Korean J Physiol Pharmacol.  2022 May;26(3):145-155. 10.4196/kjpp.2022.26.3.145.

Chemosensitizing effect and mechanism of imperatorin on the anti-tumor activity of doxorubicin in tumor cells and transplantation tumor model

• Liang Xl
• Ji Mm
• Liao Zg
• Zhao Gw
• Tang Xl
• Dong W

• KMID: 2529397
• DOI: http://doi.org/10.4196/kjpp.2022.26.3.145

Abstract

Multidrug resistance of tumors has been a severe obstacle to the success of cancer chemotherapy. The study wants to investigate the reversal effects of imperatorin (IMP) on doxorubicin (DOX) resistance in K562/DOX leukemia cells, A2780/Taxol cells and in NOD/SCID mice, to explore the possible molecular mechanisms. K562/ DOX and A2780/Taxol cells were treated with various concentrations of DOX and Taol with or without different concentrations of IMP, respectively. K562/DOX xenograft model was used to assess anti-tumor effect of IMP combined with DOX. MTT assay, Rhodamine 123 efflux assay, RT-PCR, and Western blot analysis were determined in vivo and in vitro. Results showed that IMP significantly enhanced the cytotoxicity of DOX and Taxol toward corresponding resistance cells. In vivo results illustrated both the tumor volume and tumor weight were significantly decreased after 2-week treatment with IMP combined with DOX compared to the DOX alone group. Western blotting and RT-PCR analyses indicated that IMP downregulated the expression of P-gp in K562/DOX xenograft tumors in NOD/SCID mice. We also evaluated glycolysis and glutamine metabolism in K562/DOX cells by measuring glucose consumption and lactate production. The results revealed that IMP could significantly reduce the glucose consumption and lactate production of K562/DOX cells. Furthermore, IMP could also remarkably repress the glutamine consumption, α-KG and ATP production of K562/DOX cells. Thus, IMP may sensitize K562/DOX cells to DOX and enhance the antitumor effect of DOX in K562/DOX xenograft tumors in NOD/SCID mice. IMP may be an adjuvant therapy to mitigate the multidrug resistance in leukemia chemotherapy.

Keyword

Doxorubicin; Imperatorin; Leukemia; Multidrug resistance; P-glycoprotein

Figure

• 

  Fig. 1 The chemical structure of imperatorin (IMP) .

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  Fig. 2 The resistance index of resistant cell line of K562/DOX and A2780/Taxol. (A) Cell survival rate of different concentration of DOX on K562 and K562/DOX. (B) IC50 (µmol/L) values were calculated from survival curves using the Bliss method and then RI was calculated by dividing the IC50 for K562/DOX cells by that of K562 cells. (C) Cell survival rate of different concentration of Taxol on A2780 and A2780/Taxol. (D) IC50 (µM) values were calculated from survival curves using the Bliss method and then RI was calculated by dividing the IC50 for A2780/DOX cells by that of A2780 cells. Taxol, Taxinol; DOX, doxorubicin; IC50, 50% inhibiting concentration; RI, resistance index.

• 

  Fig. 3 Chemosensitizing effect analysis of IMP on various tumor-resistant cell lines. (A) Cell viability of different concentrations of DOX combined with IMP (2.78, 5.56, and 11.10 µM) on K562 cells. (B) Cell viability of different concentrations of DOX combined with IMP ( 2.78, 5.56, and 11.10 µM) on K562/DOX cells. (C) Cell viability of different concentrations of Taxol combined with IMP (7.40, 18.50, and 37.00 µM) on A2780 cells. (D) Cell viability of different concentrations of Taxol combined with IMP (7.40, 18.50, and 37.00 µM) on A2780/Taxol cells. Representative data from three independent experiments. IMP, imperatorin; DOX, doxorubicin; Taxol, Taxinol.

• 

  Fig. 4 Effect of IMP on intracellular accumulation of Rho 123. (A) Effect of time on the fluorescence intensity of Rho 123 in K562/DOX cells. (B) Effect of the coincubation time of Rho 123 and drugs on the fluorescence intensity of Rho 123 in K562/DOX cells. (C) HPLC method specificity. (C-a) K562/DOX cells solution. (C-b) K562/DOX cells solution containing Rho 123 reference solution. (C-c) K562/DOX cells solution after incubation with Rho 123 for 75 min. (D) Effect of IMP on intracellular accumulation of Rho 123 in K562 cells and K562/DOX cells. K562/DOX cells treated with 10.2 and 20.4 µmol/L VER, which used as the positive control. Data are shown as means ± SD, n = 3. IMP, imperatorin; DOX, doxorubicin; Taxol, Taxinol; HPLC, high-performance liquid chromatography; VER, verapamil. **p < 0.01 vs. K562 cells. ##p < 0.01 vs. K562/DOX cells untreated group.

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  Fig. 5 IMP restrained the glycolysis and glutamine metabolism of K526/DOX cells in vitro. (A) DOX promoted the expression of P-gp. (B, C) The glucose consumption, lactate production and ECAR were measured to assess cell glycolysis. (D, E) Glutamine consumption, α-KG production and ATP production were determined using corresponding Assay Kits, respectively. K562/DOX cells treated with 0 and 2.8 µM IMP. IMP, imperatorin; DOX, doxorubicin; P-gp, P-glycoprotein; ECAR, extracellular acidification rate; α-KG, α-ketoglutaric acid; 2-DG, 2-deoxy-D-glucose. **p < 0.01.

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  Fig. 6 Effect of IMP combined with DOX on tumor growth of K562/DOX xenograft tumors in NOD/SCID mice. (A) The tumor volumes were measured at 0, 7, and 14 days. (B) The tumor weights were analyzed after 14 days. (C) Representative images of tumor tissues subjected to different treatments of DOX and IMP (1, untreated model group; 2, DOX 2.5 mg/kg alone group; 3, IMP 10 mg/kg + DOX 2.5 mg/kg group; 4, IMP 20 mg/kg + DOX 2.5 mg/kg group). Data are shown as means ± SD, n = 8. IMP, imperatorin; DOX, doxorubicin. *p < 0.05; **p < 0.01 vs. untreated model group. ##p < 0.01 vs. DOX 2.5 mg/kg alone group.

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  Fig. 7 Effect of IMP combined with DOX on pathological abnormalities of K562/DOX xenograft tumors in NOD/SCID mice. (A) Tumor tissues were fixed, sectioned, and stained with H&E (magnification, ×50). The blue arrows indicate that the cell structure was seriously damaged and the cell density was enhanced. (B) Electron microscope observation of tumor tissues subjected to different treatments with IMP (10 and 20 mg/kg) and DOX (2.5 mg/kg). The red arrows indicated mitochondria indicates that the cells have shrinkage, necrosis, cytoplasmic vacuoles. The orange arrows were more obvious mitochondrial structure. The green arrows pointed out the mitochondrial morphology of normal cells. IMP, imperatorin; DOX, doxorubicin.

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  Fig. 8 Effect of IMP combined with DOX on P-gp expression in K562/DOX xenograft tumors in NOD/SCID mice. (A) Representative Western blots of P-gp protein. Quantified data showing IMP at dosage of 10 mg/kg and 20 mg/kg combined with DOX (2.5 mg/kg) treatments inhibited P-gp protein expression (B) and mRNA level (C) in K562/DOX xenograft tumor tissues as compared with DOX (2.5 mg/kg) alone treatment. Data are presented as the means ± SD, n = 8. IMP, imperatorin; DOX, doxorubicin; P-gp, P-glycoprotein. **p < 0.01 vs. untreated model group. ##p < 0.01 vs. DOX (2.5 mg/kg) alone group.

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