Abstract:
Uses of exogenous ganglioside to inhibit the propagation of prostate cancer cells are 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&lt;0.01). Ganglioside can have therapeutic benefit in prostate cancer as demonstrated by decreased growth of malignant PC-3 cells.

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
       [0001]    This application claims the benefit of U.S. Provisional Application No. 61/388,701, filed on Oct. 1, 2010, and said earlier application is incorporated herein by reference in its entirety for continuity of disclosure. 
     
    
     TECHNICAL FIELD 
       [0002]    This disclosure relates to methods of inhibiting the propagation of prostate cancer cells. 
       BACKGROUND 
       [0003]    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. 
         [0004]    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. 
         [0005]    Accordingly, there is a need to provide methods of inhibiting the propagation of prostate cancer cells that overcome the shortcomings of the prior art. 
       BRIEF SUMMARY 
       [0006]    Uses of exogenous ganglioside to inhibit the propagation of prostate cancer cells are 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&lt;0.01). Ganglioside can have therapeutic benefit in prostate cancer as demonstrated by decreased growth of malignant PC-3 cells. 
         [0007]    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. 
         [0008]    Broadly stated, in some embodiments, a use of exogenous ganglioside is provided to inhibit the propagation of prostate cancer cells. 
         [0009]    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. 
         [0010]    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. 
         [0011]    Broadly stated, in some embodiments, a method of treatment to inhibit the propagation of prostate cancer cells is provided, the method comprising the administration of exogenous ganglioside to an individual in need of such treatment. 
         [0012]    Broadly stated, in some embodiments, a method of inhibiting the propagation of prostate cancer cells is provided, the method comprising contacting the prostate cancer cells with exogenous ganglioside. 
         [0013]    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. 
         [0014]    Broadly stated, in some embodiments, a kit is provided to inhibit the propagation of prostate cancer cells, the kit comprising exogenous ganglioside and instructions for use of the exogenous ganglioside. 
         [0015]    In some embodiments, methods and compositions of exogenous ganglioside to inhibiting the propagation of prostate cancer cells are provided wherein the exogenous ganglioside comprises GD3-enriched zeta dairy lipid powder which is administered to a subject as a dietary supplement in a dosage of at least 30 mg/mL, the inhibition of the propagation of prostate cancer cells is accomplished by apoptosis, the prostate cancer cells comprising PC-3 cells, the propagation of normal prostate cells is not inhibited, and the normal prostate cells comprising RWPE-1 cells. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWING 
         [0016]      FIG. 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 
       [0017]    Uses and compositions of exogenous ganglioside to inhibit the propagation of prostate cancer cells are disclosed herein. 
         [0018]    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 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. 
         [0019]    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. 
         [0020]    In some embodiments, the exogenous ganglioside can comprise GD3. In some embodiments, the exogenous ganglioside can comprise 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. 
         [0021]    In some embodiments, the prostate cancer cells inhibited can comprise PC-3 cells. As would be understood by one skilled in the art, PC-3 cells can serve as classical models for human prostate cancer. 
         [0022]    Referring now to  FIG. 1 , 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. In some embodiments, the normal prostate cells can comprise RWPE-1 cells. 
         [0023]    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. 
         [0024]    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. 
         [0025]    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 
       [0026]    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 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 μL/100 mL) and human recombinant epidermal growth factor (0.591 μL/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&#39;s F-12 Nutrient Mixture containing L-glutamine and supplemented with NaHCO 3  (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 CO 2 . All cell cultures were supplemented with 1% (v/v) pooled human serum and 1% (v/v) antibiotic/antimycotic (penicillin, streptomycin, amphotericin B). 
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Cell lines employed in current study 
               
             
          
           
               
                   
                   
                 Androgen 
                 PSA 
                   
               
               
                 Cell Line 
                 Type 
                 Responsiveness 
                 Producing 
                 Origin 
               
               
                   
               
               
                 RWPE-1 
                 Normal 
                 Yes 
                 Yes 
                 Normal Human 
               
               
                   
                   
                   
                   
                 Prostate Cells 
               
               
                 PC 3 
                 Tumour 
                 No 
                 No 
                 Human Bone Metastasis 
               
               
                   
               
             
          
         
       
     
       Example 2 
     Ganglioside Extraction 
       [0027]    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 (&gt;2 hr). 0.025% (w/v) CaCl 2 /double-distilled (dd) H 2 O 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 ddH 2 O before eluting ganglioside with 2 mL of methanol, followed by 10 mL of C/M (2:1 v/v). C/M was removed under N 2  gas before ganglioside was redissolved in 500 μL 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). 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Ganglioside composition of zeta dairy lipid powder 
               
             
          
           
               
                   
                 Ganglioside 
                 Relative % 
               
               
                   
                   
               
             
          
           
               
                   
                 GM3 
                 3.86 
               
               
                   
                 GD3 
                 92.2 
               
               
                   
                 Unknown 
                 3.92 
               
               
                   
                   
               
             
          
         
       
     
       Example 3 
     Quantification 
       [0028]    Samples were redissolved in C/M (2:1 v/v) after drying under N 2  gas. Aliquots (10 μL) were taken in duplicate and dried under N 2  gas before adding 500 μL ddH 2 O and vortexing. Resorcinol-HCl (500 μL) was added to test tubes; then capped, vortexed, and heated (8 min at 160° C.). After cooling to room temperature, 500 μL 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 μm). 
       Example 4 
     Cell Growth Assay 
       [0029]    Cells were grown to 60% confluence before replacing medium with fresh medium containing 0, 10, 20, or 30 μg/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 (&gt;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. 
         [0030]    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 μg/mL. At 30 μg/mL, a 30% reduction (p&lt;0.01) in the number of PC-3 cells was observed ( FIG. 1 ). 
         [0031]    Ganglioside can decrease growth of PC-3 cells in vitro. Treatment group counts were calculated as a percentage of control group counts (n≧5). The asterisk in  FIG. 1  indicates significant (p&lt;0.05) difference from 100%. Results of  FIG. 1  are summarized in Table 3. 
         [0000]                                                                        TABLE 3                   Summary of cell growth data                Dose (μg/mL)   RWPE-1   p-value   PC-3   p-value                            10   89.28   NS   95.07   NS           20   88.00   NS   103.6    NS           30   105.9   NS   69.84   &lt;0.01                        
p-values denote significant difference from 100%, NS=not significant.
 
       Example 5 
     Statistics 
       [0032]    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&#39;s paired t-test. 
         [0033]    Although a few embodiments have been shown and described, it will be appreciated by those skilled in the art that various changes 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 
       [0034]    The following documents are hereby incorporated by reference into this application in their entirety.
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