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. 2019 May 13:12:3581-3593.
doi: 10.2147/OTT.S195205. eCollection 2019.

The reversal of MRP1 expression induced by low-frequency and low-intensity ultrasound and curcumin mediated by VEGF in brain glioma

Lei Yao  1 , Zhen Zhang  1
Affiliations

The reversal of MRP1 expression induced by low-frequency and low-intensity ultrasound and curcumin mediated by VEGF in brain glioma

Lei Yao et al. Onco Targets Ther. .

Abstract

Purpose: To explore the effect of curcumin and low-frequency and low-intensity ultrasound (LFLIU) on C6 and U87 cell, and whether LFLIU could inhibit multidrug resistance protein 1 (MRP1) by increasing the sensitivity of curcumin via vascular epithelial growth factor (VEGF)/PI3K/Akt signaling pathway targeting. Methods: C6 and U87 cells were treated with various doses of curcumin and/or different intensities of LFLIU for 60 s. After 24 hrs, the effects of curcumin and/or LFLIU on the proliferation of C6 and U87 cells were examined. Real-time PCR and western blot analysis were used to detect the expression of VEGF and MRP1 at both mRNA and protein levels. The expression of MRP1 in C6 and U87 cells was also determined following stimulation with recombinant human VEGF and/or LY294002. Results: Curcumin and LFLIU inhibited the proliferation of glioma cells in an intensity- or dose-dependent manner. Furthermore, survivin was significant after combined treatment compares with that of curcumin or LFLIU treatment alone. VEGF and MRP1 were highly expressed in C6 and U87 cells, curcumin and LFLIU alone or in combination could decrease the expression of both VEGF and MRP1. MRP1 expression was down-regulated following LY294002 treatment, which blocked after exposure to VEGF. Conclusion: The synergistic effects, such as a higher inhibition rate, and lower expressions of MRP1 and VEGF, of combined curcumin and LFLIU against glioma was much better than that of a single treatment. The down-regulation of MRP1 may be related with the VEGF/PI3K/Akt pathway in glioma.

Keywords: LFLIU; MRP1; VEGF; curcumin; glioma cells.

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Conflict of interest statement

The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
Curcumin inhibited C6 and U87 cells proliferation in a dose-dependent manner. The growth-inhibitory effect in C6 and U87 cells that are treated with curcumin was evaluated by cck-8. There was no significant difference between DMSO group and control group (*P>0.05)，and there was a significant difference when curcumin ≥10 μmol/L compared with control group (n=3, *P<0.05). Notes: 5, 10, 15 and 20: the concentration of curcumin at 5, 10, 15 and 20 μmol/L, respectively. Abbreviation: DMSO, dulbecco's modification of Eagle's Medium.
Figure 2
Figure 2
LFLIU inhibited C6 and U87 cells proliferation in an intensity-dependent manner. The growth-inhibitory effect in C6 and U87 cells that are treated with LFLIU was evaluated by cck-8. For C6 cells, there was a significant difference when LFLIU ≥142 mW/cm2 compare with control group; for U87 cells, there was a significant difference when LFLIU ≥83.4 mW/cm2 compare with control group (n=3, *P<0.05). Notes: 3, 50.4, 83.4, 142, 290 and 474, the intensity of low-frequency and low-intensity ultrasound (LFLIU) at 3, 50.4, 83.4, 142, 290 and 474 mW/cm2, respectively.
Figure 3
Figure 3
LFLIU and curcumin in combination inhibited C6 and U87 cells proliferation in a synergistic manner. The growth-inhibitory effect in C6 and U87 cells that are treated with the combination of LFLIU and curcumin was evaluated by cck-8. Curcumin alone or in combination with LFLIU induced glioma cell death that is synergistic in effect. *P<0.05 vs control group, †P<0.05 vs corresponding LFLIU group, and ‡P<0.05 vs corresponding curcumin group. Notes: U142, the intensity of low-frequency and low-intensity ultrasound (LFLIU) at 142 mW/cm2; C10 and C15,  the concentration of curcumin at 10 and 15 μmol/L, respectively.
Figure 4
Figure 4
LFLIU and/or curcumin down-regulates the expressions of survivin by western blot analysis. C6 and U87 cells were treated with LFLIU and curcumin alone or in combination. GAPDH was used as an internal reference. *P<0.05 vs control group, †P<0.05 vs corresponding LFLIU group, and ‡P<0.05 vs corresponding curcumin group. Notes: U142, the intensity of low-frequency and low-intensity ultrasound (LFLIU) at 142 mW/cm2; C10 and C15,  the concentration of curcumin at 10 and 15 μmol/L, respectively. Abbreviation: GAPDH, glyceraldehyde-3- phosphate dehydrogenase.
Figure 5
Figure 5
Cell morphological changes of C6 and U87 cells induced by curcumin or/and LFLIU detected by the microscope. Cells morphology was examined under a light microscope (Bar=100 μm). Morphological changes and decreases in cell density were associated with treatment with curcumin and LFLIU. Decreasing of the cell intensity (C6 and U87) was observed after treatment of curcumin (10, 15 μmol/L) and LFLIU (142 mW/cm2). And the combination of curcumin and LFLIU decreased the cell intensity significantly in a synergistic manner. Notes: U142, the intensity of low-frequency and low-intensity ultrasound (LFLIU) at 142 mW/cm2; C10 and C15,  the concentration of curcumin at 10 and 15 μmol/L, respectively.
Figure 6
Figure 6
LFLIU and/or curcumin down-regulates the expressions of VEGF and MRP1 at mRNA. The relative expressions of VEGF and MRP1 at mRNA level of glioma cell are reduced significantly after LFLIU and/or curcumin. *P<0.05 vs control group, †P<0.05 vs corresponding LFLIU group and ‡P<0.05 vs corresponding curcumin group. Notes: U142, the intensity of low-frequency and low-intensity ultrasound (LFLIU) at 142 mW/cm2; C10 and C15,  the concentration of curcumin at 10 and 15 μmol/L, respectively.
Figure 7
Figure 7
LFLIU and/or curcumin down-regulates the expressions of VEGF and MRP1 at the protein level. After treated with LFLIU and/or curcumin, the expressions of VEGF and MRP1 of (A) C6 and (B) U87 cells at protein levels are shown by western blot. The analysis of bands of gray intensity(C and D) shows a significant down-regulation after treatment, and GAPDH is detected as the loading control. *P<0.05 vs control group, †P<0.05 vs corresponding LFLIU group and ‡P<0.05 vs corresponding curcumin group. Notes: U142, the intensity of low-frequency and low-intensity ultrasound (LFLIU) at 142 mW/cm2; C10 and C15,  the concentration of curcumin at 10 and 15 μmol/L, respectively. Abbreviations: VEGF, vascular epithelial growth factor; MRP1, multidrug resistance protein 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Figure 8
Figure 8
VEGF increased the expressions of MRP1 in glioma cells, but LY294002 reduced this effect. C6 and U87 cells were treated with medium (control group), rhVEGF (rhVEGF group), LY294002 (LY294002 group) or both rhVEGF and LY294002 (rhVEGF ＋ LY294002 group) for 24 hrs. Western blot analysis detected the expression of VEGF, p-Akt and MRP1 in (A) C6 and (B) U87 cells. Protein expressions of the displayed in A and B were quantified and the results are shown in C and D, respectively. VEGF can increase MRP1 expression, and LY294002 could diminish this effect of VEGF on MRP1GAPDH was detected as the loading control. *p<0.05 vs control group. Abbreviations: VEGF, vascular epithelial growth factor; MRP1, multidrug resistance protein 1; p-Akt, phospho-protein kinase B; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; rhVEGF, recombinant human vascular epithelial growth factor.
Figure 9
Figure 9
Effects of VEGF in MRP1 expression. LFLIU and curcumin acted synergistically in their antitumor effects by down-regulating the expressions of MRP1, in which the VEGF/PI3K/Akt/NF-kB signaling pathway may be involved in this regulation. Abbreviations: VEGFR2, Vascular Endothelial Growth Factor Receptor 2; ATP, Adenosine triphosphate; ADP, Adenosine diphosphate; DNA, Deoxyribonucleic acid;)
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