Note: Descriptions are shown in the official language in which they were submitted.
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TITLE: USE OF GANGLIOSIDE TO DECREASE PROPAGATION OF
MALIGNANT PROSTATE CELLS
INVENTORS: Michael Thomas Clandinin, John Miklavcic, and
Vera Mazurak
TECHNICAL FIELD:
[0001] This disclosure relates to inhibiting the propagation of prostate
cancer cells and more specifically, to the use of exogenous ganglioside
to inhibit the propagation of prostate cancer cells.
BACKGROUND:
[0002] Prostate cancer ("CaP") is the most prevalent cancer in North
American men. The diagnosis of CaP evokes patient anxiety and
subsequently, 90% of men elect for immediate treatment. About half of
treated cases elect for radical prostatectomy. This treatment requires a
patient to accept diminished quality of life thereafter and does not
guarantee freedom from recurrence.
[0003] Watchful waiting and active surveillance are CaP management
strategies that monitor tumor characteristics over time and prompt
intervention at signs of increased growth. This period of surveillance
provides opportunity for nutritional intervention. Due to the long latency
period of CaP, it would be beneficial to determine if bioactive
components in foods can reduce cancer cell growth. Many dietary
supplements analyzed to date (ex. lycopene, vitamin E) have not
scientifically demonstrated significant decreases in CaP cell growth.
[0004] Gangliosides are a class of glycosphingolipids present in
mammalian plasma membrane and are a major component of lipid
rafts. Synthesis of ganglioside GD3 is catalyzed by the addition of sialic
acid to a-Neu5Ac-(2-3)-(3-Gal-(1-4)-[3-Glc-(1-1)-Cer ("GM3"), which is
synthesized from a lactosylceramide precursor. Ganglioside is also
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available in small quantities from a limited number of exogenous
dietary sources including whole milk and colostrum.
[0005] It is known that treatment with GD3 stimulates cell death via
apoptosis. However, the potential of GD3's stimulation of apoptosis
has not been explored as an anti-cancer effect in prostate
malignancies.
[0006] Accordingly, there is a need to assess the role of ganglioside on
prostate cancer cell propagation in CaP.
SUMMARY:
[0007] Uses of exogenous ganglioside to inhibit the propagation of
prostate cancer cells is provided. Gangliosides regulate many cellular
processes including cell death. The present disclosure assesses the
role of ganglioside in prostate cell growth. Malignant prostate ("PC-3")
and control ("RWPE-1") cells can be cultured with or without
ganglioside treatment. Cells can be assayed for differences in cell
growth. Supplementation with ganglioside ("GD3") can decrease
growth of PC-3 cells by 30% compared to controls (p<0.01).
Ganglioside can have therapeutic benefit in prostate cancer as
demonstrated by decreased growth of malignant PC-3 cells.
[0008] Incorporated by reference in its entirety into this application is a
paper written by the within inventors/applicants entitled, "Effects of
Ganglioside on Growth and Cell Surface Ganglioside Densities in
Prostate Cancer In Vitro", published as Chapter 3 of the thesis entitled
Ganglioside Increases Metastatic Potential and Susceptibility of
Prostate Cancer to Gene Therapy In Vitro by John Miklavcic, University
of Alberta, Department of Agricultural, Food and Nutritional Science;
Edmonton, Alberta, Canada, Fall 2009. A copy of this paper is
attached to this application as Appendix "A".
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[0009] Broadly stated, in some embodiments, a use of exogenous
ganglioside is provided to inhibit the propagation of prostate cancer
cells.
[0010] Broadly stated, in some embodiments, a dietary supplement is
provided comprising exogenous ganglioside wherein the dietary
supplement is used to inhibit the propagation of prostate cancer cells.
[0011] Broadly stated, in some embodiments, a composition is provided
comprising exogenous ganglioside wherein the composition is used to
inhibit the propagation of prostate cancer cells.
[0012] Broadly stated, in some embodiments, the use of exogenous
ganglioside is provided in the manufacture of a medicament to be used
to inhibit the propagation of prostate cancer cells.
[0013] Broadly stated, in some embodiments, a kit is provided to inhibit
the propagation of prostate cancer cells, the kit comprising a dietary
supplement comprising exogenous ganglioside and instructions for use
of the dietary supplement.
BRIEF DESCRIPTION OF THE DRAWINGS:
[0014] Figure 1 is a bar graph depicting the decrease of growth of PC-3
cells in vitro, as compared to control cell lines as a function of dosage
of a ganglioside treatment;
DETAILED DESCRIPTION:
[0015] Uses and compositions of exogenous ganglioside to inhibit the
propagation of prostate cancer cells are disclosed herein.
[0016] In some embodiments, exogenous ganglioside can be used to
inhibit the propagation of prostate cancer cells. In some embodiments,
exogenous ganglioside can be provided to an individual as a dietary
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supplement. The dietary supplement can be provided in any
appropriate form, mode, or method as would be understood by one
skilled in the art. In some embodiments, exogenous ganglioside can be
provided to an individual diagnosed with prostate cancer to control
tumor growth. In some embodiments, exogenous ganglioside can be
provided to an individual not diagnosed with prostate cancer to control
propagation of prostate cancer cells as a preventative measure.
[0017] In some embodiments, exogenous ganglioside can be provided
to an individual as a daily dietary supplement. In some embodiments,
exogenous ganglioside can be provided to an individual at a
concentration slightly higher than physiologically relevant. As one
skilled in the art would appreciate, the concentration of exogenous
ganglioside provided can be varied appropriately to inhibit the
propagation of prostate cancer cells.
[0018] In some embodiments, the exogenous ganglioside can be GD3.
In some embodiments, the exogenous ganglioside can be GD3-
enriched zeta dairy lipid powder. In some embodiments, the
exogenous ganglioside can be taken up by prostate cancer cells as
would be understood by those skilled in the art. In some embodiments,
the exogenous ganglioside can induce apoptosis as would be
understood by those skilled in the art. In some embodiments,
apoptosis can inhibit the propagation of prostate cancer cells. In some
embodiments, the inhibition of the propagation of prostate cancer cells
can prevent or reduce prostate tumor growth.
[0019] In some embodiments, the prostate cancer cells inhibited can be
bone metastasized cells. In some embodiments, the prostate cancer
cells inhibited can be PC-3 cells.
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[0020] In some embodiments, exogenous ganglioside can be used to
inhibit the propagation of prostate cancer cells while the propagation of
normal prostate cells is not inhibited.
[0021] In some embodiments, a dietary supplement for use to inhibit
the propagation of prostate cancer cells can comprise exogenous
ganglioside. In some embodiments, a composition comprising
exogenous ganglioside can be used for use to inhibit the propagation
of prostate cancer cells.
[0022] In one embodiment, the use of exogenous ganglioside to inhibit
the propagation of prostate cancer cells could be presented as a kit
comprising a dietary supplement and instructions for use of the dietary
supplement.
[0023] The following examples and figure are provided to aid the
understanding of the present disclosure, the true scope of which is set
forth in the claims. It is understood that modifications can be made in
the procedures set forth without departing from the spirit or scope of
the invention.
EXAMPLE 1 - Growth Conditions
[0024] RWPE-1 and PC-3 cell lines were obtained from the American
Type Culture Collection. Cell line characteristics are outlined in Table
1. Cultures were maintained in CostarTM 3516 six-well tissue culture
treated plates. RWPE-1 cells were cultured in Keratinocyte-SFM
containing L-glutamine, supplemented with bovine pituitary extract (193
pL/100 mL) and human recombinant epidermal growth factor (0.591
pL/100 mL). Medium was replaced two-three times/week and cells
were subcultivated (1:6) every seven days. PC-3 cells were cultured in
Ham's F-12 Nutrient Mixture containing L-glutamine and supplemented
with NaHCO3 (1.18 g/L). Medium was replaced three times/week and
cells were subcultivated (1:5) every 7 days. To passage cells,
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monolayers were rinsed once with phosphate-buffered saline (PBS)
before being dislodged by cell lifter into fresh medium. Cells were
grown in standard culture incubation conditions of 37 C and 5%
atmospheric CO2. All cell cultures were supplemented with 1% (v/v)
pooled human serum and 1% (v/v) antibiotic/antimycotic (penicillin,
streptomycin, amphotericin B).
TABLE 1
Cell lines employed in current study
Cell Line Type Androgen PSA Origin
Responsiveness Producing
RWPE-1 Normal Yes Yes Normal Human Prostate Cells
PC 3 Tumour No No Human Bone Metastasis
EXAMPLE 2 - Human Serum
[0025] Blood was drawn from six healthy males aged 18-35 having no
history of cancer, autoimmune, or other disease. Subjects using
steroidal medications, hormone therapies, or having had surgery within
3 months were excluded. Blood (100 mL) was drawn from the
subcubital vein and was spun (365 g for 30 min at 37 C) to obtain
serum fractions. Serum samples from all subjects were pooled,
aliquoted, and frozen. Serum was thawed immediately prior to use.
EXAMPLE 3 - Ganglioside Extraction
[0026] Ganglioside was extracted from GD3-enriched zeta dairy lipid
powder (Fonterra, Cambridge NZ) by modified Folch method (12, 13).
Powder (0.50 g) was added to 30 mL of chloroform/methanol (C/M; 2:1
v/v), vortexed (30 sec), and shaken (>2 hr). 0.025% (w/v)
CaCl2/double-distilled (dd) H2O was added to samples before inversing
several times. Samples were spun (1,000 rpm for 10 min at room
temperature) and the upper layer was withdrawn and filtered through a
Sep-Pak Classic C18 cartridge. Cartridges were rinsed with 10 mL
ddH2O before eluting ganglioside with 2 mL of methanol, followed by
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mL of C/M (2:1 v/v). C/M was removed under N2 gas before
ganglioside was redissolved in 500 pL C/M (2:1 v/v) and stored at 4 C
before quantifying. Ganglioside extract is composed of 4% GM3, 92%
GD3, and 4% unknown (Table 2).
TABLE 2
Ganglioside composition of zeta dairy lipid powder
Ganglioside Relative %
GM3 3.86
GD3 92.2
Unknown 3.92
EXAMPLE 4 - Quantification
[0027] Samples were redissolved in C/M (2:1 v/v) after drying under N2
gas. Aliquots (10 pL) were taken in duplicate and dried under N2 gas
before adding 500 pL ddH2O and vortexing. Resorcinol-HCI (500 pL)
was added to test tubes; then capped, vortexed, and heated (8 min at
160 C). After cooling to room temperature, 500 pL of
butylacetate/butanol (85:15 v/v) was added to test tubes and vortexed.
The upper layer was withdrawn and read by a spectrophotometer
(8452A, Hewlett Packard) at 580 nm. Ganglioside quantitification was
determined by relating absorbance values to an authentic N-acetyl
neuraminic acid standard curve. Samples were dried under nitrogen
gas and suspended in appropriate cell culture medium to the desired
concentration before filter sterilization (0.22 pm).
EXAMPLE 5 - Cell Growth Assay
[0028] Cells were grown to 60% confluence before replacing medium
with fresh medium containing 0, 10, 20, or 30 pg/mL of ganglioside.
After 48 hr, cells were rinsed once with PBS and harvested with 0.25%
trypsin-EDTA for 5-10 min before inactivation with FBS. Cell counts
were estimated by trypan blue exclusion using a haemacytometer.
High viability (>95%) was obtained for each experiment. Cell counts in
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ganglioside-supplemented groups were computed as a percentage
relative to cell counts in the non-supplemented group.
[0029] There was no difference in cell viability between treatment and
control groups. Ganglioside did not alter cell growth in RWPE-1 cells at
concentrations of 10, 20, or 30 pg/mL. At 30 pg/mL, a 30% reduction
(p<0.01) in the number of PC-3 cells was observed (Figure 1).
[0030] Ganglioside can decrease growth of PC-3 cells in vitro.
Treatment group counts were calculated as a percentage of control
group counts (n). The asterisk in Figure 1 indicates significant
(p<0.05) difference from 100%. Results of Figure 1 are summarized in
Table 3.
TABLE 3
Summary of cell growth data
Dose (pg/mL) RWPE-1 pvalue PC-3 p-value
89.28 NS 95.07 NS
88.00 NS 103.6 NS
105.9 NS 69.84 <0.01
p-values denote significant difference from 100%, NS=not significant.
EXAMPLE 7 - Statistics
[0031] Observations were made in duplicate or triplicate across
microtitre plate wells. Each observation was obtained from a
consecutive cell passage. A t-test for proportion means was conducted
to determine whether ganglioside treatment altered cell growth relative
to untreated control. Means for cell surface ganglioside absorbance
were computed for treatment and control groups and compared using
Student's paired t-test.
[0032] Although a few embodiments have been shown and described,
it will be appreciated by those skilled in the art that various changes
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and modifications might be made without departing from the spirit or
scope of the invention. Unless defined otherwise, all technical and
scientific terms used herein have the same meaning as commonly
understood to one of ordinary skill and the art to which this invention
belongs. In addition, the terms and expressions used in this
specification have been used herein as terms of description and not of
limitation, and there is no intention in the use of such terms and
expressions of excluding equivalents of the features shown and
described or portions thereof, it being recognized that the scope of the
invention is defined and limited only by the appended claims.
REFERENCES
[0033] The following documents are hereby incorporated by reference
into this application in their entirety.
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Promotion of neuritogenesis in mouse neuroblastoma cells by
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association of ganglioside GM1. J Neurochem 1984;42(2):299-305.
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5. Vihko P, Herrala A, Harkonen P, et al. Control of cell proliferation by
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10. Hurdle HL. Examination of in vitro prostate cancer models
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12. Schnabl KL, Larcelet M, Thomson AB, Clandinin MT. Uptake and
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16. Tempera I, Buchetti B, Lococo E, et al. GD3 nuclear localization
after apoptosis induction in HUT-78 cells. Biochem Biophys Res
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17. Omran OM, Saqr HE, Yates AJ. Molecular mechanisms of GD3-
induced apoptosis in U-1242 MG glioma cells. Neurochem Res
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18. Alessandri G, Filippeschi S, Sinibaldi P, et al. Influence of
gangliosides on primary and metastatic neoplastic growth in human
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19. Malisan F, Franchi L, Tomassini B, et al. Acetylation suppresses
the proapoptotic activity of GD3 ganglioside. J Exp Med
2002;196(12):1535-41.
20. Kniep B, Kniep E, Ozkucur N, et al. 9-O-acetyl GD3 protects tumor
cells from apoptosis. Int J Cancer 2006;119(1):67-73.
21. Furukawa K, Thampoe IJ, Yamaguchi H, Lloyd KO. The addition of
exogenous gangliosides to cultured human cells results in the cell type-
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process. J Immunol 1989;142(3):848-54.
22. Popa I, Pons A, Mariller C, et al. Purification and structural
characterization of de-N-acetylated form of GD3 ganglioside present in
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23. Park EJ, Suh M, Clandinin MT. Dietary ganglioside and long-chain
polyunsaturated fatty acids increase ganglioside GD3 content and alter
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24. Prinetti A, Basso L, Appierto V, et al. Altered sphingolipid
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25. Sorensen LK. A liquid chromatography/tandem mass spectrometric
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26. Cheresh DA, Pytela R, Pierschbacher MD, Klier FG, Ruoslahti E,
Reisfeld RA. An arg-gly-asp-directed receptor on the surface of human
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27. Ohkawa Y, Miyazaki S, Miyata M, Hamamura K, Furukawa K,
Furukawa K. Essential roles of integrin-mediated signaling for the
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28. Sun J, Shaper NL, Itonori S, Heffer-Lauc M, Sheikh KA, Schnaar
RL. Myelin-associated glycoprotein (siglec-4) expression is
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complex gangliosides. Glycobiology 2004;14(9):851-7.
29. Ladisch S, Kitada S, Hays EF. Gangliosides shed by tumor cells
enhance tumor formation in mice. J Clin Invest 1987;79(6):1879-82.
30. Valentino LA, Ladisch S. Localization of shed human tumor
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31. Li RX, Ladisch S. Shedding of human neuroblastoma gangliosides.
Biochim Biophys Acta 1991;1083(1):57-64.
32. Radsak K, Schwarzmann G, Wiegandt H. Studies on the cell
association of exogenously added sialo-glycolipids. Hoppe Seylers Z
Physiol Chem 1982;363(3):263-72.
33. Ravindranath MH, Muthugounder S, Presser N, Ye X, Brosman S,
Morton DL. Endogenous immune response to gangliosides in patients
with confined prostate cancer. Int J Cancer 2005;116(3):368-77.
34. Ravindranath MH, Tsuchida T, Morton DL, Irie RF. Ganglioside
GM3:GD3 ratio as an index for the management of melanoma. Cancer
1991;67(12):3029-35.
35. Hyuga S, Yamagata S, Tai T, Yamagata T. Inhibition of highly
metastatic FBJ-LL cell migration by ganglioside GD1 a highly expressed
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36. Hyuga S, Yamagata S, Takatsu Y, et al. Suppression by
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1999;83(5):685-91.
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APPENDIX "A"
Attached to this page is the inventors' paper entitled, "EFFECTS OF
GANGLIOSIDE ON GROWTH AND CELL SURFACE GANGLIOSIDE
DENSITIES IN PROSTATE CANCER IN VITRO" published as Chapter
3 of the thesis entitled Ganglioside Increases Metastatic Potential and
Susceptibility of Prostate Cancer to Gene Therapy In Vitro by John
Miklavcic, University of Alberta, Department of Agricultural, Food and
Nutritional Science; Edmonton, Alberta, Canada, Fall 2009.
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3.0 EFFECTS OF GANGLIOSIDE ON GROWTH AND CELL SURFACE
GANGLIOSIDE DENSITIES IN PROSTATE CANCER IN VITRO
3.1 INTRODUCTION
Several cell lines have been established representing various
stages of CaP. Cultured cells are typically grown in nutrient media
supplemented with fetal bovine serum (FBS). The albumin component
of serum inhibits ganglioside uptake in vitro (1, 2); therefore, serum-
free media is commonly used when incubating cultures with
ganglioside. Interpretations from these studies may be obscured since
use of serum-deficient media deviates from optimal culture conditions.
Albumin concentration is approximately two times higher in human
serum (HS) than FBS (3). This research model in this study employs
1% (v/v) HS as opposed to the typical 10% (v/v) FBS; equating to
approximately one-fifth the amount of albumin. This model minimizes
the inhibitory effect of albumin and other serum components (4) on
ganglioside uptake and minimizes misinterpretation that may result
from using serum-deficient media.
Prostate Cell Culture Model
Use of HS in place of FBS bears additional advantages for
prostate cell culture. FBS is devoid of many nutrients and growth-
regulating hormones found in HS derived from males. HS provides a
source of testosterone, which is absent in FBS. Normal and malignant
prostate epithelial cell growth is influenced by testosterone in nutrient
medium (5, 6). Evidence suggests that HR CaP is not dependent on
testosterone for growth, but still responds to presence of androgens
(7). The high sympathetic innervation of prostate and preponderance of
al and (32 adrenergic receptors suggests necessity of testosterone for
growth and maturation of the gland (8). The lipid profiles of both sera
also differ. Species of fatty acids vary greatly among phospholipid,
triacylglycerol, and cholesterol ester fractions between FBS and HS.
There is a strong trend indicating higher n-6/n-3 fatty acid ratio and
higher quantities of total saturated, monounsaturated, and
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polyunsaturated fats in HS compared to FBS (9, 10). In order to better
represent in vivo conditions, HS is arguably a more appropriate serum
source for use in culture of human prostate cells in vitro.
Ganglioside Uptake, Metabolism, and Effect on Cell Growth
Ganglioside has been investigated for its influence mainly in
melanoma and osteosarcoma, but not in CaP. The role of GD3 as both
an inducer and mediator of apoptosis has been extensively reviewed
(11) (Section 1.3.4). The effect of GD3 on growth of CaP cells has not
yet been determined. GD3 uptake has been described in many
biological models (Section 1.5.1), but has not been investigated in
CaP. This research is also among the first to explore whether CaP cells
natively express 9-0-acetyl GD3 and whether treatment with GD3 can
induce its appearance (Section 1.5.2). GD1a is synthesized in a
parallel ganglioside metabolism pathway, and is particularly abundant
in DU-145 and PC-3 cells (Section 1.5.3). The effect of GD3 on cell
surface GD1a density remains to be established. This research also
highlights differences in ganglioside uptake and metabolism between
models of healthy and malignant prostate.
3.2 HYPOTHESIS AND OBJECTIVES
It is hypothesized that ganglioside decreases growth and
increases metastatic potential of CaP. This hypothesis was explored in
an in vitro model of CaP. Cell counts were performed to test whether
supplemental ganglioside decreases growth of CaP cells; and cell
surface markers were assayed by ELISA to determine whether
treatment increases metastatic potential of CaP cells.
3.3 MATERIALS AND METHODS
Growth Conditions
RWPE-1, DU-145, and PC-3 cell lines were obtained from the
American Type Culture Collection (ATCC). Cell line characteristics are
outlined in Table 3-1. Cultures were maintained in Costar 3516 six-well
tissue culture treated plates. RWPE-1 cells were cultured in
Keratinocyte-SFM containing L-glutamine, supplemented with bovine
pituitary extract (193 pL/100 mL) and human recombinant epidermal
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growth factor (0.591 pL/100 mL). Medium was replaced two-three
times/week and cells were subcultivated (1:6) every seven days. DU-
145 cells were cultured in Minimum Essential Medium containing
Earle's Salts, L-glutamine, non-essential amino acids; and
supplemented with NaHCO3 (2.2 g/L), and sodium pyruvate (1.0 mM).
Medium was replaced three times/week and cells were subcultivated
(1:10) every five days. PC-3 cells were cultured in Ham's F-12 Nutrient
Mixture containing L-glutamine and supplemented with NaHCO3 (1.18
g/L). Medium was replaced three times/week and cells were
subcultivated (1:5) every 7 days. To passage cells, monolayers were
rinsed once with phosphate-buffered saline (PBS) before being
dislodged by cell lifter into fresh medium. Cells were grown in standard
culture incubation conditions of 37 C and 5% atmospheric C02. All cell
cultures were supplemented with 1% (v/v) pooled human serum and
1% (v/v) antibiotic/antimycotic (penicillin, streptomycin, amphotericin
B).
Human Serum
Ethics approval was obtained from the University of Alberta
Faculty of Agricultural, Life, and Environmental Sciences Human
Research Ethics Board (Biomedical Panel) to draw blood from six
healthy males aged 18-35 having no history of cancer, autoimmune, or
other disease. Subjects using steroidal medications, hormone
therapies, or having had surgery within 3 months were excluded. Blood
(100 mL) was drawn from the subcubital vein and was spun (365 g for
30 min at 37 C) to obtain serum fractions. Serum samples from all
subjects were pooled, aliquoted, and frozen. Serum was thawed
immediately prior to use.
Ganglioside Extraction
Ganglioside was extracted from zeta dairy lipid powder
(Fonterra, Cambridge NZ) by modified Folch method (12, 13). Powder
(0.50 g) was added to 30 mL of chloroform/methanol (2:1 v/v), vortexed
(30 sec), and shaken (>2 hr). 0.025% (w/v) CaCl2/double-distilled (dd)
H2O was added to samples before inversing several times. Samples
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were spun (1,000 rpm for 10 min at room temperature) and the upper
layer was withdrawn and filtered through a Sep-Pak Classic C18
cartridge. Cartridges were rinsed with 10 mL ddH2O before eluting
ganglioside with 2 mL of methanol, followed by 10 mL of C/M (2:1 v/v).
C/M was removed under N2 gas before ganglioside was redissolved in
500 pL C/M (2:1 v/v) and stored at 4 C before quantifying. Ganglioside
extract is composed of 4% GM3, 92% GD3, and 4% unknown (Table 3-
2).
Quantification
Samples were redissolved in C/M (2:1 v/v) after drying under N2
gas. Aliquots (10 pL) were taken in duplicate and dried under N2 gas
before adding 500 pL ddH2O and vortexing. Resorcinol-HCI (500 pL)
was added to test tubes; then capped, vortexed, and heated (8 min at
160 C). After cooling to room temperature, 500 pL of
butylacetate/butanol (85:15 v/v) was added to test tubes and vortexed.
The upper layer was withdrawn and read by a spectrophotometer
(8452A, Hewlett Packard) at 580 nm. Ganglioside quantitification was
determined by relating absorbance values to an authentic N-acetyl
neuraminic acid (Neu5Ac) standard curve. Samples were dried under
nitrogen gas and suspended in appropriate cell culture medium to the
desired concentration before filter sterilization (0.22 pm).
Cell Growth Assay
Cells were grown to 60% confluence before replacing medium
with fresh medium containing 0, 10, 20, or 30 pg/mL of ganglioside.
After 48 hr, cells were rinsed once with PBS and harvested with 0.25%
trypsin-EDTA for 5-10 min before inactivation with FBS. Cell counts
were estimated by trypan blue exclusion using a haemacytometer.
High viability (>95%) was obtained for each experiment. Cell counts in
ganglioside-supplemented groups were computed as a percentage
relative to cell counts in the non-supplemented group.
Cell Surface Ganglioside Measures
Cells were grown to 60% confluence before adding fresh
medium with or without 10 pg/mL of ganglioside. After 24 hr, respective
{E5846533. DOC;1 }
CA 02716264 2010-10-01
19
control and treatment media were replaced. After another 24 hr, cells
were rinsed once with PBS and harvested by cell lifter since
trypsinization effects ganglioside detection (14). Cells (0.2 - 0.5 x106)
were added to individual wells in Costar 3894 96-well tissue culture
treated v-bottom plates and incubated with either 1:100 mouse anti-9-
O-acetyl GD3 monoclonal antibody (clone 7H2, Abcam Inc.), 1:2000
mouse anti-GD3 monoclonal antibody (clone MB3.6, Millipore Corp.),
or 1:2000 mouse anti-GD1a monoclonal antibody (clone GD1a-1,
Millipore Corp.) in 180 pL of PBS-4% human serum albumin (HSA) and
put on a water bath shaker (60 min at <10 C). Negative controls were
incubated with PBS-4% HSA alone, IGG3 or IGG1 isotype control
mouse antibodies (clones B10, 15H6, SouthernBiotech Corp.). Cells
were spun (>1730 g for 2 min at 4 C) and rinsed three times in PBS-
4% HSA after primary antibody incubation. Cells were then incubated
with 1:5000 peroxidase-conjugated goat anti-mouse antibody (115-
036-062, Jackson ImmunoResearch Laboratories Inc.) in PBS-4% HSA
(150 pL) and put on a water bath shaker (60 min at <10 C). Cells were
washed three times as before, then transferred to a new plate and
incubated with of o-phenylenediamine dihydrochloride peroxidise
substrate (50 pL) for 40 min in the dark. Cells were spun (>1730 g for 5
min at 4 C) before supernatant was added to Costar 3598 96-well flat-
bottom plates containing of 6 N H2SO4 (60 pL) in each well. Finally,
dual absorbance was read at 490-650 nm using a microtitre plate
reader (SPECTRAmax 190, Molecular Devices).
Statistics
Observations were made in duplicate or triplicate across
microtitre plate wells. Each observation was obtained from a
consecutive cell passage. A t-test for proportion means was conducted
to determine whether ganglioside treatment altered cell growth relative
to untreated control. Means for cell surface ganglioside absorbance
were computed for treatment and control groups and compared using
Student's paired t-test.
{E5846533.DOC;1 }
CA 02716264 2010-10-01
3.4 RESULTS
Cell Growth
There was no difference in cell viability between treatment and
control groups. Ganglioside did not alter cell growth in RWPE-1 or DU-
145 cells at concentrations of 10, 20, or 30 pg/mL. At 30 pg/mL, a 30%
reduction (p<0.01) in the number of PC-3 cells was observed (Figure 3-
1). Results of Figure 3-1 are summarized in Table 3-3.
Cell Surface Ganglioside
Cell surface 9-0-acetyl GD3 was not detected in control or
treatment groups in any of the cell lines (Figure 3-2). Cell surface GD3
density increased (p<0.03) by 12% in RWPE-1 cells with treatment, but
did not significantly change in DU-145 or PC-3 cells (Figure 3-3). Cell
surface GD1 a density was reduced by half in DU-1 45 (p<0.01) and PC-
3 cells (p<0.01) in the treatment groups, but did not change in RWPE-1
cells (Figure 3-4).
3.5 DISCUSSION
The effect of ganglioside was tested on growth of healthy and
malignant prostate cells in vitro. Growth was expected to decrease as
ganglioside has demonstrated an apoptotic effect in numerous cancer
cells (15-17). Although no effect was shown at physiologically-relevant
doses, mixed ganglioside treatment (92% GD3) decreased the number
of viable, adherent PC-3 cells at the highest dose (30 pg/mL) tested. A
similar finding was shown in B16/BL6 melanoma and 3LL lung
carcinoma cells; low dose (-same concentration used in this study) did
not affect (or increased) cell growth, but very high dose (-10 times
higher concentration than used in this study) inhibited growth (18).
Although the mechanism was not investigated, this effect is believed to
have occurred via apoptosis. A peculiar finding in the present research,
however, was that GD3 failed to reduce growth of RWPE-1 and DU-
145 cells.
GD3 is a proven facilitator of apoptosis, but some tissue types
employ methods to subvert agents of cell death. Some cell/tumour
types exhibit the capacity to nullify the pro-apoptotic effect of GD3 via
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CA 02716264 2010-10-01
21
9-0-acetylation (19). RWPE-1 and DU-145 were expected to stain
positively for 9-0-acetyl GD3, as growth in these cell lines was
unaffected by treatment. Interestingly, cell surface 9-0-acetyl GID3 was
not detected in any cell line tested, and treatment was insufficient to
induce its appearance. Therefore, conversion of GD3 to 9-0-acetyl
GD3 does not explain why growth of RWPE-1 and DU-145 cells is
unchanged by treatment. However, Kniep et al. report appearance of 9-
O-acetyl GD3 in Jurkat-R cells after 3 weeks of culturing with GD3 (20).
In the present experiment, dose or time exposures may have been
insufficient to induce acetylation. Other GD3 metabolites (N-glycolyl
GD3, de-N-acetyl GD3) have been documented in cancer (21, 22).
Conversion to a GD3 metabolite or downstream ganglioside like GD2
or GT3 may explain why GD3 did not decrease growth in RWPE-1 or
DU-145 cells, but was not assessed in the present study. Furthermore,
9-0-acetyl GD3 density was assayed only at the cell surface. Intra- or
total cellular 9-O-acetyl GD3 may be functionally implicated in
resistance to apoptosis. PC-3 cells may be sensitive to GD3-induced
apoptosis, and may be unable to counteract (via 9-0-acetylation) the
pro-apoptotic influence of GD3. Therefore, GD3 may be exploited as a
potential anti-CaP agent at a concentration slightly higher than
physiologically-relevant.
Many cells and tissues have been shown to take up ganglioside
when provided in vitro (12, 14) and in vivo (23). Ganglioside uptake in
prostate cell lines has not been documented. When provided in culture,
ganglioside treatment increased cell surface GD3 density in RWPE-1,
but not malignant prostate cells. Elevated GD3 density in RWPE-1 cells
is believed to have occurred via GD3 uptake, and not increased
ganglioside synthesis. It is unknown whether ganglioside is taken up in
a dose-dependent manner in RWPE-1 cells, or whether a higher dose
would have resulted in increased GD3 density in malignant cell lines.
Changes in cell surface GD3 density were explored, but changes in
GD3 composition/structure were not investigated. Treatment may have
reasonably induced a change in ganglioside composition, as
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CA 02716264 2010-10-01
22
ganglioside from diet has more variation in structure than endogenous
ganglioside (24, 25). This study suggests differential ganglioside
uptake between healthy and malignant prostate cells, but further
investigation in more models of CaP is required to confirm this
suggestion.
Conversion of GD3 to another ganglioside may explain failure to
detect increased GD3 in malignant prostate cell lines. As previously
indicated, treatment did not induce appearance of 9-0-acetyl GD3.
However, conversion of GD3 to GD3 metabolites or downstream
gangliosides was not assessed. Gangliosides are known to co-localize
with other components of cell membrane (26-28). Thus, ganglioside
uptake and organization in the cell membrane may affect display of the
ganglioside antibody-recognition site and subsequent detection. For
example, trypsinization has been shown to increase display/detection
of GD2 (14). This may occur via digestion of proteins than block GD2
exposure at the cell surface. On the other hand, GD3 taken up by
malignant cells may be shed before being assayed 48 hr after
incubation. Evidence suggests that ganglioside shedding may be
implicated in tumour cell growth (29-31). It has been shown that
ganglioside uptake can occur in as little as five min (32). Therefore, an
acute increase in GD3 in CaP cells may be blunted by shedding of
GD3 over 48 hr. Though, some cell lines simply do not noticeably
incorporate ganglioside into the cell membrane when provided in
culture. This study suggests that RWPE-1, but not malignant prostate
cells experience significantly increased cell surface GD3 density after
treatment with a physiologically-relevant concentration of ganglioside.
GD1 a is the most abundant cell surface ganglioside in DU-145
and PC-3 cells (33). In the present study, treatment with ganglioside
mixture decreased GD1 a in malignant prostate, but not in RWPE-1
cells. This is among the first work to explore the effect of simple
ganglioside on level/density of a complex ganglioside. Since GD3 is
positively (27, 34) and GD1a is negatively related to metastatic
potential of tumour cells (35, 36), the net effect of treatment may
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CA 02716264 2010-10-01
23
promote metastasis. The results of this study highlight distinct
differences in uptake and subsequent metabolism of ganglioside
between healthy and malignant prostate cells. The findings indicate
that a physiologically-relevant concentration of ganglioside may
increase the metastatic potential of CaP.
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24
3.6 FIGURES
Table 3-1 Cell lines employed in current study
Cell Line Type Androgen PSA Origin
Responsiveness Producing
RWPE-1 Normal Yes Yes Normal Human Prostate Cells
DU145 Tumour No No Human Brain Metastasis
PC 3 Tumour No No Human Bone Metastasis
Table 3-2 Ganglioside composition of zeta dairy lipid powder
Ganglioside Relative %
GM3 3.86
GD3 92.2
Unknown 3.92
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CA 02716264 2010-10-01
Figure 3-1 Ganglioside decreases growth of PC-3 cells in vitro
0RWPE-1
0DU-146
PC-3
100
t.7
0
10 20 30
Dose (p g/mL)
Cell cultures were incubated with ganglioside for 48 hr. Adherent cells
were harvested and cell counts were estimated by trypan blue
exclusion using a haemacytometer. Treatment group counts were
calculated as a percentage of control group counts (n ). Asterisk
indicates significant (p<0.05) difference from 100%.
Table 3-3 Summary of cell growth data
Dose
(pg/mL) RWPE-1 p-value DU-145 p-value PC-3 p-value
10 89.28 NS 95.85 NS 95.07 NS
20 88.00 NS 100.4 NS 103.6 NS
105.9 NS 101.8 NS 69.84 <0.01
Cell cultures were incubated with ganglioside for 48 hr. Adherent cells
were harvested and cell counts were estimated by trypan blue
exclusion using a haemacytometer. Values are reported as treatment
group counts calculated as percentage of control group counts (n ).
p-values denote significant difference from 100%, NS=not significant.
{E5846533.DOC;1}
CA 02716264 2010-10-01
26
Figure 3-2 9-0-acetyl GD3 is undetectable in prostate cells in vitro
D.4
Control
13 Tre acme nt
D3
3
`02
u
A
41
0.1
0
Background RWPE-1 P0.3 011146
Cell Line
Cell cultures were incubated with 10 pg/mL ganglioside for 48 hr. Cells
were harvested and stained for 9-0-acetyl GD3 prior to analysis by
csELISA (n a4).
{E5846533. DOC;1 }
CA 02716264 2010-10-01
27
Figure 3-3 Ganglioside increases cell surface GD3 density in RWPE-1
but not malignant prostate cells in vitro
0.B
0Control
^ Treatment
0.5-
0.4-
03 `r
cJ r
02}"i xY;
cE3 ^t~
0.1
Yr ~;
0
RWPE-1 DU=148 PC-3
Cell Line
Cell cultures were incubated with 10 pg/mL ganglioside for 48 hr. Cells
were harvested and stained for GD3 prior to analysis by csELISA
(n=5). Asterisk indicates significant (p<0.05) difference between treated
and untreated control group.
{E5846533. DOC;1)
CA 02716264 2010-10-01
28
Figure 3-4 Ganglioside decreases cell surface GD1 a density in
malignant prostate but not RWPE-1 cells in vitro
2.5-
M C ontro I
^Treatment
2
ii
1.5
as v
as t; 7
U 0.5-
t
gTl
0
RWPE-1 DU-146 PC-3
Cell Line
Cell cultures were incubated with 10 pg/mL ganglioside for 48 hr. Cells
were harvested and stained for GD1 a prior to analysis by csELISA
(n). Asterisk indicates significant (p<0.05) difference between treated
and untreated control group.
{E5846533.DOC;1 }
