[Show abstract][Hide abstract] ABSTRACT: A polyphenol constituent of green tea, epigallocatechin gallate (EGCG), has anti-carcinogenic properties. A growing number of studies document EGCG-mediated induction of apoptotic pathways and inhibition of pro-survival factors when combined with chemotherapy or radiation. We evaluated the efficacy of EGCG in modulating photofrin (PH)-mediated photodynamic therapy (PDT) responses.
Mouse mammary carcinoma (BA) cells and transplanted BA tumors growing in C3H mice were treated with PH-mediated PDT. Select groups of treated cells and mice also received EGCG and then cytotoxicity, tumor response, and expression of survival molecules were evaluated in all experimental groups.
EGCG increased apoptosis and cytotoxicity in BA cells exposed to PH-mediated PDT. The initial pro-survival phase of the unfolded protein response (UPR), characterized by increased expression of the 78 kDa glucose-regulated protein (GRP-78), was induced by PDT. The second pro-apoptotic phase of the UPR, characterized by phospho-c-Jun N-terminal kinase (p-JNK) expression, activation of caspases-3 and 7, poly ADP ribose polymerase (PARP) cleavage, and expression of C/EBP homologous protein was observed when PDT was combined with EGCG. EGCG also decreased the expression of the pro-survival proteins GRP-78 and survivin, and attenuated PDT-induced prostaglandin E2 (PGE2 ) expression in PDT-treated cells. Comparable responses also were observed when BA tumors were treated with PDT and EGCG. In addition, PDT-induced expression of metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF) was down-regulated in treated tumor tissue by EGCG.
The polyphenol EGCG improves PDT efficacy by increasing tumor apoptosis and decreasing expression of pro-survival and angiogenic molecules within the tumor microenvironment.
Full-text · Article · Sep 2011 · Lasers in Surgery and Medicine
[Show abstract][Hide abstract] ABSTRACT: Photodynamic therapy (PDT) effectiveness can be improved by employing combined modality approaches involving pharmaceuticals targeting the tumor microenvironment and/or tumor cell death pathways. In one approach, combining PDT with celecoxib improves long-term tumoricidal activity without increasing normal tissue photosensitization. However, side effects arising from the use of coxib based cyclooxygenase-2 (COX-2) inhibitors, including cardiovascular injury, decreases the clinical applications of this class of compounds. A growing number of studies demonstrate that the tumoricidal actions of coxibs such as celecoxib involve non-COX-2 mediated mechanisms. The celecoxib analog, 2,5-dimethyl celecoxib (DMC), lacks COX-2 inhibitory activity but exhibits cytotoxic properties comparable to the COX-2 inhibitor celecoxib. We compared the effectiveness of DMC and celecoxib in modulating PDT response at both the in vitro and in vivo level using a C3H/BA murine mammary carcinoma model. Both DMC and celecoxib blocked PDT induced expression of the pro-survival protein survivin, enhanced the endoplasmic reticulum stress (ERS) response of PDT, and increased both apoptosis and cytotoxicity in BA cells exposed to combination protocols. DMC enhanced the in vivo tumoricidal responsiveness of PDT without altering PGE2 levels. Our data demonstrates that DMC improved PDT by increasing apoptosis and tumoricidal activity without modulating COX-2 catalytic activity. Our results also suggest that celecoxib mediated enhancement of PDT may involve both COX-2 dependent and independent mechanisms.
[Show abstract][Hide abstract] ABSTRACT: Photodynamic therapy (PDT) using the photosensitizer Photofrin is approved for the clinical treatment of solid tumors. PDT causes cytotoxic oxidative stress, but additionally induces prosurvival molecules such as cyclooxygenase-2 (COX-2). Combining PDT with COX-2 inhibitors increases the efficacy of in vivo treatment. Understanding mechanisms leading to prosurvival molecule induction is relevant to the design of more effective treatments. Using COX-2 promoter constructs, transcription factor-binding assays, identification of protein kinase activation, and inhibitors of transcription factor binding we were able to determine that COX-2 expression following PDT involves the p38 MAP kinase pathway.
No preview · Article · Jan 2010 · Methods in molecular biology (Clifton, N.J.)
[Show abstract][Hide abstract] ABSTRACT: Photodynamic therapy (PDT), using the porphyrin photosensitizer Photofrin (PH), is approved for the clinical treatment of solid tumors. In addition to the direct cytotoxic responses of PH-PDT-mediated oxidative stress, this procedure also induces expression of angiogenic and prosurvival molecules including cyclooxygenase-2 (COX-2). In vivo treatment efficacy is improved when PH-PDT is combined with inhibitors of COX-2. In the current study we evaluated the signaling pathways involved with PH-PDT-mediated COX-2 expression in a mouse fibrosarcoma cell line. COX-2 promoter reporter constructs with mutated transcription elements documented that the nuclear factor kappa B (NFkappaB) element, cyclic-AMP response element 2 (CRE-2), CCAAT/enhancer binding protein (C/EBP) element and activator binding protein-1 (AP-1) element were responsive to PH-PDT. Transcription factor binding assays demonstrated that nuclear protein binding to NFkappaB, CRE-2, c-fos and c-jun elements were elevated following PH-PDT. Kinase phosphorylation upstream of COX-2 expression was also examined following PH-PDT. Stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and c-Jun were phosphorylated following PH-PDT but the SAPK/JNK inhibitor SP600125 failed to attenuate COX-2 expression. In contrast, p38 mitogen-activated protein kinase (MAPK), which activates CRE-2 binding, was phosphorylated following PH-PDT and inhibitors of p38 MAPK, SB203580 and SB202190, decreased PH-PDT-induced COX-2 expression at both the mRNA and protein levels. Extracellular signal-regulated kinase (ERK1/2) phosphorylation, which also increases CRE-2 binding activity, was initially high in untreated cells, decreased immediately following PH-PDT and then rapidly increased. MEK1/2 is immediately upstream of ERK1/2 and the MEK1 inhibitor PD98059 failed to attenuate COX-2 expression while the MEK1/2 inhibitor U0126 induced a slight decrease in COX-2 expression. The NFkappaB inhibitor SN50 failed to reduce COX-2 expression. These results demonstrate that multiple protein kinase cascades can be activated by oxidative stress and that the p38 MAPK signaling pathway and CRE-2 binding are involved in COX-2 expression following PH-PDT.
No preview · Article · Mar 2008 · Photochemistry and Photobiology
[Show abstract][Hide abstract] ABSTRACT: A growing number of clinically relevant molecular and cellular responses are observed following photodynamic therapy (PDT). PDT-mediated oxidative stress and PDT-induced tissue hypoxia can elicit the transcriptional and/or translational expression of genes associated with cellular stress, inflammation, angiogenesis, immuno-modulation, apoptosis and signal transduction. One of the signaling molecules activated by oxidative stress is Akt/protein kinase B. Phosphorylation of Akt/protein kinase B activates this signaling molecule and induces a survival response in effected cells and tissue. We hypothesized that PDT using Photofrin (PH) as the photosensitizer could also induce increased levels of Akt phosphorylation. Results from our initial set of experiments demonstrated that in vitro and in vivo PDT treatments induced Akt phosphorylation. Interestingly, incubation of mouse and human breast cancer cells with the porphyrin-based photosensitizer, PH, increased the expression of Akt phosphorylation in the absence of light. Exposure of the corresponding mouse and human-derived breast cancer tumors growing in mice to 630 nm light in the absence of PH administration also induced Akt phosphorylation. These results demonstrate that individual components of the PDT process, photosensitizer alone and light alone, as well as the complete PDT procedure can activate the Akt signaling pathway.
No preview · Article · Sep 2007 · Photochemistry and Photobiology
[Show abstract][Hide abstract] ABSTRACT: We observed that photodynamic therapy (PDT) induces the expression and phosphorylation of the inhibitor of apoptosis (IAP) protein survivin in murine and human cancer cells and tumors. Survivin inhibits caspase-9, blocks apoptosis, and is associated with resistance to chemotherapy and radiation. Survivin is a client protein for the 90-kDa heat shock protein (Hsp-90), and the binding of survivin to Hsp-90 assists in the maturation, proper folding, assembly, and transport of this IAP protein. A derivative of the antibiotic geldanamycin, 17-allylamino-17-demethoxygeldanamycin (17-AAG), interferes with proper binding of client proteins, such as survivin, to Hsp-90 and leads to misfolding of client proteins, ubiquination, and proteasome degradation. We hypothesized that PDT efficacy may be reduced by treatment-mediated expression and phosphorylation of survivin, and therefore, targeting the survivin pathway could increase PDT responsiveness. To address this hypothesis, we examined cellular and molecular responses following exposure to PDT, 17-AAG, and the combination of PDT plus 17-AAG in human BT-474 breast cancer cells using Photofrin and NPe6 as photosensitizers. Cells treated with the combination of PDT and 17-AAG exhibited decreased expression of the Hsp-90 client proteins phosphorylated survivin, phosphorylated Akt, and Bcl-2. The decreased expression of these client proteins was accompanied by higher apoptotic indexes and increased cytotoxicity. To confirm a specific role for survivin in modulating PDT, we used a human melanoma cell line, YUSAC2/T34A-C4, stably transfected with an inducible dominant-negative survivin gene under the control of a tetracycline-regulated (tet-off) promoter. PDT treatment of melanoma cells expressing the dominant-negative survivin resulted in increased cleavage of the caspase substrate poly(ADP-ribose) polymerase, apoptosis, and cytotoxicity when compared with results following PDT of the same melanoma cell line expressing wild-type survivin. These results show for the first time that targeting survivin and possibly other Hsp-90 client proteins improves in vitro PDT responsiveness and suggest that manipulation of the antiapoptotic pathway maintained by survivin may enhance PDT-mediated cancer therapy.
[Show abstract][Hide abstract] ABSTRACT: Photodynamic therapy causes direct cytotoxicity to malignant cells within a tumor. Photodynamic therapy (PDT) can also have both direct and indirect effects upon various non-malignant components of the tumor microenvironment. This action can lead to PDT-mediated angiogenesis and inflammation, which are emerging as important determinants of PDT responsiveness.
Preclinical studies have been performed to document how PDT modulates the tumor microenvironment. The expression, function, and treatment relevance of angiogenic growth factors, proteinases, and inflammatory molecules have been monitored following PDT using mouse tumor models.
Photofrin-mediated PDT was shown to be a strong activator of VEGF, MMPs, and COX-2 derived prostaglandins within the tumor microenvironment. Inhibitors that target these angiogenic and pro-survival molecules can enhance the effectiveness of PDT.
Improvements in PDT tumor responsiveness may be achieved by employing combined modality regimens targeting malignant cells as well as treatment-induced angiogenesis and/or inflammation.
No preview · Article · Jun 2006 · Lasers in Surgery and Medicine
[Show abstract][Hide abstract] ABSTRACT: C/EBP homologous protein (CHOP) is an endoplasmic reticulum (ER) stress inducible transcription factor involved in the development of apoptosis, growth arrest, and differentiation. CHOP deficient (chop - / - ) mouse embryonic fibroblasts (MEFs) exposed to ER stresses such as tunicamycin exhibit decreased apoptosis and reduced toxicity when compared to chop + / + control cells. Overexpression of the 70 kDa heat shock stress protein (HSP-70) can inhibit apoptotic pathways. The biological significance of photodynamic therapy (PDT) protocols that induce cellular damage resulting in differential CHOP and stress protein expression patterns was examined.
Wild type mouse radiation induced fibrosarcoma (RIF) cells as well as MEFs with chop + / + and chop - / - genotypes were used with either a mitochondrial and ER localizing porphyrin (PH) photosensitizer or a lysosomal localizing chlorin (NPe6) photosensitizer. PDT induced cytotoxicity, apoptosis, and stress protein expression patterns were determined as a function of cell type and photosensitizer.
PH mediated PDT induced expression of CHOP and 78 kDa glucose regulated protein (GRP-78), but not HSP-70 while NPe6 mediated PDT induced protein expression of HSP-70 but did not activate CHOP or GRP-78 expression. Enhanced apoptosis and toxicity were observed in chop + / + cells following exposure to tunicamycin or PH mediated PDT when compared to identical treatments in chop - / - cells. NPe6 mediated PDT induced minimally detectable apoptosis in both chop + / + and chop - / - cells and only a modest increase in survival for chop - / - cells.
These results demonstrate that PDT activation of CHOP, GRP-78, and HSP-70 varied as a function of photosensitizer subcellular localization and that a single oxidative stress response was not observed following PDT. We also show that CHOP expression increased apoptosis following PH mediated PDT and that increased CHOP expression is associated with enhanced PDT photosensitization.
No preview · Article · Dec 2004 · Lasers in Surgery and Medicine
[Show abstract][Hide abstract] ABSTRACT: GRP78 is a stress-inducible chaperone protein with antiapoptotic properties that is overexpressed in transformed cells and cells under glucose starvation, acidosis, and hypoxic conditions that persist in poorly vascularized tumors. Previously we demonstrated that the Grp78 promoter is able to eradicate tumors using murine cells in immunocompetent models by driving expression of the HSV-tk suicide gene. Here, through the use of positron emission tomography (PET) imaging, we provide direct evidence of spontaneous in vivo activation of the HSV-tk suicide gene driven by the Grp78 promoter in growing tumors and its activation by photodynamic therapy (PDT) in a controlled manner. In this report, we evaluated whether this promoter can be applied to human cancer therapy. We observed that the Grp78 promoter, in the context of a retroviral vector, was highly activated by stress and PDT in three different types of human breast carcinomas independent of estrogen receptor and p53. Complete regression of sizable human tumors was observed after prodrug ganciclovir treatment of the xenografts in immunodeficient mice. In addition, the Grp78 promoter-driven suicide gene is strongly expressed in a variety of human tumors, including human osteosarcoma. In contrast, the activity of the murine leukemia virus (MuLV) long-terminal repeat (LTR) promoter varied greatly in different human breast carcinoma cell lines, and in some cases, stress resulted in partial suppression of the LTR promoter activity. In transgenic mouse models, the Grp78 promoter-driven transgene is largely quiescent in major adult organs but highly active in cancer cells and cancer-associated macrophages, which can diffuse to tumor necrotic sites devoid of vascular supply and facilitate cell-based therapy. Thus, transcriptional control through the use of the Grp78 promoter offers multiple novel approaches for human cancer gene therapy.
No preview · Article · Jul 2004 · Human Gene Therapy
[Show abstract][Hide abstract] ABSTRACT: Photodynamic therapy (PDT) continues to be used in the treatment of solid tumors. Clinical results are promising, but the therapy has not been optimized, and tumor recurrences can occur. Recently, it has been shown that inhibitors of cyclooxygenase (COX)-2 can be effective in combination with conventional chemotherapy and radiation therapy. In the current study, we examined the parameters of PDT-mediated activation of COX-2 expression. We also examined the tumoricidal effectiveness of combining PDT with the selective COX-2 inhibitor NS-398. PDT induced the transcriptional activation of COX-2. Prolonged expression of COX-2 protein was observed in PDT-treated mouse sarcoma and carcinoma cell lines, whereas COX-1 was not inducible by PDT. Prostaglandin (PG) E2 synthesis was also increased in PDT-treated cells, and PGE2 levels were attenuated in cells coincubated with NS-398, indicating that PDT induced the expression of biologically active COX-2. Both porphyrin- and chlorin-based photosensitizers were able to elicit PDT-mediated COX-2 expression. COX-2 was also elevated in radiation-induced fibrosarcoma (RIF) tumors after treatment with PDT. We also observed that systemic administration of NS-398 decreased PDT induction of both PGE2 and vascular endothelial growth factor in treated RIF tumors. Additionally, we demonstrated that NS-398 enhanced PDT responsiveness in RIF tumors without increasing toxicity to normal tissue. These results provide strong evidence that combination procedures involving selective COX-2 inhibitors may improve the therapeutic effectiveness of PDT.
[Show abstract][Hide abstract] ABSTRACT: Photodynamic therapy (PDT) is a promising cancer treatment involving the administration of a tumor-localizing photosensitizer followed by the photochemical generation of cytotoxic singlet oxygen. PDT elicits strong transcriptional activation of a variety of genes including stress response genes belonging to the glucose-regulated protein (grp) family. Oxidative stress and hypoxia can activate GRP-78, and both of these physiological insults occur in treated tissue during and/or after PDT. In the current study, we evaluated the grp promoter as a PDT-inducible molecular switch for controlled expression of the herpes simplex virus-thymidine kinase (HSV-tk) suicide gene in mouse mammary adenocarcinoma (TSA) cells and tumors stably transduced with the G1NaGrpTk retroviral expression vector. We also examined whether PDT-inducible expression of HSV-tk, together with systemic administration of ganciclovir, could enhance the tumoricidal responsiveness of PDT. Inducible expression of HSV-tk was observed after PDT in stably transduced TSA cells grown in culture and in TSA tumors growing in BALB/c mice. We also observed enhanced tumoricidal activity in mice with TSA tumors containing the G1NaGrpTk expression vector treated with PDT plus ganciclovir when compared with either treatment alone. Our results confirm that the grp promoter was able to effectively function as a molecular switch for the inducible expression of the HSV-tk gene after exposure to PDT.
[Show abstract][Hide abstract] ABSTRACT: Neuroblastomas can acquire a sustained high-level drug resistance during chemotherapy and especially myeloablative chemoradiotherapy. p53 mutations are rare in primary neuroblastomas, but a loss of p53 function could play a role in multidrug resistance. We determined p53 function by measuring induction of p21 and/or MDM2 proteins in response to melphalan (L-PAM) in seven L-PAM-sensitive and 11 L-PAM-resistant neuroblastoma cell lines. p53 was functional in seven/seven drug-sensitive but in only 4/11 drug-resistant cell lines (P = 0.01). In four of the seven cell lines lacking p53 function, mutations of p53 were detected by the microarray GeneChip p53 Assay and automated sequencing, whereas six cell lines with functional p53 had no evidence of p53 mutations. All of the cell lines with wild-type (wt) p53 showed a strong transactivation of the p53-HBS/CAT reporter gene, whereas the four cell lines with mutant p53 failed to transactivate p53 HBS/CAT. Overexpression of MDM2 protein (relative to p53 functional lines) was seen in two p53-nonfunctional cell lines with wt p53; one showed genomic amplification of MDM2. Nonfunctional and mutated p53 was detected in a resistant cell line, whereas a sensitive cell line derived from the same patient before treatment had functional and wt p53. Loss of p53 function was selectively achieved by transduction of human papillomavirus 16 E6 (which degrades p53) into two drug-sensitive neuroblastoma cell lines with intact p53, causing high-level drug resistance to L-PAM, carboplatin, and etoposide. These data obtained with neuroblastoma cell lines suggest that the high-level drug resistance observed in some recurrent neuroblastomas is attributable to p53 mutations and/or a loss of p53 function acquired during chemotherapy. If confirmed in patient tumor samples, these data support development of p53-independent therapies for consolidation and/or salvage of recurrent neuroblastomas.
[Show abstract][Hide abstract] ABSTRACT: Oxidative stress associated with photodynamic therapy (PDT) is a transcriptional inducer of genes encoding stress proteins, including those belonging to the heat shock protein (hsp) family. The efficiency of PDT to function as a molecular switch by initiating expression of heterologous genes ligated to the human hsp promoter was examined in the present study. Selective and temporal reporter gene expression was documented after PDT in mouse radiation-induced fibrosarcoma cells stably transfected with recombinant vectors containing an hsp promoter ligated to either the lac-z or CAT reporter genes and in transfected radiation-induced fibrosarcoma tumors grown in C3H mice. Hyperthermia treatments were included as a positive control for all experiments. Expression vectors containing either human p53 or tumor necrosis factor (TNF)-alpha cDNA under the control of an hsp promoter were also constructed and evaluated. A p53 null and TNF-alpha-resistant human ovarian carcinoma (SKOV-3) cell line was stably transfected with either the p53 or TNF-alpha constructs. Inducible expression and function of p53 as well as inducible expression, secretion, and biological activity of TNF-alpha were documented after PDT or hyperthermia in transfected SKOV cells. These results demonstrate that PDT-mediated oxidative stress can function as a molecular switch for the selective and temporal expression of heterologous genes in tumor cells containing expression vectors under the control of an hsp promoter.
[Show abstract][Hide abstract] ABSTRACT: Photodynamic therapy (PDT) is an experimental treatment for malignant diseases (1,2). This procedure involves the systemic administration of a tumor-localizing photosensitizer and subsequent exposure of the
malignant lesion to tissue penetrating red light (3). Properties of photosensitizer localization in tumor tissue and photochemical generation of reactive oxygen species are
combined with precise delivery of laser generated light to produce a procedure offering effective local tumoricidal activity
(3). Tumors of the bronchus, bladder, esophagus, head and neck, brain and skin are being treated with PDT in clinical trials
(4). The clinical results are encouraging and PDT recently received FDA approval for the treatment of advanced esophageal carcinoma
in the United States. Additional regulatory approval of PDT has been obtained in Canada, the Netherlands, and Japan (5).
[Show abstract][Hide abstract] ABSTRACT: The positive clinical results associated with photodynamic therapy (PDT) have led to an expanded need to identify the cellular targets and molecular responses associated with this treatment. Increased knowledge regarding the mechanisms of action associated with PDT-mediated cytotoxicity should contribute to the continued advancement of this therapy. This report focuses on recent studies analyzing PDT resistance and examining stress protein and early response gene activation induced by photosensitizer mediated oxidative stress. Recurring observations from these studies indicate that subcellular targets and cellular responses associated with PDT can vary significantly for different photosensitizers.
No preview · Article · Nov 1996 · Journal of Clinical Laser Medicine & Surgery
[Show abstract][Hide abstract] ABSTRACT: Parental and photodynamic therapy (PDT)-resistant mouse, radiation-induced fibrosarcoma cell lines were evaluated using mRNA differential display in an attempt to identify unique transcripts. We detected one transcript that was consistently present in the parental cells but absent in PDT-resistant cells. The transcript was cloned, sequenced, and identified as alpha-2 macroglobulin receptor/low density lipoprotein receptor-related protein (alpha-2 MR/LRP). Northern and Western immunoblot analysis confirmed that receptor expression was present in the parental cell line but barely detectable in PDT-resistant cells. Functionality of the receptor was evaluated by exposing cells to Pseudomonas exotoxin A. alpha-2 MR/LRP is responsible for Pseudomonas exotoxin A internalization, and only the parental cells exhibited toxin-mediated cytotoxicity. The binding and endocytosis of activated alpha-2 macroglobulin and lipoproteins by alpha-2 MR/LRP are consistent with modulating uptake and localization of photosensitizers. Our results demonstrate that PDT-resistant murine tumor cells exhibit minimal alpha-2 MR/LRP activity and suggest that this receptor plays a role in PDT sensitivity by modulating photosensitizer uptake and/or subcellular localization.
[Show abstract][Hide abstract] ABSTRACT: Photodynamic therapy (PDT) generates reactive oxygen species which initiate the cytotoxic events of this tumor treatment. We demonstrate that PDT mediated oxidative stress induced a transient increase in the early response genes c-fos, c-jun, c-myc, and egr-1 in murine radiation-induced fibrosarcoma cells. Incubation of exponentially growing cells with porphyrin based photosensitizers in the dark also induced an increase in mRNA levels of early response genes. However, the xanthine photosensitizer, rose bengal, produced increased c-fos mRNA levels only following light treatment. Nuclear runoff experiments confirmed that the induction of c-fos mRNA is controlled in part at the level of transcription. Likewise, a chloramphenicol acetyltransferase reporter construct containing the major c-fos transcriptional response elements was inducible by porphyrin and PDT. Signal transduction pathways associated with PDT mediated c-fos activation were examined by treating cells with protein kinase inhibitors. Staurosporine and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine inhibited PDT mediated c-fos activation while N-(2-guanidinoethyl)-5-isoquinoline-sulfonamide had no effect. In addition, quinacrine, which can inhibit phospholipase activity, blocked PDT induced c-fos mRNA expression. These results suggest that photosensitizer mediated oxidative stress acts through protein kinase-mediated signal transduction pathway(s) to activate early response genes.