Download IL-8 and Gro-α factors have the ability to shift dormant MOLT

IL-8 and Gro-α factors have the ability to shift dormant MOLT-3 cells into an aggressive or active state
Amanda Zych
Introduction
• A tumor cell is thought to stay in a state of dormancy
because the tumor cell cannot perform angiogenesis:
the formation of blood vessels from preexisting ones
• A cell’s ability to leave a state of dormancy is
thought to happen because of factors or inducers that
play an active role in causing a cell to perform
angiogenesis
• An inducer such as Vascular Endothelial Growth
Factor, or VEGF is known to cause this switch in
angiogenesis
• Short-term angiogenesis by an inducer is a reason
there is an escape from tumor dormancy
• VEGF factor is similar to KS-IMM cells by causing
cells to become active and aggressive
• KS or Kaposi sarcoma is a tumor that can overcome
the inhibition to different cells it comes into contact
with
• It activates other cells by inducing angiogenesis and
therefore become non-dormant
• VEGF is found in KS-IMM and MOLT-3 cells, however,
it is weakly present in MOLT-3 cells so the cells remain
dormant.
• It is uncertain whether the VEGF factor is the only
inducer or if there are others, with which many papers
have suggested, such as Interleukin-8 (IL-8) and (Groα).
Flowchart
Grow MOLT-3 and
KS-IMM cells
Treatment
with different
gene factors
After 6 days isolate
100ng/mL RNA from
treatments: IL-8, Gro-α, IL8/Gro-α, IL-8/VEGF, Groα/VEGF, IL-8/Gro-α/VEGF,
control no treatment (-),
and control treatment VEGF
(+)
After 6 days (constant with
paper) isolate 100mg/mL
Protein from treatments: IL-8,
Gro-α, IL-8/Gro-α, IL-8/VEGF,
Gro-α/VEGF, IL-8/Gro-α/VEGF,
control no treatment (-), and
control treatment VEGF (+)
Run RT-PCR using mRNA from
treatments against the genes IL8, Gro-α, MMP-1, MMP-2, and
Ang-2
Figure 3.
his electrophoresis gel was taken from an RT-PCR (PNAS, Indraccolo, 2006)
• MOLT-3 cells were coinjected with irradiated KS-IMM cells.
• The MOLT-3 cells were shown to weakly express VEGF cells and were negative
for all other factors.
•However, when MOLT-3 and KS-IMM cells were coinjected together they
showed a presence of VEGF, bFGF, Gro-α, IL-8, GM-CSF, SCF, IL-15, and β-actin
(which is the control for both cells).
Hypothesis
IL-8 and Gro-α affect the ability to shift
dormant MOLT-3 cells into an
aggressive state.
Aims:
(1) Determine that gene expression of IL-8 and
Gro-α are associated with the angiogenic
process
(2) Prove that protein expression of IL-8 and
Gro-α are associated with the angiogenic
process
Predicted Results
Methods
Figure 1.
shows the results of the RT-PCR with the genes (on left side): IL-8, Gro-α, MMP-1, MMP-2, and
Ang-2 within the treatments (on top) of IL-8, Gro-α, IL-8/Gro-α, IL-8/VEGF, Gro-α/VEGF, IL-8/Groα/VEGF, control no treatment (-), and control treatment VEGF (+) in an electrophoresis gel with
1.5% agarose gel . All the treatments increase with the genes except for the negative control with
no treatment
Extract out proteins
from treatments using
Western Blot method
Review of Literature
• Without angiogenesis tumors do not grow (Masuya 2001)
• Vascular endothelial growth factor (VEGF) is known to induce angiogenesis
(Watson 2000)
• Cancer patients are known to have more VEGF within the white blood cells
and platelets (Toi 2001)
• VEGF is known to be associated with the following types of cancers:
melanoma, ovarian carcinoma, prostate carcinoma, and colon carcinoma
(Lurje 2008)
• VEGF is also known to cause cells to become non-dormant (Montaldo 2000)
• IL-8 and VEGF are factors that are known to assist each other
• Another factor, Gro-α is an analog of IL-8 and therefore shows some of the
same characteristics as IL-8 (Baggiolini 1994)
•The structure of Gro-α is similar to IL-8 (Geiser 1993)
• Kaposi sarcoma, or KS is a tumor that has the ability to overcome inhibition
to different cells they come into contact with (Montaldo 2000)
• KS-IMM cells, or ‘feeder’ cells are inducers of angiogenesis that allow cells to
become angiogenic and therefore non-dormant (Indraccolo 2006)
• KS-IMM has been found to produce IL-8 (Albini 1997)
• MOLT-3 cells, or T lymphoblastic leukemia are resistant to apoptosis
(Indraccolo 2006)
• When KS-IMM and MOLT-3 cells were added together angiogenesis increased
(Indraccolo 2006)
• The RT-PCR analysis of the coinjection of MOLT-3 and KS-IMM showed that
IL-8 and Gro-α were positive for KS-IMM but not MOLT-3 (Indraccolo 2006)
• The coinjection of the two types of cells were needed; without KS-IMM, the
MOLT-3 cells remained dormant (Indraccolo 2006)
Conclusions
• IL-8 and Gro-α factors should be shown to change the dormancy of tumor cells.
• These factors would therefore be angiogenic nature
• They would show an increase expression of genes and protein levels.
• As a results of their increase expression, Il-8 and Gro-α activate MOLT-3 cells into an
active, aggressive state
• With these cells in left in their dormant state by these factors then the relapse of
cancer would not occur
Future Aim/Treatment:
• A future study would be to take this a step further by using an antibody to keep the
factors from turning on and aiding the IL-8 and Gro-α
• Treat an animal with the IL-8 antibody, IL-33 to detect Il-8 and test if it is possible to
stop or inactivate IL-8
• Do the same for Gro-α only use antibody Alpha-4
• The animals would then be dissected to see if the tumor cells remained inactive
when IL-8 and Gro-α were inactive
Literature Cited
Analyze bands and compare
results to + (VEGF treatment)
and – control (no treatment)
IL-8 and Gro-α were analyzed by gene expression through the
following genes: MMP-1, MMP-2, Ang-2, IL-8, and Gro-α using the
following treatments: IL-8, Gro-α, IL-8/Gro-α, IL-8/VEGF, Gro-α/VEGF,
IL-8/Gro-α/VEGF, control no treatment (-), and control treatment
VEGF (+). Then they were analyzed by protein expression using the
same genes and treatments with the addition of the antibody IL-33
for IL-8 and the antibody alpha-4 for Gro-α
Figure 2. shows the results of the Western Blot with the genes (on left side): IL-8, Gro-α, MMP1, MMP-2, and Ang-2 within the treatments (on top) of IL-8, Gro-α, IL-8/Gro-α, IL-8/VEGF, Groα/VEGF, IL-8/Gro-α/VEGF, control no treatment (-), and control treatment VEGF (+)
Albini, Adriana, Paglieri, Isabella, Orengo, Giorgia, Carlone, Sebastiano, Aluigi, Maria Grazia, DeMarchi,
Roberto, Matteucci, Cristian, Mantovani, Alberto, Carozzi, Franca, Donini, Silivia, and Benelli, Roberto. 1997 May 11.
The β-core fragment of human chorionic gonodotrophin inhibits growth of Kaposi's sarcoma-derived cells and a new
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Geiser, Thomas, Dewald, Beatrice, Ehrengruber, Markus U., Clark-Lewis, Ian, and Baggiolini, Marco. 1993. The Interleukin8-related Chemotactic Cytokines Gro-α, Gro-β, and Gro-γ Activate Human Neutrophil and Basophil Leukocytes. The
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Acknowledgements
I would like to thank Dr. Kaltreider for all his help in making this proposal the best that it can be.