CA2716264A1 - Use of ganglioside to decrease propagation of malignant prostate cells - Google Patents
Use of ganglioside to decrease propagation of malignant prostate cells Download PDFInfo
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Abstract
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.
Ganglioside can have therapeutic benefit in prostate cancer as demonstrated by decreased growth of malignant PC-3 cells.
Description
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:
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 {E5846533.DOC;1 }
available in small quantities from a limited number of exogenous dietary sources including whole milk and colostrum.
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:
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.
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".
{E5846533. DOC;1 }
Edmonton, Alberta, Canada, Fall 2009. A copy of this paper is attached to this application as Appendix "A".
{E5846533. DOC;1 }
[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:
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:
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 {E5846533.DOC;1) 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.
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.
{E5846533. DOC;1) [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.
{E5846533. DOC;1) [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, {E5846533. DOC;1 }
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).
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 {E5846533.DOC;1 }
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).
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 {E5846533. DOC;1 }
ganglioside-supplemented groups were computed as a percentage relative to cell counts in the non-supplemented group.
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, {E5846533. DOC;1 }
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).
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 {E5846533.DOC;1 }
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).
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 {E5846533. DOC;1 }
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.
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.
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.
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 {E5846533. DOC;1 }
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
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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Inhibition by calcium ion and dependence upon membrane protein.
Neurochem Res 1989;14(6):547-54.
2. Tomasi M, Roda LG, Ausiello C, et al. Interaction of GMI ganglioside with bovine serum albumin: Formation and isolation of multiple complexes. EurJ Biochem 1980;111(2):315-24.
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{E5846533. DOC;1 }
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22. Popa I, Pons A, Mariller C, et al. Purification and structural characterization of de-N-acetylated form of GD3 ganglioside present in human melanoma tumors. Glycobiology 2007;17(4):367-73.
23. Park EJ, Suh M, Clandinin MT. Dietary ganglioside and long-chain polyunsaturated fatty acids increase ganglioside GD3 content and alter the phospholipid profile in neonatal rat retina. Invest Ophthalmol Vis Sci 2005;46(7):2571-5.
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25. Sorensen LK. A liquid chromatography/tandem mass spectrometric approach for the determination of gangliosides GD3 and GM3 in bovine milk and infant formulae. Rapid Commun Mass Spectrom 2006;20(24):3625-33.
{E5846533.DOC;1 }
26. Cheresh DA, Pytela R, Pierschbacher MD, Klier FG, Ruoslahti E, Reisfeld RA. An arg-gly-asp-directed receptor on the surface of human melanoma cells exists in an divalent cation-dependent functional complex with the disialoganglioside GD2. J Cell Biol 1987;105(3):1163-73.
27. Ohkawa Y, Miyazaki S, Miyata M, Hamamura K, Furukawa K, Furukawa K. Essential roles of integrin-mediated signaling for the enhancement of malignant properties of melanomas based on the expression of GD3. Biochem Biophys Res Commun 2008;373(1):14-9.
28. Sun J, Shaper NL, Itonori S, Heffer-Lauc M, Sheikh KA, Schnaar RL. Myelin-associated glycoprotein (siglec-4) expression is progressively and selectively decreased in the brains of mice lacking complex gangliosides. Glycobiology 2004;14(9):851-7.
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1. Leskawa KC, Erwin RE, Leon A, Toffano G, Hogan EL. Incorporation of exogenous ganglioside GM1 into neuroblastoma membranes:
Inhibition by calcium ion and dependence upon membrane protein.
Neurochem Res 1989;14(6):547-54.
2. Tomasi M, Roda LG, Ausiello C, et al. Interaction of GMI ganglioside with bovine serum albumin: Formation and isolation of multiple complexes. EurJ Biochem 1980;111(2):315-24.
3. Tognon G, Frapolli R, Zaffaroni M, et al. Fetal bovine serum, but not human serum, inhibits the in vitro cytotoxicity of ET-743 (yondelis, trabectedin), an example of potential problems for extrapolation of active drug concentrations from in vitro studies. Cancer Chemother Pharmacol 2004;53(1):89-90.
4. Facci L, Leon A, Toffano G, Sonnino S, Ghidoni R, Tettamanti G.
Promotion of neuritogenesis in mouse neuroblastoma cells by exogenous gangliosides. relationship between the effect and the cell 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 steroids: The role of 17HSDs. Mol Cell Endocrinol 2006;248(1-2):141-8.
6. Soronen P, Laiti M, Torn S, et al. Sex steroid hormone metabolism and prostate cancer. J Steroid Biochem Mol Biol 2004;92(4):281-6.
7. Libertini SJ, Tepper CG, Rodriguez V, Asmuth DM, Kung HJ, Mudryj M. Evidence for calpain-mediated androgen receptor cleavage as a mechanism for androgen independence. Cancer Res 2007;67(19):9001-5.
8. McVary KT, McKenna KE, Lee C. Prostate innervation. Prostate Suppl 1998;8:2-13.
9. Pratt VC, Watanabe S, Bruera E, et al. Plasma and neutrophil fatty acid composition in advanced cancer patients and response to fish oil supplementation. Br J Cancer 2002;87(12):1370-8.
10. Hurdle HL. Examination of in vitro prostate cancer models supplemented with lycopene, vitamine E and fish oil. 2006.
11. Malisan F, Testi R. GD3 ganglioside and apoptosis. Biochim Biophys Acta 2002;1585(2-3):179-87.
12. Schnabl KL, Larcelet M, Thomson AB, Clandinin MT. Uptake and fate of ganglioside GD3 in human intestinal CaCo-2 cells. Am J Physiol Gastrointest Liver Physiol 2009.
13. Folch J, Lees M, Sloane M, Stanley GH. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 1957;226(1):497-509.
14. Ravindranath MH, Bauer PM, Cornillez-Ty C, Garcia J, Morton DL.
Quantitation of the density of cell surface carbohydrate antigens on cancer cells with a sensitive cell-suspension ELISA. J Immunol Methods 1996;197(1-2):51-67.
15. Ma R, Koulov A, Moulton C, et al. Apoptosis of human breast carcinoma cells in the presence of disialosyl gangliosides: II. treatment of SKBR3 cells with GD3 and GD1 b gangliosides. Glycoconj J
2004;20(5):319-30.
{E5846533. DOC;1) 16. Tempera I, Buchetti B, Lococo E, et al. GD3 nuclear localization after apoptosis induction in HUT-78 cells. Biochem Biophys Res Commun 2008;368(3):495-500.
17. Omran OM, Saqr HE, Yates AJ. Molecular mechanisms of GD3-induced apoptosis in U-1242 MG glioma cells. Neurochem Res 2006;31(10):1171-80.
18. Alessandri G, Filippeschi S, Sinibaldi P, et al. Influence of gangliosides on primary and metastatic neoplastic growth in human and murine cells. Cancer Res 1987;47(16):4243-7.
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-specific expression of novel surface antigens by a biosynthetic 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 human melanoma tumors. Glycobiology 2007;17(4):367-73.
23. Park EJ, Suh M, Clandinin MT. Dietary ganglioside and long-chain polyunsaturated fatty acids increase ganglioside GD3 content and alter the phospholipid profile in neonatal rat retina. Invest Ophthalmol Vis Sci 2005;46(7):2571-5.
24. Prinetti A, Basso L, Appierto V, et al. Altered sphingolipid metabolism in N-(4-hydroxyphenyl)-retinamide-resistant A2780 human ovarian carcinoma cells. J Biol Chem 2003;278(8):5574-83.
25. Sorensen LK. A liquid chromatography/tandem mass spectrometric approach for the determination of gangliosides GD3 and GM3 in bovine milk and infant formulae. Rapid Commun Mass Spectrom 2006;20(24):3625-33.
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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 melanoma cells exists in an divalent cation-dependent functional complex with the disialoganglioside GD2. J Cell Biol 1987;105(3):1163-73.
27. Ohkawa Y, Miyazaki S, Miyata M, Hamamura K, Furukawa K, Furukawa K. Essential roles of integrin-mediated signaling for the enhancement of malignant properties of melanomas based on the expression of GD3. Biochem Biophys Res Commun 2008;373(1):14-9.
28. Sun J, Shaper NL, Itonori S, Heffer-Lauc M, Sheikh KA, Schnaar RL. Myelin-associated glycoprotein (siglec-4) expression is progressively and selectively decreased in the brains of mice lacking 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 gangliosides: Association with serum lipoproteins. Cancer Res 1992;52(4):810-4.
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 in poorly metastatic FBJ-S1 cells. Biochem Biophys Res Commun 1997;231(2):340-3.
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36. Hyuga S, Yamagata S, Takatsu Y, et al. Suppression by ganglioside GD1A of migration capability, adhesion to vitronectin and metastatic potential of highly metastatic FBJ-LL cells. Int J Cancer 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.
{E5846533. DOC;1 }
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 {E5846533. DOC;1 }
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 {E5846533.D0C;1 }
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 (E5846533. DOC;1) 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 }
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 }
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 {E5846533. DOC;1 }
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 {E5846533. DOC;1 }
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 {E5846533. DOC;1 }
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.
{E5846533. DOC;1 }
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 {E5846533. DOC;1 }
Figure 3-1 Ganglioside decreases growth of PC-3 cells in vitro t.7 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}
Figure 3-2 9-0-acetyl GD3 is undetectable in prostate cells in vitro D.4 Control 13 Tre acme nt `02 u A
0.1 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 }
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 ~;
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) 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 ii 1.5 as v as t; 7 U 0.5-t gTl 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 }
{E5846533. DOC;1 }
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.
{E5846533. DOC;1 }
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 {E5846533. DOC;1 }
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 {E5846533.D0C;1 }
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 (E5846533. DOC;1) 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 }
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 }
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 {E5846533. DOC;1 }
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 {E5846533. DOC;1 }
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 {E5846533. DOC;1 }
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.
{E5846533. DOC;1 }
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 {E5846533. DOC;1 }
Figure 3-1 Ganglioside decreases growth of PC-3 cells in vitro t.7 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}
Figure 3-2 9-0-acetyl GD3 is undetectable in prostate cells in vitro D.4 Control 13 Tre acme nt `02 u A
0.1 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 }
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 ~;
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) 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 ii 1.5 as v as t; 7 U 0.5-t gTl 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 }
Claims (12)
1. The use of exogenous ganglioside to inhibit the propagation of prostate cancer cells.
2. The use of claim 1 wherein the exogenous ganglioside is administered to a subject as a dietary supplement.
3. The use of claim 1 wherein the exogenous ganglioside is GD3.
4. The use of claim 3 wherein the GD3 is GD3-enriched zeta dairy lipid powder.
5. The use of claim 1 wherein the prostate cancer cells are bone metasisized cells.
6. The use of claim 5 wherein the bone metasisized cells are PC-3 cells.
7. The use of claim 1 wherein the propagation of normal prostate cells is not inhibited.
8. The use of claim 1 wherein the inhibition of the propagation of prostate cancer cells is accomplished by apoptosis.
9. A dietary supplement comprising exogenous ganglioside wherein the dietary supplement is used to inhibit the propagation of prostate cancer cells.
10. A composition comprising exogenous ganglioside wherein the composition is used to inhibit the propagation of prostate cancer cells.
11. The use of exogenous ganglioside in the manufacture of a medicament to be used to inhibit the propagation of prostate cancer cells.
12. A kit 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.
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