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Hadadi et al. J Cancer Metastasis Treat 2018;4:25 I http://dx.doi.org/10.20517/2394-4722.2018.22 Page 3 of 15

The papaya (Carica papaya Linn) tree that belongs to a family Caricaceae, is originated in southern Mexico
and Costa Rica. Now it is grown all over the world including Australia, Hawaii, Philippines, Sri Lanka,
South Africa, India, and in all tropical and subtropical regions. Some counties produce papaya on a
[27]
commercial scale for export; however, in most of the tropical regions it can be grown in home gardens .
Traditionally, all parts of papaya including roots, seeds, flowers, fruit, latex, barks, and leaves have been used
to treat a number of diseases in various regions in the world. Papaya has also been studied for its anticancer

[32]
[31]
[28]
activities for colorectal , prostate [29,30] , cervical and breast cancers . The fruit, seeds, or leaves extracts
of papaya have been shown to possess cytotoxic and anti-proliferative activities for a number of cancer cells
lines including breast (MCF-7), liver (HepG2) and cervical carcinoma (Hela), lung adenocarcinoma (PC14),
oral squamous cell carcinoma (SCC25), pancreatic epithelioid carcinoma (Panc-1), mesothelioma (H2452),
and cancer of haematopoietic cell lines, including T cell lymphoma (Jurkat), plasma cell leukemia (ARH77),
Burkitt’s lymphoma (Raji), and anaplastic large cell lymphoma (Karpas-299) and human promyelocytic
+
leukaemia (HL-60) [33-35] . The lipophilic extracts of papaya pulp inhibited cell proliferation of ER breast
[36]
-
cancer MCF-7 cells but did not inhibit ER breast cancer MDA-MB-231 cells . During present investigation,
the effect of papaya extracts from leaves, skin, pulp and seeds were assessed on estrogen and Her-2-
dependent and -independent breast cancer using representative cells lines.

METHODS
Materials
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MDA-MB-231 (ER/PR/Her-2 ; triple negative), MCF-7 (ER /PR /Her-2), SK-Br-3 (ER/PR/Her-2 ), AU565
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+
+
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(ER /PR /Her-2 ), and MDA-MB-361 (ER /PR /Her-2 ) breast cancer cell were purchased from ATCC
(Manassas, VA 20110). F-12K (21127-022) media was purchased from Gibco (Grand Island, NY14072). Fetal
bovine serum was purchased from RAMBIO (Missoula, Montana). Antibiotics: penicillin and streptomycin,
and phosphate buffered saline was purchased from Fisher (Fair lawn, New Jersey). Folin-Ciocalteu,
aluminum chloride, Diphenyl-1-picrylhydrazyl (DPPH), quercetin, gallic acid, and Trolox were purchased
from Sigma Chemical Co (St Louis, MO). Green papaya was obtained from Randolph Farm at Virginia State
University. WST-1 (MK400) was purchased from Talkara (Kusatsu, Shiga, Japan).
Isolation of papaya leaves, skin, pulp, and seeds
Unripe green papaya (2-3 kg) was obtained from Randolph Farm at Virginia State University, Petersburg
VA. The papaya was washed with distilled water, then blotted dry with paper towel. The skin was peeled off
using a kitchen peeler. The unskinned papaya was cut into half to remove seeds and then the pulp was cut
into small pieces. The leaves and seeds were washed with distilled water. All fractions (leaves, skin, pulp
and seeds) were spread on a plastic trays and left for drying in a chemical hood until a constant weight was
obtained. The dried leaves, skin, pulp and seeds were ground to a fine powder using a mortar and pestle. The
o
dried powder was flash frozen with nitrogen and stored at -80 C until used.
Preparation of leaves, skin, pulp and seeds extract
A known quantity (5 g) of dried papaya powder was mixed with 200 mL of 80% methanol, 60% ethanol, or
100% distilled water. The mixtures were placed on a shaker at room temperature overnight. The next day,
the mixture was centrifuged at 1500 g for 20 min using a Thermo Scientific centrifuge (Waltham, MA). The
supernatant was collected and the residues were washed 2 times by suspending them again in the respective
solutions, mixing, and placing on shaker overnight. The collected supernatant was pooled together and
the residues were discarded. The ethanol and methanol extracts were dried in a nitrogen evaporator
(Organomation Associates, Inc, Berlin, MA) whereas the water extract was freeze dried. The dried extract
o
was stored in a -20 C freezer.
Determination of total phenolic content
The total phenolic content (TPC) of papaya extract was determined by using Folin-Ciocalteu method as