Conclusions

Results

Curly Kale Brassica oleracea var. sabellica Induces Apoptosis in Cultured Mouse Melanoma Cells

Bilal Qizilbash, Matthew Bear, Jacob Morgan, Alexandria Niswonger, Maxwell Schwam, Matthew Taitano, Elizabeth Brandon, Ph.D.

Department of Biology, Mississippi College, Clinton, Mississippi

Abstract The challenge with many cancers is not just killing the malignant cancer cells, but doing so in a non-toxic manner.

Numerous studies have been conducted on curly kale, B. oleracea sabellica, to identify some of the compounds responsible for the health benefits of consuming the plant, in its raw or juiced form. Much of this research focuses on sulforaphane, an isothiocyanate that’s also found in foods such as broccoli, brussel sprouts, and cauliflower. Sulforaphanes have been shown to decrease cell proliferation, reduce inflammation, and induce protective autophagy in vitro. Our idea is that the natural context in which sulforaphanes and other bioactive compounds exist in curly kale creates a concert of both agonist and antagonistic factors that could have significant anticancer effects. To test this hypothesis, we prepared kale juice in two forms: unfiltered and sonicated juice that was subsequently filter sterilized. Serial dilutions were tested on melanoma cells to determine the optimum dosage for a four day treatment. We found a dose-dependent decrease in cell growth and chose the lowest concentration of juice for our experiment. Melanoma cells treated with unfiltered juice were killed by day four. Cells treated with the sonicated filtered juice had significantly reduced growth (8.63e5 cells/day ± 52,700 cells for control v. 1.05e5 cells/day ± 8,660 for kale juice, p=0.01). Western blot analysis with a poly-ADP ribose polymerase (PARP) antibody show two PARP fragments in the lysates from cells treated with kale juice. This suggests that the unfiltered and sonicated filtered juices induce apoptosis. These experiments will be repeated in a normal mouse epithelial cell line to determine the toxicity in non-cancerous cells. If the sonicated filtered juice is significantly less toxic to normal cells, then we will work toward examining the safety and efficacy of kale juice for treating or augmenting the treatment of melanoma in vivo.

Background

Juicing:We utilized the Breville IKON BJE510XL for our juicing. It contains a stainless steel micromesh filter of unknown pore size (Breville would not reveal micromesh pore size). This juicer was used due to it’s consistent quality, speed, and efficacy in producing kale juice. Pulp mostly came out relatively dry resembling sawdust. Curly kale is a fibrous vegetable and can place a considerable strain on most juicer motors. The kale was acquired at a local grocery store. It was washed with Dawn dishwashing soap to remove any pesticides and/or dirt. It was then rinsed seven times with over 4 gallons of water to ensure no soap residue remained. After the juice was collected into sterile containers, it was stored at -20ºC. Several days later, it was thawed and divided into smaller aliquots of 150 µL and then frozen again.

Treatments:Three forms of kale juice were prepared. 1) Pure kale juice, which was referred to as the Unfiltered treatment. 2) Filter sterilized juice. This juice was filtered through a 0.22 micron filter into a sterile container. This treatment was referred to as Microfiltered.3) An thawed aliquot of kale juice was sonicated 7 times in 10 second bursts with a Qsonicator (125 Watts) set at 70% of 20 kHz. The sonicated juice was then filter sterilized using a 0.22 micron filter. This treatment was referred to as Sonicated.

Experiments:Dose response curvesWe plated 100,000 cells per well (4 replicates per treatment) in a 24 well plate with 1mL of our standard 5% FBS RPMI medium. After 24 hours, five dosages were applied. We tested: 100, 200, 300, 400, and 1000 µL/mL of culture medium. Cells were incubated for 24 hours and then counted using a Nexcelom AutoT4 automated cell counter. Growth Curves75 cm2 cell culture flasks were used to grow the B16F10 mouse melanoma cells because this would allow enough space for the flask to reach confluency over the course of a week. We started each flask with 200,000 cells in 13mL of RPMI with 5% FBS. The kale juice was added at a concentration of 7 µL/mL/day (0.7%). The medium was changed daily and the cells were washed with PBS before application of new treatment. Before obtaining the cell counts, each flask was visually inspected to see if live cells were present. Cells were trypsinized and counted every four days with a Nexcelom AutoT4 automated cell counter. The number of cells, mean cell diameter, and cell concentration/mL were recorded for every count. Each experiment was repeated three times.

AcknowledgementsThis work was supported by Dr. Elizabeth Brandon, Bilal Qizilbash, and the Mississippi INBRE funded by grants from the National Center for Research Resources (5P20RR016476-11) and the National Institute of General Medical Sciences (8

P20 GM103476-11) from the National Institutes of Health.

References1. ED Michelakis, The Metabolism of Cancer Cells. British Journal of Cancer (2008), 989-994. 2. Picture taken by Wikipedia User Rasbak : Curly kale Boerenkool; {{GFDL}} Brassica oleracea3. Retinoic acid slows progression and promotes apoptosis of spontaneous prostate cancer Huss WJ, Lai L, Barrios RJ, Hirschi KK, Greenberg NM. Prostate. 2004 Oct 1;61(2):142-52.4. Kaempferol induces apoptosis in glioblastoma cells through oxidative stress. Sharma V, Joseph C, Ghosh S, Agarwal A, Mishra MK, Sen E. Mol Cancer Ther. 2007 Sep;6(9):2544-53.5. Molecular iodine induces caspase-independent apoptosis in human breast carcinoma cells involving the mitochondria-

mediated pathway. Shrivastava A, Tiwari M, Sinha RA, Kumar A, Balapure AK, Bajpai VK, Sharma R, Mitra K, Tandon A, Godbole MM. J Biol Chem. 2006 Jul 14;281(28):19762-71.

6. A Review on the Dietary Flavonoid Kaempferol. J.M. Calderón-Montaño, E. Burgos-Morón, C. Pérez-Guerrero and M. López Lázaro. Mini-Reviews in Medicinal Chemistry, 2011, 11, 298-344

7. Antiproliferative Effects of Fresh and Thermal Processed Green and Red Cultivars of Curly Kale ( Brassica oleracea L. convar. acephala var. sabellica ). Olsen H, Grimmer S, Aaby K, Saha S, Borge GI. J Agric Food Chem. 2012

Aug 1;60(30):7375-83

Representative images of A) untreated melanoma cells or cells treated with 0.7% B) unfiltered, C) sonicated, or D) microfiltered kale juice for 24 hours. Phase contrast images obtained on a Nikon Inverted Eclipse Ti-E microscope with a Nikon DS-Fi2 color camera. Scale bar = 50 µm.

Western Immunoblot for cleaved PARPWe performed a Western blot check for cleaved PARP juiced curly kale treated cells. 12-well plates were used to grow the B16F10 mouse melanoma cells with 200,000 cells/well in 2mL of our standard 5% RPMI/well. We let the cells plate for a 24 hour period before treatments were administered. After the 24 hour plating period, each well was treated with 100 microliters of each respective treatment (Unfiltered, Microfiltered, Sonicated) The cells were incubated for 24 hours in their treatment and were collected and washed with phosphate buffered saline (PBS). Cells were centrifuged at 1,000 x g for 10 min. The supernatant was discarded and the cell pellet was resuspended with RIPA buffer (50mM Tris-HCl pH8, 1% NP-40, 0.5% DOC, 0.1% SDS, 150mM NaCl) with protease and phosphatase inhibitors. The cells were sonicated with three 15-second bursts at 40%, then centrifuged at 10,000 x g for 10 min. The supernatant was saved and a BCA protein assay was performed. 60 µg of samples were subjected to SDS-PAGE on a 4 to 20% gradient gel. The samples were then transferred to PVDF, blotted with rabbit α-PARP (poly-ADP ribose polymerase) (Cell Signaling Technologies) and mouse α-tubulin (Sigma). Secondary antibodies were produced in goat (Jackson Immunoresearch) and conjugated to horse-radish peroxidase. Pierce Super Signal Westpico substrate was used for detection. Images were obtained in a Bio-Rad Gel-doc system.

Cell diameters were monitored over the course of the experiment to determine if the treatments caused any osmotic effects. The mean cell diameter was obtained when cells were counted with the Auto T4 automated cell counter.

Western blot of melanoma cells treated with kale juice. Lysates (50 µg/lane) were subjected to SDA-PAGE and immunoblotting with anti-PARP and anti-tubulin antibodies. Lane 1: untreated, lane 2: filter sterilizsed, lane 3: sonicated, lane 4: unfiltered. Tubulin (~55kDA) served as the loading control. 5B Curly Kale Brassica oleracea var. sabellica (2)

A B

A. Untreated B. Unfiltered

C. Sonicated D. Microfiltered

Melanoma cells were treated with kale juices (7 µL/mL/day) until there were no live cells left in the flasks. Results are reported as the number of live cells. Growth rates are the slopes of the linear regression lines for data gathered at time zero and on day 4. Data from cell counts on day 8 are not shown, since the cells in all groups were beginning to decrease in number. Student’s T-test for control versus sonicated groups gave a p value of 0.03. Error bars represent the standard error of the mean.

tubulin

PARP

• The unfiltered treatment induces cell death within 8 hours. All cells in the flasks died by day 2.5 of treatment.

• Kale juice that is sonicated prior to filter sterilization is more effective in killing the cells than the juice that is only filter sterilized.

• The use of kale juice at a concentration of 0.7% did not produce an osmotic effect on the cells.

• All three kale juice treatments cause PARP cleavage.• Kale juice retains its potency after two freeze-thaw cycles.

M 1 2 3 4

1. Melanoma cells are sensitive to dilute solutions of curly kale juice.2. Curly kale juice appears to induce apoptosis, though we did not check to see if DNA

was fragmented in treated cells.3. Sonication seems to increase the activity or bioavailability of some of the chemical

compounds in the juice. 4. This is the first study to examine the effect of whole vegetable juice on melanoma

cells in vitro.

• The same procedure will be used in normal mouse epidermal and hepatocytes cells to determine the toxicity to non-cancerous cells.

• The speed with which the unfiltered juice kills melanoma cells warrants further investigation into the mechanism. Perhaps juicing releases intracellular bacteria which get strained during filter sterilization.

• If sonicated filter sterilized kale juice has a low toxicity to non-cancerous cells, we would like to test the efficacy of a topical preparation to treat melanoma on mice in vivo.

The war on cancer, which began in 1971, continues with few drugs that selectively kill tumor cells despite the wide array of molecular targets. Tumor cells’ astounding adaptability explains much of the poor performance of some of the current therapies. Drugs designed to inactivate certain receptor tyrosine kinases have a brief success that’s followed by the development of resistant cells. Similarly, drugs designed to induce enough DNA damage to trigger apoptosis are effective until the genes encoding signaling proteins required for cell death become silenced or mutated. Multi-drug resistance can occur if tumor cells increase their expression of certain ABC transporters in the plasma membrane. Genetically engineered viruses for virotherapy and tumor vaccines designed to enable MHC class I molecules to present tumor antigens to immune cells have shown efficacy in animal studies, yet few such therapies have been tested in humans in large numbers. Drug cocktails are more effective, but may worsen side effects and even the composition of these cocktails has to be adjusted as tumor cells develop resistance. Adaptability is an emergent property of cooperation among heterogeneous tumor cells and between tumor cells and stromal cells. The battle raging between clinicians and tumor cells is an arms race with escalating costs and proportionally small decreases in human suffering.

Humans have been gathering and archiving knowledge of medicinal plants for tens of thousands of years. Plants are the basis for many chemotherapies, e.g. paclitaxel, vincristine, and actinomycin D. Natural products research provides society with untold numbers of life saving drugs. One of the unavoidable downsides to this is that decades of research go into determining the mechanism of action for potential bioactive compounds extracted from plants and getting the compounds into clinical trials. This timeframe is too long for the millions of people diagnosed with a dangerous cancer each year. A useful line of investigation may be testing juices from plants considered to have high levels of anti-oxidants, sulforaphanes, and chemoprotective or cytostatic compounds, such as blueberries, broccoli, and kale.

Curly kale is a nutritious, chemical powerhouse. It contains many flavonoids, vitamins A and K, and iodine. Some of the same compounds found in curly kale have been shown to induce apoptosis in various cell cancer lines. For example, one study found that high concentrations of retinoic acid slowed the progression of prostate cancer cells (3). Kaempferol induced apoptosis in glioblastoma cells (4). Iodine has been shown to decrease the apoptotic protein Bcl-2 and increase the proapoptotic protein Bax in breast cancer cells and to decrease the rate of proliferation of peripheral blood mononuclear cells (5). This is also some evidence to suggest that kale compounds have antiproliferative effects on colon cancer cells (7). With all of these various effects of individual components within kale, we were interested to see what it did in its whole, uncooked form, since cooking or heating kale can alter its chemical properties (7).

Dose response curves with different tumor and normal cell lines are easy to generate and inexpensive to perform. This kind of research is ideal for undergraduates or even graduate students at small colleges. Promising results can be published and shared with investigators at research institutions with the capability to carry out pilot experiments in vivo.

Time course of kale juice treatments. Cells were trypsinized and counted on days 4 and 8. Untreated cells became confluent by about day 5-6 and began dying off during days 7-8. Cells treated with sonicated kale juice never reached confluency and began dying after day 4.

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Methods

Future Studies