The synergistic reversal effect of multidrug resistance by quercetin and hyperthermia in doxorubicin-resistant human myelogenous leukemia cells

Figures & data

Figure 1. Inhibitory effects of various concentrations of Que on both K562 and K562/A cell proliferation. Each point represents the mean value for three independent experiments.

Table I.  Effect of Que and HT to Dox cytotoxicity in K562 and K562/A cells.

Group	(IC50 µg/ml) (K562 cells)	IC50 µg/ml) (K562/A cells)	RF(K562/A cells)
Dox	0.31 ± 0.08	17.65 ± 0.92
Dox + HT	0.19 ± 0.05	14.14 ± 1.62	1.25
Dox + Que	0.16 ± 0.02	8.65 ± 1.19	2.04
Dox + HT + Que	0.15 ± 0.04	1.86 ± 0.89	9.49

The values represent mean ± SD of three independent experiments.

Figure 2. The expression of HSP70 and P-gp in K562/A cells. K562/A cells were heated with or without Que pretreatment and expression of HSP70 and P-gp was evaluated by FCM after 24 h recovery period at 37°C. A P-gp expression; B HSP70 expression; a-d: different groups (a, control; b, HT; c, Que; d, Que + HT, the filled area represents the normal naked cells).

Table II.  Percentage of K562/A cells with positive P-gp and HSP70 in different groups (gate%).

	Expression positive rate (gate%)
Groups	HSP70	P-gp
Control	72.09 ± 3.03	87.98 ± 2.52
HT	94.54 ± 1.81	90.73 ± 1.26
Que	55.13 ± 2.84	73.21 ± 3.67
HT+Que	71.25 ± 2.73	45.26 ± 4.83

Each value represents mean ± SD of three separate experiments.

Figure 3. RT-PCR analysis of mdr1 and HSP70 mRNA expression. (A) mdr1 gene expression; (B) HSP70 gene expression; (C) GAPDH gene expression (Lane 1, DNA maker; Lane 2, control; Lane 3, HT; Lane 4, Que + HT).

Figure 4. Effect of HT and Que on intracellular Dox accumulation in K562 and K562/A cells. Cells were incubated with medium containing 10 μg/ml Dox for 3 h with or without Que pretreatment when cells were heated. (A) Sensitive K562 cells; (B) resistant K562/A cells; a-d: different groups (a, Dox; b, HT + Dox; c, Que + Dox; d, Que + HT + Dox; the filled area represents normal naked cells).

Table III.  Intracellular Dox content of K562 and K562/A cells (gate%).

Cell line	Dox	HT+Dox	Que+Dox	Que+HT+Dox
K562	35.61 ± 4.69	76.75 ± 3.75**	34.65 ± 1.51	84.85 ± 1.73**
K562/A	4.70 ± 2.27	11.09 ± 1.95*	24.42 ± 2.14**	39.09 ± 1.54**

Each value represents mean ± SD of three separate experiments. *Compared with Dox group, P < 0.05. ***Compared with Dox group, P < 0.01.

Table IV.  Effects of Que and HT on cell cycle in K562/A cells.

Cell cycle % cells	Sub-G1	G1	S	G2/M
Control	0.02 ± 0.01	21.69 ± 2.08	65.25 ± 3.66	13.06 ± 1.59
100 µM	1.31 ± 0.21	19.02 ± 3.29	61.89 ± 3.34	19.09 ± 2.84
120 µM	3.12 ± 1.30	28.02 ± 2.34	37.31 ± 1.24	34.67 ± 3.55
140 µM	4.38 ± 0.81	23.00 ± 4.73	30.62 ± 2.85	46.37 ± 2.11
41°C	0.04 ± 0.03	25.76 ± 2.37	59.88 ± 1.55	14.35 ± 0.81
42°C	0.14 ± 0.03	25.04 ± 1.71	58.63 ± 2.76	16.33 ± 1.05
43°C	0.19 ± 0.08	32.63 ± 1.38	39.03 ± 1.21	28.35 ± 1.18
Que + HT	11.4 ± 0.94	15.25 ± 1.57	55.46 ± 3.86	29.29 ± 2.53

K562/A cells were incubated with indicated concentrations of Que (100–140 µM) or HT (41°–43°C) for 24 h, stained with PI, and analyzed by FCM. The table shows a dose-related increase of G₂/M population with a concomitant increase of Sub-G₁ population after Que treatment. G₂/M and G₁ phase cell cycle arrest was observed in heated cells. Data represent the mean ± SD of three independent experiments.

Figure 5. Apoptotic cells detected by FCM with annexin V conjugated with PI staining. K562/A cells were without any treatment (A) and treated with HT (B), Que (C) and Que + HT (D) for 24 h. (E) Viable cells, apoptotic cells, and necrotic cells were analyzed.