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Figure 6: Combination therapy promotes higher caspase-3 activity in
quiescent leukemic cells. QLCs were treated as represented in the graph.
The treated cells were harvested and subjected to caspase-3 assay. Figure 8: QLCs subjected to combination treatment generate higher
Intensity of the color was measured at 400/405 nm by using microplate levels of ROS: Quiescent cells generated from KG1a cells were incubated
spectrophotometer. In the graph, VC represents vehicle control; 10CU with low concentrations of curcumin only (10 µg/mL and 20 µg/mL), 5-FU
represents the cells treated with curcumin only (10 µg/mL); 5-FU represents only (6 µg/mL) and a combination of curcumin and 5-FU, as shown in
cells treated with 5-FU only (6 µg/mL), and 10CU + 5-FU represents the graph. The treated cells were collected and spun, and the cell pellet
combination treatment of 10 µg/mL of curcumin and 6 µg/mL of 5-FU. Serum- was incubated with 100 µL of DCFDA (10 µm) for 30 min at 37°C. After
starved KG1a cells treated with 10 µg/mL of curcumin induce minimum incubation the cells were centrifuged, and the pellet was resuspended in
caspase-3 activity as compared to VC. Cells treated with a combination of 1 × PBS. The fluorescence was measured at 495-529 nm. In the graph,
10 µg/mLof curcumin and 6 µg/mL of 5-FU induces maximum caspase-3 PC represents positive control that is hydrogen peroxide (50 µm), VC
activity as compared to the cells exposed to only 5-FU. The data represent represents vehicle control; 10CU and 20CU are cell samples treated with
mean ± S.E.M of three independent experiments (* P ≤ 0.05, *** P ≤ 0.001) curcumin only (10 µg/mL and 20 µg/mL, respectively), 5-FU represents 5-FU
only (6 µg/mL) treated cells, and 10CU + 5-FU, 20CU + 5-FU represents
combination treatment of curcumin (10 µg/mL or 20 µg/mL) and 5-FU (6 µg/mL),
respectively. Quiescent KG1a cells treated with only 10 µg/mL or 20 µg/mL of
curcumin generated minimum amount of ROS, as compared to VC. QLCs
treated with a combination of 10 µg/mL or 20 µg/mL curcumin, respectively,
and 6 µg/mL of 5-FU showed maximum ROS generation as compared to
the cells treated with only 5-FU. The data represent mean ± S.E.M of three
independent experiments (* P ≤ 0.05, ** P ≤ 0.01)
drug 5-FU.

Low concentrations of curcumin and 5-FU
together increase the levels of nitric oxide in QLCs
Since low concentrations of curcumin caused the
proliferation of QLCs that were sensitive to 5-FU
treatment, we wanted to check whether the kill seen in
QLCs was being mediated by the expression of nitric oxide
Figure 7: Combination treatment with low curcumin concentrations and
5-FU leads to higher generation of NO in quiescent KG1a cells: QLCs were (NO). NO is known to react with superoxide at a high rate
9
subjected to various treatments as shown in the graph. After treatment, (k ≥ 6.7 × 10 M s ) to form peroxynitrite, which is far
−1
−1
the cells were spun, supernatant was collected into fresh plates and to the more reactive and damaging than its precursors. The
[40]
supernatant was added an equal volume of Griess reagent. The plate was
incubated for 15 min in dark at room temperature (RT) and the intensity downstream products of superoxide, including hydrogen
of color was measured at 540 nm using microplate spectrophotometer. peroxide and peroxynitrite, are potent oxidants that induce
PC represents positive control, that is sodium nitrite solution (50 µm). VC
represents vehicle control; 10CU and 20CU represent the concentrations oxidative injury of cells, resulting in apoptosis. [41,42] Nitric
of curcumin used, that is cells treated with only 10 µg/mL and 20 µg/mL oxide assay determines nitric oxide based on the enzymatic
respectively; 5-FU represents cells treated with only 6 µg/mL of 5-FU;
10CU + 5-FU, and 20CU + 5-FU represents cells treated with combinatorial conversion of nitrate to nitrite by nitrate reductase. [43,44] The
treatment of curcumin (10 µg/mL or 20 µg/mL) and 5-FU (6 µg/mL). Serum- reaction is followed by a colorimetric detection of nitrite
starved KG1a cells treated with 10 µg/mL and 20 µg/mL of curcumin
generated minimum amount of nitrites as compared to VC, whereas cells as an azo dye product of the Griess reaction, which absorbs
treated with a combination of 10 µg/mL or 20 µg/mL of curcumin and 6 µg/mL light at 540 nm. As seen in Figure 7, cells that were treated
of 5-FU, respectively, showed maximum levels of nitrite as compared to the
cells treated with only 5-FU. The data represents mean ± S.E.M of three with a combination of both curcumin and 5-FU expressed
independed experiments (* P ≤ 0.05, ** P ≤ 0.01) higher levels of NO than cells that were treated with only
curcumin or only 5-FU.
QLCs that were being targeted by 5-FU were undergoing
apoptosis. To check the apoptotic profile of QLCs treated Combination treatment of quiescent KG1a cells
with both curcumin and 5-FU, we performed caspase-3 with low concentrations of curcumin and 5-FU
assay as per manufacturer’s instructions. As seen in Figure stimulates higher generation of reactive oxygen
6, there was indeed a high caspase-3 activity in cells treated species
with combination therapy when compared to those treated Generally, the production of ROS by mitochondria is a
with only 5-FU. This confirms that curcumin sensitized the consequence of the blockade of the electron transfer chain.
QLCs to undergo apoptosis in presence of the antimitotic It has been well documented that NO can inhibit the activity
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Journal of Cancer Metastasis and Treatment ¦ Volume 2 ¦ July 8, 2016 ¦