Crocin Increases Gastric Cancer Cells' Sensitivity to Doxorubicin

Abstract

Background: Crocin is one of the substantial constituents of saffron extract. It has multiple clinical effects including anti-cancer effects. The development of the multidrug resistance (MDR) phenotype is one of the principal causes of cancer chemotherapy failure. The multidrug resistance protein 1 (MDR1) is one of the MDR-related protein and is often overexpressed in different cancers. In the present study, we aimed to evaluate the influence of crocin on the expression and function of MDR1 protein in EPG85-257 and EPG85-257RDB gastric cancer cell lines.

Methods: The cytotoxicity effect of crocin was evaluated by the MTT assay. The impacts of crocin on the expression and function of MDR1 were assessed by Real-time RT-PCR and MTT assay, respectively.

Results: The results demonstrated that crocin decreased cell viability in a dose-dependent manner with higher intensity on the EPG85-257 than the EPG85-257RDB cells. Crocin did not make any significant changes in the MDR1 gene expression level in EPG85-257 and EPG85-257RDB cell lines. In contrast, crocin increased doxorubicin cytotoxicity in drug-resistant cells, which might be induced by reduced MDR1 activity.

Conclusion: In summary, although crocin did not affect mRNA expression of MDR1, results of MTT assay suggest that it might inhibit the MDR1 function.

Keywords: Crocin; Gastric cancer; MDR1; Multidrug Resistance; doxorubicin.

Figure 1

The Effects of Crocin on the Cell Viability of EPG85-257 (a) and EPG85-257RDB (b) cell lines. The cells were incubated with various concentrations of crocin at 37 °C for 4, 24, 48 and 72 h. Cell viability was measured by the MTT assay. Each experiment was repeated independently three times in triplicate tests and data are shown as mean ± SD. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001

Figure 2

Basal Expression of MDR1 in Gastric Cancer Cell Lines was Analyzed by Real Time RT- PCR. MDR1 mRNA level of EPG85-257RDB drug resistance cell line was compared to EPG85-257 parental drug sensitive cells. Real-time RT-PCR was performed on total RNA and normalized to β-actin. Values is shown as mean ± SD (n = 3).

Figure 3

The Effects of Crocin on the MDR1 mRNA Expression Levels in the EPG85-257 and EPG85-257RDB Cell Lines. Cells were treated for 48 h with crocin (0–100 μM), and MDR1 gene expression was measured by real-time RT-PCR using total RNA extracted from control and treated cells. Values were normalized to the β-actin content of each samples. The results were expressed as the ratio of target/reference of the treated samples divided by the ratio of target/reference of the untreated control sample and expressed as mean ± SEM (n = 4).

Figure 4

Viability Assay of EPG85-257 (a) and EPG85-257RDB (b) cell lines under the treatment with doxorubicin. The cells were incubated with different concentrations of doxorubicin (0–500 nM) for 4, 24, 48 and 72 h. Cell viability was measured by the MTT assay. Each experiment was repeated independently three times in triplicate tests and data are shown as mean ± SD. ***P ≤ 0.001

Figure 5

The Cytotoxicity Effects of Cotreatment of the EPG85-257 Cells with Crocin and Doxorubicin was Analyzed by MTT Assay. EPG85-257 cells treated with different concentrations of crocin and doxorubicin for 24 hours (A), 48 hours (B) and 72 hours (C). The results are expressed as mean ± SD (n = 3); *, p < 0.05; **, p< 0.01; ***, p < 0.001

Figure 6.

The Cytotoxicity Effects of Cotreatment of the EPG85-257 RDB Cells with Crocin and Doxorubicin was Analyzed Using MTT Assay. EPG85-257 cells treated with different concentrations of crocin and doxorubicin for 24 hours (A), 48 hours (B) and 72 hours (C). The results are expressed as mean ± SD (n = 3); *, p < 0.05; **, p< 0.01; ***, p < 0.001