Photosensitizer Pc Research Articles

Increased killing of SCCVII squamous cell carcinoma cells after the combination of Pc 4 photodynamic therapy and dasatinib is associated with enhanced caspase-3 activity and ceramide synthase 1 upregulation

Photodynamic therapy (PDT) is not always effective as an anticancer treatment, therefore, PDT is combined with other anticancer agents for improved efficacy. The combination of dasatinib and PDT with the silicone phthalocyanine photosensitizer Pc 4 was assessed for increased killing of SCCVII mouse squamous cell carcinoma cells, a preclinical model of head and neck squamous cell carcinoma, using apoptotic markers and colony formation as experimental end-points. Because each of these treatments regulates the metabolism of the sphingolipid ceramide, their effects on mRNA levels of ceramide synthase, a ceramide-producing enzyme, and the sphingolipid profile were determined. PDT + dasatinib induced an additive loss of clonogenicity. Unlike PDT alone or PDT + dasatinib, dasatinib induced zVAD-fmk-dependent cell killing. PDT or dasatinib-induced caspase-3 activation was potentiated after the combination. PDT alone induced mitochondrial depolarization, and the effect was inhibited after the combination. Annexin V+ and propidium iodide+ cells remained at control levels after treatments. In contrast to PDT alone, dasatinib induced upregulation of ceramide synthase 1 mRNA, and the effect was enhanced after the combination. Dasatinib induced a modest increase in C20:1-and C22-ceramide but had no effect on total ceramide levels. PDT increased the levels of 12 individual ceramides and total ceramides, and the addition of dasatinib did not affect these increases. PDT alone decreased substantially sphingosine levels and inhibited the activity of acid ceramidase, an enzyme that converts ceramide to sphingosine. The data suggest that PDT-induced increases in ceramide levels do not correlate with ceramide synthase mRNA levels but rather with inhibition of ceramidase. Cell killing was zVAD-fmk-sensitive after dasatinib but not after either PDT or the combination and enhanced cell killing after the combination correlated with potentiated caspase-3 activation and upregulation of ceramide synthase 1 mRNA but not the production of ceramide. The data imply potential significance of the combination for cancer treatment.

Optimization of a Nanomedicine-Based Silicon Phthalocyanine 4 Photodynamic Therapy (Pc 4-PDT) Strategy for Targeted Treatment of EGFR-Overexpressing Cancers

The current clinical mainstays for cancer treatment, namely, surgical resection, chemotherapy, and radiotherapy, can cause significant trauma, systemic toxicity, and functional/cosmetic debilitation of tissue, especially if repetitive treatment becomes necessary due to tumor recurrence. Hence there is significant clinical interest in alternate treatment strategies like photodynamic therapy (PDT) which can effectively and selectively eradicate tumors and can be safely repeated if needed. We have previously demonstrated that the second-generation photosensitizer Pc 4 (silicon phthalocyanine 4) can be formulated within polymeric micelles, and these micelles can be specifically targeted to EGFR-overexpressing cancer cells using GE11 peptide ligands, to enhance cell-specific Pc 4 delivery and internalization. In the current study, we report on the in vitro optimization of the EGFR-targeting, Pc 4 loading of the micellar nanoformulation, along with optimization of the corresponding photoirradiation conditions to maximize Pc 4 delivery, internalization, and subsequent PDT-induced cytotoxicity in EGFR-overexpressing cells in vitro. In our studies, absorption and fluorescence spectroscopy were used to monitor the cell-specific uptake of the GE11-decorated Pc 4-loaded micelles and the cytotoxic singlet oxygen production from the micelle-encapsulated Pc 4, to determine the optimum ligand density and Pc 4 loading. It was found that the micelle formulations bearing 10 mol % of GE11-modified polymer component resulted in the highest cellular uptake in EGFR-overexpressing A431 cells within the shortest incubation periods. Also, the loading of ∼ 50 μg of Pc 4 per mg of polymer in these micellar formulations resulted in the highest levels of singlet oxygen production. When formulations bearing these optimized parameters were tested in vitro on A431 cells for PDT effect, a formulation dose containing 400 nM Pc 4 and photoirradiation duration of 400 s at a fluence of 200 mJ/cm(2) yielded close to 100% cell death.

Fluence Rate‐Dependent Photobleaching of Intratumorally Administered Pc 4 Does not Predict Tumor Growth Delay

We examined effects of fluence rate on the photobleaching of the photosensitizer Pc 4 during photodynamic therapy (PDT) and the relationship between photobleaching and tumor response to PDT. BALB/c mice with intradermal EMT6 tumors were given 0.03 mg kg(-1) Pc 4 by intratumor injection and irradiated at 667 nm with an irradiance of 50 or 150 mW cm(-2) to a fluence of 100 J cm(-2). While no cures were attained, significant tumor growth delay was demonstrated at both irradiances compared with drug-only controls. There was no significant difference in tumor responses to these two irradiances (P = 0.857). Fluorescence spectroscopy was used to monitor the bleaching of Pc 4 during irradiation, with more rapid bleaching with respect to fluence shown at the higher irradiance. No significant correlation was found between fluorescence photobleaching and tumor regrowth for the data interpreted as a whole. Within each treatment group, weak associations between photobleaching and outcome were observed. In the 50 mW cm(-2) group, enhanced photobleaching was associated with prolonged growth delay (P = 0.188), while at 150 mW cm(-2) this trend was reversed (P = 0.308). Thus, it appears that Pc 4 photobleaching is not a strong predictor of individual tumor response to Pc 4-PDT under these treatment conditions.

Cutaneous penetration of the topically applied photosensitizer Pc 4 as detected by intravital 2-photon laser scanning microscopy

The fundamental mechanism of photodynamic therapy (PDT)-induced cell death has been characterized, but early critical PDT events in vivo remain incompletely defined. With the recent development in advanced fluorescence imaging modalities, such as intravital 2-photon laser scanning microscopy (2P-LSM), researchers are now able to investigate and visualize biological processes with high resolution in real time. This powerful imaging technology allows deep tissue visualization with single-cell resolution, thus providing dynamic information on the 3-dimensional architectural makeup of the tissue. The main goal of this study was to determine the cutaneous penetration of a topically applied photosensitizer, the silicon phthalocyanine Pc 4, into the skin of live animals and to assess the effective absorption of Pc 4 through the skin barrier. Our 2P-LSM images indicate that Pc 4 penetrates to the epidermal/dermal junction of mouse skin. The data also indicate that the degree of Pc 4 absorption is dose dependent. These findings represent initial steps that may help in improving the clinical utilization of topical Pc 4-PDT.

EGFR-mediated intracellular delivery of Pc 4 nanoformulation for targeted photodynamic therapy of cancer: in vitro studies

In photodynamic therapy (PDT), the light activation of a photosensitizer leads to the generation of reactive oxygen species that can trigger various mechanisms of cell death. Harnessing this process within cancer cells enables minimally invasive yet targeted cancer treatment. With this rationale, here we demonstrate tumor-targeted delivery of a highly hydrophobic photosensitizer Pc 4 loaded within biocompatible poly(ethylene glycol)–poly(ɛ-caprolactone) block co-polymer micelles. The micelles were surface-modified with epidermal growth factor receptor (EGFR)–targeting GE11 peptides for active targeting of EGFR-overexpressing cancer cells, in vitro. Pc 4–loaded EGFR-targeted micelles were incubated with EGFR-overexpressing A431 epidermoid carcinoma cells for various time periods, to determine Pc 4 uptake by epifluorescence microscopy. The cells were subsequently photoirradiated, and PDT-induced cell death for various incubation periods was determined by MTT assay and fluorescence Live/Dead assay. Our results indicate that active EGFR targeting of the Pc 4–loaded micelles accelerates intracellular uptake of the drug. Consequently, this enhances the PDT-induced cytotoxicity within shorter time periods. From the Clinical EditorPhotodynamic cancer therapy using Pc 4, a light activated and highly hydrophobic photosensitizer is demonstrated in this paper in vitro. Pc 4 was delivered in block-copolymer micelles surface-modified with GE11 peptides targeting EGFR-overexpressing cancer cells.

Photodynamic therapy with Pc 4 induces apoptosis of Candida albicans.

The high prevalence of drug resistance necessitates the development of novel antifungal agents against infections caused by opportunistic fungal pathogens, such as Candida albicans. Elucidation of apoptosis in yeast-like fungi may provide a basis for future therapies. In mammalian cells, photodynamic therapy (PDT) has been demonstrated to generate reactive oxygen species, leading to immediate oxidative modifications of biological molecules and resulting in apoptotic cell death. In this report, we assess the in vitro cytotoxicity and mechanism of PDT, using the photosensitizer Pc 4, in planktonic C. albicans. Confocal image analysis confirmed that Pc 4 localizes to cytosolic organelles, including mitochondria. A colony formation assay showed that 1.0 μM Pc 4 followed by light at 2.0 J cm(-2) reduced cell survival by 4 logs. XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide) assay revealed that Pc 4-PDT impaired fungal metabolic activity, which was confirmed using the FUN-1 (2-chloro-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenylquinolinium iodide) fluorescence probe. Furthermore, we observed changes in nuclear morphology characteristic of apoptosis, which were substantiated by increased externalization of phosphatidylserine and DNA fragmentation following Pc 4-PDT. These data indicate that Pc 4-PDT can induce apoptosis in C. albicans. Therefore, a better understanding of the process will be helpful, as PDT may become a useful treatment option for candidiasis.

Preliminary clinical and pharmacologic investigation of photodynamic therapy with the silicon phthalocyanine photosensitizer pc 4 for primary or metastatic cutaneous cancers.

Photodynamic therapy (PDT) for cutaneous malignancies has been found to be an effective treatment with a range of photosensitizers. The phthalocyanine Pc 4 was developed initially for PDT of primary or metastatic cancers in the skin. A Phase I trial was initiated to evaluate the safety and pharmacokinetic profiles of systemically administered Pc 4 followed by red light (Pc 4-PDT) in cutaneous malignancies. A dose-escalation study of Pc 4 (starting dose 0.135 mg/m2) at a fixed light fluence (135 J/cm2 of 675-nm light) was initiated in patients with primary or metastatic cutaneous malignancies with the aim of establishing the maximum tolerated dose (MTD). Blood samples were taken at intervals over the first 60 h post-PDT for pharmacokinetic analysis, and patients were evaluated for toxicity and tumor response. A total of three patients (two females with breast cancer and one male with cutaneous T-cell lymphoma) were enrolled and treated over the dose range of 0.135 mg/m2 (first dose level) to 0.54 mg/m2 (third dose level). Grade 3 erythema within the photoirradiated area was induced in patient 2, and transient tumor regression in patient 3, in spite of the low photosensitizer doses. Pharmacokinetic observations fit a three-compartment exponential elimination model with an initial rapid distribution phase (∼0.2 h) and relatively long terminal elimination phase (∼28 h), Because of restrictive exclusion criteria and resultant poor accrual, the trial was closed before MTD could be reached. While the limited accrual to this initial Phase I study did not establish the MTD nor establish a complete pharmacokinetic and safety profile of intravenous Pc 4-PDT, these preliminary data support further Phase I testing of this new photosensitizer.

Delivery of the photosensitizer Pc 4 in PEG–PCL micelles for in vitro PDT studies

The silicon phthalocyanine Pc 4 is a second-generation photosensitizer that has several properties superior to other photosensitizers currently approved by the FDA, and it has shown significant promise for photodynamic therapy (PDT) in several cancer cells in vitro and model tumor systems in vivo. However, because of the high hydrophobicity of Pc 4, its formulation for in vivo delivery and favorable biodistribution become challenging. To this end, we are studying encapsulation and delivery of Pc 4 in block copolymer micelles. Here, we report the development of biocompatible PEG–PCL micelle nanoparticles, encapsulation of Pc 4 within the micelle core by hydrophobic association with the PCL block, and in vitro PDT studies of the micelle-formulated Pc 4 in MCF-7c3 human breast cancer cells. Our studies demonstrate efficient encapsulation of Pc 4 in the micelles, intracellular uptake of the micelle-formulated Pc 4 in cells, and significant cytotoxic effect of the formulation upon photoirradiation. Quantitative estimation of the extent of Pc 4 loading in the micelles and the photocytotoxicity of the micelle-incorporated Pc 4 demonstrate the promise of our approach to develop a biocompatible nanomedicine platform for tumor-targeted delivery of Pc 4 for site-selective PDT. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99: 2386–2398, 2010

Increased ceramide accumulation correlates with downregulation of the autophagy protein ATG‐7 in MCF‐7 cells sensitized to photodamage

The purpose of this study was to determine the sphingolipid (SL) profile in cells defective in autophagy protein ATG‐7 and overall cell death after photodynamic therapy (PDT) with the photosensitizer Pc 4. MCF‐7 human breast cancer cells with downregulated ATG‐7 and their scrambled controls (Scr) were used. Exposure of ATG‐7 knockdown cells to PDT led to increased cell killing. PDT evoked an early (2 h) greater global increase in ceramides in ATG‐7 defective cells compared to Scr cells. The increases in dihydroceramide (DHceramide) were distinctly higher at 2 h than at 24 h in both cell types post‐PDT. The levels of sphingosine‐1‐phosphate (S1P) and sphingosine were decreased below resting levels at 2 h irrespective of the cell type. The data imply that ceramide might be a marker of ATG‐7 deficiency in cells sensitized to PDT.

Intratumor Administration of the Photosensitizer Pc 4 Affords Photodynamic Therapy Efficacy and Selectivity at Short Drug-Light Intervals

We evaluated intratumor (IT) versus intravenous (IV) administration of the photosensitizer Pc 4 with respect to tumor photosensitizer concentration, specificity, and responses to irradiation. BALB/c mice bearing intradermal EMT6 tumors were given 0.3 mg/kg Pc 4 injected IT or IV through the tail vein. Photosensitizer concentration was evaluated by chloroform extraction and localization assessed by fluorescence imaging and spectroscopy in vivo. Tumors were irradiated at 667 nm, 50 mW/cm2, and 100 J/cm2. Cures were defined as no palpable tumor 90 days after irradiation. Tumor Pc 4 concentrations 1 hour after IT administration were 35,000-fold higher than measured 24 hours after IV administration (0.112 vs 0.317 x 10-5µg Pc 4/mg tumor). Exquisite tumor selectivity was observed 1 hour after IT injection. Fluorescence imaging of freshly sectioned tumors revealed no regions devoid of sensitizer at this time point, with pixel intensities in a midline section within a factor of 3 of the peak intensity. For identical photosensitizer doses, IT administration significantly improved tumor responses to irradiation, with more than 70% of tumors cured with IT-Pc 4-PDT. In this model, IT-Pc 4 administration provides improved tumor control, greater selectivity, and opportunity for a short drug-light interval.

Photodynamic therapy of cottontail rabbit papillomavirus‐induced papillomas in a severe combined immunodeficient mouse xenograft system

To evaluate the efficacy of photodynamic therapy (PDT) with the phthalocyanine photosensitizer Pc 4 for treating an animal model of recurrent respiratory papillomatosis (RRP). Rabbit skin was grafted onto the dorsum of severe combined immunodeficient mice, two xenografts per animal. After the graft healed, it was inoculated with cottontail rabbit papillomavirus (CRPV). When papillomas developed, Pc 4 (0.6 or 1.0 mg/kg) was administered systemically, and 48 hours later, one papilloma of the two on each animal was exposed to 675-nm photoactivating light at either 100 or 150 J/cm(2). In addition to the contralateral tumors, which received Pc 4 but no light, other controls included animals receiving light only or neither agent. Response was assessed by measuring papilloma size with a caliper. Some papillomas and residual skin were harvested for histological assessment. For the lower-dose PDT regimens, papilloma growth rates were not significantly different from the controls. In contrast, 13 of 15 papillomas receiving the higher Pc 4 dose (1.0 mg/kg) and the higher light fluence (150 J/cm(2)) regressed completely and did not regrow within the observation period of up to 79 days. The response of these papillomas was significantly different from the controls (P < .001). Histological analysis confirmed the absence of residual tumor following complete response and replacement with near-normal epithelium. Pc 4-PDT is highly effective in treating virally induced (CRPV) papillomas in a murine model of RRP, and thus warrants further study as a treatment for HPV-induced papillomas.

Binding to and photo-oxidation of cardiolipin by the phthalocyanine photosensitizer Pc 4

Cardiolipin is a unique phospholipid of the mitochondrial inner membrane. Its peroxidation correlates with release of cytochrome c and induction of apoptosis. The phthalocyanine photosensitizer Pc 4 binds preferentially to the mitochondria and endoplasmic reticulum. Earlier Förster resonance energy transfer studies showed colocalization of Pc 4 and cardiolipin, which suggests cardiolipin as a target of photodynamic therapy (PDT) with Pc 4. Using liposomes as membrane models, we find that Pc 4 binds to cardiolipin-containing liposomes similarly to those that do not contain cardiolipin. Pc 4 binding is also studied in MCF-7c3 cells and those whose cardiolipin content was reduced by treatment with palmitate. Decreased levels of cardiolipin are quantified by thin-layer chromatography. The similar level of binding of Pc 4 to cells, irrespective of palmitate treatment, supports the lack of specificity of Pc 4 binding. Thus, factors other than cardiolipin are likely responsible for the preferential localization of Pc 4 in mitochondria. Nonetheless, cardiolipin within liposomes is readily oxidized by Pc 4 and light, yielding apparently mono- and dihydroperoxidized cardiolipin. If similar products result from exposure of cells to Pc 4-PDT, they could be part of the early events leading to apoptosis following Pc 4-PDT.

Increased ceramide accumulation correlates with downregulation of the autophagy protein ATG-7 in MCF-7 cells sensitized to photodamage

The purpose of this study was to determine the sphingolipid (SL) profile in cells defective in autophagy protein ATG-7 and overall cell death after photodynamic therapy (PDT) with the photosensitizer Pc 4. MCF-7 human breast cancer cells with downregulated ATG-7 and their scrambled controls (Scr) were used. Exposure of ATG-7 knockdown cells to PDT led to increased cell killing. PDT evoked an early (2h) greater global increase in ceramides in ATG-7 defective cells compared to Scr cells. The total increases in dihydroceramide (DHceramide) were significant at 2 and 24h in both cell types post-PDT. The levels of sphingosine-1-phosphate (S1P) and sphingosine were decreased below resting levels at both time points irrespective of the cell type. The data imply that ceramide might be a marker of ATG-7 deficiency in cells sensitized to PDT.

Structural Factors and Mechanisms Underlying the Improved Photodynamic Cell Killing with Silicon Phthalocyanine Photosensitizers Directed to Lysosomes Versus Mitochondria

The phthalocyanine photosensitizer Pc 4 has been shown to bind preferentially to mitochondrial and endoplasmic reticulum membranes. Upon photoirradiation of Pc 4-loaded cells, membrane components, especially Bcl-2, are photodamaged and apoptosis, as indicated by activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase, is triggered. A series of analogs of Pc 4 were synthesized, and the results demonstrate that Pcs with the aminopropylsiloxy ligand of Pc 4 or a similar one on one side of the Pc ring and a second large axial ligand on the other side of the ring have unexpected properties, including enhanced cell uptake, greater monomerization resulting in greater intracellular fluorescence and three-fold higher affinity constants for liposomes. The hydroxyl-bearing axial ligands tend to reduce aggregation of the Pc and direct it to lysosomes, resulting in four to six times more killing of cells, as defined by loss of clonogenicity, than with Pc 4. Whereas Pc 4-PDT photodamages Bcl-2 and Bcl-xL, Pc 181-PDT causes much less photodamage to Bcl-2 over the same dose-response range relative to cell killing, with earlier cleavage of Bid and slower caspase-3-dependent apoptosis. Therefore, within this series of photosensitizers, these hydroxyl-bearing axial ligands are less aggregated than is Pc 4, tend to localize to lysosomes and are more effective in overall cell killing than is Pc 4, but induce apoptosis more slowly and by a modified pathway.

Suppression of sphingomyelin synthase 1 by small interference RNA is associated with enhanced ceramide production and apoptosis after photodamage

We have shown that overexpression of SMS1, an enzyme that converts de novo ceramide into sphingomyelin, is accompanied by attenuated ceramide response and apoptotic resistance after photodamage with the photosensitizer Pc 4 (photodynamic therapy; PDT). To test whether SMS1 overexpression-related effects after PDT can be reversed, in this study SMS1 was downregulated in Jurkat T lymphoma/leukemia cells using small inhibitory RNA (siRNA) for SMS1. Compared to scrambled (control) siRNA-transfectants, in SMS1 siRNA-transfected cells the activity of SMS at rest was downregulated with concomitant decrease in sphingomyelin mass. In SMS1 siRNA-transfected cells increases in ceramides were higher than in control siRNA-transfectants after PDT. Similar findings were obtained for dihydroceramides suggesting the involvement of de novo ceramide pathway. PDT-induced DEVDase (caspase-3-like) activation was enhanced in SMS1 siRNA-transfected cells compared to their control counterparts. The data show that RNA interference-dependent downregulation of SMS1 is associated with increased accumulation of ceramide and dihydroceramide with concomitant sensitization of cells to apoptosis after photodamage. Similarly, in SMS2 siRNA-transfected cells, downregulation of SMS activity was accompanied by potentiated DEVDase activation post-photodamage. These findings suggest that SMS is a potential novel molecular target that can augment therapeutic efficacy of PDT.

The Death of Human Cancer Cells Following Photodynamic Therapy: Apoptosis Competence is Necessary for Bcl‐2 Protection but not for Induction of Autophagy†

Photodynamic therapy (PDT) is an efficient inducer of apoptosis in many types of cells, except in cells deficient in one or more of the factors that mediate apoptosis. Recent reports have identified autophagy as a potential alternative cell death process following PDT. Here we investigated the occurrence of autophagy after PDT with the photosensitizer Pc 4 in human cancer cells that are deficient in the pro-apoptotic factor Bax (human prostate cancer DU145 cells) or the apoptosis mediator caspase-3 (human breast cancer MCF-7v cells) and in apoptosis-competent cells (MCF-7c3 cells that stably overexpress human pro-caspase-3 and Chinese hamster ovary CHO 5A100 cells). Further, each of the cell lines was also studied with and without stably overexpressed Bcl-2. Autophagy was identified by electron microscopic observation of the presence of double-membrane-delineated autophagosomal vesicles in the cytosol and by immunoblot observation of the Pc 4-PDT dose- and time-dependent increase in the level of LC3-II, a component of the autophagosomal membrane. Autophagy was observed in all of the cell lines studied, whether or not they were capable of typical apoptosis and whether or not they overexpressed Bcl-2. The presence of stably overexpressed Bcl-2 in the cells protected against PDT-induced apoptosis and loss of clonogenicity in apoptosis-competent cells (MCF-7c3 and CHO 5A100 cells). In contrast, Bcl-2 overexpression did not protect against the development of autophagy in any of the cell lines or against loss of clonogenicity in apoptosis-deficient cells (MCF-7v and DU145 cells). Furthermore, 3-methyladenine and wortmannin, inhibitors of autophagy, provided greater protection against loss of viability to apoptosis-deficient than to apoptosis-competent cells. The results show that autophagy occurs during cell death following PDT in human cancer cells competent or not for normal apoptosis. Only the apoptosis-competent cells are protected by Bcl-2 against cell death.

The Peripheral Benzodiazepine Receptor in Photodynamic Therapy with the Phthalocyanine Photosensitizer Pc 4¶

The Peripheral Benzodiazepine Receptor in Photodynamic Therapy with the Phthalocyanine Photosensitizer Pc 4¶

Sphingomyelin synthase 1 suppresses ceramide production and apoptosis post-photodamage

The role of sphingomyelin synthase 1 (SMS1), the Golgi membrane isoform of the enzyme, in ceramide metabolism and apoptosis after photodamage with the photosensitizer Pc 4 (PDT) is unclear. In the present study, using electrospray ionization/double mass spectrometry, we show that in Jurkat cells overexpressing SMS1, increases in ceramides were lower than in empty-vector transfectants post-PDT. Similarly, the responses of dihydroceramides and dihydrosphingosine, precursors of ceramide in the de novo synthetic pathway, were attenuated in SMS1-overexpressor after photodamage, suggesting the involvement of the de novo pathway. Overexpression of SMS1 was associated with differential regulation of sphingomyelin levels, as well as with the reduced inhibition of the enzyme post-treatment. Concomitant with the suppressed ceramide response, PDT-induced DEVDase activation was substantially reduced in SMS1-overexpressors. The data show that overexpression of SMS1 is associated with suppressed ceramide response and apoptotic resistance after photodamage.

Photodynamic therapy-induced death of HCT 116 cells: Apoptosis with or without Bax expression

Cell death following photodynamic therapy (PDT) with the photosensitizer Pc 4 involves the intrinsic pathway of apoptosis. To evaluate the importance of Bax in apoptosis after PDT, we compared the PDT responses of Bax-proficient (Bax(+/-)) and Bax knock-out (BaxKO) HCT116 human colon cancer cells. PDT induced a slow apoptotic process in HCT Bax(+/-) cells following a long delay in the activation of Bax and release of cytochrome c from mitochondria. Although cytochrome c was not released from mitochondria following PDT in BaxKO cells, an alternative mechanism of caspase-dependent apoptosis with extensive chromatin and DNA degradation was found in these cells. This alternative process was less efficient and slower than the normal apoptotic process observed in Bax(+/-) cells. Early events upon PDT, such as the loss of mitochondrial membrane potential, photodamage to Bcl-2, and activation of p38 MAP kinase, were observed in both HCT116 cell lines. In spite of differences in the efficiency and mode of apoptosis induced by PDT in the Bax(+/-) and BaxKO cells, they were found to be equally sensitive to killing by PDT, as determined by loss of clonogenicity. Thus, for Pc 4-PDT, the commitment to cell death occurs prior to and independent of Bax activation, but the process of cellular disassembly differs in Bax-expressing vs. non-expressing cells.

Differential responses of Mcl-1 in photosensitized epithelial vs lymphoid-derived human cancer cells

The antiapoptotic Bcl-2-family proteins, Bcl-2 and Bcl-xL, are recognized phototargets of photodynamic therapy (PDT) with the mitochondrion-targeting phthalocyanine photosensitizer Pc 4. In the present study, we found that myeloid cell leukemia 1 (Mcl-1), another antiapoptotic member of the Bcl-2 family, was not photodamaged in Pc 4-PDT-treated human carcinoma cells MCF-7c3, MDA-MB468, DU145, and A431, although Mcl-1 turnover was observed after exposure of HeLa or MCF-7c3 cells to a supralethal dose of UVC. In contrast, when human lymphoma U937 and Jurkat cells were treated with Pc 4-PDT, staurosporine (STS) or UVC, Mcl-1 was cleaved to generate a 28-kDa fragment over a 2-4 h period. The cleavage of Mcl-1 was accompanied by the activation of caspases-3, -9, and -8. The broad-specificity caspase inhibitor z-VAD-fmk completely blocked Mcl-1 cleavage induced by PDT, STS or UVC, providing evidence for Mcl-1 as a substrate for caspases. Western blot analysis localized Mcl-1 to mitochondria, ER, and cytosol of both MCF-7c3 and U937 cells, suggesting that Mcl-1 protein, unlike Bcl-2 and Bcl-xL, is not a target for Pc 4-PDT, probably due to its localization to sites removed from those of Pc 4 binding. The 28-kDa cleaved fragment of Mcl-1, which has proapoptotic activity, was produced in PDT-treated lymphoid-derived cells, but not in cells of epithelial origin, suggesting that PDT-induced rapid and extensive apoptosis in lymphoma cells may result in part from the sensitivity of their Mcl-1 to caspase cleavage, removing an important negative control on apoptosis.