurespectra 6. (b Spectroscopic); singlet oxygen and phosphorescencephotophysical properties kinetics ofat DOXO 1275 nm in Dupon2O solutions excitation in atthe 473 absence nm ( c(green); and Figure 6. Spectroscopic and photophysical properties of DOXO in D O solutions in the absence traces)fluorescence and presence kinetics upon(blue excitationtraces) of at60 504μg/mL nm andDNA: observation absorption at spectra660 nm 2((ad);). fluorescence emission µ (green spectra traces) (b); and singlet presence oxygen (bluephosphorescence traces) of 60kineticsg/mL at 1275 DNA: nm absorptionupon excitation spectra at 473 ( anm); fluorescence(c); and emissionfluorescence spectra (kineticsb); singlet upon oxygen excitation phosphorescence at 504 nm and observation kinetics at at 1275 660 nmnm upon(d). excitation at 473 nm (c); and fluorescence kinetics upon excitation at 504 nm and observation at 660 nm (d). Cancers 2017, 9, 18 7 of 18

Cancers 2017, 9, 18 7 of 18 2.2. SubcellularCancers 2017, 9, Localization 18 Studies 7 of 18 Cancers2.2. Subcellular 2017, 9, 18 Localization Studies 7 of 18 2.2.Figure Subcellular7 shows Localization fluorescence Studies microscopy images of HeLa cells incubated with 0.2 µM DOXO Figure 7 shows fluorescence microscopy images of HeLa cells incubated with 0.2 μM DOXO and and observed immediately after 24 h of incubation. In (a), fluorescence arising from DOXO is 2.2.observed SubcellularFigure immediately 7 shows Localization fluorescence after Studies 24 h microscopy of incubation. images In (a), of HeLafluorescence cells incubated arising withfrom 0.2 DOXO μM DOXO is imaged and imaged upon green light excitation. In (b), cell’s autofluorescence spread over the cytoplasm upon observeduponFigure green immediately 7light shows excitation. fluorescence after In24 (b), h microscopy of cell’s incubation. autofluo images Inrescence (a), of HeLafluorescence spread cells overincubated arising the cytoplasm withfrom 0.2 DOXO μ uponM DOXO is UV-light imaged and UV-light irradiation is observed for the same set of cells. Finally, (c) is an overlap of previous panels, observeduponirradiation green immediately is light observed excitation. afterfor the In24 same(b),h of cell’s incubation.set ofautofluo cells. Finally,Inrescence (a), fluorescence (c) spread is an overoverlap arising the ofcytoplasm previousfrom DOXO upon panels, is UV-light imaged which which unambiguously allows localizing DOXO in the cell nucleus. uponirradiationunambiguously green islight observed allows excitation. localizingfor the In (b),same DOXO cell’s set ofautofluo in cells. the cell Finally,rescence nucleus. (c) spread is an overoverlap the ofcytoplasm previous upon panels, UV-light which irradiationunambiguously is observed allows forlocalizing the same DOXO set of in cells. the cell Finally, nucleus. (c) is an overlap of previous panels, which unambiguously allows localizing DOXO in the cell nucleus.

Figure 7. HeLa cells treated with 0.2 μM DOXO for 24 h and observed under fluorescence microscope. Figure 7. HeLa cells treated with 0.2 µM DOXO for 24 h and observed under fluorescence Figure(a) Cells 7. HeLaobserved cells treatedunder withgreen 0.2 excitation; μM DOXO ( forb) 24the h andsame observed field observed under fluorescence under UV microscope. excitation; microscope. (a) Cells observed under green excitation; (b) the same field observed under UV excitation; (ac) overlappingCells observed images under from green (a) and excitation; (b). Scale bar:(b) 10the μ M.same field observed under UV excitation; (c) overlappingFigure 7. HeLa images cells treated from ( witha) and 0.2 ( μbM). ScaleDOXO bar: for 24 10 hµ andM. observed under fluorescence microscope. ((ac) overlappingCells observed images under from green (a) and excitation; (b). Scale bar:(b) 10the μ M.same field observed under UV excitation; Fluorescence images of HeLa cells incubated for 4 h with 2 μM PhA are depicted in Figure 8. In Fluorescence(c) overlapping images images of from HeLa (a) and cells (b). incubated Scale bar: 10 for μM. 4 h with 2 µM PhA are depicted in Figure8. (a), mitochondriaFluorescence autofluorescenceimages of HeLa cells is observed incubated up foron 4UV h withexcitation. 2 μM PhA(b) shows are depicted the PhA in fluorescence Figure 8. In In (a), mitochondria autofluorescence is observed upon UV excitation. (b) shows the PhA fluorescence (a),distributed mitochondriaFluorescence in granules autofluorescenceimages of ofvariable HeLa cellssize is observed incubatedthrough upthe foron cytoplasm. 4UV h withexcitation. 2 TheμM superposition PhA(b) shows are depicted the ofPhA the in fluorescence Figuretwo images 8. In distributed in granules of variable size through the cytoplasm. The superposition of the two images (c) (a),distributed(c) rulesmitochondria out in mitochondrial granules autofluorescence of variable localization sizeis observed throughin HeLa up cells.theon cytoplasm. UV excitation. The superposition(b) shows the ofPhA the fluorescence two images rulesdistributed(c) out rules mitochondrial out in mitochondrial granules localization of variable localization size in HeLa throughin HeLa cells. thecells. cytoplasm. The superposition of the two images (c) rules out mitochondrial localization in HeLa cells.

Figure 8. HeLa cells treated with 2 μM PhA for 4 h observed in fluorescence microscopy. (a) Cells Figureobserved 8. HeLaunder cellsgreen treated light excitation;with 2 μM ( bPhA) the for same 4 h fieldobserved observed in fluorescence under UV light;microscopy. (c) overlapping (a) Cells Figure 8. HeLa cells treated with 2 µM PhA for 4 h observed in fluorescence microscopy. (a) Cells observedimages from under (a) andgreen (b ).light Scale excitation; bar: 10 μ M.(b) the same field observed under UV light; (c) overlapping observedFigure under8. HeLa green cells lighttreated excitation; with 2 μM (b PhA) the for same 4 h fieldobserved observed in fluorescence under UV microscopy. light; (c) overlapping(a) Cells observedimages from under (a) andgreen (b ).light Scale excitation; bar: 10 μ M.(b) the same field observed under UV light; (c) overlapping images from (a) and (b). Scale bar: 10 µM. imagesIn additional from (a experiments,) and (b). Scale the bar: commercial 10 μM. fluorescent probe LysoTracker was co-incubated with PhA Inin additionalHeLa cells experiments, under the same the commercialexperimental fluo conditions.rescent probe Figure LysoTracker 9 shows the was fluorescence co-incubated of withPhA In additional experiments, the commercial fluorescent probe LysoTracker was co-incubated with PhA(a), the Inin additionalHeLafluorescence cells experiments, under of the the lysosomal same the commercialexperimental marker (b) fluo coandrescentnditions. the superposition probe Figure LysoTracker 9 shows of the the was two fluorescence co-incubated images (c). of There withPhA PhA in HeLa cells under the same experimental conditions. Figure9 shows the fluorescence of PhA (a), PhA(a),is an the inalmost HeLafluorescence perfect cells under overlap of the the lysosomal between same experimental themarker PhA (b)fluorescence coandnditions. the superposition and Figure that 9of shows the of lysosomalthe the two fluorescence images marker. (c). of There PhA the fluorescence of the lysosomal marker (b) and the superposition of the two images (c). There is an (a),is an the almost fluorescence perfect overlapof the lysosomal between themarker PhA (b)fluorescence and the superposition and that of the of lysosomalthe two images marker. (c). There almostis an perfect almostoverlap perfect overlap between between the PhA the fluorescence PhA fluorescence and thatand that of the of lysosomalthe lysosomal marker. marker.

Figure 9. HeLa cells treated with 2 μM PhA and 50 nM LysoTracker Green for 4 h, observed by Figurefluorescence 9. HeLa microscopy. cells treated (a) PhA with fluorescence 2 μM PhA observedand 50 nM un derLysoTracker green light Green excitation; for 4 (h,b) observedLysoTracker by FigurefluorescenceGreen fluorescence9. HeLa microscopy. cells under treated ( ablue) PhAwith light fluorescence 2 excitation;μM PhA observedand (c) merged 50 nM un imagesderLysoTracker green from light (Greena )excitation; and for(b). 4Scale (h,b) observedLysoTracker bar: 10 μ M.by Figure 9. HeLa cells treated with 2 µM PhA and 50 nM LysoTracker Green for 4 h, observed by fluorescenceGreen fluorescence microscopy. under ( ablue) PhA light fluorescence excitation; observed (c) merged un imagesder green from light (a )excitation; and (b). Scale (b) LysoTracker bar: 10 μM. fluorescenceGreen fluorescence microscopy. under (a) blue PhA light fluorescence excitation; observed (c) merged under images green from light (a) and excitation; (b). Scale ( bbar:) LysoTracker 10 μM. Green fluorescence under blue light excitation; (c) merged images from (a) and (b). Scale bar: 10 µM.

Cancers 2017, 9, 18 8 of 18

Cancers 2017, 9, 18 8 of 18 2.3. Cell Viability Studies 2.3. Cell Viability Studies In order to reveal the effects of combined photodynamic and chemotherapeutic treatments, cell viabilityIn order studies to reveal by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H effects of combined photodynamic and chemotherapeutic tetrazolium bromide treatments, (MTT) were cell carriedviability out studies with by the MTT individual were carried components out with aiming the individual at identifying components the conditions aiming leading at identifying to 50% cellthe deathconditions for each leading component. to 50% cell death for each component.

2.3.1. Treatments withwith DoxorubicinDoxorubicin HeLa cells were incubated with different DOXO concentrations, and cell viability was assessed 24 h after treatment. treatment. As As shown shown in inFigure Figure 10, 10 concentrations, concentrations of 0.4 of 0.4μMµ andM and 0.5 0.5μM µproducedM produced a cell a cellinactivation inactivation of approximately of approximately 50%. 50%. Thus, Thus, the the lowest lowest concentration concentration (0.4 (0.4 μµM)M) was was chosen chosen for combination therapies.

Figure 10.10.Viability Viability of of HeLa HeLa cells cells treated treated with DOXOwith DOXO for 24 hfor at different24 h at concentrationsdifferent concentrations and non-treated and controlnon-treated cells (c).control *** p cells< 0.001. (c). The*** standardp < 0.001. deviation The standard (SD)is deviation the average (SD) of atis leastthe average three experiments. of at least three experiments.

Given the ability of DOXO to produce 1O upon exposure to light, we assessed whether it could be Given the ability of DOXO to produce 1O2 2 upon exposure to light, we assessed whether it could usedbe used as aas dual a dual agent, agent, namely namely cytotoxic cytotoxic and photocytotoxic.and photocytotoxic. Thus, Thus, in separate in separate experiments, experiments, cells werecells µ µ pre-incubatedwere pre-incubated with with 0.2 0.2M andμM and 0.4 0.4M μ DOXOM DOXO and and irradiated irradiated with with green green light. light. Cell Cell viabilityviability of irradiated samplessamples waswas identicalidentical toto thatthat shownshown inin FigureFigure 1010.. 2.3.2. Treatments with Pheophorbide a 2.3.2. Treatments with Pheophorbide a Figure 11 shows viability results on HeLa cells incubated with 1 µM and 2 µM PhA for 4 h. In the Figure 11 shows viability results on HeLa cells incubated with 1 μM and 2 μM PhA for 4 h. In absence of light (Figure 11a), the surviving fraction of treated cells was similar to that of control cells. the absence of light (Figure 11a), the surviving fraction of treated cells was similar to that of control On the contrary, after 4 h of incubation with 2 µM PhA and 15 min of red-light irradiation (6.4 J/cm2), cells. On the contrary, after 4 h of incubation with 2 μM PhA and 15 min of red-light irradiation the fraction of surviving cells had dropped to approximately 50% (Figure 11b). Irradiation of control (6.4 J/cm2), the fraction of surviving cells had dropped to approximately 50% (Figure 11b). Irradiation cells in the absence of PhA did not produce any measurable cytotoxicity. Hence, the above conditions of control cells in the absence of PhA did not produce any measurable cytotoxicity. Hence, the above were chosen for the combination experiments. conditions were chosen for the combination experiments.

Cancers 2017, 9, 18 9 of 18 Cancers 2017, 9, x 9 of 18

Cancers 2017, 9, 18 9 of 18

Pheophorbide a Pheophorbide a

Figure 11. Viability of HeLa cells treated with PhA at different concentrations and non-treated control Figure 11. Viability of HeLa cells treated with PhA at different concentrations and non-treated control cells (c) for 4 h in the dark (a) and after irradiation (b). *** p < 0.001. SD is the average of at least three cells (c)experiments. for 4 h in the dark (a) and after irradiation (b). *** p < 0.001. SD is the average of at least three experiments.

2.3.3. Combined Treatment of Doxorubicin and Pheophorbide a Figure 11. Viability of HeLa cells treated with PhA at different concentrations and non-treated control For the combination assays, the concentrations of DOXO and PhA that individually caused 50% 2.3.3. Combinedcells (c) Treatmentfor 4 h in the of dark Doxorubicin (a) and after andirradiation Pheophorbide (b). *** p < 0.001. a SD is the average of at least cell death each were chosen (2 μM PhA and 0.4 μM DOXO). The timing of the chemo- and three experiments. Forphoto the- combinationtherapeutic events assays, is expected the to concentrations play a major role of in the DOXO outcome and of PhAthe combined that individually treatments. caused µ µ 50% cell2.3.3.Indeath order Combined to each identify wereTreatment the chosen best of synchronizationDoxorubicin (2 M PhA and conditions, and Pheophorbide 0.4 M the DOXO). twoa compounds The timing were administered of the chemo- and photo-therapeuticaccording to three events different is expected protocols to: In play the first a major protocol role (termed in the PhA outcome-DOXO) of, cells the were combined incubated treatments. For the combination assays, the concentrations of DOXO and PhA that individually caused 50% In orderwith to identifyPhA for 4 the h, washed best synchronization to remove any unbound conditions, PhA, irradiated the two for compounds 15 min and then were further administered cellincubated death each with DOXOwere chosen for a period (2 μM of PhA24 h. and In the 0.4 second μM DOXO). protocol The (DOXO timing-PhA) of, cellsthe chemo- were first and according to three different protocols: In the first protocol (termed PhA-DOXO), cells were incubated photo-therapeutictreated with DOXO events for 24 is h, expected washed, incubatedto play a majo withr PhA role forin the4 h, outcome washed again of the and combined finally irradiated treatments. with PhAInfor order 15 for min. 4toh, Inidentify washedthe third the protocol tobest remove synchronization (DOXO any + PhA) unbound ,conditions, cells were PhA, incubatedthe irradiated two compoundswith DOXO for 15 werefor min 20 administeredh, and washed, then further incubatedaccordingco-incubated with DOXOto three with for differentPhA a and period protocols:DOXO of for 24 In4 h. h the, In washed thefirst secondprotocol again and protocol (termed finally PhA-DOXO), irradiated (DOXO-PhA), for cells15 min. cells were At were incubatedthe end first treated with DOXOwithof the PhA forthree for 24 protocols, h,4 h, washed, washed cells have incubatedto remove been incubated any with unbound PhA with forDOXO PhA, 4 h, irradiatedfor washed 24 h and for again with 15 PhAmin and forand finally 4 thenh, and irradiatedfurther the for 15 min.incubatedsame In the light thirdwith fluence DOXO protocol has been for (DOXOadelivered. period of + PhA),24 h. In cells the second were incubatedprotocol (DOXO-PhA), with DOXO cells for were 20 h,first washed, treatedFigure with 1DOXO2a shows for the 24 h,viability washed, of theincubated cells after with delivery PhA for of 4the h, twowashed compounds again and according finally irradiatedto three co-incubated with PhA and DOXO for 4 h, washed again and finally irradiated for 15 min. At the end fordifferent 15 min. protocols In the third in the protocol absence (DOXO of light. + PhA),The c ombinationcells were incubated of DOXO with DOXOPhA does for not20 h,increase washed, of the threeco-incubatedtheir protocols,intrinsic with dark cellsPhA toxicity and have (cf.DOXO beenFigures for incubated 410 h, and washed 11a) with. againCell DOXOviability and finally fordata 24irradiated after h andirradiation for with 15 min. PhAare shown At for the 4 in end h, and the same lightofFigure the fluence three 12b. protocols,While has beenone couldcells delivered. have have beenexpected incubated a cell survival with DOXO no higher for 24 than h and 25% with (50% PhA DOXO for × 4 50% h, and PhA the Figuresameand li12lightghta, showsiffluence additive thehas), viabilitythisbeen outcome delivered. of thewas cellsonly observed after delivery with the of third the protocol two compounds (DOXO + PhA). according to three different protocolsFigure 12a in shows the absencethe viability of of light. the cells The after combination delivery of the of two DOXO compounds with PhAaccording does to not three increase their intrinsicdifferent darkprotocols toxicity in the (cf. absence Figures of light.10 and The 11 combinationa). Cell viability of DOXO data with after PhA irradiation does not increase are shown in their intrinsic dark toxicity (cf. Figures 10 and 11a). Cell viability data after irradiation are shown in Figure 12b. While one could have expected a cell survival no higher than 25% (50% DOXO × 50% PhA Figure 12b. While one could have expected a cell survival no higher than 25% (50% DOXO × 50% PhA and light,and iflight, additive), if additive), this this outcome outcome was was only only observedobserved with with the the third third protocol protocol (DOXO (DOXO + PhA). + PhA).

Figure 12. Viability of HeLa cells treated with DOXO 0.4 μM and PhA 2 μM according to three different protocols. Samples were either kept in the dark (a) or irradiated for 15 min after PhA incubation (b). c: non-treated control cells. PhA-DOXO: cells incubated with PhA for 4 h, washed and incubated with DOXO for 24 h. DOXO-PhA: cells incubated with DOXO for 24 h, washed and incubated with PhA for 4 h. DOXO + PhA: cells incubated with DOXO for 20 h, washed and

Figure 12. Viability of HeLa cells treated with DOXO 0.4 μM and PhA 2 μM according to three Figure 12. Viability of HeLa cells treated with DOXO 0.4 µM and PhA 2 µM according to three different different protocols. Samples were either kept in the dark (a) or irradiated for 15 min after PhA protocols.incubation Samples (b). werec: non-treated eitherkept control in cells. the darkPhA-DOXO: (a) or irradiatedcells incubated for with 15 min PhA after for 4 h, PhA washed incubation and (b). c: non-treatedincubated control with DOXO cells. for PhA-DOXO: 24 h. DOXO-PhA: cells cells incubated incubated with with PhADOXO for for 4 24 h, h, washed washed and incubated incubated with DOXOwith for 24PhA h. for DOXO-PhA: 4 h. DOXO + cellsPhA: incubatedcells incubated with with DOXO DOXO forfor 2420 h, washed washed and and co-incubated incubated with with PhA for 4 h.PhA DOXO and DOXO + PhA: for cells 4 h. incubated*** p < 0.001. withSD is DOXOthe average for of 20 at h, least washed three separate and co-incubated experiments. with PhA and DOXO for 4 h. *** p < 0.001. SD is the average of at least three separate experiments. Cancers 2017, 9, 18 10 of 18

Cancers 2017, 9, 18 10 of 18 The results above indicate that the combination of a PhA photodynamic treatment with DOXO chemotherapyThe results is sub-additive above indicate for thethat PhA-DOXOthe combination and of DOXO-PhA a PhA photodynamic protocols. treatment In order with to better DOXO assess chemotherapy is sub-additive for the PhA-DOXO and DOXO-PhA protocols. In order to better assess the role of the photodynamic component in the combination treatments, the concentration of DOXO the role of the photodynamic component in the combination treatments, the concentration of DOXO was lowered down to 0.2 µM, maintaining invariable all other experimental conditions. At this DOXO was lowered down to 0.2 μM, maintaining invariable all other experimental conditions. At this concentration,DOXO concentration, cell viability cell was viability 84% (Figurewas 84% 10 (Figure). The results10). The of results this new of this set new of experiments set of experiments are shown in Figureare shown 13. in Figure 13.

Figure 13. Viability of HeLa cells treated with DOXO 0.2 μM and PhA 2 μM according to three Figure 13. Viability of HeLa cells treated with DOXO 0.2 µM and PhA 2 µM according to three different different protocols. Samples were either kept in the dark (a) or irradiated for 15 min after PhA protocols. Samples were either kept in the dark (a) or irradiated for 15 min after PhA incubation (b). incubation (b). c: non-treated control cells. PhA-DOXO: cells incubated with PhA for 4 h, washed and c: non-treatedincubated controlwith DOXO cells. for PhA-DOXO: 24 h. DOXO-PhA: cells cells incubated incubated with with PhA DOXO for for 4 h, 24 washed h, washed and and incubated incubated with DOXOwith for PhA 24 h.for DOXO-PhA: 4 h. DOXO + PhA: cells cells incubated incubated with with DOXO DOXO for for 24 20 h,h, washed and and coincubated incubated with with PhA PhA for 4 h. DOXOand DOXO + PhA: for 4 cells h. *** incubated p < 0.001. SD with is the DOXO average for of 20 at h, least washed three andseparate coincubated experiments. with PhA and DOXO for 4 h. *** p < 0.001. SD is the average of at least three separate experiments. In this case, there was a remarkable difference in outcome between the three protocols. For PhA-DOXO, there was no appreciable difference in cell viability between the dark and light In this case, there was a remarkable difference in outcome between the three protocols. experiments. For DOXO-PhA, the expected additive effect was observed (0.84 × 0.46 = 0.39, which For PhA-DOXO,compares well there with wasthe observed no appreciable 42%). Remarkably, difference inthe cell DOXO viability + PhA between treatment the resulted dark andin a light experiments.survival fraction For DOXO-PhA, of 18%, which theindicates expected that a additivesynergistic effect effect wascan indeed observed be achieved (0.84 × under0.46 the = 0.39, whichproper compares conditions. well with the observed 42%). Remarkably, the DOXO + PhA treatment resulted in a survival fraction of 18%, which indicates that a synergistic effect can indeed be achieved under the proper3. Discussion conditions. Chemotherapy is the most common treatment used for fighting cancer malignancies despite its 3. Discussionmajor problems, such as high toxicity and the onset of resistance [2]. Therapeutic alternatives are Chemotherapybeing highly explored, is the such most as common combined treatment treatments usedusing for drugs fighting of various cancer kinds malignancies aiming at reducing despite its majorthe problems, dose of suchthe toxic as high chemotherapeutic toxicity and the agent; onset th oferefore, resistance the [side2]. Therapeuticeffects and the alternatives acquisition are of being resistance [28]. PDT is a local treatment whose mode of action does not usually cause the emergence highly explored, such as combined treatments using drugs of various kinds aiming at reducing the dose of resistance or the inhibition of the immune system. Rather, it even activates the immune of the toxic chemotherapeutic agent; therefore, the side effects and the acquisition of resistance [28]. system [47], which makes PDT a potential partner for combined treatments [16]. In this paper, we set PDT isout a localto study treatment combinations whose mode of PhA, of action a repres doesentative not usually PS, causewith DOXO, the emergence a well-established of resistance or the inhibition of the immune system. Rather, it even activates the immune system [47], which makes PDT a potential partner for combined treatments [16]. In this paper, we set out to study combinations Cancers 2017, 9, 18 11 of 18 of PhA, a representative PS, with DOXO, a well-established chemotherapeutic drug [48], to assess whether such combinations would allow reducing the concentration of the toxic DOXO. DOXO’s properties and mode of action have been extensively characterized. It accumulates in the nucleus (Figure7), where it exerts its damage as a topoisomerase inhibitor [ 44], and its 1,4-dihydroxyanthraquinone chromophore is fluorescent under green light irradiation [49,50]. On the other hand, anthraquinones are known to produce ROS through electron transfer processes with 1 biological molecules [51–54] and can also generate O2 under irradiation [55,56]. Indeed DOXO 1 generates O2 with quantum yields Φ∆ = 0.03 in EtOH and Φ∆ = 0.01 in D2O as a consequence of its aggregation. However, HeLa cells incubated with 0.2 µM and 0.4 µM DOXO and irradiated with green light did not show any increased toxicity compared to the non-irradiated samples. 1,4-dihydroxyanthraquinone derivatives efficiently bind to DNA [57–59], which causes a marked fluorescence loss [60,61]. We have confirmed that this is the case also for DOXO (Figure6) and have 1 assessed that it leads to a 10-fold reduction in its capacity to produce O2 by photosensitization. PhA is a chlorophyll-based metabolite endowed with appealing photophysical properties, such as strong absorption in the red part of the spectrum, red fluorescence and a high capacity to generate 1 O2 when it is in monomeric form (Table1). Our values are in excellent agreement with previous literature reports (Φ∆ = 0.59 in EtOH; Φ∆ < 0.02 in water) [62,63]. PhA has been evaluated as a PDT agent against different cancer malignancies [40,64]. In our study, PhA localizes in lysosomes (Figure8). This is in line with the reports that PhA methyl ester localizes in endoplasmatic reticulum/Golgi and lysosomes [65]. Lysosomes have been previously shown to be a good target for triggering apoptosis in PDT treatments [66,67]. It was hypothesized that the combined use of drugs with different cellular localizations could increase their cytotoxic capacity and could hence be a valuable strategy to reduce their concentrations and thus their side effects [68]. The combination of PhA with DOXO seemed suitable, as damage would occur in the lysosomal compartment and the nucleus, respectively. In this sense, in vitro combinations of DOXO with different PSs and cell lines have already been explored aiming at enhancing the final cell-killing effect [26,69–72]. We determined the IC50 of DOXO in HeLa cells (0.4 µM) after an incubation time of 24 h. Likewise, we found that 15 min red-light irradiation of HeLa cells incubated for 4 h with PhA 2 µM induced also 50% cell death with a lack of toxicity in the absence of light. Combination implies sequencing of the active agents, and thus, the order is a significant factor. Indeed, it has been reported that synergic interaction between PDT and chemotherapy is dependent not only on the nature of the PS and the chemotherapeutical agent, as well as on the irradiance dose, but also on the treatment sequence [27,69,71,73]. On the one hand, we have previously mentioned that Datta et al. found that combined treatment of ALA-PDT and mitomycin C resulted in an enhanced effect when the chemotherapeutic agent was given first both for parental and mitomycin-sensitive cells [27]. On the other hand, Cowled et al. showed that, when DOXO was administered with HpD and at the time of irradiation, the photodynamic effect was potentiated, whereas DOXO administration was less effective after PDT, both in vivo [69]. In this line, Kirveliene et al. showed that combined treatment of DOXO and temoporfin was more effective if DOXO was applied right after light exposure in murine hepatoma in vitro and in vivo [71]. As a final example, Rizvi et al. observed synergism in benzoporphyrin-based treatment followed by low-dose carboplatin against ovarian cancer cells grown in a three-dimensional model, which was not achieved with the reverse treatment order [73]. Hence, the combined effect of DOXO and PhA + light on HeLa cell viability was explored for 0.4 µM DOXO and 2 µM PhA using three different delivery protocols. The outcome of the combined treatments was evaluated according to the method described by Valeriote and Lin [74], which compares the efficacy of the individual drugs (εA, εB) with that of its combination (εA+B). The treatment is synergic if εA+B < (εA × εB)/100, additive, if εA+B = (εA × εB)/100, sub-additive if (εA × εB)/100 < εA+B < εA, provided εA < εB, interference, if εA < εA+B < εB, when εA < εB, and antagonistic, if εB < εA+B, when εA < εB. Cancers 2017, 9, 18 12 of 18

Out of the three experimental conditions tested, only DOXO + PhA combined photo-treatment showed better efficacy (25% cell survival) than the individual treatments DOXO 0.4 µM (48%) or PhA + light (46%). This treatment is therefore additive, unlike other combined photodynamic and chemotherapeutic treatments reported in the literature [25,31]. The other two treatments are sub-additive, perhaps due to the quenching of PhA excited states by DOXO. Previous works have observed that DOXO can prevent drug accumulation in combination treatments [71,75]; however, this is not the case provided that the overall result was the same either with DOXO delivered before (DOXO-PhA) or after PhA (PhA-DOXO). We hypothesize that, in addition, the toxicity of 0.4 µM DOXO was so large that it masked the effects of the photodynamic component elicited by PhA. Indeed, using a lower concentration of DOXO (0.2 µM), we found that PhA-DOXO was now borderline between interference and antagonistic; DOXO-PhA was additive; and PhA + DOXO was synergic. In agreement with previous studies, it can be concluded that not only the relative concentration, but also the order in which the chemotherapeutic and the photodynamic treatments are delivered play key roles in the outcome of DOXO and PhA + light combined treatments in HeLa cells.

4. Materials and Methods

4.1. Chemicals

Phosphate buffer saline (PBS), doxorubicin (DOXO), deuterium oxide (D2O), DNA sodium salt from calf thymus, sodium azide (NaN3), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H tetrazolium bromide (MTT) and the photosensitizing agents pheophorbide a (PhA) and Rose Bengal (RB) were purchased from Sigma-Aldrich, Chemical Co. (St. Louis, MO, USA). LysoTracker Green DND-26 was purchased from Thermo Fisher (Waltham, MA, USA). Flavin mononucleotide (FMN) was from Chemodex Ltd. (St. Gallen, Switzerland).

4.2. General Spectroscopic Measurements All spectroscopic measurements were carried out in 1-cm quartz cuvettes (Hellma, Germany) at room temperature. Absorption spectra were recorded on a Cary 6000i spectrophotometer (Varian, Palo Alto, CA, USA). Fluorescence emission spectra were recorded using a Spex Fluoromax-4 spectrofluorometer (Horiba Jobin-Ybon, Edison, NJ, USA). Fluorescence decays were recorded with a time-correlated single photon counting system (Fluotime 200, PicoQuant GmbH, Berlin, Germany) equipped with a red sensitive photomultiplier. Excitation was achieved by either a 504-nm picosecond LED or a 654-nm laser working at a 10-MHz repetition rate. The counting frequency was kept always below 1%. Fluorescence decays were analyzed using the PicoQuant FluoFit v4.6.5 data analysis 1 software. For O2 phosphorescence measurements, an AO-Z-473 solid state AOM Q-switched laser (Changchun New Industries Optoelectronics Technology Co., Changchun, China) working at a 4-kHz repetition rate (<1.5 mW average power) was used for excitation at 473 nm, and a diode-pumped Nd:YAG laser (FTSS355-Q, Crystal Laser, Berlin, Germany) working at a 1-kHz repetition rate (1.2 µJ per pulse, 1-ns pulse-width) was used for excitation at 532 nm. A 1064-nm rugate notch filter (Edmund Optics Ltd., York, UK) was placed in the laser path to remove any NIR emission. The luminescence emitted from the sample was collected at 80 degrees, filtered by a long-pass filter with the cut-off at 1100 nm in order to remove any scattered laser radiation and by a narrow bandpass filter at 1270 nm to isolate the NIR emission. A TE-cooled near-IR sensitive photomultiplier tube assembly (H9170−45, Hamamatsu Photonics Hamamatsu City, Shizuoka Prefacture, Japan) in combination with a multichannel scaler (NanoHarp 250, PicoQuant Gmbh, Berlin, Germany) was used as the 1 photon-counting detector [76]. O2 decays were analyzed using the GraphPad Prism 5.0 software to fit the data to Equation (1).

τ∆ S(t) = S0 × × (exp(−t/τ∆) − exp(−t/τT)) (1) τ∆ − τT Cancers 2017, 9, 18 13 of 18

where S0 is a quantity proportional to the singlet oxygen quantum yield, Φ∆. For the determination of Φ∆ values, optically-matched solutions of DOXO, PhA and suitable reference PSs (Flavin mononucleotide (FMN), Φ∆ = 0.56 for D2O and Rose Bengal (RB), Φ∆ = 0.75 for EtOH [55,56]) were excited and their S0 values scaled using Equation (2).

S0,Sample Φ∆,Sample = Φ∆, Re f × (2) S0,Re f

4.3. Cell Cultures Cell cultures from cervical adenocarcinoma human uterus (ATCC® CCL2™, LGC Standards S.L.U., Manassas, VA, USA) HeLa cells were used [77]. The cells were grown with minimum essential medium modified by Dulbecco (DMEM) to which 10% (v/v) fetal bovine serum, 50 units/mL penicillin, 50 µg/mL streptomycin and 1% (v/v) 0.2 M L-glutamine were added. All of these products were supplied by Invitrogen. Cell were cultured in a 200 SteriCult (Hucoa-Erlöss, Thermo Fisher) incubator ◦ 2 with humid atmosphere at 37 C and 5% CO2. Cells were seeded in 25-cm flasks (F 25) or in multi-well plates (6 or 24 wells rack) from Corning Inc. (New York, NY, USA). Cell manipulation was carried out in a vertical laminar flow hood SUPACRIS 12 (Labconco, Kansas City, MO, USA).

4.4. Doxorubicin and Pheophorbide a Subcellular Localization To characterize the subcellular localization of drugs used in this study, observations on live cells in culture were made in fluorescence microscopy. Immediately after incubation with DOXO or PhA, cells on coverslips were washed 3 times with PBS and mounted on a slide, removing excess liquid. In the case of treatment with PhA plus co-localization, experiments using LysoTracker Green were performed. The probe was prepared at a concentration of 50 nM in complete medium without serum. Cells previously treated with PhA for 4 h were incubated with LysoTracker Green for 5 min, washed with PBS (×3) and observed in fluorescence microscopy. Microscopic observations and photographs were performed with an Olympus photomicroscope IMT-2, equipped with a mercury lamp HBO 100 W and the corresponding filter sets for fluorescence microscopy: UV (365–390 nm), blue (450–490 nm) and green (510–550 nm). The images were processed using Adobe Photoshop 5.0 software (Adobe Systems, Inc., San José, CA, USA). The routine observation of cells was performed on an inverted Olympus microscope X31 CK (Barcelona, Spain).

4.5. Chemotherapeutic, Photodynamic and Combined Treatments For photodynamic treatments, a stock solution of PhA was prepared at a concentration of 10 mM in DMSO and stored at −20 ◦C. The PS was diluted in culture medium to the final desired concentration, added to the cells and incubated for 4 h. The wells were washed 3 times with PBS, and fresh culture medium was added prior to irradiation. Then, they were irradiated for 15 min with red light from an LED Par 64 Short V2 lamp (Showtec, Kerkrade, The Netherlands) at 632 nm and 7.1 mW/cm2. After photodynamic treatments, the plates were kept in the incubator until further processing. For chemotherapeutic treatments, a 100 mM stock solution of DOXO in water was prepared and stored at −20 ◦C. Cells were incubated for 24 h with DOXO previously diluted in culture medium, washed three times with PBS and left with culture medium in the incubator until further processing. For combined treatments, DOXO and PhA were delivered according to three different protocols as described in Section 2.3.3.

4.6. Cell Viability Evaluation of cell survival was performed 24 h after each treatment, using the MTT assay. A 1-mg/mL stock solution of MTT in water was prepared and filtered. From this initial solution, a 0.05-mg/mL dilution was prepared in culture medium, and a 0.5-mL aliquot of this dilution was added to each well of a 24-well rack. After 3 h, DMSO was added to dissolve the formazan precipitate. Cancers 2017, 9, 18 14 of 18

The absorbance of formazan was then measured in a plate reader (Tecan Spectra Fluor, MTX Lab Systems, Bradenton, FL, USA) at 540 nm. The survival of the cells was represented as the percentage absorbance of the treated cells with respect to the absorbance of control cells.

4.7. Statistical Analysis Statistical analyses were performed using the R 3.2.5 program. By ANOVA, test the mean factor experiments (each condition vs. control) were compared. It was considered that there were significant differences between means when: * p < 0.05; ** p < 0.01 and *** p < 0.001.

5. Conclusions Our study supports the trend towards the development of combination therapies by evaluating the combined use of the photosensitizing agent PhA and the chemotherapeutic drug DOXO to kill HeLa cells. Both compounds show different subcellular accumulation in this cell line and exert damage by different modes of action. Photophysical studies of the compounds alone and in combination have revealed that DOXO quenches the excited states of PhA, detracting from its photosensitizing ability. On the other hand, 1 while DOXO can itself photosensitize the production of O2, this is largely suppressed by aggregation and by binding to DNA. Three different combined treatment protocols have been assayed. The order of drug and light delivery plays a crucial role in the final outcome of the treatments, ranging from antagonistic to synergic for the same concentrations, which is supported by several previous reports. Thus, while combined photodynamic and chemotherapeutic treatments show strong potential to decrease the dose of the toxic chemotherapeutic agent and therefore its side effects, the delivery protocols must be very finely tuned to achieve this desired outcome.

Acknowledgments: The research described herein has been supported by the Ministerio de Economía y Competitividad through the grants CTQ2013-48767-C3-1-R and CTQ2013-48767-C3-3-R (Spain) and by Obra Social “la Caixa” through Universitat Ramon Llull with a research grant for seed projects (ref. 2016-URL-Trac-013). Roger Bresolí-Obach thanks the European Social Funds and the Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya for his predoctoral fellowship (2016 FI_B1 00021). Author Contributions: Rubén Ruiz-González, Santi Nonell and Magdalena Cañete conceived of and designed the experiments. Rubén Ruiz-González, Paula Milán and Roger Bresolí-Obach performed the experiments. Rubén Ruiz-González, Santi Nonell and Magdalena Cañete wrote the manuscript. All the authors contributed in the final discussion of the paper. Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript; nor in the decision to publish the results.

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