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. 2019 May 3;20(1):22.
doi: 10.1186/s40360-019-0301-2.

Apigenin and hesperidin augment the toxic effect of doxorubicin against HepG2 cells

Agnieszka Korga  1 , Marta Ostrowska  2 , Aleksandra Jozefczyk  3 , Magdalena Iwan  4 , Rafal Wojcik  5 , Grazyna Zgorka  3 , Mariola Herbet  2 , Gemma Gomez Vilarrubla  4 , Jaroslaw Dudka  2
Affiliations

Apigenin and hesperidin augment the toxic effect of doxorubicin against HepG2 cells

Agnieszka Korga et al. BMC Pharmacol Toxicol. .

Abstract

Background: Hepatocellular carcinoma (HCC) is one of the most common malignancies, with an increasing incidence. Despite the fact that systematic chemotherapy with a doxorubicin provides only marginal improvements in survival of the HCC patients, the doxorubicin is being used in transarterial therapies or combined with the target drug - sorafenib. The aim of the study was to evaluate the effect of natural flavonoids on the cytotoxicity of the doxorubicin against human hepatocellular carcinoma cell line HepG2.

Methods: The effect of apigenin and its glycosides - cosmosiin, rhoifolin; baicalein and its glycosides - baicalin as well as hesperetin and its glycosides - hesperidin on glycolytic genes expression of HepG2 cell line, morphology and cells' viability at the presence of doxorubicin have been tested. In an attempt to elucidate the mechanism of observed results, the fluorogenic probe for reactive oxygen species (ROS), the DNA oxidative damage, the lipid peroxidation and the double strand breaks were evaluated. To assess impact on the glycolysis pathway, the mRNA expression for a hexokinase 2 (HK2) and a lactate dehydrogenase A (LDHA) enzymes were measured. The results were analysed statistically with the one-way analysis of variance (ANOVA) and post hoc multiple comparisons.

Results: The apigenin and the hesperidin revealed the strongest effect on the toxicity of doxorubicin. Both flavonoids simultaneously changed the expression of the glycolytic pathway genes - HK2 and LDHA, which play a key role in the Warburg effect. Although separate treatment with doxorubicin, apigenin and hesperidin led to a significant oxidative DNA damage and double strand breaks, simultaneous administration of doxorubicin and apigenin or hesperidin abolished these damage with the simultaneous increase in the doxorubicin toxicity.

Conclusion: The obtained results indicate the existence of a very effective cytotoxic mechanism in the HepG2 cells of the combined effect of doxorubicin and apigenin (or hesperidin), not related to the oxidative stress. To explain this synergy mechanism, further research is needed, The observed intensification of the cytotoxic effect of doxorubicin by this flavonoids may be a promising direction of the research on the therapy of hepatocellular carcinoma, especially in a chemoembolization.

Keywords: Apigenin; Doxorubicin; Flavonoids; Hepatocellular carcinoma; Hesperidin.

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Figures

Fig. 1
Fig. 1
Chemical structures of tested flavonoids
Fig. 2
Fig. 2
The relative HepG2 cell viability determined by MTT assay. The results were calculated as % of control cultures which were averaged to define the 100%. Values were presented as mean ± SD value of three independent experiments. To compare more than two groups, the one-way analysis of variance (ANOVA) and post hoc multiple comparisons on a basis of Tukey’s HSD test were used. DOX – 1 μM doxorubicin, A – 100 μM apigenin, H – 200 μM hesperidin, DOX A – 1 μM doxorubicin and 100 μM apigenin, DOX H – 1 μM doxorubicin and 200 μM hesperidin
Fig. 3
Fig. 3
Morphological changes of HepG2 cells. The control cells showed a normal morphology, they were closely arranged and well adherent in large numbers. After treatment with tested compounds (DOX, apigenin, hesperidin or combined) the number of normal cells was significantly reduced, the cells became round and had poor adherence, especially the DOX A and DOX H treated cultures. The results present one representative experiment of three independently performed that showed similar patterns. HepG2 cell morphology was analysed under a phase-contrast microscope Nikon Eclipse Ti, magnification x200, scale bar = 100 μm. C – control, DOX – 1 μM doxorubicin, A – 100 μM apigenin, H – 200 μM hesperidin, DOX A – 1 μM doxorubicin and 100 μM apigenin, DOX H – 1 μM doxorubicin and 200 μM hesperidin
Fig. 4
Fig. 4
The detection of ROS generation using CellROX Green Reagent. In cells treated with DOX there was a high fluorescent signal deriving from mitochondria. In the case of A cultures the signal came from the nuclei. In DOX A, the signal came only from the nuclei. H showed oxidative signal in nuclei, however DOX and H combination – in both nuclei and mitochondria. The results present one representative experiment of three independently performed that showed similar patterns. HepG2 cells morphology was analysed under a phase-contrast microscope Nikon Eclipse Ti, magnification x300, scale bar = 100 μm. C – control, DOX – 1 μM doxorubicin, A – 100 μM apigenin, H – 200 μM hesperidin, DOX A – 1 μM doxorubicin and 100 μM apigenin, DOX H – 1 μM doxorubicin and 200 μM hesperidin
Fig. 5
Fig. 5
AP sites’ number per 100,000 bp in HepG2 cell line. Values were presented as mean ± SD. To compare more than two groups, the one-way analysis of variance (ANOVA) and post hoc multiple comparisons on a basis of Tukey’s HSD test were used. C – control, DOX – 1 μM doxorubicin, A – 100 μM apigenin, H – 200 μM hesperidin, DOX A – 1 μM doxorubicin and 100 μM apigenin, DOX H – 1 μM doxorubicin and 200 μM hesperidin
Fig. 6
Fig. 6
The content of DSB in tested cell’s DNA (based on phosphorylated H2AX level) presented as a % of a control. Values were presented as mean ± SD. To compare more than two groups, the one-way analysis of variance (ANOVA) and post hoc multiple comparisons on a basis of Tukey’s HSD test were used. C – control, DOX – 1 μM doxorubicin, A – 100 μM apigenin, H – 200 μM hesperidin, DOX A – 1 μM doxorubicin and 100 μM apigenin, DOX H – 1 μM doxorubicin and 200 μM hesperidin
Fig. 7
Fig. 7
Lipid peroxidation level in HepG2 cells on the basis of MDA and 4-HAE concentration, presented as a % of a control. Values were presented as mean ± SD. To compare more than two groups, the one-way analysis of variance (ANOVA) and post hoc multiple comparisons on a basis of Tukey’s HSD test were used. C – control, DOX – 1 μM doxorubicin, A – 100 μM apigenin, H – 200 μM hesperidin, DOX A – 1 μM doxorubicin and 100 μM apigenin, DOX H – 1 μM doxorubicin and 200 μM hesperidin
Fig. 8
Fig. 8
Relative mRNA expression level of HK2 (a) and LDH-A (b) in tested cells. GAPDH was used as a reference gene. The results were calculated as RQ values and presented as mean ± SD. To compare more than two groups, the one-way analysis of variance (ANOVA) and post hoc multiple comparisons on a basis of Tukey’s HSD test were used. C – control, DOX – 1 μM doxorubicin, A – 100 μM apigenin, H – 200 μM hesperidin, DOX A – 1 μM doxorubicin and 100 μM apigenin, DOX H – 1 μM doxorubicin and 200 μM hesperidin
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References

    1. Minotti G, Menna P, Salvatorelli E, Cairo G, Gianni L. Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity. Pharmacol Rev. 2004;56:185–229. doi: 10.1124/pr.56.2.6. - DOI - PubMed
    1. Gabizon AA, Patil Y, La-Beck NM. New insights and evolving role of pegylated liposomal doxorubicin in cancer therapy. Drug Resist Updat. 2016;29:90–106. doi: 10.1016/j.drup.2016.10.003. - DOI - PubMed
    1. Agudelo D, Bourassa P, Bérubé G, Tajmir-Riahi HA. Review on the binding of anticancer drug doxorubicin with DNA and tRNA: structural models and antitumor activity. J Photochem Photobiol B. 2016;158:274–279. doi: 10.1016/j.jphotobiol.2016.02.032. - DOI - PubMed
    1. Lin S, Hoffmann K, Schemmer P. Treatment of hepatocellular carcinoma: a systematic review. Liver Cancer. 2012;1(3–4):144–158. doi: 10.1159/000343828. - DOI - PMC - PubMed
    1. Kudo M. Systemic therapy for hepatocellular carcinoma: 2017 update. Oncology. 2017;93(Suppl 1):135–146. doi: 10.1159/000481244. - DOI - PubMed

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