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Cam Ha's Presentation MS2 10-03-12
1. Nanosuspension-formulation Genistein
Inhibits Radiation-Induced Inflammation and
Protects Mouse Hematopoietic Tissue
Cam Ha, MD, Ph.D.
Senior Research Associate
National Research Council
Armed Forces Radiobiology Research Institute (AFRRI)
RRS, Puerto Rico, October 03, 2012
2. 2
Introduction
• Nanotechnology investment from NIH: $200 M each year
• A great benefit of this technology: Novel materials can be
designed to enhance medicine delivery; used as a diagnostic
tool; as biomarker detectors…
• Our study, the 1st
ever attempt to evaluate nanosuspension-
formulation Genistein (Nano Genistein, for short):
Determine the radiation protective effects of genistein in
hematopoiesis and in the regulation of inflammatory
factors (IL-1β, IL-6, COX-2) following exposure to gamma-
radiation
3. Plant polyphenols Flavonoid Isoflavones Genistein
3
• Non-toxic, natural occurring isoflavone
• Major source: Soybeans, green beans, black beans, chickpeas …
• Natural form: insoluble in water, low bioavailability, high absorption
• Oral and parenteral administration
• Bioavailability enhancement for clinical application: Saline-based nano genistein
(Humanetics Pharmaceuticals)
4. Nano Genistein
Injectable Suspension and an Improved Bioavailability
4
• High degree of chemical stability; physically stable at 6 months;
radiation sterilization without deterioration, particle size < 1
micron (mean = 0.2 microns)
• Enhancement of BA:
- high dissolution rates
- supersaturation
- minimal recrystallization
• Saline based vehicle, pH 7.0 - 7.4,
5. Experimental Design
5
Experimental Implementation:
•Gamma radiation-induced cell death and hematopoietic recovery:
- Cell viability by trypan blue staining
- Clonogenic assay (CFU)
- Flow analysis of apoptosis
- HE pathological examination of sternal BM
•Gamma radiation-induced cytokine production:
- Cytokine array
- ELISA
•Gamma radiation-induced protein expression:
- Immunoblotting
- Immunohistochemistry
9.25 Gy: 30-day survival
Genistein IM
150 mg/kg
Vehicle
IM
Control
• Serum
• BM
• Sternum
• Spleen
7 Gy Day 1
7 Gy Day 7
7 GY Day 14 24 h
60
Co Total Body
Irradiation(TBI),
0.6 Gy/min
CD2F1 Male, 12-14 week (N=8-18)
in vivo studies of hematopoietic tissue
6. 6
Results
Nano Genistein
50, 100, 150 mg/kg, IM, 9.25 Gy TBI, LD 70/30
Mouse survival study of saline-based nano genistein
Nano genistein protected mice from a lethal dose of TBI
Optimized dose of genistein (regular form, s.c.) for animal protection was 200 mg/kg
7. 7
Viability Assessment of Mouse BM by Trypan Blue Staining
* p<0.05
** p<0.01
Enhanced viable BM cells from mice treated by genistein were observed at 1 day, 7
days, and 14 days after radiation
BM cells were collected from femurs and humuri of individual mice, N=6 mice/group
8. Genistein Promotes Mouse BM Hematopoietic Recovery After 7 Gy 60
Co
Sham Untreated, 7Gy Vehicle, 7 Gy Genistein, 7 Gy
7 days
14 days
* p<0.05
** p<0.01
Genistein treatment significantly improved BM cellularity starting at day 7 postirradiation
8
50 µM, x 400HE staining of sternal BM, N=6 mice/group
9. Flow Analysis of Genistein’s Effect on Cell Viability of Mouse BM at 7 Gy
Sham 7 Gy, 1 day after 60
Co TBI 7 Gy, 7 days after 60
CoTBI
7AAD positive 7AAD positive 7AAD positive
o BM cells were collected from femurs and humuri of individual mice, N=6 mice/group
o A combination of staining with: 7AAD (positive: cell death), Lineage APC, cKit FITC
o Population of Lineage-
cKit+
: Hematopoietic stem and progenitor cells in mouse BM
9
**
**
* p<0.05
** p<0.01
10. Genistein Accelerated Mouse Hematopoietic Recovery by Day 7 Postirradiation
10day 7 postirradiation
**
p<0.01
**
**
Genistein promotes the recovery
of hematopoietic progenitors
Genistein accelerates the recovery
of hematopoietic precursors
11. **
*
**
*
*
11
* p<0.05
** p<0.01
Colony forming assay using BM cells collected from femurs and humuri
of individual mice, N=6 mice/group
Genistein Enhances Survival of HSPCs in 60
Co mouse BM
12. 12
Genistein Inhibits 7 Gy Radiation-Induced Synthesis of IL-1β
and IL-6 after 24 hrs in Bone Marrow and Mouse Spleen
* p<0.05
**
p<0.01
murine IL-1β production in BM homogenate
murine IL-1β production in spleen homogenatemurine IL-6 production in spleen homogenate
• “Inflammation recurs in cycle for months after radiation”
Prof. W. McBride, Inflammation and Immunity in Radiation, RRS Puerto Rico
• Radiation-induced cytokine storm can impair hematopoietic recovery
and advance pathological immune responses
13. β Actin
COX-2
COX-2
β Actin
BM
Spleen
1 2 3 1 2 3 1 2 31 2 3
Sham
Untreated
7 Gy
Vehicle
7 Gy
Genistein
7 Gy
13
Fold induction of COX 2 over sham at day 1
Untreated, 7 Gy Vehicle, 7 Gy Genistein, 7 Gy
BM 2.2 ± 0.3 2.5± 0.6 1.2 ± 0.5 *
Spleen 1.8 ± 0.4 1.7 ± 0.3 1.2 ± 0.1 *
*p <0.05 compared to irradiated control and vehicle, Mean ± SEM
7 Gy,
Genistein
7 Gy,
Vehicle
sham
COX2DAPI D+C
Sectioned mouse sternae were stained
with fluorescent anti COX-2 and DAPI
Genistein Reduces COX-2 Expression in BM and Spleen after 7Gy
COX 2
COX 2
β Actin
β Actin
1 2 3 1 2 3
Sham 7 Gy
Lineage
-
BM
Lineage+
BM
14. Summary
14
• Nanosuspension-formulation genistein enhances the
survival of mice from TBI and protects HSPCs from
radiation exposure.
• Genistein suppresses radiation-induced pro-inflammatory
factor release, which may protect hematopoietic cells from
bone marrow failure.
15. Acknowledgments
Radiation Countermeasures Program
Mang Xiao, MD PI
Michael Landauer, Ph.D. Co-PI
Xiang Hong Li, MS
Dadin Fu, Ph.D.
Program Advisor
Mark H. Whitnall, Ph.D.
AFRRI Cobalt Facility Staff
Vitaly Nagy, Ph.D.
William A. Melendez-Cruz
Veterinary Sciences Department
Maj. Eric D. Lombardini, VMD
LTC Steven Tobias, DVM
VSD staff
15
AFRRI Scientific Directorate
Christopher R. Lissner, Ph.D.
Capt. John R. Gilstad, MD
Capt. David R. Lesser, Ph.D
Lt. Col. Oswald L. Johnson, Ph.D
OPEN LAB and outside OPEN LAB
Thank you for coming to my presentation.
Today, I am going to present the findings from our study that focuses on Genistein, a promising radiation counter-measure candidate.
Currently the INH invests about $200 M each year for biomedical nanotechnology research.
Novel materials developed by nanotechnology can be designed to deliver medicines directly to diseased or damaged tissues or can be used as diagnostic tools
Our study is the first attempt ever made to evaluate the radiation protective effect of the new nanosuspension formulation of genistein. For short, we will call it nano Genistein
In particular, we wanted to determine the radiation protective effects of genistein in hematopoiesis and in the regulation of inflammatory factors (IL-1, IL-6, COX-2) following exposure to gamma-radiation.
Some of you may wonder, What is genistein?”.
Allow me to give you a short background on the nanogenistein
Genistein is a non toxic natural occurring isoflavone, which is mainly found in soybeans, and other plant products such as green beans, chickpeas …
If derived from soybean, the natural form of genistein is insoluble in water, and has low bioavailability.
To tackle this issue, the nanosuspension form of genistein has been generated successfully.
We were very excited to be able to use one version of the new form, which was kindly provided by the company Humanetics Pharmaceuticals.
This new formulation of genistein, indeed, has an Improved Bioavailability, which includes:
High degree of chemical stability and a very particle size.
Nano genistein also has a higher dissolution rate, a super-saturation and minimal recrystalization
Importantly, with the saline based vehicle, genistein can be injected intramuscularly.
Other major properties of genistein will be discussed later.
This schematic describes how we carried out our project.
CD2F1, 12 to 14 weeks old, in a sample size of 8 to 12 mice, were either injected with genistein or vehicle. The untreated group served as control.
Twenty four hours later, half of these mice were subjected to Cobalt 60 total body radiation.
The mice that received the lethal dose, 9.25 were being kept for 30-day survival study.
The rest of mice, which received a sub-lethal dose of 7 Gy were sacrificed at day 1, day 17 and day 14 post-radiation. The collected Serum, BM, Sternum and Spleen were used to study the radiation protective effect of genistein in hematopoietic system.
(Click)
The assays that we used here will be discussed in more detail in the result section.
First of all, the survival data was very encouraging. They show that, in a dose dependent manner, nano genistein protected mice from a lethal dose of radiation.
It also confirms the benefit of the new form: Now we can reduce the dose of genistein from 200 to 150 mg/kg while still being able to maintain the similar protective effect observed from the previous study.
The question that our group wanted to tackle next was: Can genistein protect the hematopoietic system from radiation exposure. And How?
In order to answer that, we isolated BM cells from femurs and humiri and assessed the viability of those cells by trypan blue staining.
In this graph, Genistein treated group is seen in green, vehicle in red, and sham control in blue.
We observed a massive cell death caused by radiation a day after radiation, the death became extremely severe at day 7, following by a partial restoration at day 14.
We also found that, during the course of the experiment, (Click) treatment with genistein significantly enhanced the viability of BM.
These are the images of histological staining, which show us the destruction of BM after radiation.
Consistent with the previous finding, treatment with genistein dramatically improved the cellularity of the BM as early as day 7, following by a near normal level at day14.
The results were summarized as seen in this graph.
With these findings supporting the role of genistein in protecting BM, we decided to look for the evidence that shows genistein effect in a sub-population: stem and progenitor cells.
For this purpose, we applied Flow cytometry. We used 7AAD as cell death marker, antibodies against Lineage, and cKit as the markers for HSPCs. Genistein will be in Red.
(Click)
We found, genistein treatment reduced the death of total BM cells by radiation by a half 7 days after radiation seen here in this graph.
(next slide)
Now I would like to draw your attention to the following: Looking at the data more closely, we detected a significant recovery of a sub-population which is Lineage dim, as seen here, only in genistein treated group.
We believe that this lineage dim population, which was almost wiped out 1 day after radiation, represents the hematopoietic precursors and early committed functional cells.
Finally, by gating on viable population of Lin- cKit+, we were able to determine that genistein treatment promoted the restoration of HPPC at day 7, as seen in this.
Genistein promotes hematopoiesis by protecting HSPCs, as you can see here, with a significant increase of viable Lin negative and ckit positive cells.
We confirmed the result by conducting colony forming assay using BM cells.
The left panel displayed the scores of CFU from three sham groups which showed no significant difference.
In radiated group, however, we found that, the numbers of colonies scored from genistein treated group were always significantly higher than the ones from control and vehicle groups.
Yesterday, Prof. McBride said: “Inflammation recurs in cycle for months after radiation”. To me , This is so TRUE.
Particularly, we believe the pro-inflammatory cytokine storm induced by radiation can impair hematopoietic recovery and advance pathological immune responses, leading to BM failure.
Notably, IL-1 b and Il-6 play an important role in this cytokine storm.
We decided to examine whether genistein has any effect in modulating cytokine induction by radiation.
By using cytokine array and confirming with ELISA, we have found that genistein inhibited the production of IL-1b and IL-6 in BM and spleens, which was observed at day 1.
Two noteworthy sets of findings that prompted us to study the effect of genistein in COX-2 expression:
First, COX2 inhibitor given before lethal radiation enhanced survival of mice.
Second, the ability of genistein to reduce COX2 level in cancers, causing growth repression.
What we have found was:
Radiation induced a robust production of COX-2 (as seen in the untreated and vehicle). Treatment with genistein inhibited that up-regulation (seen in green box) in a western blotting of BM and spleens. The statistical analysis are summarized in the table below.
Here are the sternal images from fluorescence staining with anti-COX and DAPI. The percentage of cells that was stained positive for COX2 was reduced remarkably in the genistein group (seen in the green box).
To summarize: what we have found are: (Read)
The take home message:
Nanosuspension-formulation genistein WORKS.
Genistein is a promising radioprotector.
I would like to express my gratitude to the PI and co-PI:
Dr. Xiao and Dr. Landauer, for their guidance.
I am grateful to the great support from my lab member, AFRRI Scientific directorate, program advisor, vet department.
And thank you for your attention.
