Combined treatment with quercetin and imperatorin as a potent strategy for killing HeLa and Hep-2 cells

Abstract

The aim of the present study was to assess the effect of quercetin and imperatorin administered separately and in combination on apoptosis and autophagy induction in human cervical carcinoma HeLa cells and laryngeal carcinoma Hep-2 cells cultured in vitro. Conducted MTT measurements proved that quercetin and imperatorin displayed a strong antiproliferative activity manifested in markedly reduction of HeLa and Hep-2 cells viability as a result of treatment with 50 μM of each compound. Further cell staining assays revealed that concentration mentioned above generated the highest percentage of apoptotic cells especially in the case of application of both drugs for 48 h. Simultaneous quercetin and imperatorin administration induced apoptosis remarkably stronger than treatment with single drugs. Experiments at the molecular level confirmed these results accompanied with the decreased Hsp27 and Hsp72 expression and, in addition, with increased caspases activity. Autophagy was not observed and no significant changes in the expression of beclin-1 were noticed. Additionally, experiments were performed on the above-mentioned cell lines with blocked Hsp27 and Hsp72 expression. In these cells, no significant changes in the sensitivity to apoptosis induction upon quercetin and imperatorin treatment were observed. The present study has provided evidence supporting the potential of the combination of quercetin and imperatorin drugs as a novel tool to be used in anticancer therapy. Our results have also demonstrated that blocking of the Hsp27 and Hsp72 gene expression is not enough to sensitize cancer cells to programmed cell death induction in HeLa and Hep-2 cells.

Fig. 1

The cytotoxic effect of quercetin, imperatorin (A, B) and combined treatment with both drugs (C, D) on viability of HeLa (A, C) and Hep-2 (B, D) cells. C control, Q quercetin, I imperatorin, Q + I cells pre-incubated with quercetin followed by imperatorin administration, I + Q cells pre-incubated with imperatorin followed by quercetin administration, QI cells treated with both drugs administered simultaneously; *p < 0.05

Fig. 2

The effect of quercetin (A, B), imperatorin (C, D) and combined treatment with both drugs (E, F) on apoptosis, necrosis, and autophagy induction in HeLa (A, C, E) and Hep-2 (B, D, F) cell lines. C control, Q quercetin, I imperatorin, Q + I cells pre-incubated with quercetin followed by imperatorin administration; I + Q cells pre-incubated with imperatorin followed by quercetin administration, QI cells treated with both drugs administered simultaneously; *p < 0.05

Fig. 3

The effect of quercetin (50 μM) and imperatorin (50 μM) administered separately or simultaneously on the activation of caspases (A, B), expression of beclin-1 (C, D), Hsp27 (E, G), and Hsp72 (F, H) in HeLa (A, C, E, F) and Hep-2 (B, D, G, H) cell lines. C control, Q quercetin, I imperatorin, Q + I cells pre-incubated with quercetin followed by imperatorin administration; I + Q cells pre-incubated with imperatorin followed by quercetin administration, QI cells treated with both drugs administered simultaneously. Representative western blots are included; *p < 0.05

Fig. 4

The expression of Hsp27 (A, C), Hsp72 (B, D) in transfected HeLa (A, B) and Hep-2 (C, D) cell lines subsequently treated with quercetin (50 μM) and imperatorin (50 μM) for 24 h. C control; TR HeLa/Hep-2 cells incubated only with Transfection Reagent (TR) introductoring siRNA into cells; si27/si72 HeLa/Hep-2 cells incubated only with specific siRNA blocking the expression of Hsp27 or Hsp72 in cells; TRsi27/TRsi72 HeLa/Hep-2 cells incubated with complex of TR and specific siRNA which blocks the expression of Hsp27 or Hsp72 in cells; Q quercetin; I imperatorin; QI drugs administered simultaneously. Representative western blots are included; *p < 0.05

Fig. 5

The level of apoptosis, necrosis, and autophagy induction in HeLa (A, B) and Hep-2 (C, D) cells after blocking the Hsp27 (A, C) and Hsp72 (B, D) expression subsequently treated with quercetin (50 μM) and imperatorin (50 μM) for 24 h. C control; TR HeLa/Hep-2 cells incubated only with Transfection Reagent (TR) introductoring siRNA into cells; si27/si72 HeLa/Hep-2 cells incubated only with specific siRNA blocking the expression of Hsp27 or Hsp72 in cells; TRsi27/TRsi72 HeLa/Hep-2 cells incubated with complex of TR and specific siRNA which blocks the expression of Hsp27 or Hsp72 in cells; Q quercetin; I imperatorin; QI drugs administered simultaneously; *p < 0.05