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Oxygen Species (ROS) by DCFDA assay. QLCs were Table 1: Doubling time of untreated versus treated
incubated with 10 µM DCFDA for 30 min at 37°C. After QLCs
incubation, 2’, 7’-dichlorofluorescein (DCF) was measured Doubling time in hours
at 495-529 nm by using a fluorometer (Fluoroskan Ascent, Day Time interval
Thermo Fisher Scientific, USA). in hours Untreated QLCs + curcumin
QLCs (10 µg/mL)
Apoptotic assay 1 0-24 23.99 11.99
This assay was performed as per manufacturer’s instructions 2 24-48 11.99 6.14
(RayBiotech) to estimate the caspase-3 activity of the 3 48-72 29.74 13.97
QLCs. The intensity of the color was measured at 400/405
nm by using a spectrophotometer reader (BioTek™ Eon™ 4 72-96 36 18.15
Microplate, USA). 5 96-120 44.8 28.2
6 120-144 35.3 20.5
Statistical analyses QLCs: quiescent leukemic cells
The data were analyzed by One-way Repeated Measure
Analysis of Variance (One-Way RM ANOVA). The
Standard Error of Mean (S.E.M.) values were used for
plotting the error bar graphs, using the SigmaPlot software
(version 13.0). Level of significance was denoted as
follows: ∗P ≤ 0.05, ∗∗P ≤ 0.01 and ∗∗∗P ≤ 0.001.

RESULTS

Low concentrations of curcumin induce
proliferation of quiescent leukemic cells
It has been shown that curcumin inhibits cell proliferation,
causes cell cycle arrest, and initiates apoptosis in several
human cancer cell lines. [37,38] We first wanted to determine Figure 1: QLCs undergo proliferation in response to low concentrations
the concentration(s) of curcumin that would be most of curcumin: Serum-starved KG1a cells were treated with varying
effective against the QLCs. Hence we cultured the serum- concentrations of curcumin (10-100 µg/mL) for 48 h and were subjected to
starved KG1a cells for 48 h with various concentrations MTT assay. Treatment with low concentrations (10 µg/mL and 20 µg/mL)
of curcumin led to proliferation of QLC cells as compared to the vehicle
(10-100 µg/mL) of curcumin. We were expecting to control (VC) cells. The data represent mean ± S.E.M. of three independent
see a dose-dependent inhibitory effect of curcumin on experiments (*** P ≤ 0.001)
the QLCs. Intrestingly, however, we observed that low
concentrations of curcumin (10 µg/mL and 20 µg/mL)
stimulated the cells to undergo proliferation, whereas at all
other concentrations of curcumin (30 µg/mL to 100 µg/mL)
imparted inhibitory effects [Figure 1]. Our proposition is
that since KG1a cells are known to contain leukemia-like
stem cells, low concentrations of curcumin could have
[16]
induced the leukemia-like stem cells to proliferate.
Curcumin results in an increased cell yield by
reducing the doubling time of QLCs
Since low concentrations (10 µg/mL and 20 µg/mL)
of curcumin led to proliferation of the QLCs, we next
wanted to know whether treatment with curcumin would
alter the cell cycle kinetics. Serum-starved KG1a cells Figure 2: Treatment with curcumin reduces the doubling time of QLCs:
were treated with 10 µg/mL of curcumin, and viable cell QLCs were treated with 10 µg/mL of curcumin. Viable cell count was taken
at an interval of 24 h for 6 days. Although minimum doubling time (6.14 h)
counts using trypan blue dye exclusion method were taken was observed at Day 2 (24-48 h), the overall doubling time of curcumin-
at every 24 h interval for a period of 6 days. As seen in treated cells was always lower than the doubling time of vehicle control
(VC). The data represent mean ± S.E.M of three independent experiments
Table 1, it was observed that till day 4, the doubling time (** P ≤ 0.01)
of QLCs treated with curcumin was reduced to almost
half that of the untreated cells. The minimum doubling 5-Fluorouracil inhibits the proliferation of
time of 6.14 h was observed on the second day. It is also QLCs in a dose-dependent manner
important to note that the doubling time of curcumin- The presence of leukemic stem cells (LSCs), also
treated cells was lower than that of untreated cells for all known as cancer stem cells (CSCs), is a major problem
6 days [Figure 2]. in the treatment of leukemia. The LSCs are refractory
Journal of Cancer Metastasis and Treatment ¦ Volume 2 ¦ July 8, 2016 ¦ 247