Safranal Prevents Liver Cancer Through Inhibiting Oxidative Stress and Alleviating Inflammation

Abstract

Despite all efforts, an effective and safe treatment for liver cancer remains elusive. Natural products and their derived biomolecules are potential resources to mine for novel anti-cancer drugs. Chemopreventive effects of safranal, a major bioactive ingredient of the golden spice "saffron", were evaluated in this study against diethylnitrosamine (DEN)-induced liver cancer in rats. Safranal's mechanisms of action were also investigated in the human liver cancer line "HepG2". When administered to DEN-treated rats, safranal significantly inhibited proliferation (Ki-67) and also induced apoptosis (TUNEL and M30 CytoDeath). It also exhibited anti-inflammatory properties where inflammatory markers such as NF-kB, COX2, iNOS, TNF-alpha, and its receptor were significantly inhibited. Safranal's in vivo effects were further supported in HepG2 cells where apoptosis was induced and inflammation was downregulated. In summary, safranal is reported here as a potent chemopreventive agent against hepatocellular carcinoma that may soon be an important ingredient of a broad-spectrum cancer therapy.

Keywords: inflammation; liver cancer; oxidative stress; prevention; safranal.

FIGURE 1

Schematic diagram showing the experimental design to induce and treat liver cancer in vivo study.

FIGURE 2

Safranal inhibits DEN/2-AAF–induced induction of AHF (A) and of GST-p expression (B–D). (A) Figures representing the livers of all groups with a magnification of ×100 and ×400 (scale bars = 100 and 20 μm) using H&E staining. AHF is indicated by arrows at ×100 magnification (scale bar = 100 μm). (B) Representative Images of immunohistochemical stains with GST-p of in all groups studied. The brown color represented those cells and is indicated by arrows. (C, D) Present quantitative analyses in 10 fields of each section of the GST-p–positive foci and quantitative region analysis of the GST-p–positive foci ×100 magnifications. Treatment of HCC rats with safranal decreased the number and area (mm²) of GST-positive foci. The value was evaluated by one-way ANOVA followed by Dunnett’s t-test: ^a p < 0.05 vs. HCC group. Data are represented as mean ± SEM of six animals per group.

FIGURE 3

Effects of safranal on proliferation (Ki-67) and apoptotic cell death (TUNEL and with M30) and HDAC activity in rat livers. (A) The upper panel includes representative images of immunohistochemical staining with Ki-67, TUNEL, and M30 in liver sections from all the groups (scale bar = 20 µm). The bottom panel represents quantitative analysis of Ki-67 (B), TUNEL (C), and M30 (D) positive cells as well as HDAC activity (E), which is expressed in mmol/mg protein. The positive expression in each section was calculated by counting the number of brown staining in 10 fields at ×400 magnifications, then the number of positive cells/field. Values expressed as mean ± SEM for six animals in each group. Significance was determined by one-way ANOVA followed by Dunnett’s t-test: ^a p < 0.05 vs. control group, ^b p < 0.05 vs. HCC group.

FIGURE 4

Safranal reduces the upregulation of ED-1, ED-2, and p-TNF-R1 in liver and TNF-α in serum of HCC rats. (A) The upper panel are representative images of immunohistochemical staining with ED-1, ED-2, and p-TNF-R1 in the liver section from all the groups. (B–D) The positive expression of cells in each section was calculated by counting the number of brown staining in 10 fields at ×400 magnifications then the number of positive cells/fields. (E) Shows quantitative analyses of TNF-α in serum. Data are represented as mean ± SEM for six animals in each group. Significance was determined by one-way ANOVA followed by Dunnett’s t-test: ^a p < 0.05 vs. control group, ^b p < 0.05 vs. HCC group.

FIGURE 5

Safranal inhibits DEN/2-AAF–induced upregulation of NF-kB-p65 and COX-2 and iNOS-positive cells expressions. (A) The upper panel are representative images of immunohistochemical staining with NF-kB-p65, COX-2, and iNOS in the liver section from all the groups (scale bar = 20 µm). (B, C, D) show quantitative analyses of NF-kB-p65, COX-2, and iNOS-positive cells. The positive expression of cells in each section was calculated by counting the number of brown staining in 10 fields at ×400 magnifications then the number of positive cells/fields. Data are represented as mean ± SEM of six rats per group. Significance was determined by one-way ANOVA followed by Dunnett’s t-test: ^a p < 0.05 vs. control group, ^b p < 0.05 vs. HCC group.

FIGURE 6

In vitro analysis. (A) Viability of HepG2 cells after safranal treatment for 24 h. HepG2 cells were treated with 0.01, 0.03, 0.1, 0.3, and 1 mM of safranal. (B) Caspase-3/7 activities after safranal treatment for 48 h. HepG2 cells were treated with 0.5, 0.7, and 1 mM of safranal. (C) IL-8 secretion after safranal treatment. HepG2 cells were treated with 2 mM for 6 and 12 h, and subsequently, the supernatants were analyzed by IL-8 ELISA.