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Resistance of oral squamous cell carcinoma cells to 5-aminolevulinic acid-mediated photodynamic therapy
A70
Abstracts / Photodiagnosis and Photodynamic Therapy 17 (2017) A4–A78
Poster PH-070
Poster PH-071
Targeted therapy against vascular endothelial growth factor receptor (VEGFR) and epidermal growth factor receptor (EGFR) using vandetanib enhances the photodynamic therapy potential in oral squamous carcinoma cancer
Resistance of oral squamous cell carcinoma cells to 5-aminolevulinic acid-mediated photodynamic therapy
C.P. Lim 1,∗ , R. Bhuvaneswari 1 , N.Q. Feng 1 , P.S.P. Thong 1 , T.N. Chye 1,2 , S.K. Chee 1,2
School of Dentistry, University of São Paulo, São Paulo, Brazil
1
National Cancer Centre Singapore, Division of Medical Sciences, 11 Hospital Drive, Singapore 2 Duke-NUS Graduate Medical School, 8 College Road, Singapore Aims of study: Our aim is to investigate a targeted approach to treat oral cancer with photodynamic therapy (PDT) in combination with vandetanib, a multitargeted tyrosine kinase inhibitor that selectively blocks VEGFR-2, the EGFR and RET tyrosine kinases. Methods: The cytotoxic effects of vandetanib, chlorin e6 (Ce6)PDT and combination therapy on oral squamous cell carcinoma (OSCC) was studied using cell viability, reactive oxygen species (ROS) and clonogenic assays. Cell cycle arrest and cell apoptosis were also performed. The anti-angiogenic potential of vandetanib was evaluated using Transwell cell migration, invasion and endothelial tube formation assays. The expression and phosphorylation levels of receptor and downstream signaling molecules were investigated using western blot assay. Results: Our in vitro results showed that vandetanib significantly enhanced the cytotoxicity of PDT in a dose-dependent manner. The combination of PDT and vandetanib (IC50, 6 M) exhibited a 2.7 fold increase in cytotoxicity than PDT alone. Clonogenic assay showed a significant reduction in OSCC colony formation in the combination therapy group, compared to PDT treated group. Vandetanib and PDT inhibited the proliferation of OSCC cells through the induction of cell cycle arrest at G1 and G2 phase respectively and caused high apoptosis with combination therapy. Higher levels of ROS was observed in the combination therapy group, which effectively enhanced the cytotoxic efficacy of PDT in OSCC cells. Apoptosis induced by vandetanib was not inhibited by the use of caspase-inhibitor Z-VAD-FMK, suggesting that the cell death is caspase-independent, different from PDT which induced caspase-dependent apoptosis. A 2.2 fold increase in DNA fragmentation activity was noted in the combination therapy with vandetanib (IC50, 6 M), than PDT alone, that was confirmed by the down-regulation of anti-apoptotic bcl-2 protein. On the other hand, the migration and invasion efficiency of OSCC cells were reduced significantly with combination therapy when compared to monotherapies alone and control group. Furthermore, the tube formation of endothelial cells was reduced significantly in the presence of vandetanib. Combination therapy also inhibited the phosphorylation of EGFR and its downstream targets in OSCC cells. Conclusion: Vandetanib, a dual VEGFR and EGFR inhibitor, can significantly improve the efficacy of PDT by reducing the proliferation, increasing apoptosis and preventing angiogenesis in OSCC cells. http://dx.doi.org/10.1016/j.pdpdt.2017.01.158
F.C.P. Rosin ∗ , L. Corrêa
Squamous cell carcinoma of the oral cavity (OSCC) is a malignant tumor with high morbidity and mortality rates. Conventional treatment for OSCC includes surgery and radiotherapy/chemotherapy, but some cases can develop resistance to these treatments [1]. Oncologic photodynamic therapy (PDT) has been used as an adjuvant treatment for OSCC, mainly for non-invasive cases, in which this therapy has demonstrated a relative good efficacy [2]. Some studies have described that other tumors, such as cutaneous [3], colon [4], and breast [5] carcinomas, have shown PDT resistance. The hypothesis that OSCC could also develop PDT resistance has not yet been tested. The aims of this study were: (1) to investigate whether human OSCCs cells develop resistance to several cycles of 5-aminolevulinic acid-mediated PDT (5-ALA–PDT); and (2) to determine whether the expression of markers associated with cell survival (NFkB, Bcl-2, iNOS, mTOR, and Akt) is altered during this process. An OSCC cell line (SCC9) was subjected to the following conditions: (1) Control: cultured without any treatment; (2) ALA: incubated with 5-ALA (1 mM for 4 h); 3) LED: treated with LED light (630 nm, 5.86 J/cm2 , 10 J, 150 mW, 150 s); and (4) PDT: treated with 5-ALA–PDT (with the protocols of the ALA and LED groups combined), causing a cell death index of 90%. After a first PDT cycle, the survived cells were cultured and subjected to another cycle of 5-ALA–PDT, adding 2 J in the initial irradiation dose. When this process was repeated five times increasing the fluency (from 5.86 to 9.38 J/cm2 ) [4], the cell viability gradually improved. Four resistant cell populations were obtained, showing cell viability up to 4.6 times higher than that present at first PDT cycle. The most resistant population exhibited a lower intensity of protoporphyrin IX, higher migration capacity, and changes in nuclear morphology. By Western blotting analysis, there was an increase in the expression of pNFkB, iNOS, pmTOR, and pAkt, but not of anti-apoptotic Bcl-2 protein, in the resistant populations. In conclusion, OSCC cells treated with a 5-ALA–PDT exhibited increase on viability and improvement on migration capacity after six successive PDT cycles, as well as overexpression of proteins associated with cell survival. The possibility of resistance to 5-ALA–PDT should be carefully considered when initiating this therapy for OSCC, mainly for those cases in which are necessary multiple PDT cycles [6]. References [1] [2] [3] [4] [5] [6]
S.D. Silva, et al., Front. Pharmacol. 3 (2012) 1–7. W. Jerjes, et al., Head Neck Oncol. 4 (2012) 16. L.N. Milla, J. Cell. Biochem. 9 (2011) 2266–2278. G. Singh, Photochem. Photobiol. 6 (2001) 651–656. A. Casas, et al., Int. J. Oncol. 2 (2006) 397–405. A. Casas, et al., Curr. Med. Chem. 16 (2011) 2486–2515.
http://dx.doi.org/10.1016/j.pdpdt.2017.01.159
Abstracts / Photodiagnosis and Photodynamic Therapy 17 (2017) A4–A78
Poster PH-070
Poster PH-071
Targeted therapy against vascular endothelial growth factor receptor (VEGFR) and epidermal growth factor receptor (EGFR) using vandetanib enhances the photodynamic therapy potential in oral squamous carcinoma cancer
Resistance of oral squamous cell carcinoma cells to 5-aminolevulinic acid-mediated photodynamic therapy
C.P. Lim 1,∗ , R. Bhuvaneswari 1 , N.Q. Feng 1 , P.S.P. Thong 1 , T.N. Chye 1,2 , S.K. Chee 1,2
School of Dentistry, University of São Paulo, São Paulo, Brazil
1
National Cancer Centre Singapore, Division of Medical Sciences, 11 Hospital Drive, Singapore 2 Duke-NUS Graduate Medical School, 8 College Road, Singapore Aims of study: Our aim is to investigate a targeted approach to treat oral cancer with photodynamic therapy (PDT) in combination with vandetanib, a multitargeted tyrosine kinase inhibitor that selectively blocks VEGFR-2, the EGFR and RET tyrosine kinases. Methods: The cytotoxic effects of vandetanib, chlorin e6 (Ce6)PDT and combination therapy on oral squamous cell carcinoma (OSCC) was studied using cell viability, reactive oxygen species (ROS) and clonogenic assays. Cell cycle arrest and cell apoptosis were also performed. The anti-angiogenic potential of vandetanib was evaluated using Transwell cell migration, invasion and endothelial tube formation assays. The expression and phosphorylation levels of receptor and downstream signaling molecules were investigated using western blot assay. Results: Our in vitro results showed that vandetanib significantly enhanced the cytotoxicity of PDT in a dose-dependent manner. The combination of PDT and vandetanib (IC50, 6 M) exhibited a 2.7 fold increase in cytotoxicity than PDT alone. Clonogenic assay showed a significant reduction in OSCC colony formation in the combination therapy group, compared to PDT treated group. Vandetanib and PDT inhibited the proliferation of OSCC cells through the induction of cell cycle arrest at G1 and G2 phase respectively and caused high apoptosis with combination therapy. Higher levels of ROS was observed in the combination therapy group, which effectively enhanced the cytotoxic efficacy of PDT in OSCC cells. Apoptosis induced by vandetanib was not inhibited by the use of caspase-inhibitor Z-VAD-FMK, suggesting that the cell death is caspase-independent, different from PDT which induced caspase-dependent apoptosis. A 2.2 fold increase in DNA fragmentation activity was noted in the combination therapy with vandetanib (IC50, 6 M), than PDT alone, that was confirmed by the down-regulation of anti-apoptotic bcl-2 protein. On the other hand, the migration and invasion efficiency of OSCC cells were reduced significantly with combination therapy when compared to monotherapies alone and control group. Furthermore, the tube formation of endothelial cells was reduced significantly in the presence of vandetanib. Combination therapy also inhibited the phosphorylation of EGFR and its downstream targets in OSCC cells. Conclusion: Vandetanib, a dual VEGFR and EGFR inhibitor, can significantly improve the efficacy of PDT by reducing the proliferation, increasing apoptosis and preventing angiogenesis in OSCC cells. http://dx.doi.org/10.1016/j.pdpdt.2017.01.158
F.C.P. Rosin ∗ , L. Corrêa
Squamous cell carcinoma of the oral cavity (OSCC) is a malignant tumor with high morbidity and mortality rates. Conventional treatment for OSCC includes surgery and radiotherapy/chemotherapy, but some cases can develop resistance to these treatments [1]. Oncologic photodynamic therapy (PDT) has been used as an adjuvant treatment for OSCC, mainly for non-invasive cases, in which this therapy has demonstrated a relative good efficacy [2]. Some studies have described that other tumors, such as cutaneous [3], colon [4], and breast [5] carcinomas, have shown PDT resistance. The hypothesis that OSCC could also develop PDT resistance has not yet been tested. The aims of this study were: (1) to investigate whether human OSCCs cells develop resistance to several cycles of 5-aminolevulinic acid-mediated PDT (5-ALA–PDT); and (2) to determine whether the expression of markers associated with cell survival (NFkB, Bcl-2, iNOS, mTOR, and Akt) is altered during this process. An OSCC cell line (SCC9) was subjected to the following conditions: (1) Control: cultured without any treatment; (2) ALA: incubated with 5-ALA (1 mM for 4 h); 3) LED: treated with LED light (630 nm, 5.86 J/cm2 , 10 J, 150 mW, 150 s); and (4) PDT: treated with 5-ALA–PDT (with the protocols of the ALA and LED groups combined), causing a cell death index of 90%. After a first PDT cycle, the survived cells were cultured and subjected to another cycle of 5-ALA–PDT, adding 2 J in the initial irradiation dose. When this process was repeated five times increasing the fluency (from 5.86 to 9.38 J/cm2 ) [4], the cell viability gradually improved. Four resistant cell populations were obtained, showing cell viability up to 4.6 times higher than that present at first PDT cycle. The most resistant population exhibited a lower intensity of protoporphyrin IX, higher migration capacity, and changes in nuclear morphology. By Western blotting analysis, there was an increase in the expression of pNFkB, iNOS, pmTOR, and pAkt, but not of anti-apoptotic Bcl-2 protein, in the resistant populations. In conclusion, OSCC cells treated with a 5-ALA–PDT exhibited increase on viability and improvement on migration capacity after six successive PDT cycles, as well as overexpression of proteins associated with cell survival. The possibility of resistance to 5-ALA–PDT should be carefully considered when initiating this therapy for OSCC, mainly for those cases in which are necessary multiple PDT cycles [6]. References [1] [2] [3] [4] [5] [6]
S.D. Silva, et al., Front. Pharmacol. 3 (2012) 1–7. W. Jerjes, et al., Head Neck Oncol. 4 (2012) 16. L.N. Milla, J. Cell. Biochem. 9 (2011) 2266–2278. G. Singh, Photochem. Photobiol. 6 (2001) 651–656. A. Casas, et al., Int. J. Oncol. 2 (2006) 397–405. A. Casas, et al., Curr. Med. Chem. 16 (2011) 2486–2515.
http://dx.doi.org/10.1016/j.pdpdt.2017.01.159