Marian Luna

INCREASED TRANSCRIPTION AND TRANSLATION OF HEME OXYGENASE IN CHINESE HAMSTER FIBROBLASTS FOLLOWING PHOTODYNAMIC STRESS OR PHOTOFRIN II INCUBATION

Abstract— Porphyrin mediated photosensitization can enhance the transcription and translation of several oxidative stress genes. In this study, we report on the enhanced expression of the gene encoding for heme oxygenase in Chinese hamster fibroblasts by; (1) incubation in Photofrin II; (2) Photofrin II mediated photosensitization; and (3) photosensitization induced by Rose Bengal. Increased expression of heme oxygenase mRNA was accompanied by a concomitant increase in the synthesis of the 34 kDa heme oxygenase protein. Western blot analysis using antibody to heme oxygenase confirmed the immunoreactivity of the 34 kDa protein induced by Photofrin II and PDT. These results demonstrate that heme oxygenase can be activated by non‐metalloporphyrins as well as by photosensitization associated with singlet oxygen mediated subcellular injury.

Survivin, a Member of the Inhibitor of Apoptosis Family, Is Induced by Photodynamic Therapy and Is a Target for Improving Treatment Response

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.

Spontaneous and Controllable Activation of Suicide Gene Expression Driven by the Stress-Inducible Grp78 Promoter Resulting in Eradication of Sizable Human Tumors

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.

Enhancement of photodynamic therapy by 2,5-dimethyl celecoxib, a non-cyclooxygenase-2 inhibitor analog of celecoxib

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.

Multiple components of photodynamic therapy can phosphorylate akt

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.

Cyclooxygenase-2 expression induced by photofrin photodynamic therapy involves the p38 MAPK pathway.

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 (NFκB) 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 NFκB, 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 NFκB 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.

Pro-apoptotic and anti-inflammatory properties of the green tea constituent epigallocatechin gallate increase photodynamic therapy responsiveness

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.

Targeting Survivin Enhances Chemosensitivity in Retinoblastoma Cells and Orthotopic Tumors.

Treatments for retinoblastoma (Rb) vary depending on the size and location of the intraocular lesions and include chemotherapy and radiation therapy. We examined whether agents used to treat Rb induce a pro-survival phenotype associated with increased expression of survivin, a member of the inhibitor of apoptosis family of proteins. We document that exposure to carboplatin, topotecan or radiation resulted in elevated expression of survivin in two human Rb cell lines but not in normal retinal pigmented epithelial (RPE) cells. Cellular levels of survivin were attenuated in Rb cells exposed to an imidazolium-based survivin suppressant, Sepantronium bromide (YM155). Protein expression patterns of survivin in RPE cells were not altered following treatment protocols involving exposure to YM155. Including YM155 with chemotherapy or radiation increased levels of apoptosis in Rb cells but not in RPE cells. Intraocular luciferase expressing Rb tumors were generated from the Rb cell lines and used to evaluate the effects of carboplatin and YM155 on in-vivo survivin expression and tumor growth. Carboplatin induced expression of survivin while carboplatin combined with YM155 reduced survivin expression in tumor bearing eyes. The combination protocol was also most effective in reducing the rate of tumor regrowth. These results indicate that targeted inhibition of the anti-apoptotic protein survivin provides a therapeutic advantage for Rb cells and tumors treated with chemotherapy.

Identification of MAP Kinase Pathways Involved in COX-2 Expression Following Photofrin Photodynamic Therapy

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.

Rational drug design and synthesis of new α-Santonin derivatives as potential COX-2 inhibitors

Sesquiterpene compounds are widely known for their numerous pharmacological activities. Herein the focus of the authors was on α-Santonin, a sesquiterpene lactone from the Artemisia genus: the aim was to determine whether α-Santonin could be considered in the treatment of inflammation and pain. To this purpose, a small series of derivatives was designed and screened in silico against the enzyme COX-2 along with the parent compound. Drug-likeness parameters were also assessed. The compounds were eventually synthesized, and few were tested to determine their efficacy in the inhibition of COX-2 activity and expression. Overall, compound A2 was the only one with a detectable inhibitory potential of COX-2 activity whilst two of its ether derivatives demonstrated improved ability in the inhibition of COX-2 expression.