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cytokines -- particularly interleukin (IL)-6, IL-8 and IL-1 Cell culture
-- as a consequence of induced cancer cell death has been KG1a cell line was procured from National Centre for Cell
shown to stimulate replication of CSCs, [13,14] and also Science (NCCS), Pune, India and was maintained under
affect at multiple sites along CSC pathways such as Wnt, standard conditions as per the ATCC guidelines. KG1a
Notch, Hedgehog, and focal adhesion kinase (FAK). [7,12,13] is a variant sub-line of KG1 that is morphologically and
CSCs that are generated as a result of chemotherapy- functionally less mature than KG1. It does not respond to
induced tumor cell death that stimulates the release of colony-stimulating factors in soft agar assays. Cells were
inflammatory cytokines have been reported to be more starved in low serum (0.5% FBS) medium overnight to
refractory to therapy. [13,15,16] This suggests that, for therapy prepare Quiescent Leukemic Cells (QLCs) from them. [36]
to be consistently effective, it must eliminate both CSCs
and non-stem cell cancer cells. QLCs were subjected to various treatments as follows:

Currently research is being done to harness the medicinal 1. Treatment with only curcumin (CU): QLCs were
properties of natural compounds for treating leukemia. [17,18] treated with various concentrations of curcumin ranging
Natural compounds are cheap, are easily available, from 10 µg to 100 µg/mL in growth medium (IMDM
and do not cause any adverse effects. [17,18] Curcumin is supplemented with 20% FBS) for 48 h.
a well-known dietary polyphenol [19-21] and is an active 2. Treatment with only 5-fluorouracil (5-FU): QLCs
ingredient of turmeric that possesses antioxidant and anti- were exposed to varying concentrations of 5-FU (6-
inflammatory activities. [19,21] Its safety and tolerability has 100 µg/mL) in growth medium for 24 h.
been well-established by numerous clinical studies. [19,21] 3. Combinatorial treatment: QLCs subjected to curcumin
It has been shown that curcumin has significant cytotoxic treatment (10-100 µg/mL) for a period 48 h (step 1)
and apoptotic effects on the promyelocytic cell line, HL- were harvested and resuspended in fresh growth
60, suggesting that it may have a potential therapeutic medium containing 6 µg/mL of 5-FU. The cells were
role for human leukemia. [22-25] A study conducted by further incubated for a period of 24 h after which
Fong et al. showed that curcumin inhibited the side they were subjected to various biochemical assays as
[26]
population (SP) phenotype of the rat C6 glioma cell line, described below.
demonstrating for the first time in vivo that curcumin has 3-(4,5-Dimethylthiazol-2-yl)-2,
anticarcinogenic and antimetastatic activity in the brain. 5-Diphenyltetrazolium Bromide (MTT) assay
Another study demonstrated that curcumin is able to target QLCs exposed to various treatments were subjected to a
breast stem/progenitor cells, as evidenced by suppressed standard MTT assay as discussed before, and the percent
mammosphere formation along serial passage and by proliferation was determined. [36]
a decrease in the percent of aldehyde dehydrogenase
(ALDH)-positive cells. [27-29] Growth curve experiment
Growth curve experiment was performed to determine
To summarize, several cell and animal studies have the doubling time. QLCs were treated with 10 µg/mL
demonstrated and corroborated the apoptotic activity curcumin, and viable cell counts were taken at specified
and anticancer effect of curcumin in different types time intervals using trypan blue dye exclusion method.
of cancers, [30-34] and recent research has shown that
curcumin can also target CSCs. In the present work we Flow cytometric analysis of cell cycle
[35]
demonstrate that curcumin, at low concentrations, induces Cell cycle analysis using propidium iodide (PI) staining
proliferative responses in QLCs, thereby sensitizing them was performed to distinguish the cells in various stages
to the antimitotic drug, 5-fluorouracil (5-FU). of the cell cycle. Briefly, the QLCs exposed to various
treatments were stained with PI, after which analysis of the
METHODS cell cycle was performed using BD FACSCalibur TM (BD
Biosciences, USA).
Reagents
Curcumin, Fetal Bovine Serum (FBS), Griess Reagent, Nitric oxide (NO) assay
Dichloro-dihydro-fluorescein diacetate (DCFH-DA), Nitric oxide assay was performed using modified Griess
Propidium Iodide (PI), 3-(4, 5-Dimethylthiazol-2-yl)-2, reagent for the colorimetric detection of NO production
5-Diphenyltetrazolium Bromide (MTT), 5- Fluorouracil by QLCs subjected to curcumin and/or antimitotic drug
(5-FU), Dimethyl Sulfoxide (DMSO), RNase-A were treatment(s) at 540 nm using BioTek™ Eon™ Microplate
purchased from Sigma-Aldrich, USA; Iscove’s Modified Spectrophotometer (USA).
Dulbecco’s Media (IMDM), L-Glutamine, Antibiotic
Solution (Penicillin + Streptomycin), Trypan Blue Dye, 2’, 7’-dichlorofluorescein diacetate (DCFDA)
Phosphate Buffer Saline (PBS) were purchased from assay
Himedia, India; Caspase-3 Colorimetric Assay Kit was QLCs exposed to curcumin and/or antimitotic drug
purchased from RayBiotech. treatment(s) were checked for the generation of Reactive
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Journal of Cancer Metastasis and Treatment ¦ Volume 2 ¦ July 8, 2016 ¦