Michael Price

Monitoring Singlet Oxygen and Hydroxyl Radical Formation with Fluorescent Probes During Photodynamic Therapy

Singlet oxygen (1O2) is the primary oxidant generated in photodynamic therapy (PDT) protocols involving sensitizers resulting in type II reactions. 1O2 can give rise to additional reactive oxygen species (ROS) such as the hydroxyl radical (?OH). The current study was designed to assess 3′‐p‐(aminophenyl) fluorescein (APF) and 3′‐p‐(hydroxyphenyl) fluorescein (HPF) as probes for the detection of 1O2 and ?OH under conditions relevant to PDT. Cell‐free studies indicated that both APF and HPF were converted to fluorescent products following exposure to 1O2 generated by irradiation of a water‐soluble photosensitizing agent (TPPS) and that APF was 35‐fold more sensitive than HPF. Using the 1O2 probe singlet oxygen sensor green (SOSG) we confirmed that 1 mm NaN3 quenched 1O2‐induced APF/HPF fluorescence, while 1% DMSO had no effect. APF and HPF also yielded a fluorescent product upon interacting with ?OH generated from H2O2 via the Fenton reaction in a cell‐free system. DMSO quenched the fluorogenic interaction between APF/HPF and ?OH at doses as low as 0.02%. Although NaN3 was expected to quench ?OH‐induced APF/HPF fluorescence, co‐incubating NaN3 with APF or HPF in the presence of ?OH markedly enhanced fluorescence. Cultured L1210 cells that had been photosensitized with benzoporphyhrin derivative exhibited APF fluorescence immediately following irradiation. Approximately 50% of the cellular fluorescence could be suppressed by inclusion of either DMSO or the iron‐chelator desferroxamine. Combining the latter two agents did not enhance suppression. We conclude that APF can be used to monitor the formation of both 1O2 and ?OH in cells subjected to PDT if studies are performed in the presence and absence of DMSO, respectively. That portion of the fluorescence quenched by DMSO will represent the contribution of ?OH. This procedure could represent a useful means for evaluating formation of both ROS in the context of PDT.

Apoptotic and autophagic responses to photodynamic therapy in 1c1c7 murine hepatoma cells

Photodynamic therapy (PDT) is a process that can induce apoptosis, autophagy or both depending on the cell phenotype. Apoptosis is a pathway to cell death while autophagy can protect from photokilling or act as a death pathway. In a previous study, we reported a cytoprotective effect of autophagy in murine leukemia cell lines where both autophagy and apoptosis occur within minutes after irradiation of photosensitized cells. In this study, we examined the effects of mitochondrial photodamage catalyzed by low (≤1 μM) concentrations of the photosensitizing agent termed benzoporphyrin derivative (BPD, Verteporfin) on murine hepatoma 1c1c7 cells. Apoptosis was not observed until several hours after irradiation of photosensitized cells. Autophagy was clearly cytoprotective since PDT efficacy was significantly enhanced in a knockdown sub-line (KD) in which the level of a critical autophagy protein (Atg7) was markedly reduced. This result indicates that autophagy can protect from phototoxicity even when apoptosis is substantially delayed. Much higher concentrations (≥10 μM) of BPD had previously been shown to inhibit autophagosome formation. Phototoxicity studies performed with 10 μM BPD and a proportionally reduced light dose were consistent with the absence of an autophagic process in wild-type (WT) cells under these conditions.

ATG7 deficiency suppresses apoptosis and cell death induced by lysosomal photodamage

Photodynamic therapy (PDT) involves photosensitizing agents that, in the presence of oxygen and light, initiate formation of cytotoxic reactive oxygen species (ROS). PDT commonly induces both apoptosis and autophagy. Previous studies with murine hepatoma 1c1c7 cells indicated that loss of autophagy-related protein 7 (ATG7) inhibited autophagy and enhanced the cytotoxicity of photosensitizers that mediate photodamage to mitochondria or the endoplasmic reticulum. In this study, we examined two photosensitizing agents that target lysosomes: the chlorin NPe6 and the palladium bacteriopheophorbide WST11. Irradiation of wild-type 1c1c7 cultures loaded with either photosensitizer induced apoptosis and autophagy, with a blockage of autophagic flux. An ATG7- or ATG5-deficiency suppressed the induction of autophagy in PDT protocols using either photosensitizer. Whereas ATG5-deficient cells were quantitatively similar to wild-type cultures in their response to NPe6 and WST11 PDT, an ATG7-deficiency suppressed the apoptotic response (as monitored by analyses of chromatin condensation and procaspase-3/7 activation) and increased the LD50 light dose by > 5-fold (as monitored by colony-forming assays). An ATG7-deficiency did not prevent immediate lysosomal photodamage, as indicated by loss of the lysosomal pH gradient. However, unlike wild-type and ATG5-deficient cells, the lysosomes of ATG7-deficient cells recovered this gradient within 4 h of irradiation, and never underwent permeabilization (monitored as release of endocytosed 10-kDa dextran polymers). We propose that the efficacy of lysosomal photosensitizers is in part due to both promotion of autophagic stress and suppression of autophagic prosurvival functions. In addition, an effect of ATG7 unrelated to autophagy appears to modulate lysosomal photodamage.

A Role for Hydrogen Peroxide in the Pro-apoptotic Effects of Photodynamic Therapy

Although the first reactive oxygen species (ROS) formed during irradiation of photosensitized cells is almost invariably singlet molecular oxygen (1O2), other ROS have been implicated in the phototoxic effects of photodynamic therapy (PDT). Among these are superoxide anion radical (•O2−), hydrogen peroxide (H2O2) and hydroxyl radical (•OH). In this study, we investigated the role of H2O2 in the pro‐apoptotic response to PDT in murine leukemia P388 cells. A primary route for detoxification of cellular H2O2 involves the peroxisomal enzyme catalase. Inhibition of catalase activity by 3‐amino‐1,2,4‐triazole led to an increased apoptotic response. PDT‐induced apoptosis was impaired by addition of an exogenous recombinant catalase analog (CAT‐skl) that was specifically designed to enter cells and more efficiently localize in peroxisomes. A similar effect was observed upon addition of 2,2′‐bipyridine, a reagent that can chelate Fe+2, a co‐factor in the Fenton reaction that results in the conversion of H2O2 to •OH. These results provide evidence that formation of H2O2 during irradiation of photosensitized cells contributes to PDT efficacy.

Effects of the Oxygenation Level on Formation of Different Reactive Oxygen Species During Photodynamic Therapy

We examined the effect of the oxygenation level on efficacy of two photosensitizing agents, both of which target lysosomes for photodamage, but via different photochemical pathways. Upon irradiation, the chlorin termed NPe6 forms singlet oxygen in high yield while the bacteriopheophorbide WST11 forms only oxygen radicals (in an aqueous environment). Photokilling efficacy by WST11 in cell culture was impaired when the atmospheric oxygen concentration was reduced from 20% to 1%, while photokilling by NPe6 was unaffected. Studies in a cell‐free system revealed that the rates of photobleaching of these agents, as a function of the oxygenation level, were correlated with results described above. Moreover, the rate of formation of oxygen radicals by either agent was more sensitive to the level of oxygenation than was singlet oxygen formation by NPe6. These data indicate that the photochemical process that leads to oxygen radical formation is more dependent on the oxygenation level than is the pathway leading to formation of singlet oxygen.

Evaluation of Diethyl‐3‐3′‐(9,10‐anthracenediyl)bis Acrylate as a Probe for Singlet Oxygen Formation during Photodynamic Therapy

The cell‐permeable anthracene analog diethyl‐3‐3′‐(9,10‐anthracenediyl)bis acrylate (DADB) was recently identified as a highly selective probe for singlet oxygen (1O2). Now, we show that DADB can be used to monitor 1O2 formation in cell culture during photodynamic therapy. An atypical property of DADB is that fluorescence emission is decreased upon oxidation. Using photosensitizers that target specific organelles, we determined that DADB could detect 1O2 whether formed in ER, mitochondria or lysosomes. DADB fluorescence was not, however, significantly altered when the photosensitizing agent was the palladium bacteriopheophorbide termed WST11, an agent reported to produce mainly oxygen radicals upon irradiation in an aqueous environment, whereas singlet oxygen was formed in organic solvents.

Effects of photodynamic therapy on the endocytic pathway

In this report, we describe an effect of photodynamic therapy (PDT) on membrane trafficking in murine 1c1c7 hepatoma cells. A brief exposure of 1c1c7 cells to a 20 nM concentration of the phosphatidylinositol kinase class-3 antagonist wortmannin led to the rapid appearance of cytoplasmic vacuoles. Fluorescence monitoring of plasma membrane-associated 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TDPH) over time demonstrated that the wortmannin-induced vacuoles were derived from endocytosed plasma membrane. Low-dose photodamage catalyzed by the lysosomal photosensitizer NPe6, prior to the addition of wortmannin, prevented formation of these vacuoles. NPe6 was found to suppress for several hours the normal trafficking of TDPH-labeled plasma membrane to the cytosol, and the formation of punctate TDPH-labeled cytoplasmic vesicles. The ability of NPe6-induced photodamage to suppress wortmannin-induced vacuolization occurred under conditions that did not disrupt lysosomes and were at or below the threshold of cytostatic/cytotoxic effects. Furthermore, the suppressive effects of NPe6-PDT were not prevented by inclusion of an agent that stabilized lysosomal membranes, or by E64d, an inhibitor of lysosomal cathepsin proteases. Mitochondrial photodamage was less effective at preventing wortmannin-induced vacuole formation and PDT directed against the ER had no effect. The role of photodamage to the endocytic pathway may be a hitherto unexplored effect on cells that selectively accumulate photosensitizing agents. These results indicate that photodamage directed against endosomes/lysosomes has effects independent of the release of lysosomal proteases.

The Bcl‐2 Antagonist HA14‐1 Forms a Fluorescent Albumin Complex that Can Be Mistaken for Several Oxidized ROS Probes

The proapoptotic effects of the Bcl‐2 antagonist HA14‐1 are believed to derive from its affinity for the hydrophobic groove on Bcl‐2 and Bcl‐xL, thereby displacing proapoptotic factors, e.g. Bax and Bak. We have reported that HA14‐1 promotes the efficacy of low‐dose photodynamic therapy (PDT). A recent report proposed that the proapoptotic activity of HA14‐1 reflects its ability to generate reactive oxygen species (ROS) when incubated in an aqueous environment. This later study, like several other HA14‐1 investigations, relied on the use of fluorescent probes for ROS detection. We found that HA14‐1 reacts with the albumin in serum to yield a fluorescent product. After correcting for this effect, the putative formation of ROS by HA14‐1 could not be demonstrated with the fluorescent probes H2DCFDA, dihydroethidium or dihydrorhodamine. Indeed, the fluorescence excitation/emission spectra of HA14‐1 encompassed the excitation/emission wavelengths used to detect these ROS probes. Cells cultured in a medium supplemented with ovalbumin, instead of serum, underwent apoptosis following HA14‐1 addition, but did not exhibit fluorescence. Hence, HA14‐1 fluorescence was unrelated to its proapoptotic activity. We conclude that the enhancement of PDT by HA14‐1 reflects a pharmacologic effect, rather than its direct contribution of ROS.

The role of reactive oxygen species in PDT efficacy

While the first reactive oxygen species (ROS) formed during photodynamic therapy (PDT) is singlet molecular oxygen (1O2), other ROS are formed downstream including superoxide anion radical (•CO2 -), hydrogen peroxide (H2O2) and hydroxyl radical (•OH). In this study, we examined the role of H2O2 in the phototoxic response to PDT in murine leukemia L388 cells. Inhibition of catalase activity, a major pathway to H2O2 detoxification, led to enhanced apoptosis and cell death. Addition of exogenous catalase offered protection from phototoxicity as did chelation of Fe+2, a co-factor in •OH production from H2O2. These results indicate the H2O2 formed during PDT plays a role in PDT efficacy.

Effects of oxygenation on PDT efficacy as a function ofreactive oxygen species formation

Using two agents that preferentially target lysosomes for photodamage but produce different reactive oxygen species (ROS) upon irradiation, we examined effects of the degree of oxygenation on photokilling. Irradiation of the chlorin NPe6 results in a high yield of singlet oxygen but the bacteriopheophorbide WST11 forms only oxygen radicals. We found that PDT efficacy of WST11 was impaired when the ambient oxygen concentration was reduced to 1%, while that of NPe6 was essentially unaffected. These result were correlated with photobleaching effects. Use of fluorescent probes for singlet oxygen vs. OH radical revealed that photobleaching was correlated with 1O2 but not .OH formation during irradiation of solutions containing NPe6. Photoproduct formation could be followed by changes in absorbance spectra. In the presence of mercaptoethanol, different photoproducts were formed, showing that environmental factors could influence photochemical reactions.