Transcript n - York College of Pennsylvania

n-Hexane
n-Hexane Increases Blood Vessel Formation in a Human U87 Glioblastoma
In Vivo Chicken Chorioallantoic Membrane (CAM) Culture System
Ryan J. Carter and Ronald Kaltreider, Ph. D.
Department of Biological Sciences, York College of Pennsylvania
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Angiogenesis and tumorshttp://cisncancer.org/research/images/angiogenesisrew.jpg
Materials & Methods
Introduction
• n-Hexane is commonly found in commercial cleaning
agents in the printing, textile, furniture, and shoemaking
industry as well as in construction, manufacturing plants,
and the car manufacturing and mechanic industry
• Exposure is due to being employed in occupations where it
is used, living near hazardous waste sites, or its
manufacturing, processing, or storage facilities where you
could breathe in n-Hexane vapors
• It is also found in gasoline, meaning nearly everyone is
exposed to it over their lifetimes
• Research has indicated that n-Hexane exposure is directly
linked to nerve disorders (Agency for Toxic Substances and
Disease Registry 1999)
• No data has indicated a relationship between n-Hexane
and cancer, however, a study by Cheng et al. (2014)
suggested that n-Hexane exposure was uncovered to be
associated with altered expression of VEGF as well as
increase in vasculature, in other words an increase in
angiogenesis, using gastrulating chicken embryos as a
model
• Angiogenesis, which is the formation of new blood vessels
derived from preexisting ones, plays a major role in many
physiological processes but also is vital for the tumor cell
proliferation process
• In order for the proliferation, as well as metastatic spread
of cancer cells, there must be an adequate supply of oxygen
and nutrients as well as the removal of waste products,
which is provided by the induction of new growth in the
vascular network formed through angiogenesis (Kamura et
al. 2006)
• Research done by Cheng et al. (2014) led us to question if
n-Hexane could increase angiogenesis in human U87
glioblastoma tumors using a CAM assay, where chicken
embryos are used as a living model system, providing the
first evidence of n-Hexane potentially enhancing the tumor
cell proliferation process by enhancing angiogenesis
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Maintained U87 MG (ATCC HTB-14) glioblastoma cancer cell lines in
extracellular matrix culture medium (DMEM) and split at 60-80% confluency
Purchased 95% n-Hexane from Sigma-Aldrich, stored at room temperature,
and diluted with PBS using 1:10 serial dilutions to get treatment
concentrations
White horn chicken eggs purchased from Moyer’s Chicks and upon arrival
they were incubated at 98 F for the entire study
On day 10, the CAMS were candled to establish viability and location of the
air sac
CAMs were put intro treatment groups where each had 2 CAMs (n=2) and
were as follows: control receiving a carrier agent (DMSO), a low dose,
intermediate dose, and high dose (0.001 M, 0.01 M, 0.1 M) of n-Hexane
On day 11 shell surrounding the air sac was removed and we added 50,000
cells/ml (50 ul, 25% cells, 25 % extracellular matrix) U87 glioblastoma cancer
cells to viable CAMs using a syringe for injection
On day 13, n-Hexane (50 ul) was added in the desired concentrations to the
appropriate CAMs at the tumor site using a micropipette
On day 17, tissue (4x4 cm) was excised from each tumor, fixed in 4%
paraformaldehyde, and blood vessel formation and density were visually
determined in CAMs using a micro dissection-imaging microscope and the
NIKON Digital DS-03 camera software.
Results (cont’d.)
• The control showed no blood vessels in the tissue excised
that were visually apparent (Figure 1, A)
• All doses of n-Hexane dramatically increased blood vessel
formation and density
• Small, but very dense vasculature was present throughout the
entirety of the tissue in the low dosage treatment in both
CAMs (Figure 1, B). More apparent, large vessels and
arteries were seen in one of the CAMs of the intermediate
dosage treatment while the other showed little to none
vasculature (Figure 1, C)
• Blood vessel formation was both very large and dense
throughout the tissue of the high dosage treatment and in
addition to the main arteries and vessels, the branched
networks of vessels were also large in size and density
(Figure 1, D)
Conclusions
Results
• The goal of this study was to determine the effects of nHexane on angiogenesis in U87 glioblastoma tumors
• Our results strongly supported our hypothesis in that
vasculature would be increased by n-Hexane exposure
• Lack of vasculature in one of the CAMs in our intermediate
dose, could be due to some type of experimental error
• Currently, by OSHA standard, permissible n-Hexane
exposure in the work place is 500 parts per million parts of
air (ppm) (Center for Disease Control 1978)
• Our low dose of 0.001 M, being environmentally relevant, is
equal to 114.12 ppm,well under the permissible levels of nHexane by OSHA standard of 500 ppm
• Our study is the first to suggest n-Hexane could enhance the
tumor cell proliferation process, and suggest that
environmentally relevant doses of n-Hexane could pose a
threat to individuals in that although it may not cause cancer,
it could possibly play a role in the progression of cancer
A
B
C
Future Studies
Objectives & Hypothesis
• Determine the effects of n-Hexane at environmentally relevant doses on
the vascular network in human U87 glioblastoma tumors
• We suspect to see an increase in the vascular network in CAMs where nHexane is applied in comparison to controls
• Replicate this research using various cancer types, to
determine if there is any specificity associated with n-Hexane
and cancer types
• Determine n-Hexane levels in various areas across the United
States, and then directly apply those concentrations to
tumors in effort to see how angiogenesis is affected under
those conditions.
D
References
http://cellculture.meduniwien.ac.at/uploads/RTEmagicC_HET-CAMchicken-egg.jpg.jpg
Figure 1. Micro dissection imaging of tumor tissue excised from each
CAM taken at 1X. The top two images are the carrier agent control (A),
the second two are the low dosage (0.001 M) (B), the third two are the
intermediate dosage (0.01M) (C), and the bottom two are the high
dosage (0.1M) (D).
• Cheng, Xin, Rong Luo, Guang Wang, Chang-jun Xu, Xin Feng, Ren-hao Yang, E. Ding, Yan-qing He, Manli Chuai,
Kenneth Ka Ho Lee, and Xuesong Yang. "Effects of 2,5- Hexanedione on Angiogenesis and Vasculogenesis in Chicken
Embryos." Reproductive Toxicology. Elsevier, 27 Dec. 2014. Web. 2015 Fall.
<http://www.ncbi.nlm.nih.gov/pubmed/25549948>.
• Kamura, Takashi, Hirohisa Yano, Naoyo Nishida, Toshiharu Kamura, and Masamichi Kojiro. "Angiogenesis in Cancer."
Vascular Health and Risk Management. Dove Medical Press, Sept. 2006. Web. 06 Feb. 2016.
<http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1993983/>.
• "Public Health Statement for N-Hexane." Agency for Toxic Substances and Disease Registry. N.p., July 1999. Web. 06 Feb. 2016.
<http://www.atsdr.cdc.gov/phs/phs.asp?id=391&tid=68>.
Acknowledgements
I would like to thank Dr. Ronald Kaltreider for all of his help in designing this study, maintaining cell lines, and
completing this study. I would also like to thank Dr. Jeffrey Thompson for generously supplying us with the U87
cancer cells we used in this study.