Page 250 - 2014 Printable Abstract Book
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and DNA repair (flow cytometry, γH2AX) was also examined, both in the absence and presence of ionizing
radiation (4Gy). Furthermore, siRNA silencing of GLI1/2 prior to radiation was performed as a proof-of-
concept of the radiosensitization target specificity of GANT61. Results: After 72h treatment with GANT61,
cell survival was significantly decreased in both cell lines. Decreased BrdU incorporation was also detected
in 22Rv1 cells. Radiosensitization was observed after 72h pretreatment with GANT61 in PC3Luc (DEF
(SF0.5)=1,87±0,6, p <0.05) and 22Rv1 (DEF (SF0.5)=1.39±0,11, p <0.05). Western blot analyses confirm
that this effect is associated with GLI1/2 levels/activity. Experiments determining effects on gene
expression, cell cycle distribution, and DNA damage response are ongoing and will be presented at time
of the meeting as well as the results of the siRNA silencing experiments. Conclusion: GANT61 sensitizes
PCa cells to ionizing radiation in vitro. This effect could be more pronounced in vivo given the presence of
the tumor micro-environment and the potential anti-angiogenic drug effects. Further investigations in
xenograft mouse models have therefore been initiated and may provide additional evidence for the
therapeutic potential of GANT61 in combination with radiotherapy and its potential as a treatment option
for PCa patients.
(PS4-26) Raspberry polyphenol targets cancer stem cells in residual pancreatic cancer after
1
radiotherapy: A comprehensive In-vivo / Ex-vivo approach. Faizan H. Khan, PhD ; Revathi Masilamani,
1
1
1
2
PhD ; Satish K. Ramraj, PhD ; Vijayabaskar Pandian, PhD ; Sheeja Aravindan, MSc ; Mohan Natarajan,
1
1
3
PhD ; Terence S. Herman, MD ; and Natarajan Aravindan, PhD, Department of Radiation Oncology,
1
University of Oklahoma Health Sciences Center, Oklahoma city, OK ; Stephensons Cancer Center,
2
Oklahoma city, OK ; and Department of Pathology, University of Texas Health Science Center, San Antonio,
3
TX
Ascertaining the intrinsic radio-sensitivity of cancer stem cells (CSCs), particularly on
variables pertaining to therapy resistance, tumor progression and relapse, may impart
significant clinical implications. Recently, we demonstrated the anti-pancreatic cancer (PC) potential of
bioactive polyphenol from Raspberry (RSE). We investigated the effects of RSE in radiation (IR)-induced
alterations in factors that drive EMT, CSCs’ self-renewal capacity and pluripotency maintenance
in PC. Using in-vivo xeno-transplantation models utilizing human BxPC3 and Mia-PaCa-2 cells, and ex-vivo
+
+
+
tumor models utilizing ALDH CD24 CD44 PC-CSCs of Panc-1, Mia-PaCa-2, Panc-3.27 origin, we examined
for the modulation of 93 stem cell-related transcriptional regulators after IR with or without RSE
treatment. RSE associated cellular localization of EMT (E-cadherin and N-Cadherin), pluripotency
maintaining (Sox2 and Nanog) and drug resistance (ABCG2) markers were examined using TMA
construction, immunofluorescence (IF) coupled with automated high-throughput confocal scanning. As
stand-alone compound, RSE exerted significant inhibition of stem cell-related molecules both under ex-
vivo (25, Panc1; 11, MiaPaca-2; 26, Panc-3.27) and in-vivo (33, BXPC3; 78, MiaPaca-2) conditions.
Conversely, IR-exposure robustly activated the expression of 32,9,23 in Panc-1, MiaPaca-2,
Panc-3.27 respectively in ex-vivo conditions and 14 and 84 in BXPC3, MiaPaca-2 respectively in in-vivo
conditions. Evidently, RSE significantly suppressed 25 of 32 in Panc-1, 9 of 9 in MiaPaca-2 and 5 of 23 in
Panc-3.27 ex-vivo and 9 of 14 in BXPC3 and 78 of 84 in MiaPaca-2 in-vivo. TMA-IFanalysis revealed a
significant reduction in the cellular localization of ABCG2, N-Cadherin, MYC, Nanog and SOX2 and robust
increase in E-Cadherin. Together, these data demonstrate that RSE targets the IR-induced EMT, CSCs self-
renewal capacity and pluripotency maintenance in therapy resistant PC-CSCs. It could, therefore, serve as
an effective ‘drug-deliverable’ to mitigate PC resistance and subsequent relapse. Clinically feasible
248 | P a g e
radiation (4Gy). Furthermore, siRNA silencing of GLI1/2 prior to radiation was performed as a proof-of-
concept of the radiosensitization target specificity of GANT61. Results: After 72h treatment with GANT61,
cell survival was significantly decreased in both cell lines. Decreased BrdU incorporation was also detected
in 22Rv1 cells. Radiosensitization was observed after 72h pretreatment with GANT61 in PC3Luc (DEF
(SF0.5)=1,87±0,6, p <0.05) and 22Rv1 (DEF (SF0.5)=1.39±0,11, p <0.05). Western blot analyses confirm
that this effect is associated with GLI1/2 levels/activity. Experiments determining effects on gene
expression, cell cycle distribution, and DNA damage response are ongoing and will be presented at time
of the meeting as well as the results of the siRNA silencing experiments. Conclusion: GANT61 sensitizes
PCa cells to ionizing radiation in vitro. This effect could be more pronounced in vivo given the presence of
the tumor micro-environment and the potential anti-angiogenic drug effects. Further investigations in
xenograft mouse models have therefore been initiated and may provide additional evidence for the
therapeutic potential of GANT61 in combination with radiotherapy and its potential as a treatment option
for PCa patients.
(PS4-26) Raspberry polyphenol targets cancer stem cells in residual pancreatic cancer after
1
radiotherapy: A comprehensive In-vivo / Ex-vivo approach. Faizan H. Khan, PhD ; Revathi Masilamani,
1
1
1
2
PhD ; Satish K. Ramraj, PhD ; Vijayabaskar Pandian, PhD ; Sheeja Aravindan, MSc ; Mohan Natarajan,
1
1
3
PhD ; Terence S. Herman, MD ; and Natarajan Aravindan, PhD, Department of Radiation Oncology,
1
University of Oklahoma Health Sciences Center, Oklahoma city, OK ; Stephensons Cancer Center,
2
Oklahoma city, OK ; and Department of Pathology, University of Texas Health Science Center, San Antonio,
3
TX
Ascertaining the intrinsic radio-sensitivity of cancer stem cells (CSCs), particularly on
variables pertaining to therapy resistance, tumor progression and relapse, may impart
significant clinical implications. Recently, we demonstrated the anti-pancreatic cancer (PC) potential of
bioactive polyphenol from Raspberry (RSE). We investigated the effects of RSE in radiation (IR)-induced
alterations in factors that drive EMT, CSCs’ self-renewal capacity and pluripotency maintenance
in PC. Using in-vivo xeno-transplantation models utilizing human BxPC3 and Mia-PaCa-2 cells, and ex-vivo
+
+
+
tumor models utilizing ALDH CD24 CD44 PC-CSCs of Panc-1, Mia-PaCa-2, Panc-3.27 origin, we examined
for the modulation of 93 stem cell-related transcriptional regulators after IR with or without RSE
treatment. RSE associated cellular localization of EMT (E-cadherin and N-Cadherin), pluripotency
maintaining (Sox2 and Nanog) and drug resistance (ABCG2) markers were examined using TMA
construction, immunofluorescence (IF) coupled with automated high-throughput confocal scanning. As
stand-alone compound, RSE exerted significant inhibition of stem cell-related molecules both under ex-
vivo (25, Panc1; 11, MiaPaca-2; 26, Panc-3.27) and in-vivo (33, BXPC3; 78, MiaPaca-2) conditions.
Conversely, IR-exposure robustly activated the expression of 32,9,23 in Panc-1, MiaPaca-2,
Panc-3.27 respectively in ex-vivo conditions and 14 and 84 in BXPC3, MiaPaca-2 respectively in in-vivo
conditions. Evidently, RSE significantly suppressed 25 of 32 in Panc-1, 9 of 9 in MiaPaca-2 and 5 of 23 in
Panc-3.27 ex-vivo and 9 of 14 in BXPC3 and 78 of 84 in MiaPaca-2 in-vivo. TMA-IFanalysis revealed a
significant reduction in the cellular localization of ABCG2, N-Cadherin, MYC, Nanog and SOX2 and robust
increase in E-Cadherin. Together, these data demonstrate that RSE targets the IR-induced EMT, CSCs self-
renewal capacity and pluripotency maintenance in therapy resistant PC-CSCs. It could, therefore, serve as
an effective ‘drug-deliverable’ to mitigate PC resistance and subsequent relapse. Clinically feasible
248 | P a g e
