PROPOLIS OR CAFFEIC ACID PHENETHYL ESTER ( CAPE ) INHIBITS GROWTH AND VIABILITY IN MULTIPLE ORAL CANCER CELL LINES

Objective: Propolis is a natural antimicrobial resin from honeybee hives that contains caffeic acid phenethyl ester (CAPE), which has anti-proliferative activity against some human cancers, including colon, liver, lung and breast – although limited evidence has evaluated this potential in oral cancers. Based upon this information, the primary objective of this study was to evaluate the anti-tumor effects of CAPE against multiple well-characterized oral cancer cell lines. Methods: Using well-characterized oral cancer cell lines (SCC15, SCC25 and CAL27), CAPE was administered at 100 ug/mL to assess any effects on cellular viability or growth over three days. A normal, non-cancerous cell line (HGF-1) was also included. Results: The results of this pilot study demonstrated that CAPE administration significantly reduced both viability and proliferation in all three oral cancer cell lines. Viability was significantly reduced between 30.3% and 35.4% among the oral cancer cell lines (p<0.05), but remained unchanged in the HGF-1 normal cell control (p=0.878). Growth was significantly inhibited between 53.1% and 60.6% among the oral cancer cell lines (p<0.05) but was not affected in the HGF-1 normal cell control (p=0.341). Conclusions: Although the reductions in both cellular viability and proliferation were distinct for each cell line, all exhibited a similar trend and were within a narrowly defined range. These results strongly suggest that CAPE administration had a significant and immediate effect on oral cancer growth and viability and therefore should be considered as the basis for future studies as a potential complementary and alternative therapy for oral cancer.


Introduction:
Propolis is available over-the-counter and has been designated by the Food and Drug Administration (FDA) as GRAS or generally regarded as safe [1,2].It has been used as a traditional homeopathic remedy, which is well known among apiary workers or bee keepers [3,4]).In addition, propolis has been demonstrated to facilitate honeybee resistance to mites and other potential beehive hazards [5,6].
Propolis is a natural antimicrobial resin from honeybee hives that contains caffeic acid phenethyl ester (CAPE), the main active component [7,8].Propolis is reported to exhibit anti-bacterial and free radical scavenging properties [9][10][11].Although some evidence has found propolis and extractions containing CAPE have anti-proliferative activity against some human cancers, including colon, liver, lung and breast -limited evidence has evaluated this potential in oral cancers [12][13][14][15][16].
Although some studies have used oral cancer cell lines, each of these studies utilized only one cell line or cell lines derived from only one patient [17][18][19][20].Based upon this preliminary evidence, the primary goal of this study was to evaluate the anti-tumor potential of propolis -and more specifically the primary active agent caffeic acid phenethyl ester or CAPE against multiple wellcharacterized oral cancer cell lines in tandem.The working hypothesis for this pilot study was that CAPE would exhibit similar effects on oral cancer viability and growth inhibition.

Cell
culture Three oral cancer cell lines were obtained from American Tissue Culture Collection (ATCC), which included SCC15 (CRL-1623), SCC25 (CRL-1628) and CAL27 (CRL2095).CAL27 cells were grown in in Dulbecco's Modified Eagle's Medium (DMEM) with the addition of 10% fetal bovine serum (FBS) obtained from Fisher Scientific (Fair Lawn, NJ).SCC15 cells were grown in in a 1:1 mixture of Dulbecco's Modified Eagle's Medium and Ham's F12 (DMEM:F12) containing 1.2 g/L sodium bicarbonate, 2.5 mM L-glutamine, 15mM HEPES and 0.5 mM sodium pyruvate, which was also supplemented with 10% FBS.A non-cancerous, normal cell control line HGF-1 (CRL-2014) was also obtained and cultured in DMEM with 10% FBS.All cells were maintained in a humidified tissue culture incubator supplemented with 5% C0 2 .

Cell viability and proliferation
All assays were done in triplicate with each experimental condition containing eight wells, resulting in a combined data set of n=24 for each cell line and variable.Cell viability was measured at baseline (starting time point) and all subsequent time points using the Trypan Blue exclusion assay and the TC20 Cell Counter from Bio-Rad (Hercules, CA).Proliferation data was also obtained from the Trypan Blue exclusion assay, but was then subsequently quantified and confirmed using a BioTek ELx808 Absorbance Microplate Reader from Fisher Scientific (Fair Lawn, NJ).

Statistical analysis
Differences in viability and proliferation between the starting time point and all subsequent time points were measured using two-tailed Students t-Because multiple two-tailed t-tests may have a higher possibility of Type I error, analysis of variance (ANOVA) was also performed to verify each result.

Results
Baseline measurements of cell viability revealed similar, non-significant differences among each cell line, which ranged from 79.3% to 84.2% (Figure 1).More specifically, SCC15 (81.3%),SCC25 (79.3%),CAL27 (81.1%) and HGF-1 (84.2%) exhibited comparable levels of viability that were not statistically significant (p>0.3).The addition of CAPE at a concentration of 100 ug/ML significantly reduced cellular viability by the first time point, day one (d1) in all oral cancer cell lines; SCC15 -17.4% (63.9% viability), SCC25 -22.2% (57.1% viability), CAL27 -21.3% (59.8%), which was significantly lower than the initial baseline measurements, p<0.05.In addition, these effects were also observed at two additional time points (day two, d2 and day three, d3), which were also significantly lower than the baseline measurements and negative controls (without the addition of CAPE), p<0.05.However, the addition of CAPE did not exhibit any significant effect on the normal, noncancerous oral cell line HGF-1 at any time point of this assay, p=0.878.To determine if the significant reductions in cellular viability induced by CAPE administration among the oral cancer cell lines also affected cell growth, 96-well proliferation assays were also conducted using CAPE at these concentrations (Figure 2).The results of this assay revealed that HGF-1 proliferation was similar under both the negative control and experimental (CAPE) assay conditions, p=0.341.However, these results also clearly demonstrated that CAPE administration was sufficient to significantly reduce oral cancer growth among all three cell lines tested.

Discussion
The working hypothesis for this pilot study was that CAPE would exhibit similar effects on oral cancer viability and growth inhibition.The results of this pilot study demonstrated that CAPE administration significantly reduced both viability and proliferation in three, wellcharacterized oral squamous cell carcinoma cell lines.Although the reductions in both cellular viability and proliferation were distinct for each cell line, all exhibited a similar trend and were within a narrowly defined range, which provides support for the initial hypothesis.
Due to the similar response from all three oral cancer cell lines, an investigation into the mechanisms that may be responsible for these observations is warranted.Although this initial pilot study did not seek to determine the mechanisms of action, previous reports of the anti-oxidant, anti-mitogenic, and anticarcinogenic properties of CAPE suggest that some of the mechanisms responsible for these effects include the inhibition of NFkB, lipid peroxidation, protein tyrosine kinase, ornithine decarboxylase, and matrix metalloproteinase (MMP)-9 catalytic activity. 21-23Other studies have revealed that enhanced expression and activation of the tumor suppressor p53 and activation of pro-apoptotic Bax and caspase-3 may also be induced by CAPE administration. 24,25 These results must also be viewed in context of the limitations that were intrinsic to this type of pilot study.First, and most importantly, this study was limited to only three commercially available oral cancer cell lines.Future studies should include additional oral cancer cell lines, as well as other normal oral cell lines, which should provide additional insights and information about the potential anti-oral cancer properties of CAPE administration.In addition, this study involved a relatively short time interval (three days), although the significant reductions in cell proliferation over this short time period may suggest CAPE administration may provide a promising complementary and alternative therapy to traditional oral cancer treatments.

Conclusion
Although these data are preliminary, the results clearly demonstrated CAPE administration had a significant and immediate effect on oral cancer growth and viability and therefore should be considered as the basis for future studies as a potential therapeutic agent for oral cancer.

Figure 1 :
Figure 1: Effects of CAPE (100 ug/mL) on cellular viability.The addition of CAPE (100 ug/mL) significantly reduced viability significantly among the oral cancer cell lines SCC15, SCC25 and CAL27 at each time point (days one, two and three: d1, d2, d3), compared with baseline measurements and parallel non-CAPE (negative) controls, p<0.05.The normal gingival fibroblast cell line HGF-1 did not exhibit any significant changes to viability under CAPE administration, p=0.878*.