JesseColeASSIPPoster_Version2_080814
1
Common Chemotherapy Drugs Linked with Cognitive Decline in Cancer Survivors Jesse Cole, Grace Lawrence, Virginia Espina Abstract Results Methods Conclusions •T0 min •T1 min •T3 min •T5 min •T7 min Introduction 2014 Two widely-administered chemotherapy drugs may be linked to memory loss and other forms of cognitive decline in cancer survivors. A large subset of cancer survivors have shown this decline, a phenomenon known as "chemobrain." Up to 75% of cancer patients that undergo chemotherapy report developing cognitive impairment after treatment. Common problems include loss of memory, inability to multitask, and general inability to focus. The molecular mechanism causing these problems is not yet understood, but it was recently hypothesized to be due to chemotherapy agents crossing the blood/brain barrier. To investigate the effects of chemotherapy on neurologic functions, we developed a rat model, with or without chemotherapy, to evaluate changes in the brain proteome. Eight healthy adult rats were administered either saline injections or one or both of the chemotherapy drugs of interest. Each rat brain was fixed in a novel molecular fixative to preserve histomorphology and phosphoproteins. Cell signaling proteins involved in the inflammatory response, apoptosis, proliferation and oxidative stress were evaluated by immunohistochemistry and Reverse Phase Protein Microarray (RPPA). Cells in the olfactory bulb, hippocampus, and cerebellum were procured by Laser Capture Microdissection, lysed and printed on the RPPA. High levels of HIF-1α were found in the olfactory bulb and hippocampus via IHC. HIF-1 indicates hypoxic conditions. PCNA, a proliferation marker, showed intense staining in the periventricular zone. Additional proteins involved in oxidative stress and the immune response were evaluated via RPPA, and found to be expressed at differential levels The results of this ongoing study have the potential to vastly improve the lives of cancer patients. Our findings could help medical professionals better recommend cancer treatments by elucidating some of the mechanisms behind the chemobrain phenomenon and making the risks of chemotherapy clearer. Additionally, the data may help identify specific brain regions and signaling pathways for drug intervention to halt or prevent cognitive decline. SAMPLE TREATMENT OLFACTORY BULB HIPPOCAMPUS SID 002 Combo (Cyclo . + Docet) SID 003 Cyclo. Only SID 004 Cyclo. only SID 005 Docet. Only SID 007 Saline SID 008 Saline Figure 3: HIF-1 ɑ staining in two brain locations. This was intriguing, but RPPA analysis revealed HIF-1 ɑ to be independent of treatment. Immunohistochemistr y Laser Capture Microdissection Reverse Phase Protein Microarray LCM Reagent s Pixcell IIe LCM Instrument •A common combination regimen of chemotherapy, using the drugs cyclophosphamide and docetaxel, were investigated via a rat model, to elucidate the proteomic effects in brain tissue. •We were interested in the mechanism behind the cognitive decline that these drugs may cause in cancer survivors. •We hypothesize that the combination drug dose affected proteins in the brain related to neurogenesis, oxidative stress, autophagy, and growth. •Immunohistochemistry was used to detect selected proteins in rat brain sections. •Cells were procured from these sections via Laser Capture Microdissection. •Protein concentrations in these cells were quantified via Reverse Phase Protein Microarrays. Proteins Investigate d: Neurogenesis •CC3 •CC7 ASP198 •CD44 •Doublecorti n •PCNA •Sox2 •Survivin Stress •AMPKBSer108 •Cu/Zn SOD •HIF-1 ɑ •IL-6 •Osteopontin •Stat1 Y701 •Stat3Ser727 Autophagy •Beclin-1 •LC3B •mTOR Ser2448 Growth •B-Raf S445 •EGFR Y1045 •EGFR Y1148 •ERKT202Y204 •Hexokinase II •RasGRF Ser916 Other •ß-Actin •PMAC2 •Prolactin Receptor Immunohistochemistry: •Tissue is dewaxed via passage through graded alcohols •Paraffin cross-links are broken with heat •Tissue is stained by applying, amplifying, and coloring the primary antibody LCM: •Tissue dehydrated and rehydrated with xylenes and graded alcohols •LCM instrument sticks tissue onto a polymer by firing a laser through it •Laser is moved via a joystick and tissue is collected throughout the sample RPPA: •Samples are micropipetted into a microtiter plate •The arrayer prints the samples onto a nitrocellulose paper, which is stained for proteins in a similar manner as IHC •The resulting slide is scanned, and analyzed via Microsoft Excel Figure 1: Sox2 concentration by brain region. Sox2 is a protein involved in pluripotency and post-embryonic neurogenesis (PEN). High levels were observed in the hippocampus, one of the few brain regions where PEN has been shown to occur. Figure 2: Sox2 concentration by treatment. Sox2 concentrations increased compared to the control value with each individual drug, but the increase was the strongest when the rat was treated with the standard combination regimen. Figure 4: Stat1 Y701 concentrations by brain region and by treatment . Stat1 Y701 is a protein that upregulates genes in response to interferons as part of the inflammatory response. Cyclophosphamide and Docetaxel have both been shown to promote the inflammatory response. We hypothesize that the combination dose of drugs aggravates patient suffering in part by activating the inflammatory response with help from proteins like Stat1 Figure 5: Copper/Zinc Superoxidedismutase (Cu/Zn SOD) concentrations by brain region and by treatment. Cu/Zn SOD is an enzyme with antioxidative properties that scavenges the cytoplasm for free radicals. It has been shown in a mouse model that mice without Cu/Zn SOD died after three days due to oxidative stress. While less Cu/Zn SOD was observed in the two experimental brain regions than in the control region (cerebellum), more of the protein was present in the rats that received the combination regimen (compared to the control). While neither proved anything definitively, the combined results of HIF-1 ɑ and Cu/Zn SOD staining warrant further investigation into oxidative stress pathways. Deparaffinizat ion Heat- Induced Epitope Retrieva l Dako Autostainer Microscopic Visualization Loading samples into microtiter plate Printing on the arrayer Freshly- printed slide Dako Auto- stain er Scanned slide after staining Data Analysi s Graphs of protein concentrati ons •HIF-1 ɑ staining via immunohistochemistry showed high levels of HIF-1, but RPPA analysis showed no difference in concentration between treated and control groups. • Cu/Zn SOD concentrations quantified by RPPA lead us to think that there may be a relationship between drug regimen and oxidative stress. •Sox2 concentrations from the RPPA indicate that neurogenesis and proliferation are also areas of interest in the study of chemobrain. •The concentrations of the inflammatory response protein Stat1 Y701 did not show clear effects from our treatment as strongly as other proteins. We will investigate further the role of Stat1 and the inflammatory response in the chemobrain mechanism. Acknowledgements: We would like to thank Dr. Ted Dumas and Dr. Lance Liotta for their contributions to this project References: •Kaiser, Jochen, Bledowski, Christof, Dietrich, Jorg. “Neural correlates of chemotherapy-related cognitive impairment.” Cortex. 54 (2014): 33-50. Web 7/6/2014 •Wigmore, Peter. “The Effect of Systemic Chemotherapy on Neurogenesis, Plasticity, and Memory.” Current Topics in Behavioral Neuroscience. 15 (2013): 211-240. Web. 7/19/2014. Average Relative Sox2 Concentration in DifferentRatBrain Regions 0 0.2 0.4 0.6 0.8 1 O lfactory Bulb (n=6) Hippocam pus (n=6) Cerebellum (Control;n=4) R atBrain R egion Average Relative Concentration SEM = +/- 0.135 SEM = +/- 0.071 SEM = +/- 0.250 Average Relative Sox2 Concentration by Treatm ent 0 0.2 0.4 0.6 0.8 1 Saline (Control) Cyclophospham ide O nly DocetaxelOnly Combination Tream ent Average Relative Concentration SEM = +/- 0.256 SEM = +/- 0.140 SEM = +/- 0.506 SEM = +/- 0.272 Average R elative Stat1 Y701 C oncentration in D ifferentR at B rain R egions 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 O lfactory B ulb (n=6) H ippocam pus (n=6) C erebellum (C ontrol;n=4) R atB rain R egion A verag e R ela t C o n cen tratio SEM = +/- 0.188 SEM = +/- 0.108 SEM = +/- 0.289 A verage R elative Stat1 Y 701 C oncentration by Treatm ent 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 S aline (C ontrol) C yclophospham ide O nly D ocetaxelO nly C om bination Treatm ent A v e ra g e R ela C o n cen tratio SEM = +/- 0.329 SEM = +/- 0.320 SEM = +/- 0.511 SEM = +/- 0.115 Average R elat ive C oncentration ofC u/ZN SO D in R atB rain R egions -0.1 0.1 0.3 0.5 0.7 0.9 O lfactory B ulb (n=6) H ippocam pus (n=6) C erebellum (C ontrol;n=4) R atB rain R egion A verag e R elativ C o n cen tratio n SEM = +/- 0.130 SEM = +/- 0.096 SEM = +/- 0.221 A verage R elative C oncentration ofC u/Zn SO D by Treatm ent 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 S aline (C ontrol) C yclophospham ide O nly D ocetaxelO nly Com bination Treatm ent A verage R elativ C o ncen tratio n SEM = +/- 0.159 SEM = +/- 0.248 SEM = +/- 0.302 SEM = +/- 0.225
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Transcript of JesseColeASSIPPoster_Version2_080814
Common Chemotherapy Drugs Linked with Cognitive Decline in Cancer SurvivorsJesse Cole, Grace Lawrence, Virginia Espina
Abstract
Results
Methods
Conclusions
•T0 min•T1 min•T3 min•T5 min•T7 min
Introduction
2014
Two widely-administered chemotherapy drugs may be linked to memory loss and other forms of cognitive decline in cancer survivors. A large subset of cancer survivors have shown this decline, a phenomenon known as "chemobrain." Up to 75% of cancer patients that undergo chemotherapy report developing cognitive impairment after treatment. Common problems include loss of memory, inability to multitask, and general inability to focus. The molecular mechanism causing these problems is not yet understood, but it was recently hypothesized to be due to chemotherapy agents crossing the blood/brain barrier. To investigate the effects of chemotherapy on neurologic functions, we developed a rat model, with or without chemotherapy, to evaluate changes in the brain proteome.
Eight healthy adult rats were administered either saline injections or one or both of the chemotherapy drugs of interest. Each rat brain was fixed in a novel molecular fixative to preserve histomorphology and phosphoproteins. Cell signaling proteins involved in the inflammatory response, apoptosis, proliferation and oxidative stress were evaluated by immunohistochemistry and Reverse Phase Protein Microarray (RPPA). Cells in the olfactory bulb, hippocampus, and cerebellum were procured by Laser Capture Microdissection, lysed and printed on the RPPA.
High levels of HIF-1α were found in the olfactory bulb and hippocampus via IHC. HIF-1 indicates hypoxic conditions. PCNA, a proliferation marker, showed intense staining in the periventricular zone. Additional proteins involved in oxidative stress and the immune response were evaluated via RPPA, and found to be expressed at differential levels
The results of this ongoing study have the potential to vastly improve the lives of cancer patients. Our findings could help medical professionals better recommend cancer treatments by elucidating some of the mechanisms behind the chemobrain phenomenon and making the risks of chemotherapy clearer. Additionally, the data may help identify specific brain regions and signaling pathways for drug intervention to halt or prevent cognitive decline.
SAMPLE TREATMENT OLFACTORY BULB
HIPPOCAMPUS
SID 002 Combo (Cyclo.
+ Docet)
SID 003 Cyclo.
Only
SID 004 Cyclo.
only
SID 005 Docet.
Only
SID 007 Saline
SID 008 Saline
Figure 3: HIF-1 ɑ staining in two brain locations. This was intriguing, but RPPA
analysis revealed HIF-1 ɑ to be independent of treatment.
Immunohistochemistry Laser Capture Microdissection
Reverse Phase Protein Microarray
LCM Reagents
Pixcell IIe LCM
Instrument
•A common combination regimen of chemotherapy, using the drugs cyclophosphamide and docetaxel, were investigated via a rat model, to elucidate the proteomic effects in brain tissue.
•We were interested in the mechanism behind the cognitive decline that these drugs may cause in cancer survivors.
•We hypothesize that the combination drug dose affected proteins in the brain related to neurogenesis, oxidative stress, autophagy, and growth.
•Immunohistochemistry was used to detect selected proteins in rat brain sections.
•Cells were procured from these sections via Laser Capture Microdissection.
•Protein concentrations in these cells were quantified via Reverse Phase Protein Microarrays.
Proteins Investigated:Neurogenesis
•CC3•CC7 ASP198•CD44•Doublecortin•PCNA•Sox2•Survivin
Stress•AMPKBSer108•Cu/Zn SOD•HIF-1 ɑ•IL-6•Osteopontin•Stat1 Y701•Stat3Ser727
Autophagy•Beclin-1•LC3B•mTOR Ser2448
Growth•B-Raf S445•EGFR Y1045•EGFR Y1148•ERKT202Y204•Hexokinase II•RasGRF Ser916
Other•ß-Actin•PMAC2•Prolactin Receptor
Immunohistochemistry:
•Tissue is dewaxed via passage through graded alcohols
•Paraffin cross-links are broken with heat
•Tissue is stained by applying, amplifying, and coloring the primary antibody
LCM:
•Tissue dehydrated and rehydrated with xylenes and graded alcohols
•LCM instrument sticks tissue onto a polymer by firing a laser through it
•Laser is moved via a joystick and tissue is collected throughout the sample
RPPA:
•Samples are micropipetted into a microtiter plate
•The arrayer prints the samples onto a nitrocellulose paper, which is stained for proteins in a similar manner as IHC
•The resulting slide is scanned, and analyzed via Microsoft Excel
Figure 1: Sox2 concentration by brain region. Sox2 is a protein involved in pluripotency and post-embryonic neurogenesis (PEN).
High levels were observed in the hippocampus, one of the few brain regions where PEN has been shown to occur.
Figure 2: Sox2 concentration by treatment. Sox2 concentrations increased compared to the control
value with each individual drug, but the increase was the strongest when the rat was treated with the
standard combination regimen.
Figure 4: Stat1 Y701 concentrations by brain region and by treatment . Stat1 Y701 is a protein that upregulates genes in response to interferons as part of the inflammatory response. Cyclophosphamide and Docetaxel have both been shown to promote the
inflammatory response. We hypothesize that the combination dose of drugs aggravates patient suffering in part by activating the inflammatory response with help
from proteins like Stat1 Y701.
Figure 5: Copper/Zinc Superoxidedismutase (Cu/Zn SOD) concentrations by brain region and by treatment. Cu/Zn SOD is an enzyme with antioxidative properties that scavenges the cytoplasm for free radicals. It has been shown in a mouse model that
mice without Cu/Zn SOD died after three days due to oxidative stress. While less Cu/Zn SOD was observed in the two experimental brain regions than in the control region (cerebellum), more of the protein was present in the rats that received the combination regimen (compared to the control). While neither proved anything
definitively, the combined results of HIF-1 ɑ and Cu/Zn SOD staining warrant further investigation into oxidative stress pathways.
DeparaffinizationHeat-Induced Epitope Retrieval
Dako Autostainer Microscopic Visualization
Loading samples into microtiter
plate
Printing on the arrayer
Freshly-printed slide Dako
Auto- stainer
Scanned slide after staining
Data
Analysis
Graphs of protein
concentrations
•HIF-1 ɑ staining via immunohistochemistry showed high levels of HIF-1, but RPPA analysis showed no difference in concentration between treated and control groups.
• Cu/Zn SOD concentrations quantified by RPPA lead us to think that there may be a relationship between drug regimen and oxidative stress.
•Sox2 concentrations from the RPPA indicate that neurogenesis and proliferation are also areas of interest in the study of chemobrain.
•The concentrations of the inflammatory response protein Stat1 Y701 did not show clear effects from our treatment as strongly as other proteins. We will investigate further the role of Stat1 and the inflammatory response in the chemobrain mechanism.
Acknowledgements: We would like to thank Dr. Ted Dumas and Dr. Lance Liotta for their contributions to this projectReferences:
•Kaiser, Jochen, Bledowski, Christof, Dietrich, Jorg. “Neural correlates of chemotherapy-related cognitive impairment.” Cortex. 54 (2014): 33-50. Web 7/6/2014
•Wigmore, Peter. “The Effect of Systemic Chemotherapy on Neurogenesis, Plasticity, and Memory.” Current Topics in Behavioral Neuroscience. 15 (2013): 211-240. Web. 7/19/2014.
Average Relative Sox2 Concentration in Different Rat Brain Regions
0
0.2
0.4
0.6
0.8
1
Olfactory Bulb (n=6) Hippocampus (n=6) Cerebellum (Control; n=4)
Rat Brain Region
Avera
ge R
elativ
e Co
ncen
tratio
n
SEM = +/- 0.135
SEM = +/- 0.071
SEM = +/- 0.250
Average Relative Sox2 Concentration by Treatment
0
0.2
0.4
0.6
0.8
1
Saline (Control) Cyclophosphamide Only Docetaxel Only Combination
Treament
Avera
ge Re
lative
Co
ncen
tratio
n SEM = +/- 0.256
SEM = +/- 0.140
SEM = +/- 0.506
SEM = +/- 0.272
Average Relative Stat1 Y701 Concentration in Different Rat Brain Regions
00.10.20.30.40.50.60.70.80.9
Olfactory Bulb (n=6) Hippocampus (n=6) Cerebellum (Control; n=4)
Rat Brain Region
Avera
ge R
ela
tive
Co
ncen
trati
on
SEM = +/- 0.188SEM = +/- 0.108
SEM = +/- 0.289
Average Relative Stat1 Y701 Concentration by Treatment
00.10.20.30.40.50.60.70.80.9
Saline (Control) Cyclophosphamide Only Docetaxel Only Combination
Treatment
Avera
ge R
ela
tive
Co
ncen
trati
on
SEM = +/- 0.329
SEM = +/- 0.320 SEM = +/- 0.511
SEM = +/- 0.115
Average Relative Concentration of Cu/ZN SOD in Rat Brain Regions
-0.1
0.1
0.3
0.5
0.7
0.9
Olfactory Bulb (n=6) Hippocampus (n=6) Cerebellum (Control; n=4)
Rat Brain Region
Avera
ge R
ela
tive
Co
ncen
trati
on
SEM = +/- 0.130
SEM = +/- 0.096 SEM = +/- 0.221
Average Relative Concentration of Cu/Zn SOD by Treatment
00.10.20.30.40.50.60.70.80.9
Saline (Control) Cyclophosphamide Only Docetaxel Only Combination
Treatment
Avera
ge R
ela
tive
Co
ncen
trati
on
SEM = +/- 0.159
SEM = +/- 0.248
SEM = +/- 0.302
SEM = +/- 0.225
