Proteins In Cell Culture Medium Research Articles

Targeting a therapeutically relevant concentration of alendronate for in vitro studies on osteoblasts

Objective Bisphosphonates like alendronate mainly exert their effects on osteoclasts. However, osteoblasts are also affected, but exposed to a much lower concentration in vivo than the osteoclasts. Given that the effects are dose-dependent, the intention of the study was to identify a therapeutically relevant concentration of alendronate for in vitro studies on osteoblasts. Materials and methods Primary human osteoblasts were incubated with alendronate (5, 20 and 100 µM) for 1, 3, 7 and 14 days. Proliferation and viability were assessed, and the effects on cellular growth and function were evaluated by multianalyte profiling of selected proteins in cell culture media using the Luminex 200TM. Results The viability was not affected by any of the dosages. Exposure to 5 µM alendronate had a neutral effect on osteoblast proliferation, and on secretion of osteogenic and inflammatory markers, while enhancing synthesis of a marker of angiogenesis. 20 µM alendronate induced a decline in proliferation and affected angiogenic and osteogenic biomarkers adversely. 100 µM alendronate reduced proliferation dramatically, and this dosage was excluded from further experiments. Conclusion A concentration of 5 µM alendronate exerted effects on human osteoblasts that may translate to those observed in vivo and could therefore be relevant for in vitro studies.

Alendronate and omeprazole in combination reduce angiogenic and growth signals from osteoblasts

ObjectiveDue to gastrointestinal side effects of oral bisphosphonates (BPs), proton pump inhibitors (PPIs) are often prescribed. PPIs may enhance the risk of osteonecrosis of the jaw, a rare side effect of BPs. Therefore, the objective of this study was to evaluate the effects of the oral BP alendronate (ALN) and the PPI omeprazole (OME) alone and in combination on primary human osteoblasts and gingival fibroblasts in vitro. MethodsHuman gingival fibroblasts and normal human osteoblasts were incubated with either 5 μM of ALN or 1 μM of OME, or ALN + OME for 1, 3, 7 or 14 days. Effect on viability was evaluated by the lactate dehydrogenase activity in the medium and on proliferation by quantifying 3H-thymidin incorporation. Multianalyte profiling of proteins in cell culture media was performed using the Luminex 200TM system to assess the effect on selected bone markers and cytokines. ResultsThe proliferation of osteoblasts and fibroblasts was reduced upon exposure to ALN + OME. ALN induced an early, temporary rise in markers of inflammation, and OME and ALN + OME promoted a transient decline. An initial increase in IL-13 occurred after exposure to all three options, whereas ALN + OME promoted IL-8 release after 7 days. OME and ALN + OME promoted a transient reduction in vascular endothelial growth factor (VEGF) from osteoblasts, whereas ALN and ALN + OME induced a late rise in VEGF from fibroblasts. Osteoprotegerin release was enhanced by ALN and suppressed by OME and ALN + OME. ConclusionsALN + OME seemed to exaggerate the negative effects of each drug alone on human osteoblasts and gingival fibroblasts. The anti-proliferative effects, modulation of inflammation and impairment of angiogenesis, may induce unfavorable conditions in periodontal tissue facilitating development of osteonecrosis.

Investigating the Interaction of Nanodiamonds with Human Serum Albumin and Induced Cytotoxicity

Nanodiamonds (NDs) have been recognized as emerging carbon-based delivery vehicles due to their biocompatibility. NDs were reported to be nontoxic and suited for biomedical applications in the complete cell culture medium. However, in this study, the cytotoxicity of NDs in serum-free medium was studied and indicated that serum proteins in cell culture medium played significant effect on the toxicity of NDs. Therefore, the interaction mechanism between NDs and a serum protein (human serum albumin, HSA) was first investigated by fluorescence quenching technique and circular dichroism (CD) spectrometry. The results suggest that HSA strongly bonds on the NDs surface to form “protein corona,” which not only prevents the aggregation of NDs and improves its stability but also inhibits the cytotoxicity of NDs. In serum-free medium, NDs exhibited obvious toxicity toward the human lung epithelial cell line (BEAS-2B) and showed concentration-dependent cytotoxicity. In the presence of bovine serum albumin (BSA), which shares structural homology and similar properties with HSA, toxicity of NDs was apparently inhibited. Therefore, the interaction between serum protein and NDs should be considered in the understanding of the biological effects of NDs exposure in biomedical applications.

Surface characterization of insulin-coated Ti6Al4V medical implants conditioned in cell culture medium: An XPS study

Surface characterization of insulin-coated Ti6Al4V medical implants, after incubation in α-minimum essential medium (α-MEM), was done by X-ray photoelectron spectroscopy (XPS), in order to analyze the insulin behavior at the implant – α-MEM interface. In the absence of serum proteins in cell culture medium, the coated insulin layer remained intact, but experienced a time-dependent structural transformation exposing hydrophobic parts of the protein toward the solution. The presence of fetal bovine serum (FBS) in the medium resulted in partial substitution of insulin by serum proteins. In spite of some insulin release, the remaining coated layer demonstrated a direct surface effect by stabilizing the structure of protein competitors, and by supporting the accumulation of calcium and phosphate ions at the interface. A structurally stable protein layer with incorporated calcium and phosphate ions at the implant–tissue interface could be an important prerequisite for enhanced bone formation.

Serum adsorption, cellular internalization and consequent impact of cuprous oxide nanoparticles on uveal melanoma cells: implications for cancer therapy.

To investigate the biological fate of cuprous oxide nanoparticles (Cu2O-NPs) and to evaluate their potential in uveal melanoma therapy. The protein corona, cellular uptake mechanism and localization of Cu2O-NPs were investigated. Furthermore, the effect of Cu2O-NPs on uveal melanoma cell proliferation, migration and invasion, and possible mechanisms were studied in detail. Cu2O-NPs are able to adsorb serum proteins in cell culture medium, which are then internalized by uveal melanoma cells mainly through lipid raft-mediated endocytosis. Furthermore, Cu2O-NPs selectively inhibit cancer cell growth and impair the ability of uveal melanoma cell migration, invasion and the cytoskeleton assembly. The mechanism may be that Cu2O-NPs located in and damage mitochondria, autophagolysosomes and lysosomes, leading to elevated reactive oxygen species level and over-stimulated apoptosis and autophagy. The data provide detailed information of Cu2O-NPs for further application and indicate that Cu2O-NPs could be a potential agent for uveal melanoma therapy.

Enhancement of human erythropoietin production in Chinese hamster ovary cells through supplementation of 30Kc19-30Kc6 fusion protein

The importance of anti-apoptosis in mammalian cell culture has been widely recognized. We reported previously that expression of Bombyx mori 30K genes in Chinese hamster ovary (CHO) cells increases recombinant protein production by inhibiting apoptosis and enhancing specific productivity. However, previous studies have shown expression of the anti-apoptotic 30Kc6 protein as inclusion bodies in Escherichia coli. 30Kc19 protein, another silkworm hemolymph protein, has cell-penetrating and recombinant protein productivity-improving properties, and we found that it improves soluble expression of its partner. In this study, we fused 30Kc6 with 30Kc19 as an expression partner to express a soluble fused protein and to deliver the protein to cells. Supplementing the recombinant 30Kc19-30Kc6 fusion protein in cell culture medium increased viability, effectively penetrated the cells, and inhibited CHO cell apoptosis by 68%. Moreover, the mitochondrial membrane potential and ATP generation also increased by 50% and 33%, respectively. Erythropoietin (EPO) productivity increased by>30% because of the anti-apoptotic effect and increased specific productivity. These results demonstrate the potential use of this fusion protein as a supplement in mammalian cell culture during production of biopharmaceutical proteins.

Protein impurities from cell culture dramatically impact transduction efficiency of polymer/virus hybrid vectors

Polyethylenimine (PEI) was used recently with murine leukemia virus-like particles (MLV-VLPs) to produce a hybrid vector that possesses advantages over the native virus; the transduction efficiency of this vector, however, was less than the transduction efficiency of the native virus. The cause of the reduced efficiency was hypothesized to be related to the involvement of proteins in PEI/MLV-VLP complex formation and overall complex size. To test the hypothesis and potentially improve the efficiency of the hybrid vector, ultracentrifugation and size exclusion chromatography were used to purify MLV-VLP and to study the effect of proteins in cell culture medium on complex formation. Based on dynamic light scattering and electron microscopy, complexes formed from the purified MLV-VLPs were smaller, but surprisingly, less efficient than complexes formed from unpurified MLV-VLPs. The addition of protein to purified MLV-VLPs showed that the initial efficiency could be restored and that the purification strategy was not inactivating the MLV-VLPs. Further, by optimizing the amount of protein added to the purified MLV-VLPs, the level of transduction by PEI/MLV-VLP improved 1.6-fold. Particle characterization showed a correlation between the size of the PEI/MLV-VLP complex and the transduction efficiency, which is likely a result of greater sedimentation and cell contact during in vitro studies.

Identifying the CHO Secretome using Mucin-type O-Linked Glycosylation and Click-chemistry

Chinese hamster ovary cells (CHO) are the most common cell line used in the production of therapeutic proteins. Understanding the complex pattern of secreted host cell proteins (HCP) that are released by CHO cells will facilitate the development of new recombinant protein production processes. In this study, we have adapted the N-azido-galactosamine (GalNAz) metabolic labeling method to enable the mass spectrometry identification and quantification of secreted proteins in cell culture media. CHO DG44 and CHO-S cells were cultured in media containing GalNAz, which was metabolically incorporated into mucin-type O-linked glycans of secreted proteins. These proteins were effectively enriched using click-chemistry from the cell culture media, allowing for the analysis of secreted proteins across multiple days of cell growth. When compared to the standard method for secretome analysis, the GalNAz method not only increased the total number of proteins identified but dramatically improved the quality of data by decreasing the number of background proteins (cytosolic or nuclear) to essentially zero.

Biofunctionalized bacterial cellulose membranes by cold plasmas

Bacterial cellulose (BC) membranes were modified with oxygen (O2), nitrogen (N2), and tetrafluoromethane (CF4) plasmas in order to enhance cell affinity. The surface properties of the pristine and plasma-treated BCs were analyzed through water contact angles, electron spectroscopy for chemical analysis (ESCA), and scanning electron microscopy. O2 and N2 plasmas changed the surface of BCs to more hydrophilic while CF4 plasma altered BCs to be very hydrophobic. ESCA analyses indicated that O2, N2, and CF4 plasmas incorporated the functionalities of carbon–oxygen, amides and amino, and carbon-fluoride on BCs, respectively. The effects of the plasma treatments on the adhesion of L-929 fibroblast and Chinese hamster ovary cell lines showed that the cell adhesion and proliferation of both cells was significantly improved on BC-CF4, in contrast with that on the pristine BC, BC-O2, and BC-N2, revealing a functionality-specific effect resulted from different plasmas. Moreover, protein adsorption tests indicated that a higher quantity of proteins in cell culture medium was adsorbed on the CF4 plasma-treated BCs which presumably played the role of enhancing the subsequent cell growth. This work highlights the great potential of plasma treatments on the improvement of biocompatibility and surface property of BCs for biomedical applications.

Detection of weak receptor–ligand interactions using IgM and J‐chain‐based fusion proteins

Detection of weak receptor–ligand interactions using <scp>I</scp>g<scp>M</scp> and <scp>J</scp>‐chain‐based fusion proteins

Abstract 3609: Generation and characterization of a novel monoclonal antibody recognizing both the blood and tissue form of human PCBP-1

Abstract Background: Poly(rC)-binding protein-1 (PCBP-1) is a nucleic-acid-binding protein that localizes in nucleus and/or cytoplasm of cells. Shuttling between nucleus and cytoplasm has been related to its function in regulation of RNA transcription, translation and also implicated its involvement in cancer metastasis, progression and chemoresistance. In the present study, we investigated the levels and expression of PCBP-1 in breast carcinoma and its potential use as a biomarker in breast carcinoma differential diagnosis. Methods/Results: We generated a novel monoclonal antibody (mAb), AB-PCBP-1 using hybridoma technology and native protein in cell culture medium as immunogen. This mAb is of IgG1 (kappa) type. It specifically recognized both native and denatured human PCBP-1 protein and displayed high affinity as assessed by ELISA, immunohistochemistry assay, western blot and flow cytometry applications. Using this mAb in competitive ELISA, we showed for the first time that soluble PCBP-1 level is significantly increased in the plasma of metastatic breast cancer patients’ plasma. PCBP-1 is also expressed in metastatic breast carcinoma tissue by immunohistochemistry. Seventy percent of normal breast ducts and lobules expressed strong nuclear staining whereas cytoplasmic staining was less specific and found in most cancer tissues. We examined 90 cases of breast carcinoma tissues classified as either ductal and lobular carcinoma. To evaluate the immunoreactivity of PCBP-1, the staining intensity and intracellular distribution were compared and scored as 0, 1+, 2+ and 3+ based on intensity. Seventy nine % (60 out of 76) of ductal carcinomas showed strong cytoplasmic staining (2+ and 3+) and 45% also exhibited strong nuclear localization for PCBP-1. Lobular carcinomas (n=14), on the other hand, showed an exclusively nuclear staining pattern.Conclusion: AB-PCBP-1 mAb showed high performance in blood and tissue assays. PCBP-1 levels were significantly increased in blood and demonstrated a distinct pattern of expression related to histological tumor types, suggesting that PCBP-1 may be involved in a regulatory mechanism that differs in various subtypes of breast carcinoma. The results suggest the potential clinical use of PCBP-1 as a therapeutic target and/or a biomarker in blood and tissue for the diagnosis and prognosis of breast carcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3609. doi:1538-7445.AM2012-3609

Identification of serum proteins bound to industrial nanomaterials

Nanoparticles (NPs) are decorated with proteins and other biomolecules when they get into contact with biological systems. The presence of proteins in cell culture medium can therefore have effects on the biological outcome in cell-based tests. In this study, the manufactured nanomaterials silicon dioxide (SiO 2), titanium dioxide (TiO 2), iron-III-oxide (Fe 2O 3), and carbon black (CB) were used to study their interaction with single proteins from bovine and human plasma (albumin, fibrinogen and IgG) as well as with complete human serum. The protein binding capacity of the material was investigated and 1D gel electrophoresis was used to separate the bound proteins and to identify the bands by matrix-assisted laser desorption/ionisation-time-of-flight (MALDI-TOF) mass spectrometry. We found that the NP surface chemistry had a great impact on the amount of bound protein with distinct ligands for each of the tested particles. The hydrophobic CB NPs bound much more protein than the hydrophilic metal oxide NPs. Among the single proteins investigated, fibrinogen showed the strongest affinity for SiO 2, TiO 2 and CB NPs. The identified proteins from human serum adsorbed to these NPs were very different. Only apolipoprotein A1 was found to be adsorbed to all NPs. These studies will help to explain the different degree of biological responses observed after in vitro exposure of cells in the absence or presence of serum and might also support the interpretation of in vivo experiments were NPs come directly into contact with blood plasma.

Sequentially Modified, Polymer-Stabilized Gold Nanoparticle Libraries: Convergent Synthesis and Aggregation Behavior

This manuscript describes a versatile, yet experimentally facile, method for producing libraries of polymer-coated (core−shell type) gold nanoparticles. The synthetic principle relies on two, sequential postmodification reactions, which ensures homogeneity across each series. First, poly(pentafluorophenyl methacrylate) synthesized by RAFT polymerization is used here as a reactive precursor, which can be modified, postpolymerization, to create a library of functional polymers each bearing a ω-thiol end-group. In a second step, these well-defined polymers are then tethered by their ω-thiol group to the surface of prefabricated citrate-stabilized gold nanoparticles to give a library of 75 unique, yet sequentially modified organic−inorganic hybrid particles. The optical properties of the gold core were exploited to create a high-throughput assay for investigating the role of nanoparticle size and surface coating on aggregation in various biologically relevant media. These experiments demonstrated the importance of the type of dissolved salts present and also the strong influence of serum proteins in cell-culture media and their interactions with nanoparticles surfaces, which in turn might affect their biological profiles. Therefore, this method presents a powerful, yet accessible tool for creating model nanoparticle libraries with intrinsic sensing properties.

Nanodiamonds as intracellular transporters of chemotherapeutic drug

To explore the application of nanodiamonds (NDs) as anticancer drug delivery vehicles, firstly we examined the toxicity of NDs to three kinds of mammalian cells. The studies indicated that serum proteins in cell culture medium had significant effect. When cells were exposed to NDs dispersed in complete cell culture medium, no cytotoxicity was detected. However, severe cell death was found after exposed to NDs dispersed in serum-free medium. Possible reasons for serum-dependent cytotoxicity were discussed and the potential influence of serum on the test of efficacy was pointed for anticancer drug delivery system based on NDs. Then adsorption of anticancer drug 10-hydroxycamptothecin (HCPT) by NDs was studied. Experiments indicated that diluted NaOH solution could promote the loading of HCPT on NDs. And a slow release of HCPT from the HCPT-ND complex was established in low pH media. Assessments of cell viability and imaging with transmission electron microscopy demonstrated a much higher efficacy of the HCPT-ND complex compared with HCPT alone. On the basis of adsorption feature of ND aggregates, new strategy for the design of targeting drug delivery systems of NDs was proposed.

Incorporation of proteins within alginate fibre-based scaffolds using a post-fabrication entrapment method

In this study, a physical entrapment process was explored for the incorporation of proteins within preformed fibrous alginates and the release profile was tuned by varying the processing parameters. The entrapment process was carried out in a series of aqueous solutions at room temperature and involved pre-swelling of the fibrous alginate within a Na(+)-rich solution, followed by exposure to the protein of choice and entrapping it by re-establishing cross-links of alginate with BaCl2. Entrapment and release of fluorescein isothiocyanate-labelled bovine serum albumin (FITC-BSA), a model protein, was studied. It was found that a sustained release of the incorporated protein in cell culture medium for about 6 days was achieved. The main factors determining the release profile included the NaCl/CaCl2 ratio in the pre-swelling solution, protein concentration, and the exposure time. To retard protein release, alginate fibres with entrapped FITC-BSA were processed together with poly(D, L-lactide) (PDLLA) into porous alginate fibre/PDLLA composites using supercritical CO2. In this manner, release of the protein for up to 3 months was achieved.

Modular scaffolds assembled around living cells using poly(ethylene glycol) microspheres with macroporation via a non-cytotoxic porogen

Modular, bioactive, macroporous scaffolds were formed by crosslinking poly(ethylene glycol) (PEG) microspheres around living cells. Hydrogel microspheres were produced from reactive PEG derivatives in aqueous sodium sulfate solutions without the use of surfactants or copolymers. Microspheres were formed following thermally induced phase separation if the gel point was reached prior to extensive coarsening of the PEG-rich domains. Three types of PEG microspheres with different functionalities were used to form scaffolds: one type provided mechanical support, the second type provided controlled delivery of the angiogenesis-promoting molecule, sphingosine 1-phosphate (S1P) and the third type served as a slowly dissolving non-cytotoxic porogen. Scaffolds were formed by centrifuging microspheres in the presence of HepG2 hepatoma cells, resulting in a homogenous distribution of cells. During overnight incubation at 37°C, the microspheres reacted with serum proteins in cell culture medium to stabilize the scaffolds. Within 2days in culture, macropores formed due to the dissolution of the porogenic PEG microspheres, without affecting cell viability. Gradients in porosity were produced by varying the buoyancy of the porogenic microspheres. Conjugated RGD cell adhesion peptides and the delivery of S1P promoted endothelial cell infiltration through macropores in the scaffolds. The scaffolds presented here differ from previous hydrogel scaffolds in that: (i) cells are not encapsulated in hydrogel; (ii) macropores form in the presence of cells; and (iii) scaffold properties are controlled by the modular assembly of different microspheres that perform distinct functions.

Effects of serum proteins on intracellular uptake and cytotoxicity of carbon nanoparticles

To explore the effects of the novel properties of carbon nanoparticles (CNPs) on cytotoxicity, the adsorption of serum proteins in cell culture medium on multi-walled carbon nanotubes and three kinds of carbon blacks was investigated. The uptake of CNPs by Hela cells was measured quantitatively using 99mTc radionuclide labeling and tracing techniques, and the dependence of CNPs uptake on serum proteins was examined. The cytotoxicity of CNPs in medium with and without serum was assayed by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method. It was found that cellular uptake was much higher in cells exposed to CNPs in serum-free culture medium than those in culture medium with serum. Serum proteins adsorbed on CNPs attenuated the inherent cytotoxicity of CNPs, and the extent of toxicity attenuation increased with increasing amounts of serum proteins adsorbed on CNPs. The possible reasons responsible for the considerable influence of serum proteins on cytotoxicity were indicated.

Estrogen Receptor β Inhibits Angiogenesis and Growth of T47D Breast Cancer Xenografts

Estrogens, which are stimulators of growth of both the normal breast and malignant breast, mediate their effects through two estrogen receptors (ER), namely ERalpha and ERbeta. ERalpha mediates the proliferative effect of estrogen in breast cancer cells, whereas ERbeta seems to be antiproliferative. We engineered ERalpha-positive T47D breast cancer cells to express ERbeta in a Tet-Off-regulated manner. These cells were then injected orthotopically into severe combined immunodeficient mice, and the growth of the resulting tumors was compared with tumors resulting from injecting the parental T47D cells that do not express ERbeta. The presence of ERbeta resulted in a reduction in tumor growth. Comparison of the ERbeta-expressing and non-ERbeta-expressing tumors revealed that the expression of ERbeta caused a reduction in the number of intratumoral blood vessels and a decrease in expression of the proangiogenic factors vascular endothelial growth factor (VEGF) and platelet-derived growth factor beta (PDGFbeta). In cell culture, with the Tet-Off-regulated ERbeta-expressing cells, expression of ERbeta decreased expression of VEGF and PDGFbeta mRNA under normoxic as well as hypoxic conditions and reduced secreted VEGF and PDGFbeta proteins in cell culture medium. Transient transfection assays with 1,026 bp VEGF and 1,006 bp PDGFbeta promoter constructs revealed a repressive effect of ERbeta at the promoter level of these genes. Taken together, these data show that introduction of ERbeta into malignant cells inhibits their growth and prevents tumor expansion by inhibiting angiogenesis.

Synthetic DNA aptamers to detect protein molecular variants in a high-throughput fluorescence quenching assay.

Real-time protein detection in homogeneous solutions is necessary in many biotechnology and biomedical studies. The recent development of molecular aptamers, combined with fluorescence techniques, may provide an easy and efficient approach to protein elucidation. This report describes the development of a fluorescence-based assay with synthetic DNA aptamers that can detect and distinguish molecular variants of proteins in biological samples in a high-throughput process. We used an aptamer with high affinity for the B chain of platelet-derived growth factor (PDGF), labeled it with a fluorophore and a quencher at the two termini, and measured fluorescence quenching by PDGF. The specific quenching can be used to detect PDGF at picomolar concentrations even in the presence of serum and other cell-derived proteins in cell culture media. This is the first successful application of a synthetic aptamer for the detection of tumor-related proteins directly from the tumor cells. We also show that three highly related molecular variants of PDGF (AA, AB, and BB dimers) can be distinguished from one another in this single-step assay, which can be readily adapted to a microtiter plate assay for high-throughput analysis. The use of fluorescence quenching as a measure of binding between the DNA probe and the target protein eliminates potential false signals that may arise in traditional fluorescence enhancement assays as a result of degradation of the DNA aptamer by contaminating nucleases in biological specimens. This assay is applicable to proteins that are not naturally DNA binding. The excellent specificity, ultrahigh sensitivity, and simplicity of this one-step assay addresses a growing need for high-throughput methods that detect changes in the expression of gene products and their variants in cell cultures and biological specimens.

Involvement of Fas (APO-1/CD-95) during Photodynamic-Therapy-Mediated Apoptosis in Human Epidermoid Carcinoma A431 Cells

Photodynamic therapy is a promising treatment modality for a variety of cutaneous neoplasms and other skin disorders. Studies suggest an involvement of multiple pathways during photodynamic-therapy-mediated cell death. A complete knowledge of the mechanisms involved in photodynamic therapy may lead to an improvement in its therapeutic efficacy. In vitro as well as in vivo studies have shown the involvement of apoptosis during photodynamic- therapy-mediated cell death. The pathways by which photodynamic therapy causes this are not fully understood. In this study, employing human epidermoid carcinoma (A431) cells and silicon phthalocyanine 4 photodynamic therapy, we show that the cell surface death receptor Fas (also known as APO-1 or CD-95) pathway is an important contributor to photodynamic-therapy-mediated apoptosis. Employ- ing flow cytometric analysis and confocal microscopy we first established that silicon phthalocyanine 4 photodynamic therapy results in a significant induction of apoptosis in A431 cells. Immunoblot analysis revealed a significant time-dependent increase in the protein expression of Fas at 5, 15, 30, and 60 min post-photodynamic therapy followed by a decrease at later time-points (2 and 3 h post-photodynamic therapy). A Fas enzyme-linked immunosorbent assay demonstrated an increase in this protein in cell culture medium starting at 1 h post-photodynamic therapy and showing a time-dependent response up to 3 h following therapy, suggesting a diffusion of soluble Fas from cells into the medium from 1 h after photodynamic therapy. Silicon phthalocyanine 4 photodynamic therapy also resulted in a time-dependent increase in (i) the multimerization of Fas protein, (ii) the protein expression of Fas ligand, (iii) FADD, an adapter molecule for Fas, and (iv) the binding of FADD with Fas. Silicon phthalocyanine 4 photodynamic therapy also caused a significant activation of FLICE, as evident from the appearance of cleaved products of pro-caspase 8. Further, a pretreatment of cells with rhFas:Fc fusion protein or general caspase inhibitor Z-VAD-FMK followed by silicon phthalocyanine 4 photodynamic therapy resulted in a significantly enhanced cell survival. Taken together, our data, for the first time, delineate an involvement of the Fas pathway as an important contributor to photodynamic-therapy-mediated apoptosis of cancer cells. These observations may be important for improving the efficacy of photodynamic therapy for the treatment of skin cancer and possibly other skin disorders. J Invest Dermatol 115:1041-1046 2000