- Email: [email protected]
Hexyl aminolevulinate and light induces posttranslational modifications of proteins in different cell pathways
Oral Abstracts O004 PDT for drug delivery
127 tion of cell rescue under both conditions heavy metal exposure and ALA-PDT.
H. van den Bergh, E. Debefve, P. Nowak-Sliwinska
doi:10.1016/j.pdpdt.2011.03.020
Medical Photonics Group, EPFL, Lausanne, Switzerland
O006
Introduction: Low dose photodynamic therapy (PDT) targeting the vasculature can induce an increased vascular leakage. By using combination therapies and optimizing the treatment modalities we enhance this phenomenon with the final goal being to use PDT for drug delivery. For this purpose, we investigated the mechanisms of the PDT-induced vascular permeability. Methods: For the screening of the different drugs to be combined with PDT for enhanced leakage, we used the chorio-allantoic membrane (CAM) of the chicken embryo. Anti-platelet factor (Aspirin® ), vasodilator (Flolan® ), anti-tyrosine kinase (Sutent® ) and a snake venom-derived disintegrin (kistrin) were assessed in combination with Visudyne® -PDT, for their potential to enhance the PDT-induced leakage of different sizes of FITC-Dextran. We also investigated the role of leukocyte recruitment in this photosensitization-induced vascular permeabilisation. Results: Vascular PDT induces the destruction of tight junctions, platelet aggregation, vasoconstriction of the arteries, selectin expression and activation of leukocytes in veins, leading to vascular occlusion and/or leakage. Inhibition of these mechanisms separately or in combination influences the efficacy of PDT drug delivery. Different drugs delaying hemostasis, dilating the vessel or inhibiting the tyrosine kinase receptors, when combined with PDT improved FITC-Dextran leakage. Conclusion: In oncology, in a tri-therapy modality (photosensitizer + enhancer of leakage + chemotherapeutic), a single PDT can be used first as a drug delivery for the cytostatic drugs, and subsequently for the occlusion of the blood vessels feeding the tumour.
Amplification of PDT-induced cancer cell apoptosis by adjuvant sphingolipid mediators Mladen Korbelik1 and Duska Separovic2
doi:10.1016/j.pdpdt.2011.03.019 O005 The roles heme oxygenase-1 in activation of cellular rescue mechanism during PDT oxidative stress and cell death protection Z. Malik
M. Korbelik 1 , D. Separovic 2 1
British Columbia Cancer Agency, Vancouver, BC, Canada Department of Pharmacological Sciences, Wayne State University, Detroit, MI, USA 2
Background: A central position in sphingolipid metabolism is occupied by ceramide, which has been shown to participate in signaling that leads to cell death by apoptosis. We have shown that PDT affects de novo biosynthesis of ceramide resulting in its increased accumulation in treated cancer cells in vitro and tumors in vivo, and also that this pro-death sphingolipid species promotes apoptosis in PDT-treated cells in vitro. Our recent work has revealed that adjuvant treatment with ceramide analogue LCL29 markedly improves the cures of PDT-treated tumors in mice. Methods: Using SCCVII squamous cell carcinoma model with tumors growing in syngeneic C3H/HeN mice, we have examined the effect of foscan-based PDT with or without adjuvant LCL29 treatment on the extent of apoptosis in cancer cells in treated tumors. This was investigated using a selection of apoptosis-related stains followed by flow cytometry analysis. Results: Adjuvant LCL29 treatment markedly enhanced the numbers of apoptotic cancer cells in PDT-treated tumors. Loss of mitochondrial transmembrane potential was evident after both PDT and LCL29 alone treatments and was exhibited at a similar level with LCL29 plus PDT group. The raise in the intracellular calcium levels detected with PDT alone was further elevated with adjuvant LCL29 treatment. Conclusion: Therapeutic gain with adjuvant LCL29-medaited modulation of sphingolipid levels can be at least in part attributed to potentiation of apoptotic cancer cell death, which seems to be attained by a positive feedback loop induced by ceramide-enhanced cytochrome c release (resulting from mitochondrial depolarization) augmenting intracellular calcium release triggered by PDT.
Life Sciences Faculty, Bar-Ilan University Ramat-Gan, Israel The underlying mechanism of PDT leading to oxidative stress and activation of cancer cell death is light absorption by a sensitizer; the energy is transferred to oxygen which in turn is converted into singlet oxygen and ROS. The immediate physiological reaction to oxidative stress is up-regulation of heme oxygenase-1 (HO-1) activity as an immediate rescue response. Up-regulation of HO-1 is reveled as an immediate response to ALA-PDT. On the other hand heavy metal exposure Pb2+ , Ga3+ and Cu2+ induces over expression of HO-1 as a specific stress reaction that induces cell rescue similar to the response to ALA-PDT. The ALA-PDT or heavy metal stress responses stimulate HO-1 nuclear translocation pointing to protection mechanisms of HO-1 in the nucleus. The induction of HO1 activity is directly correlated with ROS generation in both cases of heavy metals exposure or PDT activation. Heme breakdown by HO-1 was described to generate a variety of anti-oxidant responses which explain partially its physiological role to stress. We show that heme degradation due to elevated levels of ROS in directly correlated with activation of protein degradation by the proteasome. It was suggested that proteasomal proteolytic activity is controlled by the cellular heme that bind to regulatory proteasomal proteins and inhibit its activity. We propose that heme degradation by HO-1 diminish the repression proteasomal proteolytic activity, consequently induces breakdown of oxidized proteins and activa-
doi:10.1016/j.pdpdt.2011.03.021 O007 Hexyl aminolevulinate and light induces posttranslational modifications of proteins in different cell pathways O.A. Gederaas, Y. Baglo, M. Mittelstedt Leal de Sousa, S. Havag, K.A. Zub, L. Helander, T. Arset, G. Slupphaug, H.E. Krokan Department of Cancer Research and Molecular Medicine,Norwegian University of Science and Technology (NTNU), N-7006 Trondheim, Norway Introduction: Photodynamic therapy (PDT) is an effective treatment involving light and a photosensitizer used in conjunction with molecular oxygen to elicit cell death. The purpose of this study is search for changes in protein expression induced by HAL-PDTmediated treatment of cytotoxic effect in rat bladder carcinoma cells (AY-27). Materials and methods: AY-27 cells were cultured in RPMI-1640 medium for one day and then incubated with hexyl-aminolevulinate (HAL), 10 M for 3.5 h in the dark in humidified atmosphere of 95% air and 5% CO2 at 37 ◦ C. At room temperature, the HAL-treated cells were exposed to blue light (435 nm, 0.45 J/cm2 , LD70 ) in PBS and
128 further incubated (2 h) in serum free medium. Protein lysates from non-, HAL- and HAL-PDT treated cells were analyzed by 2D-DIGE (2-dimensional difference gel electrophoresis). Proteins of interest were then identified by mass spectrometry (MS). Results and conclusions: Difference in protein expression between HAL-treated and HAL-PDT samples was clearly displayed in scanning images. 40 proteins were subjected to MS-based peptide map fingerprinting. Of these, 15 proteins were either posttranslationally modified (pI-shift) or differentially expressed (downor up-regulation) due to light treatment. Pre-purification methodologies are now being optimized to allow an in depth study of proteome changes due to PDT. These results have extended our understanding of the clinic effects associated with ALA-PDT, and may pave the way for potential new adjuvant drugs.
Oral Abstracts Conclusions: This work demonstrates that methylene-blue mediated PDT can downregulate proteins critical in the metastasis of head-and-neck cancer. Additionally, these results illuminate one aspect of the mechanism of PDT and suggest that MMP-9 may even serve as a biomarker for the efficacy of photodynamic therapy. doi:10.1016/j.pdpdt.2011.03.023 O009 Photofrin photosensitizer inhibits the expression of EGFR independent of light irradiation in cancer cells P.-W. Yang 1 , M.-C. Hung 2,3 , C.-Y. Wang 1 , Y.-C. Lee 1,4 , J.-M. Lee 1
Hsieh 1 , E.-C.
Tung 1 , Y.-H.
1
doi:10.1016/j.pdpdt.2011.03.022 O008 Photodynamic therapy downregulates matrix metalloproteinases in oral carcinoma M. Biel, M. Le, B. Wuertz
Department of Surgery, National Taiwan University Hospital, Taipei, Taiwan 2 Department of Molecular and Cellular Oncology, University of Texas MD Anderson, USA 3 Center for Molecular Medicine and Graduate Institute of Cancer Biology, CMUH, Taiwan 4 College of Medicine, National Taiwan University, Taipei, Taiwan
Objective: To test the hypothesis that photodynamic therapy downregulates MMP-9 in cell cultures of oral squamous cell carcinoma and leukoplakia. Design: Molecular biologic analysis. Intervention: Cells were cultured to 50—90% confluence from cell lines of oral squamous cell carcinoma (CA-9-22), oral leukoplakia (MSK-Leuk1), and immortalized oral keratinocytes (Rhek-1A). These were photosensitized with 0.5—1 mg/mL methylene blue, and treated with a 665 nm laser at a fluence of 6.3 J/cm2 . Twenty-four hours after photodynamic therapy (PDT), cells were harvested for isolation of total RNA, which was converted to first-strand cDNA, then probed for target sequences via qRT-PCR. Concentrated, conditioned media were analyzed by gelatin zymography to confirm the qRT-PCR findings at a protein and functional level. Results: MMP-9 gene expression was significantly decreased in carcinoma and in leukoplakia, as well as in immortalized oral
Introduction: Photodynamic therapy (PDT) has been demonstrated as an effective minimally invasive treatment modality for early esophageal cancer. EGFR plays an important role in the resistance to many anti-cancer treatments. However, its association with PDT in treating esophageal cancer is still unclear. Methods and results: In this study, we investigate the effect of PDT on the expression of EGFR in esophageal cancer cells by western blotting and Q-PCR. We found the EGFR expression in esophageal cancer cells CE48T/VGH was significantly downregulated after PDT treatment. Interestingly, this down-regulation was mainly attributed to the treatment with the photosensitizer, Photofrin. Expression of EGFR was inhibited both in the translation and transcription level. Irradiation of red light, however, did not further decrease the EGFR in the transcription level. This Photofrinassociated down-regulation of EGFR can be repeated in various cancer cells including lung cancer cell lines A549 and CL1-5 as well as human cervical cancer line HeLa and glioma cell line T98G. Conclusion: Our study demonstrates the photosensitizerassociated EGFR down-regulation in esophageal cancer cells during PDT, and suggests the possible mechanism of anti-neoplasm effect of PDT in addition to its photo-reactivation on the esophageal cancer tissue.
keratinocytes, following the administration of PDT (P < 0.05). MMP-9 enzymatic activity also decreased in all cell lines after PDT.
doi:10.1016/j.pdpdt.2011.03.024
University of Minnesota, Department of Otolaryngology, Minneapolis, USA
127 tion of cell rescue under both conditions heavy metal exposure and ALA-PDT.
H. van den Bergh, E. Debefve, P. Nowak-Sliwinska
doi:10.1016/j.pdpdt.2011.03.020
Medical Photonics Group, EPFL, Lausanne, Switzerland
O006
Introduction: Low dose photodynamic therapy (PDT) targeting the vasculature can induce an increased vascular leakage. By using combination therapies and optimizing the treatment modalities we enhance this phenomenon with the final goal being to use PDT for drug delivery. For this purpose, we investigated the mechanisms of the PDT-induced vascular permeability. Methods: For the screening of the different drugs to be combined with PDT for enhanced leakage, we used the chorio-allantoic membrane (CAM) of the chicken embryo. Anti-platelet factor (Aspirin® ), vasodilator (Flolan® ), anti-tyrosine kinase (Sutent® ) and a snake venom-derived disintegrin (kistrin) were assessed in combination with Visudyne® -PDT, for their potential to enhance the PDT-induced leakage of different sizes of FITC-Dextran. We also investigated the role of leukocyte recruitment in this photosensitization-induced vascular permeabilisation. Results: Vascular PDT induces the destruction of tight junctions, platelet aggregation, vasoconstriction of the arteries, selectin expression and activation of leukocytes in veins, leading to vascular occlusion and/or leakage. Inhibition of these mechanisms separately or in combination influences the efficacy of PDT drug delivery. Different drugs delaying hemostasis, dilating the vessel or inhibiting the tyrosine kinase receptors, when combined with PDT improved FITC-Dextran leakage. Conclusion: In oncology, in a tri-therapy modality (photosensitizer + enhancer of leakage + chemotherapeutic), a single PDT can be used first as a drug delivery for the cytostatic drugs, and subsequently for the occlusion of the blood vessels feeding the tumour.
Amplification of PDT-induced cancer cell apoptosis by adjuvant sphingolipid mediators Mladen Korbelik1 and Duska Separovic2
doi:10.1016/j.pdpdt.2011.03.019 O005 The roles heme oxygenase-1 in activation of cellular rescue mechanism during PDT oxidative stress and cell death protection Z. Malik
M. Korbelik 1 , D. Separovic 2 1
British Columbia Cancer Agency, Vancouver, BC, Canada Department of Pharmacological Sciences, Wayne State University, Detroit, MI, USA 2
Background: A central position in sphingolipid metabolism is occupied by ceramide, which has been shown to participate in signaling that leads to cell death by apoptosis. We have shown that PDT affects de novo biosynthesis of ceramide resulting in its increased accumulation in treated cancer cells in vitro and tumors in vivo, and also that this pro-death sphingolipid species promotes apoptosis in PDT-treated cells in vitro. Our recent work has revealed that adjuvant treatment with ceramide analogue LCL29 markedly improves the cures of PDT-treated tumors in mice. Methods: Using SCCVII squamous cell carcinoma model with tumors growing in syngeneic C3H/HeN mice, we have examined the effect of foscan-based PDT with or without adjuvant LCL29 treatment on the extent of apoptosis in cancer cells in treated tumors. This was investigated using a selection of apoptosis-related stains followed by flow cytometry analysis. Results: Adjuvant LCL29 treatment markedly enhanced the numbers of apoptotic cancer cells in PDT-treated tumors. Loss of mitochondrial transmembrane potential was evident after both PDT and LCL29 alone treatments and was exhibited at a similar level with LCL29 plus PDT group. The raise in the intracellular calcium levels detected with PDT alone was further elevated with adjuvant LCL29 treatment. Conclusion: Therapeutic gain with adjuvant LCL29-medaited modulation of sphingolipid levels can be at least in part attributed to potentiation of apoptotic cancer cell death, which seems to be attained by a positive feedback loop induced by ceramide-enhanced cytochrome c release (resulting from mitochondrial depolarization) augmenting intracellular calcium release triggered by PDT.
Life Sciences Faculty, Bar-Ilan University Ramat-Gan, Israel The underlying mechanism of PDT leading to oxidative stress and activation of cancer cell death is light absorption by a sensitizer; the energy is transferred to oxygen which in turn is converted into singlet oxygen and ROS. The immediate physiological reaction to oxidative stress is up-regulation of heme oxygenase-1 (HO-1) activity as an immediate rescue response. Up-regulation of HO-1 is reveled as an immediate response to ALA-PDT. On the other hand heavy metal exposure Pb2+ , Ga3+ and Cu2+ induces over expression of HO-1 as a specific stress reaction that induces cell rescue similar to the response to ALA-PDT. The ALA-PDT or heavy metal stress responses stimulate HO-1 nuclear translocation pointing to protection mechanisms of HO-1 in the nucleus. The induction of HO1 activity is directly correlated with ROS generation in both cases of heavy metals exposure or PDT activation. Heme breakdown by HO-1 was described to generate a variety of anti-oxidant responses which explain partially its physiological role to stress. We show that heme degradation due to elevated levels of ROS in directly correlated with activation of protein degradation by the proteasome. It was suggested that proteasomal proteolytic activity is controlled by the cellular heme that bind to regulatory proteasomal proteins and inhibit its activity. We propose that heme degradation by HO-1 diminish the repression proteasomal proteolytic activity, consequently induces breakdown of oxidized proteins and activa-
doi:10.1016/j.pdpdt.2011.03.021 O007 Hexyl aminolevulinate and light induces posttranslational modifications of proteins in different cell pathways O.A. Gederaas, Y. Baglo, M. Mittelstedt Leal de Sousa, S. Havag, K.A. Zub, L. Helander, T. Arset, G. Slupphaug, H.E. Krokan Department of Cancer Research and Molecular Medicine,Norwegian University of Science and Technology (NTNU), N-7006 Trondheim, Norway Introduction: Photodynamic therapy (PDT) is an effective treatment involving light and a photosensitizer used in conjunction with molecular oxygen to elicit cell death. The purpose of this study is search for changes in protein expression induced by HAL-PDTmediated treatment of cytotoxic effect in rat bladder carcinoma cells (AY-27). Materials and methods: AY-27 cells were cultured in RPMI-1640 medium for one day and then incubated with hexyl-aminolevulinate (HAL), 10 M for 3.5 h in the dark in humidified atmosphere of 95% air and 5% CO2 at 37 ◦ C. At room temperature, the HAL-treated cells were exposed to blue light (435 nm, 0.45 J/cm2 , LD70 ) in PBS and
128 further incubated (2 h) in serum free medium. Protein lysates from non-, HAL- and HAL-PDT treated cells were analyzed by 2D-DIGE (2-dimensional difference gel electrophoresis). Proteins of interest were then identified by mass spectrometry (MS). Results and conclusions: Difference in protein expression between HAL-treated and HAL-PDT samples was clearly displayed in scanning images. 40 proteins were subjected to MS-based peptide map fingerprinting. Of these, 15 proteins were either posttranslationally modified (pI-shift) or differentially expressed (downor up-regulation) due to light treatment. Pre-purification methodologies are now being optimized to allow an in depth study of proteome changes due to PDT. These results have extended our understanding of the clinic effects associated with ALA-PDT, and may pave the way for potential new adjuvant drugs.
Oral Abstracts Conclusions: This work demonstrates that methylene-blue mediated PDT can downregulate proteins critical in the metastasis of head-and-neck cancer. Additionally, these results illuminate one aspect of the mechanism of PDT and suggest that MMP-9 may even serve as a biomarker for the efficacy of photodynamic therapy. doi:10.1016/j.pdpdt.2011.03.023 O009 Photofrin photosensitizer inhibits the expression of EGFR independent of light irradiation in cancer cells P.-W. Yang 1 , M.-C. Hung 2,3 , C.-Y. Wang 1 , Y.-C. Lee 1,4 , J.-M. Lee 1
Hsieh 1 , E.-C.
Tung 1 , Y.-H.
1
doi:10.1016/j.pdpdt.2011.03.022 O008 Photodynamic therapy downregulates matrix metalloproteinases in oral carcinoma M. Biel, M. Le, B. Wuertz
Department of Surgery, National Taiwan University Hospital, Taipei, Taiwan 2 Department of Molecular and Cellular Oncology, University of Texas MD Anderson, USA 3 Center for Molecular Medicine and Graduate Institute of Cancer Biology, CMUH, Taiwan 4 College of Medicine, National Taiwan University, Taipei, Taiwan
Objective: To test the hypothesis that photodynamic therapy downregulates MMP-9 in cell cultures of oral squamous cell carcinoma and leukoplakia. Design: Molecular biologic analysis. Intervention: Cells were cultured to 50—90% confluence from cell lines of oral squamous cell carcinoma (CA-9-22), oral leukoplakia (MSK-Leuk1), and immortalized oral keratinocytes (Rhek-1A). These were photosensitized with 0.5—1 mg/mL methylene blue, and treated with a 665 nm laser at a fluence of 6.3 J/cm2 . Twenty-four hours after photodynamic therapy (PDT), cells were harvested for isolation of total RNA, which was converted to first-strand cDNA, then probed for target sequences via qRT-PCR. Concentrated, conditioned media were analyzed by gelatin zymography to confirm the qRT-PCR findings at a protein and functional level. Results: MMP-9 gene expression was significantly decreased in carcinoma and in leukoplakia, as well as in immortalized oral
Introduction: Photodynamic therapy (PDT) has been demonstrated as an effective minimally invasive treatment modality for early esophageal cancer. EGFR plays an important role in the resistance to many anti-cancer treatments. However, its association with PDT in treating esophageal cancer is still unclear. Methods and results: In this study, we investigate the effect of PDT on the expression of EGFR in esophageal cancer cells by western blotting and Q-PCR. We found the EGFR expression in esophageal cancer cells CE48T/VGH was significantly downregulated after PDT treatment. Interestingly, this down-regulation was mainly attributed to the treatment with the photosensitizer, Photofrin. Expression of EGFR was inhibited both in the translation and transcription level. Irradiation of red light, however, did not further decrease the EGFR in the transcription level. This Photofrinassociated down-regulation of EGFR can be repeated in various cancer cells including lung cancer cell lines A549 and CL1-5 as well as human cervical cancer line HeLa and glioma cell line T98G. Conclusion: Our study demonstrates the photosensitizerassociated EGFR down-regulation in esophageal cancer cells during PDT, and suggests the possible mechanism of anti-neoplasm effect of PDT in addition to its photo-reactivation on the esophageal cancer tissue.
keratinocytes, following the administration of PDT (P < 0.05). MMP-9 enzymatic activity also decreased in all cell lines after PDT.
doi:10.1016/j.pdpdt.2011.03.024
University of Minnesota, Department of Otolaryngology, Minneapolis, USA