First, we explored the role of Crithmum maritimum in regulating lipid and metabolic health homeostasis in HCC. Figure 1 shows that Crithmum maritimum 0.5 mM ethyl acetate extract prevents lipid accumulation in two HCC cell lines, HepG2 and HepaRG. These two cell lines were chosen because they have been shown to be good models of lipid accumulation in vitro [20]. To evaluate lipid accumulation, we used Oil Red O staining, which stains neutral lipids [9].
*** p < 0.001 compared to vehicle; ** p < 0.01 compared to Vehicle ### p < 0.001 compared to OA
Next, we analysed the expression of two major genes involved in the lipid biosynthetic pathway, namely fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC). Overexpression of these two genes is associated with hepatocarcinogenesis [21, 22]. Additionally, we considered the expression of HMG-CoA reductase (HMG-CoA Red), a key gene involved in cholesterol biosynthesis, whose overexpression is associated with the development of cancer [23]. Finally, we also evaluated the expression of CD36, a fatty acid transporter whose overexpression has been shown to be associated with the development of hepatic steatosis and HCC [24, 25]. Our results show that Crithmum maritimum is effective in reducing lipid dysregulation (Fig. 2), which promotes the development of HCC. Since these genes are overexpressed in HCC, Crithmum maritimum can be used to normalise the transformed lipid phenotype in well-established HCC.
As a successive step, we evaluated the nutraceutical potential of Crithmum maritimum in the regulation of genes that are considered reliable indicators of the metabolic status of cells. In particular, we analysed the expression of Lactate Dehydrogenase A (LDHA), Lactate Dehydrogenase B (LDHB), AMP-activated protein kinase (AMPK), Sirtuin 1 (SIRT1), Sirtuin 3 (SIRT3). Each of these genes controls key steps in the regulation of metabolic homeostasis, and their expression is dysregulated in tumour cells and also in HCC. In particular, LDHA is usually upregulated [26], whereas LDHB is downregulated [27]. All the other genes are downregulated [28]. In particular, the activation of AMPK, SIRT1 and SIRT3 has been suggested as a preventative and therapeutic opportunity for HCC [29–31]. The data reported in Fig. 3 demonstrate that Crithmum maritimum effectively reverses the pathogenic expression pattern of these genes (Fig. 3a-d). Quantitative differences can be observed in the different cell lines used, which model different degrees of transformation, differentiation, and invasiveness of HCC. For example, HLE are less differentiated and relay more on lactic acid fermentation compared with Huh7 [18], and Huh7 are in turn more aggressive and more fermentative compared to HepG2 and HepaRG, which are the least aggressive and less tumorigenic. In particular, HepaRGs are considered a good model for non-transformed hepatocytes, even though they cannot be compared with primary hepatocytes. Notably, the effect of Crithmum maritimum was evident despite the different characteristics of the cell lines. The effect on gene expression has been substantiated by Western Blot analyses, by the evaluation of the activation by phosphorylation at Thr172 of AMPK. The results demonstrated that the AMPK signalling pathway was activated in HepG2 and Huh7 (Fig. 3e). Figure 3f summarises the main concepts expressed in the reported results.
Finally, we investigated the role of Crithmum maritimum in the regulation of the insulin signaling pathway, whose dysregulation has been implicated in the development and progression of HCC. In particular, we measured the expression of the three main genes involved in the control of the pathway, namely Insulin Receptor (IR), Insulin Receptor Substrate 1 (IRS-1) and Insulin Receptor Substrate 2 (IRS-2). Moreover, we evaluated the activation by phosphorylation of Akt at Ser473, which is a well-recognised marker of activation of the insulin signalling pathway. The results reported in Fig. 4 depicts a very interesting scientific picture, as Crithmum maritimum shows a differential effect that varies according to the degree of aggressiveness and Warburg phenotype of the cell line used. In fact, in HepG2, which display a “low tumorigenic” phenotype, both gene expression and Western blot analyses show a moderate activation of insulin signalling (Fig. 4a, e), while in HepaRG no significative effect was observed (Fig. 4b). In contrast, in the more tumorigenic and invasive Huh7 and HLE, Crithmum maritimum inhibited insulin signalling (Fig. 4c-e). These results are graphically summarised in Fig. 4f. Of note, it has been reported that both IRS-1 and IRS-2 are upregulated in HCC [32].
We previously reported that Crithmum maritimum inhibits HCC cell growth [13] and modulates metabolic [14] and bioenergetic characteristics by activating OXPHOS [15]. We also demonstrated that Crithmum maritimum reduced the expression of HCC markers α-fetoprotein (α-FP) and α1-antitrypsin (α1-AT) and induced a shift in the HCC bioenergetic profile from lactic acid fermentation to OXPHOS [33, 34]. Indeed, we recently showed that OXPHOS inhibition is associated with drug resistance in HCC cell lines [35]. In our future studies, we will continue to explore OXPHOS-mediated drug sensitivity promotion and explore the mechanisms behind the drug sensitization effects of Crithmum maritimum. This may provide additional translational applications for the nutritional properties of Crithmum maritimum.
Overall, Crithmum maritimum shows a strong multifaceted bioactive action, which represents a very important resource in the management of HCC and possibly of other types of tumours. It is worth to underline here the systemic and multi-targeted action exerted by the blend of compounds found in Crithmum maritimum (Fig. S1), which guarantees a full-spectrum action on several of the central bioenergetic traits characteristic of hepatocytes. Finally, it is important to emphasize that the effects of Crithmum maritimum appear to be modulated according to the metabolic phenotype of the interacting cells, as shown in Fig. 5 below.