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. 2021 Feb 5:9:629182.
doi: 10.3389/fcell.2021.629182. eCollection 2021.

Anti-tumor Effect of Oleic Acid in Hepatocellular Carcinoma Cell Lines via Autophagy Reduction

Federico Giulitti  1 , Simonetta Petrungaro  1 , Sara Mandatori  1 , Luana Tomaipitinca  1 , Valerio de Franchis  1 , Antonella D'Amore  1 , Antonio Filippini  1 , Eugenio Gaudio  1 , Elio Ziparo  1 , Claudia Giampietri  1
Affiliations

Anti-tumor Effect of Oleic Acid in Hepatocellular Carcinoma Cell Lines via Autophagy Reduction

Federico Giulitti et al. Front Cell Dev Biol. .

Abstract

Oleic acid (OA) is a component of the olive oil. Beneficial health effects of olive oil are well-known, such as protection against liver steatosis and against some cancer types. In the present study, we focused on OA effects in hepatocellular carcinoma (HCC), investigating responses to OA treatment (50-300 μM) in HCC cell lines (Hep3B and Huh7.5) and in a healthy liver-derived human cell line (THLE-2). Upon OA administration higher lipid accumulation, perilipin-2 increase, and autophagy reduction were observed in HCC cells as compared to healthy cells. OA in the presence of 10% FBS significantly reduced viability of HCC cell lines at 300 μM through Alamar Blue staining evaluation, and reduced cyclin D1 expression in a dose-dependent manner while it was ineffective on healthy hepatocytes. Furthermore, OA increased cell death by about 30%, inducing apoptosis and necrosis in HCC cells but not in healthy hepatocytes at 300 μM dosage. Moreover, OA induced senescence in Hep3B, reduced P-ERK in both HCC cell lines and significantly inhibited the antiapoptotic proteins c-Flip and Bcl-2 in HCC cells but not in healthy hepatocytes. All these results led us to conclude that different cell death processes occur in these two HCC cell lines upon OA treatment. Furthermore, 300 μM OA significantly reduced the migration and invasion of both HCC cell lines, while it has no effects on healthy cells. Finally, we investigated autophagy role in OA-dependent effects by using the autophagy inducer torin-1. Combined OA/torin-1 treatment reduced lipid accumulation and cell death as compared to single OA treatment. We therefore concluded that OA effects in HCC cells lines are, at least, in part dependent on OA-induced autophagy reduction. In conclusion, we report for the first time an autophagy dependent relevant anti-cancer effect of OA in human hepatocellular carcinoma cell lines.

Keywords: autophagy; cancer; cell death; fatty acids; lipid droplets.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Neutral lipid accumulation upon OA treatment. Control (THLE-2), Hep3B and Huh7.5 cell lines treated with OA. In the left panels: images of the three cell lines: Control, Hep3B and Huh7.5 stained with Oil-Red O after treatment with increasing doses of OA (50, 150, and 300 μM). In the right end panels: quantification of Oil-Red O eluates/cell number, upon treatment with increasing doses of OA (n = 3; *p < 0.05).
Figure 2
Figure 2
Autophagic flux and perilipin-2 modulation upon OA treatment in Hep3B, Huh7.5 and THLE-2 cell lines. (A) Control (THLE-2), Hep3B and Huh7.5 and cell lines treated with OA increasing doses in the presence of bafilomycin A1. LC3II quantification reveals a significant reduction of autophagic flux upon high OA doses in both HCC cell lines, but not in the healthy hepatocyte cell line. (B) Western blot analyses for perilipin-2, were performed. Perilipin-2 levels in both HCC cell lines are increased in a dose dependent manner, while in Control cells perilipin-2 levels do not significantly increase upon 48 h OA treatment (n = 3; *p < 0.05; **p < 0.01).
Figure 3
Figure 3
OA reduces viability, cyclin D1 and PCNA in HCC cell lines. Cell viability assays and western blot analyses for Cyclin D1 and PCNA were performed. (A) OA induced a significant dose-dependent reduction of cellular viability in HCC cell lines but not in healthy hepatocyte cell line, measured by Alamar Blue assay. (B) Western blot Cyclin D1 and PCNA analyses showed that OA treatment induced a significant reduction of Cyclin D1 and PCNA levels in HCC cell lines, but not in healthy hepatocytes cell line (n = 3; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).
Figure 4
Figure 4
OA increases cell death in HCC cell lines but not in healthy hepatocyte cell line. Dead/total cells percentage ratio, western blot analysis for the cleaved forms of caspase-3 and PARP, cytofluorimetric analysis for Ann V/PI as well as β-galactosidase staining were performed. (A) Trypan blue staining showed that OA treatment induced cell death in both HCC cell lines, but not in healthy hepatocytes cell line. (B) Western blot analysis for the cleaved forms of caspase-3 and PARP proteins showed that 300 μM OA induced apoptotic cell death in Huh7.5 cell line. (C,D) Cytofluorimetric analysis for Ann V/PI staining of Hep3B and Huh7.5 cell lines cultured with different concentration of OA (50, 150, and 300 μM). The strategy of cytometric analysis is showed on the left: representative dot plots from five different experiments, by using PI staining alone for gating Ann V + / PI + cells. On the right, histograms of Ann V – / PI + necrotic cells (fold increase) of Hep3B and Huh7.5 cell lines showed that 300 μM OA significantly induces necrosis in Hep3B (n = 3; *p < 0.05; **p < 0.01). (E) β-galactosidase staining for Control cell line and Hep3B was performed. Images and graphs revealed that 300 μM OA treatment induced significant increase of senescence phenotype in Hep3B but not in Control (n = 3; *p < 0.05).
Figure 5
Figure 5
OA significantly reduces ERK phosphorylation and anti-apoptotic proteins in HCC cell lines. (A) Western blot analyses show that p-ERK levels are significantly decreased upon 300 μM OA treatment in both HCC cell lines, but not in Control cell line. (B) c-Flip levels are significantly reduced in both HCC cell lines, but not in the healthy cells. (C) Bcl-2 protein expression is significantly decreased in both HCC cell lines but not in the healthy cells. (n = 3; *p < 0.05).
Figure 6
Figure 6
OA reduces migration and invasion of both hepatocarcinoma cell lines. Wound-healing assay on Control (A), Hep3B (B), and Huh7.5 (C) cell lines were performed. Representative phase-contrast images wound-healing assay (scratch test) taken at different time points (0, 24, and 48 h) after 300 μM OA treatment are shown. Quantitative analysis of the percentage of uncovered area at 48 h revealed a statistical significance difference in both HCC cell lines after OA treatment, while no differences in Control cell line were observed upon OA treatment (n = 3; *p < 0.05; **p < 0.01). (D) Invasion assay of Hep3B and Huh7.5 cell lines was performed. Top: significant reduction of invading cells percentage after 48 h OA treatment in both HCC cell lines. Bottom: Representative images of Hep3b and Huh7.5 DAPI-stained nuclei after 300 μM OA treatment are shown (n = 3; ***p < 0.001; ****p < 0.0001).
Figure 7
Figure 7
Torin-1 partially reverts the OA-induced effects in HCC cell lines. (A) Autophagic flux modulation upon combined OA and torin-1 treatment in both HCC cell lines. Western blot analyses for HCC cell lines treated with 300 μM OA plus torin-1 in the presence of bafilomycin A1 were performed. LC3II quantification confirms that torin-1 activates the autophagic flux after OA treatment in both HCC cell lines. (B) Hep3B and Huh7.5 cell lines were treated with OA (300 μM) and torin-1. Oil-Red O-eluates/cell number of Hep3B and Huh7.5 cell lines are shown. OA induced a significant accumulation of neutral lipids in both HCC cell lines; the simultaneous presence of torin-1 significantly decreased the accumulation in both HCC cell lines. (C) Cell counts with trypan blue staining were performed. Dead/total cell percentage ratio showed that OA treatment induced cell death in both HCC cell lines; the simultaneous presence of torin-1 significantly reduced the percentage of dead cells in both HCC cell lines (n = 3; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).
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