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RITA Enhances Radiosensitivity Mainly Via S100A9 through a p53-Independent Way in Squamous Cell Cervical Cancer Cells
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Volume 99 Number 2S Supplement 2017 J. Yue: None. Z. Jiang: None. X. Zhang: None. Q. Hou: None. M. Huang: None. W. Li: None. S. Wu: None.
3506 The Inhibitory Effect of Minocycline on RadiationInduced Neuronal Apoptosis Via AMPKa1 Signaling-Mediated Autophagy L. Zhang,1 P. Huang,2 Y. Tian,3 and H. Yang4; 1Department of Radiotherapy and Oncology, Second Affiliated Hospital, Soochow University, Suzhou, China; , 2Department of Radiotherapy and Oncology, Second Affiliated Hospital, Suzhou, China; , 3Department of Radiotherapy and Oncology, Second Affiliated Hospital, Soochow University, Suzhou, China; , 4School of Radiation Medicine and Protection, Medical College of Soochow University/ Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Suzhou, China Purpose/Objective(s): In our previous study, we have found that minocycline, a clinical available antibiotics that can easily cross the blood brain barrier, mitigates radiation-induced long-term memory loss in rats, accompanied by decreased hippocampal neuron apoptosis shortly after radiation. Thus, in the present study, we aimed to investigate the detailed mechanisms underlying the protective effect of minocycline on neurons from radiation-induced apoptosis. Materials/Methods: The immortalized mouse hippocampal neuron HT22 cell line was used. The protective effect of minocycline on X-irradiated HT22 cells was demonstrated using clonogenic assay and apoptosis analysis. The underlying mechanisms were explored by using methods such as flow cytometry, immunofluorescence, shRNA knockdown, Western blot et al. Results: We found that minocycline protected HT22 hippocampal neurons from radiation-induced cell death, manifest as increased clonogenic cell survival and decreased apoptosis in irradiated cells. This protective effect of minocycline might involve cell cycle perturbation after radiation, but not DNA damage and repair. Further investigation showed that X-irradiation activated AMPKa1-mediated autophagy, and minocycline significantly enhanced AMPKa1 activation and autophagy, resulting in decreased apoptosis. Additionally, the antioxidant potential of minocycline might not be involved in the enhancing effect of minocycline on radiation-induced autophagy, but it might at least partially contribute to the inhibitory effect of minocycline on radiation-induced apoptosis. Conclusion: These results indicate that minocycline protects neurons from radiation-induced apoptosis mainly through targeting radiation-induced AMPKa1-mediated autophagy. Author Disclosure: L. Zhang: None. P. Huang: None. Y. Tian: None. H. Yang: None.
3507 RITA Enhances Radiosensitivity Mainly Via S100A9 through a p53-Independent Way in Squamous Cell Cervical Cancer Cells H. Zhu, H. Liu, L. Liu, F. He, and Q. Liu; Xiangya Hospital, Central South University, Changsha, China Purpose/Objective(s): This study is to explore the relationship among small-molecule reactivation of p53 and induction of tumor cell apoptosis (RITA), p53 and S100A9, and its impact on radiotherapy (RT) on squamous cell cervical cancer cells. Materials/Methods: Several published p53 mutations were introduced into Caski cells. Expression of wild type p53 (wtp53) was measured. Caski cells under different p53 status were respectively treated with RITA, radiotherapy (RT), RITA+RT, with untreated cells as the control. Cell apoptosis, proliferation and invasion were measured with or without S100A9 knockdown. Results: We found that mup53 273 R-H but not mup53 183 S-A could inhibit the expression of wtp53. RITA alone or in synergy with RT induced apoptosis and inhibition of cell proliferation and invasion in Caski cells. The effects were not significantly affected by knockdown of p53 by
p53-shRNA or mup53 273 R-H. On the other hand, the expression of S100A9 at both the RNA and the protein levels was significantly enhanced by RITA and wtp53, and inhibited by p53 273 R-H and p53-shRNA but not p53 183 S-A. Knockdown of S100A9 abolished RITA-induced apoptosis and inhibition of cell proliferation and invasion in the RITA group and markedly decreased the enhancing effect of RITA on RT in the RITA+RT group. Promoter/luciferase reporter assays revealed that while wtp53 significantly enhanced the S100A9 gene promoter activity, mup53 273 RH but not mup53 183 S-A significantly inhibited it. Although having no significant effect on the S100A9 gene promoter activity, RITA markedly increased the stability of S100A9 mRNA in Caski cells. Conclusion: RITA alone or in synergy with RT induces apoptosis and inhibition of cell proliferation and invasion in Caski cells mainly through inducing the expression of S100A9 by increasing its mRNA stability. This is a p53-independent mechanism that does not rely on p53-induced transactivation of the S100A9 gene promoter/transcription. It adds new insights into the pharmacological potential of RITA alone or in combination with radiotherapy on treatment of cervical cancer. Author Disclosure: H. Zhu: None. H. Liu: None. L. Liu: None. F. He: None. Q. Liu: None.
3508 Impact of Proteins MCM7 and P16 on Oral Cancer Patient Prognosis D.M. Zielecka-De˛bska,1 A. Hao,2 R. Matkowski,1 J. Kornafel,3 and J. Szelachowska1; 1Lower Silesia Oncology Center, Wroclaw, Poland., Wroclaw, Poland; , 2Department of Pathomorphology and Oncological Cytology, Wroclaw Medical University, Wroclaw, Poland; , 3Department of Oncology, Wroclaw Medical University, Wroclaw, Poland, Wroclaw, Poland Purpose/Objective(s): 6000 new cases and 3800 deaths caused by head & neck cancer are reported every year in Poland. Most often patients are diagnosed in advanced stages of the disease. In those cases qualification to the treatment method can be difficult and has to be personalized. MCM7 protein is a marker of proliferation. P16 protein plays a pivotal role in cell cycle progression, cell aging and the development of cancer. In addition, there are reports showing that MCM7 and P16 are a good indicators of oncoprotein E7 activity in HPV 16 virus-infected cells. They can be used to distinguish head and neck tumors of viral etiology. The aim of the study is to evaluate prognostic significance of proteins MCM7 and P16 on oral cancer patients. Materials/Methods: The study included 129 patients, suffering from squamous cell carcinoma of mouth floor (C04) or anterior parts of tongue (C02), treated in the Lower Silesian Oncology Center in the years 19962011. 39 (30%) patients were diagnosed in stage II, 37 (29%) in stage III and 53 (41%) in stage IVa. All patients underwent microscopically radical resection of the primary tumor with simultaneous resection of regional lymph nodes and postoperative radiotherapy. MCM7 and P16 proteins expression was evaluated using a immunoreactivity Remmele scale (IRS). Results: In all 129 patients MCM7 and P16 proteins expression was observed. 43 (33%) patients with a high expression of the p16 IRS8 protein and MCM7 IRS4 were isolated as a group of potential viral Abstract 3508; Table 1 The percentage of 5-year locoregional recurrencefree survival (LRFS), disease-free survival (DFS), disease specific survival (DSS) and overall survival (OS) of patients based on the level of MCM7 and p16 expression in cells of squamous cell carcinoma IRS p168 i MCM7<4 p16<8 i MCM7<4 p168 i MCM74 p16<8 i MCM74
Numer of 5 year LRFS 5 year DFS 5 year DSS 5 year OS patients pZ0,013 pZ0,0029 pZ0,0027 pZ0,079 22
89%
85%
85%
67%
27
82%
78%
82%
58%
43
78%
73%
73%
53%
37
60%
50%
50%
43%
Volume 99 Number 2S Supplement 2017 J. Yue: None. Z. Jiang: None. X. Zhang: None. Q. Hou: None. M. Huang: None. W. Li: None. S. Wu: None.
3506 The Inhibitory Effect of Minocycline on RadiationInduced Neuronal Apoptosis Via AMPKa1 Signaling-Mediated Autophagy L. Zhang,1 P. Huang,2 Y. Tian,3 and H. Yang4; 1Department of Radiotherapy and Oncology, Second Affiliated Hospital, Soochow University, Suzhou, China; , 2Department of Radiotherapy and Oncology, Second Affiliated Hospital, Suzhou, China; , 3Department of Radiotherapy and Oncology, Second Affiliated Hospital, Soochow University, Suzhou, China; , 4School of Radiation Medicine and Protection, Medical College of Soochow University/ Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Suzhou, China Purpose/Objective(s): In our previous study, we have found that minocycline, a clinical available antibiotics that can easily cross the blood brain barrier, mitigates radiation-induced long-term memory loss in rats, accompanied by decreased hippocampal neuron apoptosis shortly after radiation. Thus, in the present study, we aimed to investigate the detailed mechanisms underlying the protective effect of minocycline on neurons from radiation-induced apoptosis. Materials/Methods: The immortalized mouse hippocampal neuron HT22 cell line was used. The protective effect of minocycline on X-irradiated HT22 cells was demonstrated using clonogenic assay and apoptosis analysis. The underlying mechanisms were explored by using methods such as flow cytometry, immunofluorescence, shRNA knockdown, Western blot et al. Results: We found that minocycline protected HT22 hippocampal neurons from radiation-induced cell death, manifest as increased clonogenic cell survival and decreased apoptosis in irradiated cells. This protective effect of minocycline might involve cell cycle perturbation after radiation, but not DNA damage and repair. Further investigation showed that X-irradiation activated AMPKa1-mediated autophagy, and minocycline significantly enhanced AMPKa1 activation and autophagy, resulting in decreased apoptosis. Additionally, the antioxidant potential of minocycline might not be involved in the enhancing effect of minocycline on radiation-induced autophagy, but it might at least partially contribute to the inhibitory effect of minocycline on radiation-induced apoptosis. Conclusion: These results indicate that minocycline protects neurons from radiation-induced apoptosis mainly through targeting radiation-induced AMPKa1-mediated autophagy. Author Disclosure: L. Zhang: None. P. Huang: None. Y. Tian: None. H. Yang: None.
3507 RITA Enhances Radiosensitivity Mainly Via S100A9 through a p53-Independent Way in Squamous Cell Cervical Cancer Cells H. Zhu, H. Liu, L. Liu, F. He, and Q. Liu; Xiangya Hospital, Central South University, Changsha, China Purpose/Objective(s): This study is to explore the relationship among small-molecule reactivation of p53 and induction of tumor cell apoptosis (RITA), p53 and S100A9, and its impact on radiotherapy (RT) on squamous cell cervical cancer cells. Materials/Methods: Several published p53 mutations were introduced into Caski cells. Expression of wild type p53 (wtp53) was measured. Caski cells under different p53 status were respectively treated with RITA, radiotherapy (RT), RITA+RT, with untreated cells as the control. Cell apoptosis, proliferation and invasion were measured with or without S100A9 knockdown. Results: We found that mup53 273 R-H but not mup53 183 S-A could inhibit the expression of wtp53. RITA alone or in synergy with RT induced apoptosis and inhibition of cell proliferation and invasion in Caski cells. The effects were not significantly affected by knockdown of p53 by
p53-shRNA or mup53 273 R-H. On the other hand, the expression of S100A9 at both the RNA and the protein levels was significantly enhanced by RITA and wtp53, and inhibited by p53 273 R-H and p53-shRNA but not p53 183 S-A. Knockdown of S100A9 abolished RITA-induced apoptosis and inhibition of cell proliferation and invasion in the RITA group and markedly decreased the enhancing effect of RITA on RT in the RITA+RT group. Promoter/luciferase reporter assays revealed that while wtp53 significantly enhanced the S100A9 gene promoter activity, mup53 273 RH but not mup53 183 S-A significantly inhibited it. Although having no significant effect on the S100A9 gene promoter activity, RITA markedly increased the stability of S100A9 mRNA in Caski cells. Conclusion: RITA alone or in synergy with RT induces apoptosis and inhibition of cell proliferation and invasion in Caski cells mainly through inducing the expression of S100A9 by increasing its mRNA stability. This is a p53-independent mechanism that does not rely on p53-induced transactivation of the S100A9 gene promoter/transcription. It adds new insights into the pharmacological potential of RITA alone or in combination with radiotherapy on treatment of cervical cancer. Author Disclosure: H. Zhu: None. H. Liu: None. L. Liu: None. F. He: None. Q. Liu: None.
3508 Impact of Proteins MCM7 and P16 on Oral Cancer Patient Prognosis D.M. Zielecka-De˛bska,1 A. Hao,2 R. Matkowski,1 J. Kornafel,3 and J. Szelachowska1; 1Lower Silesia Oncology Center, Wroclaw, Poland., Wroclaw, Poland; , 2Department of Pathomorphology and Oncological Cytology, Wroclaw Medical University, Wroclaw, Poland; , 3Department of Oncology, Wroclaw Medical University, Wroclaw, Poland, Wroclaw, Poland Purpose/Objective(s): 6000 new cases and 3800 deaths caused by head & neck cancer are reported every year in Poland. Most often patients are diagnosed in advanced stages of the disease. In those cases qualification to the treatment method can be difficult and has to be personalized. MCM7 protein is a marker of proliferation. P16 protein plays a pivotal role in cell cycle progression, cell aging and the development of cancer. In addition, there are reports showing that MCM7 and P16 are a good indicators of oncoprotein E7 activity in HPV 16 virus-infected cells. They can be used to distinguish head and neck tumors of viral etiology. The aim of the study is to evaluate prognostic significance of proteins MCM7 and P16 on oral cancer patients. Materials/Methods: The study included 129 patients, suffering from squamous cell carcinoma of mouth floor (C04) or anterior parts of tongue (C02), treated in the Lower Silesian Oncology Center in the years 19962011. 39 (30%) patients were diagnosed in stage II, 37 (29%) in stage III and 53 (41%) in stage IVa. All patients underwent microscopically radical resection of the primary tumor with simultaneous resection of regional lymph nodes and postoperative radiotherapy. MCM7 and P16 proteins expression was evaluated using a immunoreactivity Remmele scale (IRS). Results: In all 129 patients MCM7 and P16 proteins expression was observed. 43 (33%) patients with a high expression of the p16 IRS8 protein and MCM7 IRS4 were isolated as a group of potential viral Abstract 3508; Table 1 The percentage of 5-year locoregional recurrencefree survival (LRFS), disease-free survival (DFS), disease specific survival (DSS) and overall survival (OS) of patients based on the level of MCM7 and p16 expression in cells of squamous cell carcinoma IRS p168 i MCM7<4 p16<8 i MCM7<4 p168 i MCM74 p16<8 i MCM74
Numer of 5 year LRFS 5 year DFS 5 year DSS 5 year OS patients pZ0,013 pZ0,0029 pZ0,0027 pZ0,079 22
89%
85%
85%
67%
27
82%
78%
82%
58%
43
78%
73%
73%
53%
37
60%
50%
50%
43%