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Alotaibi et al. Papaya seeds effect prostate cancer

Cell culture slightly by 20% (non-significant) at 25 g/mL. The
The PC-3 prostate cancer cells were cultured in F-12K cells viability did not change further on increasing the
media whereas 3T3L1 cells were cultured in DMBM papaya seeds extract [Figure 1A]. When cells were
media. Both media were supplemented with 10% FBS treated with methanol extract of papaya black seeds,
and 1% penicillin and Streptomycin. The cells were the cells viability initially decreased significantly to
incubated in a humidified incubator at 37 C with 5% 60% (P < 0.05) in a concentration depends manner
o
CO . Media was changed every 3 days and cells were up to 25 µg/mL; however, the cells viability was not
2
subcultured when they became confluent.
Cell proliferation assay
Effect of papaya seeds extract on cell proliferation
was determined using a WST-1 assay as per
manufacturer instructions. The assay is based on the
reduction of WST-1 dye (brown color) by mitochondrial
dehydrogenases in viable cells. The reduced dye
changes to an orange color and the intensity of color
is proportional to number of living cells, which can be
estimated by reading at 420 nm in a spectrophotometer.
Cells (10,000/well) were initially incubated for 24 h in
a 96 well plate as described above. For treatment with
extracts, media was replaced by serum-free media
containing varying amounts of papaya seeds extract.
The dried water extract was dissolved in serum-free
F-12 or DMEM media whereas dried methanol and
hexane extracts were initially suspended in 50%
DMSO. The concentration of stock solution was
250 mg/mL. The extracts were diluted with serum-
free F-12 or DMEM media to make 0-250 µg/mL
concentrations for treatment. The concentration of
DMSO did not exceed to 0.05% and has no effect on
cell viability.

Determination of total polyphenols
The extracts of papaya seed was used to determine
the total polyphenols as described previously. [26]
Briefly, the extracts was incubated with Folin
Ciocalteu reagent (Sigma Chem. Co., St. Louis, MO)
and the formation of a blue chromophore from the
reduction of phosphotungstic phosphomolybdenum
was determined at 765 nm. The total phenolic content
was calculated from a calibration curve using Gallic
acid as a standard, and the result are expressed as
mg Gallic acid equivalent per g dry weight of sample.
Data analysis
The data is expressed as mean ± SD for at least 3
replicates. All comparisons were made by one-way
ANOVA with Tukey’s-HSD-post-hoc test using SPSS Figure 1: The effect of black papaya seeds on PC-3 prostate
Statistics 20 software. All significant differences are cancer cells. Cells (10,000/well) were incubated with different
reported at P < 0.05 and indicated by “*”. concentration of water (A), methanol (B), and hexane (C) extracts
of papaya black seeds in a CO 2 incubator at 37 C for 24 h. After
o
treatment, cell viability was determined using a WST-1 assay.
RESULTS Results are expressed as mean ± SD for at least 3 replicates.
All comparisons were made by one-way ANOVA with Tukey’s-
When cells were treated with water extract of papaya HSD-post-hoc test using SPSS Statistics 20 software. *Significant
differences between treated and untreated groups were reported as
black seeds, the cells viability initially decreased *P < 0.05
Journal of Cancer Metastasis and Treatment ¦ Volume 3 ¦ August 28, 2017 163