HCC is the sixth most common cancer all over the world [20]. Several cellular and molecular mechanisms are involved in the pathogenesis of HCC including necroinflammation, hypoxia, oxidative stress, mitochondrial damage, changes in the tumor microenvironment, activation of cytokines and growth factors, and genetic alterations [21]. The emergence of immune- and genomic- based therapies has been transformed the treatment strategy of many types of cancer and these therapies are starting to be applied to HCC [22]. Therefore, a deep exploration of these different mechanisms involved in HCC is urgently needed to find effective biomarkers and specific therapeutic targets to improve the treatment efficacy [23].

One key step in the inflammatory response, generated by innate immunity to protect the liver against various pathogens and danger signals, is the activation of inflammasomes. The NLRP3 inflammasome has a double-edged sword effect in cancer which is dependent on many factors such as the expression of its components, the presence of mutations that affect its expression, cancer type, and stages of tumorigenesis [24]. However, its direct role in HCC remains poorly described as reported by García-Pras et al. [8].

The present study involved 20 patients with different grades and stages of HCC. Seventeen of these patients developed HCC in a background of liver cirrhosis. The NLRP3, CASP1, and GSDMD expression levels were evaluated in HCC tissues and adjacent non-cancerous liver tissues obtained from these HCC patients aiming to clarify their role in the development and progression of HCC. Our results showed a statistically significant under-expression of NLRP3 and CASP1 genes in HCC tissues when compared to the adjacent non-cancerous tissues.

These results correspond with the data of Wei et al. who observed that the NLRP3 inflammasome components (NLRP3, ASC, and CASP1) were significantly decreased at both mRNA and protein levels in the HCC tissues in comparison with the adjacent non-cancerous liver tissues suggesting that lack of this multi-protein complex was involved in HCC development [14].

Fujikawa et al. firstly reported that caspase-1 protein levels were significantly lower in the HCC tissues than those levels in the adjacent non-cancerous tissues indicating its association with HCC development [25]. Moreover, Chu et al. assessed both protein and mRNA expression levels of CASP1 in Human HCC tissues and in HCC cell lines using Western blot and RT-qPCR. They found that CASP1 levels were significantly lower in HCC tissues and HCC cell lines than those levels in adjacent non-cancerous tissues and normal hepatocyte cell lines suggesting that loss of CASP1expression is involved in the pathogenesis of HCC [26].

Inflammation was defined as one of the hallmarks of tumorigenesis. In its early phases, the immune system can suppress it via NLRP3 inflammasome activation and IL-18 secretion which in turn can trigger the NK cells to exert their cytotoxic potential against cancer cells. Nevertheless, continuous unprovoked activation of this inflammasome and presence of IL-1β in the tumor microenvironment can attract immunosuppressive cells including tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs) as well as regulatory T cells (Tregs) which promote tumor invasion and metastasis [27]. HCC typically originates on the top of chronic hepatic inflammation which is almost accompanied by a massive loss of hepatocytes and irreversible liver damage. In turn, dying hepatocytes release DAMPs which aggravate inflammation and then induce further liver damage generating a highly hepatotoxic cycle of inflammation, cell death, and compensatory liver regeneration increasing the risk of liver cancer. Thus, hepatocytes proliferating under conditions of continuous liver injury may accumulate mutations leading to dysplasia and finally to tumor development [28]. Once HCC formed, the NLRP3 inflammasome components were significantly under-expressed in the transformed liver cancer cells demonstrating that these malignant cells originated from NLRP3 inflammasome-deficient precursor cells [14].

Parallel previous findings in colorectal cancer mice models [29,30,31,32], multiple myeloma patients [33], and lung cancer patients [34] suggesting a protective role of the NLRP3 inflammasome against cancer development as they showed that NLRP3 and/or CASP1 levels were significantly downregulated in these different cancers.

In contrast, several studies suggested that NLRP3 inflammasome components were overexpressed in many types of cancer such as gastric cancer, endometrial carcinoma, laryngeal squamous cell carcinoma and colorectal cancer demonstrating that NLRP3 inflammasome activation may contribute to the development and progression of these different cancers [35,36,37,38]. All these reported data suggest that a united mechanism for activating of NLRP3 inflammasome and its contribution to cancer has not emerged yet, so further studies are needed to clarify its role in each kind of tumor.

In the present study, NLRP3 and CASP1 expression levels are lower in poorly differentiated HCC (grade III) when compared with mild/moderately differentiated HCC (grade I-II) which are statistically significant for NLRP3, but not for CASP1. Also, there is a statistically insignificant decrease in NLRP3 and CASP1 expression levels in late HCC stages when compared with early ones suggesting that the HCC patients with lower NLRP3 and CASP1 expression levels are prone to have more advanced stages (stages III and IV) and poorer cancer cell differentiation (Grade III). These findings are in line with Wei et al. who demonstrated that the patients with advanced HCC stage were likely to have lower expression levels of the NLRP3 inflammasome components, whereas HCC patients with higher grade had weaker immunoreactivity to these components [14].

Gasdermin-D has a pore-forming activity and is well-recognized as an executor for pyroptosis [39]. However, the role of GSDMD itself in HCC development and progression remains unclear. Hence, the present study also analyzed the mRNA expression levels of GSDMD showing that its expression levels were statistically significant under-expressed in HCC tissues suggesting that deficiency of GSDMD may be involved in HCC development. Similarly, other previous studies revealed that GSDMD expression was significantly downregulated in other types of cancer such as gastric cancer and colorectal cancer [40, 41].

In contrast, Lv et al. found that HCC tissues and metastatic HCC tissues exhibited overexpression of GSDMD mRNA level when compared with normal and non-cancerous tissues hypothesizing that GSDMD might induce HCC progression. They also found that “the high mobility group box 1/toll-like receptor 4/caspase-1 activation pathway” may contribute to GSDMD over-expression and its cleavage which then can promote HCC tumorigenesis [42]. Similar studies also demonstrated that GSDMD expression was significantly upregulated in other cancers such as lung cancer and glioma [43, 44].

In the current study, statistically significant lower GSDMD mRNA levels are noticed in grade III HCC when compared with grades I-II as well as in late HCC stages when compared with early ones. These findings suggest that GSDMD under-expression is closely related to late HCC stages and poorly differentiated carcinoma.

According to the current study, a correlation analysis was performed showing that NLRP3 mRNA expression had a statistically significant negative correlation with HCC grades, but not with stages of HCC. Also, CASP1 mRNA expression levels were negatively correlated with different grades and stages of HCC but without a statistical significance. In addition, there was a statistically significant negative correlation between the mRNA expression levels of GSDMD and different HCC grades. Also, there was a negative correlation between the mRNA expression levels of GSDMD and HCC staging but without a statistically significance. These findings suggested that the lower the mRNA expression level, the higher the grade and the later the stage of HCC.

These results are in agreement with Wei’s findings which revealed that the expression of NLRP3 inflammasome components was correlated inversely with the pathological grades as well as advanced stages of HCC indicating that loss of NLRP3 inflammasome was involved in HCC progression [14].

Our data also reveals that NLRP3, CASP1 and GSDMD mRNA expression levels in HCC patients had statistically significant strong positive correlations with each other. Also, Wei et al. observed that the NLRP3, CASP1, and ASC expression levels had significantly positive correlations with each other suggesting that these components cooperated to contribute to HCC development [14]. Pyroptosis is dependent on caspase-1 activation and gasdermin cleavage to induce cancer cell death. It is involved in the pathologic process of various kinds of cancers and its beneficial effect against HCC was reported by Chu et al. [26]. The present study provided evidence for the involvement of NLRP3 inflammasome and pyroptosis in HCC as revealed by the statistically significant under-expression of NLRP3 (the sensor of inflammasome), CASP1 (the effector of inflammasome), and GSDMD (the downstream molecule of NLRP3 inflammasome activation and the executioner of pyroptotic cell death) in the cancerous tissues obtained from HCC patients.

Similarly, Zhang et al. found that CASP1 is downregulated in HCC tissues when compared to the normal liver tissues indicating that the canonical pyroptotic pathway is possibly repressed in the pathogenesis of HCC [45]. On the other hand, inhibition of the pyroptotic signaling pathways has been reported to possess anti-HCC effects. Fan et al. showed that the proliferation, invasion, and metastasis of HCC cells are suppressed through NLRP3 inflammasome inhibition [46]. This difference is probably related to the degree of activation of pyroptosis. Minor activation of pyroptosis induces a minority production of IL-1β which promotes the proliferation of liver cancer, while severe activation of this pathway causes the death of HCC cells [47].

Epidemiological studies have revealed that females have a lower incidence and reduced mortality from HCC than males suggesting a protective role of the estrogen signaling against liver cancer [1]. In 2019, Wei et al. found that 17β-estradiol exerted anticancer effects in HCC by targeting the NLRP3 inflammasome however, its precise role in HCC development remains to be investigated [13].

So in the present work, we treated the HepG2 HCC cell line with 17β-estradiol for 48 and 72 h. Then, the NLRP3, CASP1, and GSDMD mRNA expression levels were analyzed to define if there is a potential link between the NLRP3 inflammasome and estrogen. Also, to clarify the potential molecular mechanism by which E2 can be used as a protective factor against HCC development.

By inverted microscope, our results revealed that E2 inhibits cell proliferation and causes morphological changes in the treated HepG2 HCC cells. Also, we found that E2 can activate NLRP3 inflammasome and induce pyroptosis in HCC cells as demonstrated by the statistically significantly higher mRNA expression levels of NLRP3, CASP1, and GSDMD in each of the two treated groups when compared with the untreated group. All these findings indicate a protecting role of estrogen against HCC development and its progression.

In partial agreement with these findings, Wei et al. demonstrated a significant positive correlation between the expression level of NLRP3 inflammasome components and estrogen receptors beta (ERβ). They also showed that after treatment with E2, proliferation, migration, and colony formation capabilities of HCC cells were significantly inhibited in an ERβ/MAPK/ERK pathway-dependent manner which is consistent with a significantly upregulated expression and activation of NLRP3 inflammasome [12]. Mitogen-activated protein kinase pathway is one of the signaling pathways that regulate the NLRP3inflammasome activation [48]. MAPKs mainly include p38, extracellular signal-regulated kinase (ERK1/2), and several c-Jun N terminal kinases (JNKs) [49]. Phosphorylation of NLRP3 at the Ser198 residue by JNK1 is critical for its deubiquitination facilitating its self-association as well as subsequent inflammasome complex assembly and activation [50].

Also, Wei et al. showed that E2-induced activation of the NLRP3 inflammasome promoted caspase-1-dependent pyroptotic cell death in HCC cells which may be a potential neoplastic target for the treatment of HCC [13]. In addition, Zhang et al. revealed a tumor-suppressor function of the NLRP3 inflammasome activation in HCC represented by induction of pyroptosis indicated by the elevated expression levels of NLRP3, cleaved caspase-1, and also an increased level of GSDMD-NT domain [51].

The protecting role of 17β-estradiol in HCC has been attributed to the anti-proliferative and anti-inflammatory activities brought by E2 through binding to and activation of ERβ [52, 53]. Initiation of both genomic and non-genomic estrogen signaling pathways depends on E2 binding to its receptor. In the genomic pathway, E2 binds to nuclear ERs activating them which in turn stimulate the expression of target genes either by direct binding to specific DNA sequences called estrogen response elements (EREs) or tethering where ER interacts with DNA-bound transcription factor in a way that stabilizes the DNA binding of that transcription factor resulting in enhanced transcription of target genes [54]. In the non-genomic pathway, E2 binding to membrane-associated ERs may exert rapid actions which start with the activation of a variety of signal transduction pathways such as MAPK/p38, MAPK/ERK, PLC/PKC, and PI3K/AKT leading to regulation of gene expression and cytoplasmic alterations [55].

Regarding all these data, we suggest that after treating HepG2 HCC cells with 17β-estradiol, it binds to and activates ERs which in turn can upregulate the expression of NLRP3 inflammasome components and also can activate the MAPK signaling pathway resulting in phosphorylation of NLRP3 protein. This phosphorylation may be responsible for the NLRP3 inflammasome activation. Once it becomes activated, active caspase-1 can trigger the pyroptotic cell death via cleavage of gasdermin-D which perforates the cell membrane by its amino-terminal domain [56].

In conclusion, we found that in combination with each other, NLRP3, CASP1, and GSDMD possibly serve as promising therapeutic targets for HCC based on the under-expression of the NLRP3 inflammasome components (NLRP3 and CASP1) and the executioner of pyroptosis (GSDMD) in the studied HCC patients. In addition, our study suggests that 17β-estradiol may be effective in HCC treatment as it can inhibit tumor cell growth and proliferation by activating the NLRP3 inflammasome and the caspase-1-dependent pyroptosis in HCC cells.