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. 2021 Jun 24;26(13):3855.
doi: 10.3390/molecules26133855.

Saffron and Its Major Ingredients' Effect on Colon Cancer Cells with Mismatch Repair Deficiency and Microsatellite Instability

Amr Amin  1   2 , Aaminah Farrukh  1 , Chandraprabha Murali  1 , Akbar Soleimani  3 , Françoise Praz  4 , Grazia Graziani  5 , Hassan Brim  3 , Hassan Ashktorab  3
Affiliations

Saffron and Its Major Ingredients' Effect on Colon Cancer Cells with Mismatch Repair Deficiency and Microsatellite Instability

Amr Amin et al. Molecules. .

Abstract

Background: Colorectal cancer (CRC) is one of the most common cancers worldwide. One of its subtypes is associated with defective mismatch repair (dMMR) genes. Saffron has many potentially protective roles against colon malignancy. However, these roles in the context of dMMR tumors have not been explored. In this study, we aimed to investigate the effects of saffron and its constituents in CRC cell lines with dMMR.

Methods: Saffron crude extracts and specific compounds (safranal and crocin) were used in the human colorectal cancer cell lines HCT116, HCT116+3 (inserted MLH1), HCT116+5 (inserted MSH3), and HCT116+3+5 (inserted MLH1 and MSH3). CDC25b, p-H2AX, TPDP1, and GAPDH were analyzed by Western blot. Proliferation and cytotoxicity were analyzed by MTT. The scratch wound assay was also performed.

Results: Saffron crude extracts restricted (up to 70%) the proliferation in colon cells with deficient MMR (HCT116) compared to proficient MMR. The wound healing assay indicates that deficient MMR cells are doing better (up to 90%) than proficient MMR cells when treated with saffron. CDC25b and TDP1 downregulated (up to 20-fold) in proficient MMR cells compared to deficient MMR cells, while p.H2AX was significantly upregulated in both cell types, particularly at >10 mg/mL saffron in a concentration-dependent manner. The reduction in cellular proliferation was accompanied with upregulation of caspase 3 and 7. The major active saffron compounds, safranal and crocin reproduced most of the saffron crude extracts' effects.

Conclusions: Saffron's anti-proliferative effect is significant in cells with deficient MMR. This novel effect may have therapeutic implications and benefits for MSI CRC patients who are generally not recommended for the 5-fluorouracil-based treatment.

Keywords: DNA damage and repair; HCT116; MLH1; MSH3; apoptosis; colorectal cancer; crocin; saffron; safranal.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Chemical structure of saffron, safranal and crocin.
Figure 2
Figure 2
Cell viability assay of saffron-treated cells. Percentage of viability shown vs. concentration of saffron. (a–d) show the effect of saffron treatment on the viability of HCT116, HCT116+3, HCT116+5 and HCT116+3+5 cells respectively. (* p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001).
Figure 3
Figure 3
Cell viability assay of safranal-treated cells. Percentage of viability shown vs. concentration of safranal. (a–d) show the effect of safranal treatment on the viability of HCT116, HCT116+3, HCT116+5 and HCT116+3+5 cells respectively. (* p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001).
Figure 4
Figure 4
Cell viability assay of crocin-treated cells. Percentage of viability shown vs. concentration of crocin. (a–d) show the effect of crocin treatment on the viability of HCT116, HCT116+3, HCT116+5 and HCT116+3+5 cells respectively. (* p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001).
Figure 5
Figure 5
Quantification histogram of the wound healing assay. The assay was performed in triplicates. (* p < 0.05, ** p < 0.01).
Figure 6
Figure 6
Saffron’s effect on cell cycle and DNA repair machinery [15]. Cells were treated with saffron 5, 10, and 15 mg/mL for 24 h. (a–c) show the effect of Saffron treatment on CDC25b, p.H2AX and TDP1 respectively. (d). GAPDH was used as loading control. Red font represents the fold change value.
Figure 7
Figure 7
Safranal’s effect on cell cycle and DNA repair machinery. (a–d) Cells were treated with safranal 300, 500, and 700 µM for 24 h. (a–c) show the effect of Safranal treatment on CDC25b, p.H2AX and TDP1 respectively. (d). GAPDH was used as loading control. Red font represents the fold change value.
Figure 8
Figure 8
Crocin’s effect on cell cycle and DNA repair machinery. (a–d) Cells were treated with crocin 300, 600, and 900 µM for 24 h. (a–c) show the effect of Crocin treatment on CDC25b, p.H2AX and TDP1 respectively. (d). GAPDH was used as loading control. Red font represents the fold change value.
Figure 9
Figure 9
Saffron activates the caspase pathway. A Western blot analysis was performed to determine the expression of pro-caspase 3 in saffron-treated cells. The indicated cells were treated with 5, 10, and 15 mg/mL of saffron for 24 h. Pro-caspase 3, which cleaves to caspase 3, was analyzed. The saffron treatment led to a decrease in the expression of pro-caspase 3, which was visible in the control cells. The fold change of relative expression compared to the control is mentioned below each band. Red font represents the fold change value. (a,b) show the effect of saffron treatment on caspase pathway in HCT116, HCT116+3 and HCT116+5, HCT116+3+5 cells respectively.
Figure 10
Figure 10
Effect of safranal on the caspase pathway (caspase activity measured in the relative light unit (RLU). (a–d) show the effect of Safranal on the caspase activity in HCT116, HCT116+3, HCT116+5 and HCT116+3+5 cells respectively. An ANOVA (Analysis of Variance) test was carried out (≥0.05 NS, ≤0.01 *, <0.01 **, ≤0.001 ***).
Figure 11
Figure 11
Effect of crocin on caspase pathway (caspase activity measured in the relative light unit (RLU). (a–d) show the effect of crocin on the caspase activity in HCT116, HCT116+3, HCT116+5 and HCT116+3+5 cells respectively. An ANOVA (Analysis of Variance) test was carried out (≥0.05 NS).
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