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The Mechanism of Metformin-Induced Radiosensitization of Pancreatic Cancer Cells
Poster Viewing Abstracts S653
Volume 87 Number 2S Supplement 2013 Purpose/Objective(s): Malignant glioma, the most common type of brain tumor in adults, remains resistant to current treatment strategies. The purpose of this work was to investigate the possible application of silicon nanoparticles (Si-NPs) as therapeutic tools based in its capability of increase the yield of reactive species upon irradiation (IR) of C6 glioma cells. Materials/Methods: Si-NPs uptake by C6 cells was evaluated by spectrofluorometry (SF), confocal biphotonic microscopy and transmission electron microscopy. Cell viability was measured by Neutral Red Assay. IR experiments were carried out employing a 4 MeV nominal accelerating potential Varian Clinac electron lineal accelerator. IR setup was adjusted for the cell culture to absorb 1-3 Gy. Reactive oxygen species (ROS) production was assessed by an oxidation-sensitive probe, CM-H2DCF-DA. Results: Internalization of Si-NPs measured by SF was observed after a lag period of 2 h incubation, reaching a threefold increase after 6 h. Under these conditions, Si-NPs were localized mainly in liposomes. Cytotoxic analysis of Si-NPs performed at different concentrations (0-50 mg/mL) during 2-12 revealed a 50% decrease in cell viability for 50 mg/ mL, 12 h (p < 0.001) suggesting Si-NPs cytotoxicity was significantly lower than numerous other nanoparticles used in biological studies. Then, Si-NPs treated cells (25, 50 mg/mL and 6 h incubation) underwent IR at variable doses (0-2 Gy) and cell viability was measured. C6 cells exposed to 50 mg/mL of Si-NPs for 6 h showed a 36% decrease in cell viability; a further 15% diminution was observed when irradiated with 1 Gy dose, lower than the standard dose delivered to the cancer patients (2 Gy)(p < 0.01). Finally, ROS generation evaluated in C6 cells exposed to 50 mg/mL of Si-NPs for 6 h and irradiated at 1-3 Gy indicated that ROS generation increases with IR dose, reaching a 700% at 3 Gy. Conclusions: These data suggest Si-NPs can be applied for to advance IR cytotoxicity via generation of ROS. With optimization, Si-NPs have the potential to be used as radiosensitizers to improve the outcomes of existing cancer radiation therapy by employing lower IR doses with a consequent decrease in damage to healthy tissue. Future work will address specific targeting of Si-NPs to tumoral tissues to further potentiate these beneficial properties. Author Disclosure: M.L. Kotler: None. N.I. Garabano: None. P. David Gara: None. O.R. Casas: None. D. Dodat: None. S.E. Finkelstein: None. C. Mantz: None. E. Fernandez: None. M.C. Gonzalez: None.
3178 The Mechanism of Metformin-Induced Radiosensitization of Pancreatic Cancer Cells A. Fasih, H.A. Elbaz, M. Huttemann, A.A. Konski, and S.P. Zielske; Wayne State University and Karmanos Cancer Institute, Detroit, MI Purpose/Objective(s): Local control of pancreatic cancer with radiation therapy is poor and the development of radiosensitizers can positively impact treatment. We have previously shown that the anti-diabetic drug metformin radiosensitizes pancreatic cancer cells. Here we investigate the mechanism of metformin induced radiosensitization. Materials/Methods: DNA damage signaling following metformin and radiation treatment was analyzed by immunofluorescent staining of gamma-H2AX foci. Reactive oxygen species (ROS) generation was measured with CM-H2DCFDA. To determine the role of AMPK in radiosensitization, we used Compound C (CC) to inhibit AMPK kinase activity and RNAi to knockdown AMPK-alpha expression before performing clonogenic experiments to measure radiosensitization. AMPK inhibition was determined by Western blotting. Results: Metformin radiosensitized KRas mutant pancreatic cancer cells at micromolar concentrations with radiation enhancement factors of up to 1.40 according to clonogenic assays, but failed to radiosensitize a wild type K-ras cell line. When we assessed DNA damage signaling by quantifying gamma-H2AX foci in K-ras mutant cells 1 hour following treatment, we found 56% more foci following 6 Gy plus metformin than in 6 Gy-alone treated cells. Furthermore, cells treated with metformin alone showed 60% more gamma-H2AX foci than untreated cells. ROS levels in irradiated
cells treated with metformin were 20% higher than in irradiated cells not exposed to metformin. We then investigated the role of AMPK in mediating radiosensitization by inhibiting AMPK kinase activity using CC. CC treatment of cells abrogated metformin-induced radiosensitization by clonogenic assay, suggesting AMPK is required for this effect. To confirm specificity, we used siRNA to knockdown AMPK-alpha before metformin and radiation treatment. Clonogenic survival of control siRNA-transfected cells was reduced by metformin plus radiation to indicate radiosensitization, however, AMPK-alpha siRNA-transfected cells showed no radiosensitization. Conclusions: We show that metformin radiosensitizes KRas mutant pancreatic cancer cells, increases DNA damage signaling and ROS, and inhibition of AMPK abrogates radiosensitization. Thus, radiosensitization may be mediated through increased DNA damage or oxidative stress and activation of the AMPK pathway. Author Disclosure: A. Fasih: None. H.A. Elbaz: None. M. Huttemann: None. A.A. Konski: None. S.P. Zielske: None.
3179 Influence of ATM on DNA Methyltransferase Inhibitor-Induced Radiosensitization J. Kim,1 H. Kim,1 D. Park,2 I. Kim,3 and I. Kim1; 1Radiation Oncology, Seoul National University Hospital, Seoul, Korea, Republic of Korea, 2 Cancer Research Institute, Seoul National University College of Medicine, Seoul, Korea, Republic of Korea, 3Radiation Oncology, Seoul National University Bundang Hospital, Seongnam, Korea, Republic of Korea Purpose/Objective(s): Preclinical studies have demonstrated that inhibition of DNA methyltransferase (DMNT) induces radiosensitization in human cancer cells. The current study is to investigate the role of ATM in DNMT inhibitor-mediated radiosensitization. Materials/Methods: A pair of isogenic cell lines was constructed from AT fibroblasts: one without ATM and the other expressing functional ATM. The effects of DNMT inhibitor treatment on radiosensitivity were evaluated in these cell lines using two DNMT inhibitors: psammaplin A (PSA) and 5-aza-2’-deoxycytidine (DAC). DNA damage repair was analyzed using gH2AX foci. Western blot of DNA damage foci proteins was done. Results: DNMT inhibition induced radiosensitization of ATM-expressing cells, but had no effect in ATM-null cells. DNMT inhibitor treatment prolonged expression of both gH2AX foci and phospho-BRCA1 after irradiation in cells with ATM, whereas neither was observed in ATM-null cells treated with DNMT inhibitors. Expression of MRN complex subunits (MRE11, Rad50, and NBS1) was not altered by either of DNMT inhibition or irradiation. DNMT inhibitor treatment or irradiation induced MRE11 phosphorylation in both ATM-null and ATM-expressing cell lines. Conclusions: DNMT inhibitors enhance radiosensitivity by delaying DNA damage repairs. Radiosensitization by DNMT inhibition requires functional ATM. Author Disclosure: J. Kim: None. H. Kim: None. D. Park: None. I. Kim: None. I. Kim: None.
3180 Metformin Potentiates the Effects of Radiation on Cancer Cells and Cancer Stem Cells by Activating Ampk C.W. Song,1 H. Park,2 K. Dusenbery,1 and C. Cho1; 1University of Minnesota Medical School, Minneapolis, MN, 2Inha University, Incheon, Korea, Republic of Korea Purpose/Objective(s): To investigate the anti-cancer effects of metformin (Met), the most widely used drug for type 2 diabetes, in combination with radiation therapy. Materials/Methods: Human fibrosarcoma (FS), synovial sarcoma (SS), breast cancers (MCF-7 and MDA-MB-231) and pancreatic cancer (Miapaca 2), and mouse fibrosarcoma (FSaII) were used. Effects of Met alone and in combination with radiation on cancer cells were determined using
Volume 87 Number 2S Supplement 2013 Purpose/Objective(s): Malignant glioma, the most common type of brain tumor in adults, remains resistant to current treatment strategies. The purpose of this work was to investigate the possible application of silicon nanoparticles (Si-NPs) as therapeutic tools based in its capability of increase the yield of reactive species upon irradiation (IR) of C6 glioma cells. Materials/Methods: Si-NPs uptake by C6 cells was evaluated by spectrofluorometry (SF), confocal biphotonic microscopy and transmission electron microscopy. Cell viability was measured by Neutral Red Assay. IR experiments were carried out employing a 4 MeV nominal accelerating potential Varian Clinac electron lineal accelerator. IR setup was adjusted for the cell culture to absorb 1-3 Gy. Reactive oxygen species (ROS) production was assessed by an oxidation-sensitive probe, CM-H2DCF-DA. Results: Internalization of Si-NPs measured by SF was observed after a lag period of 2 h incubation, reaching a threefold increase after 6 h. Under these conditions, Si-NPs were localized mainly in liposomes. Cytotoxic analysis of Si-NPs performed at different concentrations (0-50 mg/mL) during 2-12 revealed a 50% decrease in cell viability for 50 mg/ mL, 12 h (p < 0.001) suggesting Si-NPs cytotoxicity was significantly lower than numerous other nanoparticles used in biological studies. Then, Si-NPs treated cells (25, 50 mg/mL and 6 h incubation) underwent IR at variable doses (0-2 Gy) and cell viability was measured. C6 cells exposed to 50 mg/mL of Si-NPs for 6 h showed a 36% decrease in cell viability; a further 15% diminution was observed when irradiated with 1 Gy dose, lower than the standard dose delivered to the cancer patients (2 Gy)(p < 0.01). Finally, ROS generation evaluated in C6 cells exposed to 50 mg/mL of Si-NPs for 6 h and irradiated at 1-3 Gy indicated that ROS generation increases with IR dose, reaching a 700% at 3 Gy. Conclusions: These data suggest Si-NPs can be applied for to advance IR cytotoxicity via generation of ROS. With optimization, Si-NPs have the potential to be used as radiosensitizers to improve the outcomes of existing cancer radiation therapy by employing lower IR doses with a consequent decrease in damage to healthy tissue. Future work will address specific targeting of Si-NPs to tumoral tissues to further potentiate these beneficial properties. Author Disclosure: M.L. Kotler: None. N.I. Garabano: None. P. David Gara: None. O.R. Casas: None. D. Dodat: None. S.E. Finkelstein: None. C. Mantz: None. E. Fernandez: None. M.C. Gonzalez: None.
3178 The Mechanism of Metformin-Induced Radiosensitization of Pancreatic Cancer Cells A. Fasih, H.A. Elbaz, M. Huttemann, A.A. Konski, and S.P. Zielske; Wayne State University and Karmanos Cancer Institute, Detroit, MI Purpose/Objective(s): Local control of pancreatic cancer with radiation therapy is poor and the development of radiosensitizers can positively impact treatment. We have previously shown that the anti-diabetic drug metformin radiosensitizes pancreatic cancer cells. Here we investigate the mechanism of metformin induced radiosensitization. Materials/Methods: DNA damage signaling following metformin and radiation treatment was analyzed by immunofluorescent staining of gamma-H2AX foci. Reactive oxygen species (ROS) generation was measured with CM-H2DCFDA. To determine the role of AMPK in radiosensitization, we used Compound C (CC) to inhibit AMPK kinase activity and RNAi to knockdown AMPK-alpha expression before performing clonogenic experiments to measure radiosensitization. AMPK inhibition was determined by Western blotting. Results: Metformin radiosensitized KRas mutant pancreatic cancer cells at micromolar concentrations with radiation enhancement factors of up to 1.40 according to clonogenic assays, but failed to radiosensitize a wild type K-ras cell line. When we assessed DNA damage signaling by quantifying gamma-H2AX foci in K-ras mutant cells 1 hour following treatment, we found 56% more foci following 6 Gy plus metformin than in 6 Gy-alone treated cells. Furthermore, cells treated with metformin alone showed 60% more gamma-H2AX foci than untreated cells. ROS levels in irradiated
cells treated with metformin were 20% higher than in irradiated cells not exposed to metformin. We then investigated the role of AMPK in mediating radiosensitization by inhibiting AMPK kinase activity using CC. CC treatment of cells abrogated metformin-induced radiosensitization by clonogenic assay, suggesting AMPK is required for this effect. To confirm specificity, we used siRNA to knockdown AMPK-alpha before metformin and radiation treatment. Clonogenic survival of control siRNA-transfected cells was reduced by metformin plus radiation to indicate radiosensitization, however, AMPK-alpha siRNA-transfected cells showed no radiosensitization. Conclusions: We show that metformin radiosensitizes KRas mutant pancreatic cancer cells, increases DNA damage signaling and ROS, and inhibition of AMPK abrogates radiosensitization. Thus, radiosensitization may be mediated through increased DNA damage or oxidative stress and activation of the AMPK pathway. Author Disclosure: A. Fasih: None. H.A. Elbaz: None. M. Huttemann: None. A.A. Konski: None. S.P. Zielske: None.
3179 Influence of ATM on DNA Methyltransferase Inhibitor-Induced Radiosensitization J. Kim,1 H. Kim,1 D. Park,2 I. Kim,3 and I. Kim1; 1Radiation Oncology, Seoul National University Hospital, Seoul, Korea, Republic of Korea, 2 Cancer Research Institute, Seoul National University College of Medicine, Seoul, Korea, Republic of Korea, 3Radiation Oncology, Seoul National University Bundang Hospital, Seongnam, Korea, Republic of Korea Purpose/Objective(s): Preclinical studies have demonstrated that inhibition of DNA methyltransferase (DMNT) induces radiosensitization in human cancer cells. The current study is to investigate the role of ATM in DNMT inhibitor-mediated radiosensitization. Materials/Methods: A pair of isogenic cell lines was constructed from AT fibroblasts: one without ATM and the other expressing functional ATM. The effects of DNMT inhibitor treatment on radiosensitivity were evaluated in these cell lines using two DNMT inhibitors: psammaplin A (PSA) and 5-aza-2’-deoxycytidine (DAC). DNA damage repair was analyzed using gH2AX foci. Western blot of DNA damage foci proteins was done. Results: DNMT inhibition induced radiosensitization of ATM-expressing cells, but had no effect in ATM-null cells. DNMT inhibitor treatment prolonged expression of both gH2AX foci and phospho-BRCA1 after irradiation in cells with ATM, whereas neither was observed in ATM-null cells treated with DNMT inhibitors. Expression of MRN complex subunits (MRE11, Rad50, and NBS1) was not altered by either of DNMT inhibition or irradiation. DNMT inhibitor treatment or irradiation induced MRE11 phosphorylation in both ATM-null and ATM-expressing cell lines. Conclusions: DNMT inhibitors enhance radiosensitivity by delaying DNA damage repairs. Radiosensitization by DNMT inhibition requires functional ATM. Author Disclosure: J. Kim: None. H. Kim: None. D. Park: None. I. Kim: None. I. Kim: None.
3180 Metformin Potentiates the Effects of Radiation on Cancer Cells and Cancer Stem Cells by Activating Ampk C.W. Song,1 H. Park,2 K. Dusenbery,1 and C. Cho1; 1University of Minnesota Medical School, Minneapolis, MN, 2Inha University, Incheon, Korea, Republic of Korea Purpose/Objective(s): To investigate the anti-cancer effects of metformin (Met), the most widely used drug for type 2 diabetes, in combination with radiation therapy. Materials/Methods: Human fibrosarcoma (FS), synovial sarcoma (SS), breast cancers (MCF-7 and MDA-MB-231) and pancreatic cancer (Miapaca 2), and mouse fibrosarcoma (FSaII) were used. Effects of Met alone and in combination with radiation on cancer cells were determined using