Omega-3 Polyunsaturated Fatty Acids Provoke Apoptosis in Hepatocellular Carcinoma through Knocking Down the STAT3 Activated Signaling Pathway: In Vivo and In Vitro Study

Abstract

Hepatocellular carcinoma (HCC) is a common type of liver cancer and is a leading cause of death worldwide. Signal transducer and activator of transcription 3 (STAT3) is involved in HCC progression, migration, and suppression of apoptosis. This study investigates the apoptotic effect of the dietary antioxidant (n-3 PUFAs) on HepG2 cells and analyzes the underlying molecular mechanisms of this effect both in vivo and in vitro. In vivo study: Seventy-five adult male albino rats were divided into three groups (n = 25): Group I (control): 0.9% normal saline, intraperitoneal. Group II: N-Nitrosodiethylamine (200 mg/kg b.wt) intraperitoneal, followed by phenobarbital 0.05% in drinking water. Group III: as group II followed by n-3 PUFAs intubation (400 mg/kg/day). In vivo study: liver specimens for biochemical, histopathological, and immunohistochemical examination. In vitro study: MTT assay, cell morphology, PCR, Western blot, and immunohistochemical analysis. n-3 PUFAs significantly improved the histopathologic features of HCC and decreased the expression of anti-apoptotic proteins. Further, HepG2 cells proliferation was suppressed through inhibition of the STAT3 signaling pathway, cyclin D1, and Bcl-2 activity. Here we report that n-3 PUFAs may be an ideal cancer chemo-preventive candidate by targeting STAT3 signaling, which is involved in cell proliferation and apoptosis.

Keywords: Bcl-2; HepG2; STAT3; apoptosis; cyclin D1; n-3 polyunsaturated fatty acids (PUFAs).

Figure 1

Activation of STAT3 induces the transcription of the target genes that promote cell growth, anti-apoptosis, migration, invasion, metastasis, and angiogenesis.

Figure 2

H&E-stained sections showing histopathological changes in liver tissue of all research groups. (A) Control group showing normal hepatic architecture formed of cords of acidophilic hepatocytes with uniform nuclei (arrow) radiating from the central vein (C.V), separated by blood sinusoids (S). (B) DEN group showing lost architecture with focal necrosis and hyalinosis (stars), atypical pleomorphic hepatocytes (arrows) with pleomorphic hyperchromatic nuclei and distinct nucleoli (arrowheads), markedly vacuolated cytoplasm (V), and dilated sinusoids (S). (C) DEN group showing large nodule with central necrosis (arrow), marked inflammatory infiltrates (arrowheads), and dilated central vein (C.V). A pseudo-glandular pattern can be noticed (rectangle). (D) n-3 PUFAs group showing nearly normal liver architecture with regular hepatocytes’ cords except for mildly dilated sinusoids (S) and congested central vein (C.V). (magnification A,B,D × 200, C × 100).

Figure 3

Immunohistochemical staining for p-STAT3, Cyclin D1, and Bcl-2 antibodies in different research groups, magnification 400, scale bar = 50 μm. (A–C) Control group showing a positive reaction for p-STAT3, cyclin D1, and Bcl-2 antibodies. (D–F) DEN group showing strong positive expression of all antibodies. (G–I) n-3 PUFAs group showing regression of immune reactions for all antibodies. Outcomes are expressed as mean ± SD; one-way ANOVA followed by post hoc Tukey’s test for intergroup comparison. * significant from the control group, # significant from the DEN group, p < 0.001.

Figure 4

Influence of n-3 PUFAs on DEN-induced hepatic DNA. Lane (A): no DNA fragmentation in normal control. Lane (B): no DNA fragmentation in DEN-treated HCC rats. Lane (C): weak DNA fragmentation in HCC rats treated with n-3 PUFAs.

Figure 5

Effect of n-3 PUFAs on the viability of HepG2 cells measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Data were from at least three separate experiments. IC₅₀ for HepG2 cell line at 24 h was 900 μM. Cell viability in control groups were 100% * p < 0.05, compared with the control (0 μM) group.

Figure 6

Morphological changes of HepG2 cells at 40 × 10 magnifications. (A) Normal HepG2 cells. (B) Cells treated with 450 μM (IC₂₅) n-3 PUFAs, only slight morphological changes were present. (C) Cells treated with 900 μM (IC₅₀) n-3 PUFAs are detached, and the numbers of apoptotic bodies and cell shrinkage increased. The results are from one representative experiment of the three independently performed experiments that showed similar patterns.

Figure 7

Detection of DNA fragmentation by agarose gel electrophoresis. (A) untreated cells; (0 μ M), positive control HepG2. (B) cells were exposed to n-3 PUFAs at half IC₅₀ concentration (450 μM) for 48 h; (C) DNA fragmentation at IC₅₀ (900 μM). Three experiments were performed with similar results.

Figure 8

Gene expression in HepG2 cells after n-3 PUFAs treatment of 24 h. Outcomes are expressed as mean ± SD.

Figure 9

Western blot analysis of p-STAT3, STAT3, Bcl-2, Cyclin D1, and β-actin protein expression in HepG2 after 24 h n-3 PUFAs treatment.

Figure 10

The timeline of the study.