Induction of multiple drug resistance in HMEC-1 endothelial cells after long-term exposure to sunitinib

Figures & data

Table 1 Exposure to sunitinib induces multiple drug resistance

Agents	HMEC-1 IC50 (μM)*	HMECsu IC50 (μM)*	Resistance index
Sunitinib	4.271±0.523	23.464±2.148	5.49
Doxorubicin	0.052±0.001	0.249±0.071	4.78
Vinblastine	0.158±0.032	0.571±0.085	3.61
Paclitaxel	0.215±0.045	2.968±0.254	11.82

Notes: Human microvascular endothelial cells (HMEC-1) were cultured for 72 hours in the presence of escalating concentrations of sunitinib and stabilized. MTS was used to determine half maximal inhibitory concentration (IC50) for the four drugs. The increases in IC50 for these drugs were statistically significant. Statistical analyses showed P<0.01 when comparing HMECsu cells with HMEC-1 cells in all of the tests.

* Means ± standard error.

Abbreviations: HMECsu, sunitinib-resistant HMEC-1 cells; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium.

Figure 1 Expression of ABC transporters in HMEC-1 cells and variant cell lines.

Notes: (A–C) qPCR results for P-gp, ABCG2, and MRP1 mRNA levels in HMEC-1 (C), HMECsu (or Hsu), and HMECd2 cells. Cyclosporine A (C) at 2.5 μM or verapamil (V) at 1 μM and ABCG2 inhibitors fumitremorgin C (F) at 5 μM or diethylstilbestrol (D) at 0.5 μM and MK571 (M) at 5 μM were used to treat the cells for 72 hours. The results were obtained from three independent experiments. The increase in P-gp, ABCG2, and MRP1 mRNA levels in HMECsu cells was significant (P<0.05) in comparison with HMEC-1 cells, whereas there was no statistical difference within the groups of HMECsu cells under different treatments (P>0.05). (D) Western blot of P-gp, ABCG2, and MRP1 levels in these cells. The data for the ratios were obtained from three repeated and independent blot experiments. *P<0.05 versus the control cells.
Abbreviations: ABC, adenosine triphosphate-binding cassette; HMEC-1, human microvascular endothelial cells; HMECsu, sunitinib-resistant HMEC-1 cells; qPCR, quantitative polymerase chain reaction; P-gp, P-glycoprotein; MRP1, multidrug resistance-associated protein 1; mRNA, messenger ribonucleic acid.

Table 2 Effects of ABC transporter inhibitors on IC50

Agents	HMEC-1 IC50 (μM)*	HMECsu IC50 (μM)*	Resistance index	Reversal fold
Sunitinib	4.271±0.523	23.464±2.148	5.49	1.00
+ Vrp
1 μM	4.052±0.234	21.292±1.081	5.25	1.10
+ CysA
2.5 μM	4.249±0.382	22.459±3.406	5.28	1.04
+ FumC
5 μM	3.931±0.358	20.584±2.564	5.23	1.14
+ Die
0.5 μM	4.665±0.544	19.213±3.056	4.12	1.22
+ MK571
5 μM	5.102±0.321	23.214±4.528	4.54	1.01
+ Vrp +
Fum C	4.328±0.221	22.645±3.794	5.23	1.03
Doxorubicin	0.052±0.001	0.249±0.071	4.78	1.00
+ Vrp
v1 μM	0.051±0.002	0.256±0.057	5.02	0.97
+ CysA
2.5 μM	0.049±0.004	0.221±0.082	4.51	1.08
+ FumC
5 μM	0.053±0.002	0.213±0.063	4.02	1.16
+ Die
0.5 μM	0.048±0.001	0.289±0.024	6.02	0.86
+ MK571
5 μM	0.051±0.003	0.238±0.096	4.67	0.95
Vinblastine	0.158±0.032	0.571±0.085	3.61	1.00
+ Vrp
1 μM	0.143±0.008	0.656±0.057	4.58	0.87
+ CysA
2.5 μM	0.169±0.021	0.521±0.082	3.08	1.09
+ FumC
5 μM	0.195±0.013	0.613±0.043	3.14	0.93
+ Die
0.5 μM	0.142±0.035	0.515±0.024	3.62	1.11
+ MK571
5 μM	0.151±0.023	0.506±0.066	3.35	1.12
Paclitaxel	0.215±0.045	2.968±0.254	11.82	1.00
+ Vrp
1 μM	0.203±0.065	2.651±0.412	13.05	1.11
+ CysA
2.5 μM	0.199±0.083	3.120±0.186	15.67	0.95
+ FumC
5 μM	0.235±0.051	2.756±0.169	11.72	1.07
+ Die
0.5 μM	0.183±0.059	2.581±0.368	14.10	1.14
+ MK571
5 μM	0.232±0.027	2.919±0.371	12.58	1.01

Notes: HMECsu was tested for IC50. MTS was used to determine IC50 for the four drugs in the presence or absence of ABC transporter inhibitors. The cells were treated for 72 hours. Statistical analyses showed P<0.01 when comparing HMECsu cells with HMEC-1 cells in all of the tests, and P>0.05 when comparing the data within the groups.

* Means ± standard error.

Abbreviations: ABC, adenosine triphosphate-binding cassette; IC50, half maximal inhibitory concentration; HMEC-1, human microvascular endothelial cells; HMECsu, sunitinib-resistant HMEC-1 cells; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; Vrp, verapamil; CysA, cyclosporine A; FumC, fumitremorgin C; Die, diethylstilbestrol.