Induction of Apoptosis on TRAMP-C1 Mouse Prostate Cancer Cell Line - PowerPoint PPT Presentation

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Induction of Apoptosis on TRAMP-C1 Mouse Prostate Cancer Cell Line

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Induction of Apoptosis on TRAMP-C1 Mouse Prostate Cancer Cell Line & LNCaP Human Prostate Cancer Cell Line by Chinese Medicinal Herb, Scutellaria barbata – PowerPoint PPT presentation

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Title: Induction of Apoptosis on TRAMP-C1 Mouse Prostate Cancer Cell Line


1
Induction of Apoptosis on TRAMP-C1 Mouse Prostate
Cancer Cell Line LNCaP Human Prostate Cancer
Cell Line by Chinese Medicinal Herb, Scutellaria
barbataHannah H.L. Wong, P. McHenry, Department
of Biology, Union College, Lincoln, NE 68506 N.
Greenberg, Department of Cell Biology, Baylor
College of Medicine, Houston, Tx 77030 and
B.Y.Y. Wong, Department of Biology, Union
College, Lincoln, NE 68506
Scutellaria barbata
Materials and Methods
Abstract
Preparation of herbal extract Crude SB was
purchased from Nuherbs Co.,Oakland, CA. It was
grounded into fine powder. 10g of powder was
suspended into 100ml of distilled water and
heated for 1hr at 56C. The suspension was then
centrifuged at 2000rpm for 10min, filtered and
supernatant centrifuged again at 3000rpm for
10min. The supernatant was then
filter-sterilized. The extract was lyopholized
and dry weight determined. All concentrations
used in experiment were based on dry weight of
extract. Cell Culture Preparation TRAMP-C1 cells
and LNCaP cells were obtained from Baylor College
of Medicine (from Dr.Greenbergs laboratory).
Cells were received frozen in cryo-tubes and
thawed by placing in a 37C water bath till
thawed. Cells were triturated with a 3ml, 18 gage
needle and placed in a 60mm cell culture dish
with 5ml warm media. Cells were then incubated at
37C in 5 CO2 incubator for 24 hrs. After
24hrs, cells were split by addition of 3ml of
trypsin. Cells were washed with 6ml Hanks buffer
and centrifuged at 2500 rpm for 5 minutes.
Supernatant was discarded, and 6ml of Hanks
buffer added to pellet and centrifuged for
another 5min at 2500rpm. Concentration of cells
was then found by staining with trypan blue and
hemocytometer chamber. Cells were then
re-suspended into 5ml media. Cytotoxicity and
LD50 A known concentration of cells were plated
and treated with varying concentrations of SB for
24hours. Cells were then washed with Hanks buffer
allowed to grow for 5 days. After 5 days cells
were washed with Hanks buffer and fixed with 5ml
of methanol of 3 minutes, then stained with
crystal violet. Cell colonies were then counted.
Results
Scutellaria barbata (SB) has been used in
traditional Chinese medicine for treating liver,
lung and rectal tumor. Prostate cancer is the
most common form of cancer among American men.
TRAMP C-1 is a cell line derived from transgenic
adenocarcinoma of the mouse prostate (TRAMP), an
useful model for identifying important pathways
in tumorigenesis, androgen independence, and
metastasis of prostate cancer. LNCaP is an
androgen-dependent human prostate cancer cell
line. Endonucleolysis is considered as the key
biochemical event in apoptosis that leads to the
cleavage of nuclear DNA into double-stranded, low
molecular weight DNA fragments (mono- and
oligonucleosomes) and single strand breaks
(nicks) in high molecular weight DNA. In this
study, we determined the extent of apoptosis and
necrosis by labeling the DNA strand breaks of
these two cancer cell lines on chamber slides
(after DNase I digestion of the non-treatment
control group and incubation with 1mg/ml of
aqueous extract of SB in the experimental groups
for 2 and 8 hours respectively) with Terminal
deoxynucleotidyl transferase (TdT), which
catalyzes polymerization of labeled nucleotides
to free 3'-OH DNA ends in a template-independent
manner (TUNEL assay). Incorporated fluorescein
was detected by anti-fuorescein antibody Fab
fragments from sheep, conjugated with
horse-radish peroxidase (POD). After substrate
reaction, stained cells were analyzed under light
microscope. This in situ TUNEL assay
preferentially labels apoptotic cells rather than
necrotic cells. Significant time-dependent
apoptotic and necrotic effects of SB on both
types of prostate cancer cells were observed. At
2-hour incubation, SB shown a more significant
effect in the induction of apoptosis in TRAMP-C1
than in LNCaP. (90 gt78, plt0.05) while with
8-hour incubation, induction of apoptosis by SB
was more significant in LNCaP than in TRAMP-C1
(84 gt 71.8, plt0.05). In TRAMP-C1, the amount
of necrotic cells observed increase significantly
between 2 and 8-hour of incubation time (4.8 to
23.8, (plt0.05) while difference in necrotic
cells in LNCaP between 2 and 8-hour was not
significant (6.5 to 4.5, p gt0.05). These
differences reveals that the optimum incubation
time of SB for best induction of apoptosis in
TRAMP-C1 and LNCaP is 2 hours and 8 hours
respectively. These data indicate that SB
contains phytochemical that modulates apoptosis
of mouse and human prostate cancers in vitro and
suggests a potential mechanism for the efficacy
of this extract in vivo.
TRAMP-C1 cells (250x)
TRAMP-C1 cells (250x)
LNCaP cells (250x)
Cells stained with crystal violet after the
cytotoxicity assay
Cells couterstained with DAB substrate after the
TUNEL Assay
Cells counterstained with DAB substrate after the
TUNEL Assay
TUNEL Assay Protocol for the TUNEL Assay was
based on the In Situ cell Death Detection Kit,
POD 2001 Manual from Roche Applied Science.
Cells were cultured in slides and treated with
SB. Two experimental groups (2hr and 8hr) and a
positive control were used were used. After
treatment, samples were dried and fixed with
Fixation solution for 1hr at 15-25C. Cells were
then incubated at 15-25C for 10min with Blocking
Solution. After blocking solution was removed,
Permeabilization solution added. Positive
Control was incubated with DNase, Grade I, while
50µl of TUNEL reaction mixture was added to each
sample and incubated in a humidified, dark
chamber. Signal Conversion was then conducted by
addition of 50µl Converter-POD followed by DAB
Substrate.
Student T-test and Descriptive Analysis of
Results from TUNEL Assayon TRAMP-C1 cells
treated with SBTable 1
Student T-test and Descriptive Analysis of
Results from TUNEL Assayon LNCaP cells treated
with SBTable 2
Introduction
Positive control 2hr 8hr
Apoptotic Cells 88.5 3.5 90.3 2.9 71.7 3.6
Necrotic Cells 9.0 5.0 4.8 1.3 23.8 2.0
Normal Cells 2.5 1.5 5.0 1.8 4.5 0.5
Positive Control 2hr 8hr
Apoptotic Cells 95 5 78 7 84 3
Necrotic Cells 5 3 6.5 5.5 11 2
Normal Cells 0 0 15.5 12.5 4.5 1.5
Scutellaria barbata (SB), is an erect annual
herb, 12-35cm tall found along fields and ditches
in China. SB has a mildly bitter taste and is
commonly used in Chinese Medicine for the
treatment of a variety of ailments such as
appendicitis, hepatitis, snake bites, lung, liver
and rectal cancer. Current research has found
that SB may also possess anti-mutagenic and
chemopreventative properties. Prostate Cancer
is the most common cancer in American men. It
was predicted to have killed as many as 30,200
people by the end of 2002. The incidence of
prostate cancer has found to be affected by
race, geography, diet and genetics. There is yet
no known cure for prostate cancer. Transgenic
Adenocarcinoma Mouse Prostate Model (TRAMP) is a
spontaneous Autochthonous transgenic mouse model.
It was created by incorporation of the PB SV40T
Antigen Transgene into the syngeneic C57B1/6
host. This antigen transgene Contains the rat
probasin (PB) gene and the SV40 T antigen (Tag).
SV40 is a papovavirus which produces tumors in
animals. Animals with these gene develop severe
hyperplasia and cribriform structures by 12 weeks
and 100 display tumors by 24-30 weeks. TRAMP- C1
line was established from the prostatic
adenocarcinoma of a32 week old C57B1/6 male. Two
other lines C2, -C3 were established and differ
in their growth rates and morphology. LNCaP
clone FGC was established from the cells of a
needle aspiration biopsy of the left
supraclavicularlymph node of a 50 yr old
Caucasian male with confirmed metastatic prostate
carcinoma. It was first isolated in 1977 by J.S.
Horoszewicz.
Fixed cells with Fluorescein-labeled DNA strand
breaks
Anti-fluorescein Antibody conjugated With POD
DAB Substrate for POD
(Roche Applied Science 2001)
Table 1 above shows the percentage of each type
cell present in each experimental group, compared
to the positive control where the DNA was
digested by DNAse. These values represent the
average of cell populations from two separate
experiments. The symbol suggests that the
percentage of cells of that population had a
plt0.05 when compared with the positive control.
Table 2 shows the percentage of cells in each
group, after LN CaP cells were treated with SB.
Positive control was digested by DNase, and
compared with the 2hr and 8hr groups which were
treated with SB. -indicates that the percentage
of cells in a particular population has plt0.05
compared to the positive control.
Conclusion
Apoptosis (Programmed Cell Death) and Necrosis
Involving Mitochondria Figure 1
Comparison of Results of TUNEL Assay on TRAMP-C1
and LNCaP Figure 2
The results of this experiment suggest that the
effect of SB on TRAMP-C1 and LNCaP vary. SB was
found to be most effective at inducing apoptosis
in TRAMP-C1 at 2 hours, and in LNCaP at 8hrs. The
data also suggests that SB contains
phytochemicals that modulates apoptosis of mouse
and human prostate cancers in vitro, suggesting a
potential mechanism for the efficacy of this
extract in vivo.
  • Figure 2 compares the results of TUNEL Assay on
    TRAMP-C1 and LNCaP by comparing the percentage of
    each type of cell during each hour.
  • Results suggest that
  • At 2hr the percentage of apoptotic cells in
    TRAMP-C1 is significantly greater than in LNCaP
    cells.
  • 2. At 8hr the percentage of apoptotic cells in
    LNCaP is significantly greater than TRAMP-C1.
  • 3. At 8hr the percentage of necrotic cells in
    TRAMP-C1 was also significantly greater than
    LNCaP. (p lt 0.05).


References
1. J.R. Gingrich and N.M. Greenberg, Toxicologic
Pathology 24502-504 (1996). 2. S.C. Chong L.H.
Lee, Chinese Med. Herbs of H.K., vol. 2
(1988). 3. Oncogene Research Products. The Active
Site (2002). 4. Roche Applied Science. In situ
Cell Death Detection Kit, (POD) (2002).

Acknowledgments
1. The Nebraska Academy of Sciences, Inc. for
student research award. 2. Union College, Union
Scholars Program for poster printing costs. 3.
Stephen Nazario for technical assistance with
Cell Line pictures.
Indicates the where results of TRAMP-C1 cells is
significantly different from that of LNCaP cells,
where plt0.05.
(Oncogene
Research Products 2002)
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