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REVIEW ARTICLE

An Ethanol Extract of Hawaiian Turmeric:

Extensive In Vitro Anticancer Activity Against
Human Colon Cancer Cells
Konstantinos Dimas, PhD; Chrisiida Tsimplouli, PhD; Courtney Houchen, MD; Panayotis Pantazis, PhD;
Nikos Sakellaridis, MD, PhD; George Th. Tsangaris, PhD; Ema Anastasiadou, PhD; Rama P. Ramanujam, PhD

ABSTRACT
Context • Turmeric (Curcuma longa) is a food spice and 3 dilutions—20, 10, and 5 μg/mL. For an invasion assay, 100 µL
colorant reported to be beneficial for human health. per well of Matrigel was added and allowed to polymerize
Curcumin (diferuloylmethane) is the major ingredient in prior seeding of the MDA-MB231 cells. For cultures treated
turmeric, and existing data suggest that the spice, in with the TEx, the TEx was mixed with the cell suspension
combination with chemotherapy, provides a superior prior to the seeding step. For the spheroid testing, the TEx
strategy for treatment of gastrointestinal cancer. However, was added to HCT116 cells either at the beginning of an
despite its significant effects, curcumin suffers from poor experiment (ie, before the addition of the cancer cells), which
bioavailability, due to poor absorption in the body. was a chemopreventive approach, or 48 h later, on the
Objective • The research team intended to evaluate a addition of cells to the wells to allow the generation of
liquid extract of turmeric roots (TEx) that the team had spheroids, which was a chemotherapeutic approach.
formulated for its in vitro, anticancer activity against Outcome Measures • The in vitro activities of TEx were
several human, colorectal cancer cell lines. evaluated using a 48-h-incubation, short-term assay and a
Design • The research team performed in vitro studies 2-wk, long-term (clonogenic) assay. To analyze the anti-
evaluating the anticancer efficacy via short and long-term invasive activity of the extract, images for the Matrigel
assays and also evaluated invasion using Matrigel (Corning invasion assay were taken with a camera at the 24-h time
Life Sciences, Tewksbury, MA, USA). Further, in vitro point. The in vitro, anticancer activity of TEx was also
anticancer activity of TEx was tested against 3-D cultures tested against 3-D cultures of HCT116 spheroids that
of HCT166 spheroids, which were subsequently analyzed were subsequently analyzed using flow cytometry.
by flow cytometry. Results • TEx had potently inhibited the growth of all
Setting • ADNA, Inc, Columbus, OH, USA; Foundation human colon cancer cell lines tested in a dose- and time-
for Biomedical Research of the Academy of Athens, dependent manner. TEx inhibited the formation of
Athens, Greece; and Laboratory of Pharmacology, Faculty HCT116 spheroids when the cells were incubated with the
of Medicine, University of Thessaly, Larissa, Greece. extract. The extract also disrupted the formation of
Intervention • The study used 4 human cell lines of tubules formed by MDA-MB231 cells grown on Matrigel
colorectal cancer—HT29, HCT15, DLD1, and HCT116— at concentrations that did not affect the overall viability of
and 2 breast cancer cell lines—SW480 and MDA-MB231. the cells, indicating a potent anti-invasive activity.
For a short-term assay, the extract was dissolved into culture Conclusions • These data suggest a potential therapeutic
mediums of HT29, HCT15, DLD1, HCT116, and SW480 at activity for TEx against human colon cancer, most likely
four 10-fold dilutions (100 to 0.1 μg/mL). For a long-term due to the enhanced bioavailability of the turmeric.
assay, TEx was added to the cultures of the same cell lines at (Altern Ther Health Med. 2015;21(suppl 2):46-54.)

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Konstantinos Dimas, PhD, is an assistant professor in the generation assay is an excellent model to simulate the
Laboratory of Pharmacology, Faculty of Medicine, development and microenvironmental conditions of in vivo
University of Thessaly in Larissa, Greece, and at the tumor growth. It is assumed that spheroids mimic the tumor
Laboratory of Pharmacology, Foundation for Biomedical (ie, the cells grow in a manner similar to xenografted
Research of the Academy of Athens, in Athens, Greece. tumors).13
Chrisiida Tsimplouli, PhD, is a research associate in the In this study, the research team extracted turmeric from
Laboratory of Pharmacology, Foundation for Biomedical turmeric roots using ethanol. The extract was tested for
Research of the Academy of Athens. Courtney Houchen, MD, activity against the various cells of human, colorectal cancer.
is an associate professor at the University of Oklahoma Health The team also determined the activity of the TEx on HCT116
Sciences Center, College of Medicine, in Oklahoma City. spheroid generation and an invasiveness Matrigel (Corning
Panayotis Pantazis, PhD, is a scientific consultant and and Life Sciences, Tewksbury, MA, USA) model.
Rama P. Ramanujam, PhD, is the president and founder both
at ADNA, Inc, in Columbus, Ohio. Nikos Sakellaridis, MD, METHODS
PhD, is a professor in the Laboratory of Pharmacology, Procedures
Faculty of Medicine, University of Thessaly. George Th. Reagents. All chemicals and cell culture reagents were
Tsangaris, PhD, is a researcher in the Proteomics Research purchased from Sigma-Aldrich (St Louis, MO, USA), unless
Unit, Centre of Basic Research II, Biomedical Research otherwise stated. Matrigel biomatrix was purchased from BD
Foundation of the Academy of Athens, in Soranou Ephessius, Biosciences (Becton Dickinson Hellas, Athens, Greece).
Athens. Ema Anastasiadou, PhD, is a researcher in the Cell Cultures. The 4 human cell lines of colorectal
Department of Genetics and Gene Therapy Unit, Biomedical cancer—HT29, HCT15, DLD1, and HCT116—were obtained
Research Foundation of the Academy of Athens. from the National Cancer Institute at the National Institutes
of Health (Bethesda, MD, USA), whereas the 2 breast cancer
cell lines—SW480 and MDA-MB231—were obtained from
Corresponding author: Konstantinos Dimas, PhD the American Type Culture Collection (Manassas, VA, USA).
E-mail address: [email protected] All cells were adapted to propagate in RPMI-1640 medium
containing 5% heat-inactivated, fetal bovine serum (FBS);
2 mM of L-glutamine; and 1% penicillin-streptomycin. The

T
cell cultures were grown in a 5% CO2 atmosphere of a
urmeric (Curcuma longa) is a food spice and colorant humidified incubator at 37°C.
widely used throughout India and Southeast Asia. Turmeric Extract. Turmeric roots were obtained from
Curcumin (diferuloylmethane), the major ingredient the Hawaiian Organic Farm Association (Hilo, HI, USA). The
in turmeric, is a major constituent of curry powder, to which roots were boiled in water to remove any arsenic contamination
it imparts its characteristic yellow color. For more than 4000 and blended in 100% ethanol at a ratio of 1:1 w/v. The turmeric
years, curcumin has been used in traditional Asian and was incubated at room temperature for 2 weeks in glass jugs
African medicine to treat a wide variety of ailments.1 wrapped in aluminum foil. At the end of the incubation
Experimental research has shown curcumin to be a period, the turmeric was filtered through cheesecloth. The
highly pleiotropic molecule capable of interacting with residue was stored and tested for presence of arsenic at a later
numerous molecular targets in the cell that are involved in time. The liquid portion was allowed to settle for 24 hours. The
inflammation. Based on early cell culture and animal supernatant portion was collected carefully without disturbing
research, clinical trials have indicated that curcumin may the bottom layer. The collected portion is hereafter referred to
have potential as a therapeutic agent iin diseases such as as the turmeric extract (TEx). Samples of the extract were also
inflammatory bowel disease, pancreatitis, arthritis, and subjected to high-performance liquid chromatography
chronic anterior uveitis as well as certain types of cancer.2 analysis and tested for the presence of arsenic. To concentrate
Curcumin has been shown to inhibit the growth of the TEx, the alcohol was allowed to evaporate overnight in a
transformed cells and colon carcinogenesis at the initiation, rotator. The residue was suspended in dimethyl sulfoxide
promotion, and progression stages in carcinogen-induced (DMSO) at a stock concentration of 20 mg/mL and kept at
rodent models. Existing data suggest that curcumin, in -20o C for further tests.
combination with chemotherapy, is a superior strategy for In Vitro Studies Against Cancer Cell Lines. After the
treatment of gastrointestinal cancer, with particular reference evaporation of the alcohol, as described earlier, and the
to colorectal cancer.3-5 Further, curcumin exhibits resuspension of the residue in DMSO, the in vitro activities
antiproliferative effects on human breast cancer cells.6,7 of TEx were evaluated using a 48-hour-incubation, short-
However, despite these significant effects, curcumin suffers term assay and a 2-week, long-term (clonogenic) assay. The
from poor bioavailability, due to poor absorption in the latter evaluates the ability of a single cell not only to survive,
body.8-12 as in the case of the short-term assay, but also to grow into a
Cells cultured as monolayers typically exhibit less colony. The 4 colorectal cell lines—HT29, HCT116, HCT15,
resistance to therapy than those grown in vivo. The spheroid and DLD1—and 1 breast cancer cell line—SW480—were

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used for both the short- and the long-term assays. Cell further removed from the 6-well plate to a 96-well microtiter
viability was assessed at the beginning of each experiment by plate, and the absorbance was measured at 540 nM using the
the trypan blue-dye exclusion method and was always EL-311 BIOTEK microplate reader. Inhibiting concentration
greater than 95%. 50 (IC50) (ie, the concentration that reduces by 50% the
For the short-term assay, cells were seeded into 96-well optical density generated in the treated wells as compared to
cell culture plates in 100 μL of medium at a density of the untreated) was further calculated as the percentage of the
5000 to 10 000 cells per well. Subsequently the plates were absorbance of the treated versus untreated cells.
incubated at standard conditions for 24 hours to allow the Matrigel Invasion Assay. The anti-invasive activity of
cells to resume exponential growth prior to addition of TEx. the extract was assessed in MDA-MB231 cells grown in
Then, to measure the starting cell population, cells in one Matrigel as previously described,17 modified for use in
plate were fixed in situ with trichloroacetic acid (TCA), 96-well, tissue culture plates. Briefly, 100 µL per well of
followed by sulforhodamine B (SRB) staining, as described Matrigel was carefully added to 96-well plates and allowed
elsewhere.14,15 to polymerize at 37°C for 30 minutes. MDA-MB231 cells
To determine the activity of TEx, the extract was dissolved were seeded at 20 000 cells per well and allowed to adhere
into the culture medium at four 10-fold dilutions—from overnight. For wells that were treated with TEx, the TEx
100 to 0.1 μg/mL—to cell culture plates, and incubation was mixed with the cell suspension prior to the seeding
continued for an additional period of 48 hours. TEx solvent step. Images were taken at the 24-hour time point using an
alone (ie, DMSO-containing cell cultures at a maximum final inverted microscope equipped with a camera (Zeiss
concentration of 0.1% v/v) was used as a negative control. That Axiovert 200, Zeiss, Germany). An SRB assay was
DMSO concentration corresponded to the highest level that performed in parallel to confirm that the concentrations
was used in the current study’s experimental set-up. DMSO used were not cytotoxic for the cells under the experimental
was found to be inert at that concentration (data not shown). conditions.
The assay was terminated by addition of cold TCA, followed Generation of Spheroids. Subconfluent, HCT116 cell
by SRB staining and absorbance measurement at 530 nM in an cultures were treated with trypsin, and the detached cells
EL-311 BIOTEK microelisa reader (BioTek, Winooski, VT, were counted and seeded into specialized 96-well plates
USA). Identifying the absorbance measurements as time (NanoCulture Plate, Scivax Corporation, Kawasaki, Japan)
0 = Tz, control growth = C, and test growth in the presence of at a seeding density of 5000 cells per well, following the
the drug at the different concentration levels = Ti, the manufacturers’ instructions. Briefly, before plating, cells
percentage growth was calculated at each of the drug were suspended in the specialized medium that was
concentrations as follows: (1) for concentrations for which provided by the manufacturer with the culture plates.
Ti ≥ Tz, ([Ti – Tz] / [C – Tz]) × 100; and (2) for concentrations According to the manufacturer, this medium (NCM
for which Ti < Tz, ([Ti – Tz] / Tz) × 100. medium) is a set of basal mediums with antibiotic added
Three dose-response parameters were calculated. and FBS. FBS was lot checked for spheroid formation on
Growth inhibition of 50% (GI50) was calculated from the nanoculture plates (ie, the microplates used for the
([Ti – Tz] / [C – Tz]) × 100 = 50 (ie, the calculation provided a seeding of the cells). Prior to performing the experiment,
concentration resulting in a 50% reduction in the net protein several cell densities were tested to select the most
increase as measured by SRB staining in control cells during appropriate for the formation of spheroids. According to
drug treatment). The drug concentration resulting in total the manufacturer, cells seeded into these specific microplates
growth inhibition (TGI) was calculated from Ti = Tz. The attach to an ultrafine, processed mesh structure on the
lethal concentration 50 (LC50) (ie, the concentration of the culture surface, in a spherical morphology. Cells then
drug resulting in a 50% reduction in the measured protein at migrate on the microstructure and continuously
the end of the treatment as compared with that of the contact/adhere to each other and form multicellular
beginning) indicates a net loss of cells following treatment clusters, spheroids that grow larger with proliferation of the
and was calculated from ([Ti – Tz] / Tz) × 100 = -50.15 cells at the same time.
For the long-treatment/clonogenic assay,16 cells from the TEx was added to the cells either at the beginning of the
same cells lines as were used in the short-term assay were experiment (ie, before the addition of the cells), which was
counted, added to 6-well culture plates at a concentration of the chemopreventive approach, or 48 hours later on the
50 cells per well, and left for an additional 24-hour adaptation addition of cells to the wells to allow the generation of
period. After the adaptation period, the TEx was added to spheroids, which was the chemotherapeutic approach. Cells
the cultures at 3 dilutions—20, 10, and 5 μg/mL. Cells were were further incubated at 37oC for up to 240 hours (5 d), and
further incubated for an additional 2-week period. At the images were taken using an inverted microscope equipped
end of that period, the experiment was terminated by adding with a camera.
TCA as for the short-term assay. Then cell colonies were Flow Cytometry. To monitor perturbations in the cell
photographed and further stained with SRB. Finally, the cycle and induction of apoptosis, identical cultures of
absorbance was measured by extracting SRB with nonbuffered HCT116 cells in spheroids were left untreated or treated, as
Tris 10 mM as described earlier. A total of 300 μL were described earlier. The spheroids were subsequently

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Figure 1. Short-term proliferation assay. Treatment of cells dissociated using the dissociation solution provided by the
with 100 μg/mL of TEx killed the cells. manufacturer, treated with ribonuclease (RNase) and stained
Concentration (μg/mL) with propidium iodide immediately before analysis.
Subsequently, the cells were analyzed by flow cytometry with
the aid of an FC500 flow cytometer equipped with a 488-
laser (blue-laser) source (Beckman Coulter, Inc, Nyon,
Switzerland).15

Statistical Analysis
% Growth Rate

All analyses were performed with the aid of Prism 5.0

software (GraphPad Software, San Diego, CA, USA).
Statistical analysis was performed using unpaired student
tests. Differences were considered significant when P < .05.

RESULTS
Effect on Cell Proliferation
The antiproliferative and cytotoxic activity of the TEx on
various human colorectal cells was determined using short-
term proliferation and long-term clonogenic assays. For the
short-term assay, the cells were treated with four 10-fold
dilution concentrations—100, 10, 1 and 0, 1 μg/mL—of TEx
Abbreviation: TEx, ethanolic extract of turmeric. for 48 hours at 37°C. Treatment of cells with 100 µg/mL of
TEx killed the cells (Figure 1), with the exception of the
Table 1. In Vitro Activity of TEx in the Short-Term Assaya DLD1 cells, which were resistant at all concentrations of TEx
tested. Concentrations that were below 10 μg/mL had no
Concentration effect on the growth of the cells.
Cell Line Parameters (μg/mL) The research team next calculated the following
parameters: (1) the GI50 (ie, the concentration that would
HT29 GI50 36.6
inhibit growth of 50% of the cells); (2) the TGI (ie, the
TGI 66.0 concentration that would completely arrest the growth of the
LC50 95.4 cells); and (3) the LC50 (ie, the concentration that would kill
HCT116 GI50 24.3b 50% of the cells) (Table 1). Among all cell lines tested,
TGI 53.5 HCT116 cells were the most sensitive, as depicted by the
corresponding GI50, which was calculated to be 24.3 μg/mL
LC50 82.8
(Table 1).
HCT15 GI50 35.2 To test in a more extended in vivo–related way, the
TGI 59.5 research team subsequently performed a long-term, modified
LC50 83.8 clonogenic assay (Figure 2; Table 2). In the clonogenic assay,
DLD1 GI50 >100 the TEx was more potent than in the short-term assay. TEx,
at concentrations as high as 20 μg/mL, could inhibit
TGI >100
proliferation of all cell lines, including the DLD1 cells, which
LC50 >100 were shown to be the most resistant in the short-term,
SW480 GI50 34.1 48-hour assay. The HCT116 cells were the most sensitive to
TGI 59.2 the TEx treatment, with an IC50 at 4.3 μg/mL, followed by
LC50 84.3 the SW480 cells at IC50 at 7.1 μg/mL (Table 2).

Abbreviations: TEx, ethanolic extract of turmeric; GI50, the Anti-invasive Activity in Matrigel
concentration that inhibits growth of 50% of the cells; TGI, To evaluate the anti-invasive potential of the TEx,
the concentration that completely arrests the growth of the MDA-MB231 cells that are able to form extensive tube
cells; LC50, the concentration that kills 50% of the cells; SRB, networks under normal conditions17 (Figure 3A) were treated
sulforhodamine B; CV, coefficient of variation. with noncytotoxic concentrations (50 and 20 μg/mL) of TEx
for 16 hours (Figure 3D). As can be seen in Figures 3B and
a
The table shows GI50, TGI, and LC50 for the HT29, 3C, both concentrations of TEx were highly active in
HCT116, HCT15, DLD1, and SW480 cell lines, as calculated preventing tubule formation in MDA-MB231 cells, suggesting
in the short-term assay using the SRB method, CV < 10%. a potent anti-invasive activity for the extract.
b
Denotes P = .05.

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Figure 2. Long-term, modified clonogenic assay. TEx was

more potent than in the short-term proliferation assay. 20 μg/mL 10 μg/mL 5 μg/mL ut

100 A 20 μg/mL HCT116

10 μg/mL
90 5 μg/mL
80
% Relative Growth Rate

70 a
HCT15
60 a

50
a
40 a
a HT29
30
20 a a
a
10 a

0 DLD1
HCT116 HCT15 HT29 DLD1 SW480
Human Colorectal Cancer Cell Lines

Abbreviations: TEx, ethanolic extract of turmeric;

ut, untreated cells. SW480

a
Denotes P =.01 compared with the corresponding control.

Figure 3. Anti-invasive activity in Matrigel.

2000
1800
1600
Arbitrary Units (OD)

1400
1200
1000
800
600
400
200
0

ut TEx 50 μg/mL TEx 20 μg/mL

Abbreviations: OD, optical density; ut, untreated cells; TEx, ethanolic extract of turmeric.

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Figure 4. Effect on the formulation of HCT116 spheroids: Table 2. In Vitro Activity of the TEx in the Long-term/
preincubation. Clonogenic Assaya

IC50
Cell Lines (μg/mL)
HCT116 4.3
HCT15 15.8
HT29 17.4
DLD1 14.6
SW480 7.1

Abbreviations: TEx, ethanolic extract of turmeric; IC50,

inhibiting concentration 50; CV, coefficient of variation.

a
The table shows the IC50 values of TEx for the HCT116,
HCT15, HT29, DLD-1, and SW480 cell lines, as calculated
by the long-term/clonogenic assay, CV < 15%.

Figure 5. Effect on the formulation of HCT116 spheroids: Figure 6. Cell count and DNA content.
postincubation.

1023
Events (Cell Counts)

1023 1023

1023 1023

DNA Content

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Effect on the Formulation of HCT116 Spheroids Table 3. FACS Analysis of HCT116 Cells Isolated From
The spheroid generation assay is a useful tool to simulate Spheroidsa
the in vivo environment under in vitro conditions. It is
assumed that spheroids mimic a tumor (ie, the cells grow in a Sub-G0/1 G0/1 S G2/M
manner similar to xenografted tumors).13 In the current study,
ut 19 73.1 5.8 21.1
HCT116 cells were incubated on a special culture plate to
initiate generation of spheroids. HCT116 cells were selected as 10 post 14, 2 73.1 7.6 19.3
they were found to be the most sensitive to TEx activity. The
10 pre 9, 46 71.3 8.1 20.6
cells were either preincubated, the chemopreventive assay, or
coincubated after the formation of spheroids, the 20 post 9, 02 67.9 8.7 23.4
chemotherapeutic assay, with various concentrations of TEx—
20 pre 11, 84 69.5 9.7 20.8
100, 50, 20, and 10 μg/mL. As is demonstrated in Figure 4, TEx
was able to abolish the formation of HCT116 spheroids. 50 post 26, 08 62.1 14.1 23.8
When cells were incubated with 100 μg/ml of the TEx,
50 pre 85, 3 ND ND ND
no spheroids were formed regardless of pre- or postincubation
(Figures 4B, 4C, 5B, and 5C). Fifty μg/mL of TEx inhibited 100 post 86, 2 ND ND ND
the formation of spheroids; however at that concentration,
100 pre 69, 8 ND ND ND
TEx was more effective when cells were preincubated with
the same concentration (Figures 4D, 4E, 5D, and 5E). Finally,
when cells were pretreated with 20 μg/mL, the spheroids that Abbreviations: FACS, fluorescence-activated cell sorting;
were formed seemed to be smaller in size as compared with CV, coefficient of variation; ut, untreated cells; ND, not
the untreated cells at 48 hours postspheroid formation d-performed due to the high percentage of dead cells.
(Figure 4F), but the effect was ultimately abolished. No effect
was observed in the presence of the 10-μg/mL concentration.
a
Analysis of the cells in various phases of the cell cycle was
Finally, the current research team performed a flow- performed only with live cells. Results are the mean of at
cytometry analysis to determine whether the cells were least 2 independent experiments, CV ≤ 12%.
propagating and still alive after 144 hours in culture, with only
1 medium change. The team was also interested in determining Various forms of turmeric extracts have been reported
whether any effect was seen on the cell cycle and type of death to show beneficial properties for human health. Turmeric
induced on TEx treatment. The team observed that the cells extract has been shown to (1) exhibit antioxidant activity,
grew well, although a relatively high percentage of sub-G0/1 protecting rabbits from atherosclerosis18,19; (2) offer
cells, indicative of apoptotic cells, were observed (Figure 6A). protection from irradiation in mouse models20,21; (3) induce
Because of the extreme culture conditions, the death percentage apoptosis of Ehlrich ascitic carcinoma in mice22; and (4) act in
for untreated and solvent-treated control cells was expected to a chemopreventive manner, protecting from cancer.23,24 In a
be high, similar to the spontaneous necrosis observed in partially blinded, randomized, 2-dose pilot study, Bundy et al25
HCT116 and other rapidly growing tumors in xenografts.15 All reported that oral administration of a standardized turmeric
cells treated with 100 μg/mL were dead and, therefore, could extract may help to reduce symptomology for irritable bowel
not be further analyzed by flow cytometry (Table 3). syndrome. However, to the current research team’s knowledge,
A sub-G0/1 population was apparent in 50 μg/mL pre- no evaluation has been done on either the chemoprotective
and posttreated spheroids, but an important difference or the anticancer activity of any type of turmeric extract with
existed between post- and pretreated cells (Figures 6D and regard to human colon cancer.
6E and Table 3). No effect on the cell-cycle phase distribution In the current study, the research team had prepared an
was observed. Finally, no differences were observed in cells ethanolic extract of turmeric (TEx), which was subsequently
treated with 20 μg/mL or 10 μg/mL. The results suggest that tested for antiproliferative and cytototoxic activity on various
the TEx exhibited a significant inhibitory activity on spheroid human colorectal and breast cancer cells, using a short-term
formation, which was time and dose dependent, and that proliferation and a long-term clonogenic assay. The results
TEx can function in a chemopreventive as well from both assays suggested that prolonged incubation times
chemotherapeutic manner. However, it appears that because can greatly improve the activity of the ethanolic extract and
of the time-dependent effect of the extract TEx, it may be that the activity of the TEx is clearly time and dose dependent
more potent in a chemopreventive manner. but also is dependent on the specific colon cancer cell line
(Tables 1 and 2).
DISCUSSION It has been previously demonstrated that cancer cell
Turmeric (Curcuma longa) is an herbaceous perennial lines can form networks on Matrigel and that this property is
plant belonging to the botanical family of Zingiberaceae, the dependent on the invasiveness of the cells.17 Accordingly, a
ginger family, with the rhizome being the most valued plant simple assay was set up in the current study to identify agents
part, used for cooking and medicine. with anti-invasive potential. In that assay, which was

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performed using MDA-MB231 cells, TEx was found to CONCLUSIONS

prevent tubule formation of those cells, thus suggesting a The current study’s findings indicate that TEx, an
potent anti-invasive activity for the extract. ethanolic extract of turmeric roots, may have potential
Finally, it is assumed that formation of spheroids mimics therapeutic benefits against colon cancer, which is a turmeric-
tumor cell behavior in xenografted tumors.13 Therefore, extract property that has been demonstrated, to the research
HCT116 cells were grown in specialized media to form team’s knowledge, for the first time in the current study. The
spheroids, and the effect of TEx on them was determined in team has currently initiated an extensive investigation to
the current study. In that assay, the cells were either optimize the process of extracting TEx at a high concentration.
preincubated or coincubated after the formation of spheroids, Future studies should address the effects of TEx in mice
a chemopreventive or a chemotherapeutic assay, respectively, bearing established human HCT116 tumors as well as other
with various concentrations of TEx. TEx was able to decrease tumors of diverse origin.
the formation of HCT116 spheroids and was more effective
when cells were preincubated with the extract. This result
may demonstrate that TEx could be more potent as a ACKNOWLEDGEMENTS
The research team thanks Fadee G. Mondalek and Sivapriya Ponnurangam for
chemopreventive than as a chemotherapeutic agent. technical assistance. The research team has included Panayotis Pantazis and Rama P.
The subsequent fluorescence-activated cell sorting Ramanujam in the list of authors as memorials: In the first case, in memory of the
(FACS) analysis of the cells that originated from spheroids team’s mentor, collaborator, and friend, who passed away on May 28, 2013, and in the
second case, in memory of the team’s friend and collaborator, who passed away
revealed a sub-G0/1 population consistent with dying cells, unexpectedly on August 28, 2014.
most probably apoptotic, but no differences were observed
with regard to cell cycle phases. Both the spheroid assay and AUTHOR DISCLOSURE STATEMENT
All the authors declare that they had no financial or commercial conflicts of interest
the FACS analysis revealed once again that the effect of TEx related to the current study. The work was partly funded by NIH grant No.
was time and dose dependent. 3R44AT004118-03S1.
In this article, the research team has presented evidence
to demonstrate a significant in vitro, antiproliferative REFERENCES
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