Ovarian cancer is a prevalent malignancy that poses a significant threat to women's lives due to its high recurrence and mortality rates. The study's findings indicate that UA can inhibit the development of ovarian cancer by suppressing tumour cell proliferation, colony formation, and migration. Additionally, UA can enhance ovarian cancer cell apoptosis. The mechanism of action involves the promotion of tumour cell endoplasmic reticulum stress and the suppression of autophagy. These results suggest that UA holds potential as an antitumour agent.
One effective mechanism for inducing tumour cell death is apoptosis, which can efficiently regulate cell number and proliferation by inducing cell membrane rupture, cell nucleus breakage, chromatin condensation, and DNA breakage [20, 21]. Promotion of apoptosis is currently the prevailing method of targeted cancer therapy [22, 23]. The colony formation assay indicated a decrease in colony numbers with an increase in the dose of UA. Additionally, the migration experiment demonstrated that UA inhibited the migration of ovarian cancer cells, and the results showed a dose-dependent effect in inhibiting the proliferation and migration of ovarian cancer cells. Numerous studies suggest that UA can regulate diverse signalling pathways to impede the proliferation and migration of various tumours. For instance, it can suppress the AKT signalling pathway to restrain oesophageal cancer proliferation [24] and inhibit the ERK signalling pathway to suppress cell adhesion and migration, thereby inhibiting the progression of breast cancer[25]. The results of the present study are consistent with previous experimental findings and establish that UA inhibits ovarian cancer cell proliferation while also reducing migration rates.
The proper balance between the antiapoptotic gene Bcl-2 and the proapoptotic gene Bax is necessary for the maintenance of cellular homeostasis [26]. Reducing Bcl-2 expression significantly increases the pro-apoptotic effects of drugs[27]. Furthermore, members of the caspase family of cysteine proteases are crucial in initiating and executing apoptosis[26, 27]. To investigate whether UA inhibits SKOV3 cell proliferation via apoptosis promotion, we detected apoptosis through flow cytometry and TUNEL staining. Additionally, Western blot analysis revealed increased expression of Bax and Caspase3 proteins, along with decreased expression of Bcl-2 protein. The results of this study were in agreement with the above expression results.
Autophagy, a metabolic process, can promote cellular homeostasis by self-phagocytosing aggregated proteins and damaged or dysfunctional organelles, which enables cellular metabolism and maintains cellular biosynthesis[28–31]. In the development of cancer, autophagy plays a bidirectional regulatory role. The inhibition of tumour progression by early autophagy is primarily dependent on the cellular microenvironment and the tumour's degree of malignancy. As tumours advance, autophagy can supply the energy and metabolites necessary for tumour growth [8, 32–34]. Autophagy transports intracellular substances to lysosomes for degradation, preserving energy for tumour cell survival and providing essential metabolites, including arginine and alanine, to facilitate tumour growth. Autophagy inhibition effectively promotes cell apoptosis.[35–38]. The expression of autophagy-related proteins, such as Beclin1, was detected, and the findings indicate that the expression of Beclin1 and LC3 decreased, whereas the protein expression of P62 considerably increased in a dose-dependent manner. These results suggest that UA could induce pronounced apoptosis by impeding autophagic flow in SKOV3 cells. However, elevated autophagy levels were found in both cisplatin-resistant and cisplatin-sensitive tumour cells after cisplatin treatment, indicating that cisplatin is not an appropriate positive control for this experiment[39]. Numerous studies have demonstrated that Beclin1 is closely linked to tumour progression and plays a significant role in cellular proliferation[40]. Some studies have indicated that decreased autophagy can promote cell proliferation and lead to the onset of malignant tumours. However, considering the bidirectional regulation of autophagy, inhibiting autophagy may effectively decrease tumour proliferation and improve survival rates. The effectiveness of this approach has been validated in experiments on various cancers, such as pancreatic ductal carcinoma, breast cancer, and non-small cell lung cancer, and the level of autophagy is associated with the degree of malignancy and the features of the tumour environment[41]. Autophagy centres on the autophagy initiation protein Beclin1, which generates the autophagy initiation complex (AIC) to aid autophagy[42, 43]. Beclin1 is part of the type III PI3 kinase complex, a crucial compound in forming autophagosomes and promoting autophagosome maturation. It generally interacts with BCL-2, which is pivotal for autophagosome maturation. This substance typically interacts with BCL-2, resulting in the inhibition of cellular autophagy[40, 44]. In contrast, upregulation of Bax induces cytochrome C release, resulting in the cleavage of Beclin1 and inhibition of its autophagy induction effect. Moreover, UA exhibited a pro-apoptotic effect on ovarian cancer, as evidenced by the results of the experiments conducted. The protein content of P62, an autophagy marker, increased, indicating inhibition of autophagy by UA in SKOV3 cells. However, degradation of autophagosomes is a separate step from the formation of autophagosomes[45]. LC3 is considered the protein that firmly binds to the autophagosome membrane. Technical abbreviations will be explained when first used. There are two variants of LC3 (LC3-I and LC3-II): LC3-I is present in the cytoplasm, and LC3-II is bound to the membrane. LC3-I is converted to LC3-II and is capable of both initiating autophagosome formation and prolonging their existence[46]. LC3 expression in SKOV3 cells was detected after UA administration, and the results showed that LC3 expression was reduced, and the expression of LC3 was further reduced with increasing doses, suggesting that UA inhibits ovarian cancer progression by inhibiting autophagy-induced apoptosis in SKOV3 cells. The PI3K-AKT-mTOR signalling pathway plays a significant role in various biological processes and serves as an effective focus for current cancer treatment[47, 48]. The PI3K family of lipid kinases primarily regulate cellular growth and modulate cellular autophagy and include three types: class I, class II, and class III PI3Ks[47, 49]. AKT belongs to the serine/threonine kinase family; when PI3K binds to AKT, it triggers the transfer of AKT from the cytoplasm to the cytosol and leads to its phosphorylation[50]. mTOR, a serine/threonine kinase, is a critical regulator of autophagy and lies downstream of the PI3K-AKT signalling pathway. It is commonly utilized in the negative regulation of autophagy[47, 50, 51]. Numerous studies have demonstrated that inhibiting the PI3K/AKT/mTOR signalling pathway efficiently stimulates autophagy activation, triggers apoptosis, and impedes tumour growth and migration. However, recent studies have reported that glycyrrhizin can inhibit autophagy-related gene expression and exert an antitumour effect by inhibiting the PI3K-AKT-mTOR signalling pathway[47]. This is consistent with the results of our experiments, suggesting that UA inhibits autophagy, promotes apoptosis, and hinders ovarian cancer cell progression by downregulating the PI3K-AKT-mTOR signalling pathway.
The endoplasmic reticulum, comprising pools, sheets, and linear tubules, is the largest organelle; it has a membranous, network structure and plays a role in the synthesis and folding of proteins[52–56]. Nevertheless, an excessive build-up of misfolded proteins beyond the threshold of endoplasmic reticulum processing is triggered by internal and external factors, leading to the unfolded protein response (UPR)[53, 54, 57, 58]. Endoplasmic reticulum stress is a crucial aspect of tumour growth and development [59] and plays a significant regulatory function in both tumorigenesis and progression. The molecular chaperone-binding immunoglobulin (BiP) typically ensures protein folding, refolding, and degradation and is a key component of this process. Also known as GRP78, this protein binds to three sensors: protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK, encoded by EIF2AK3), activating transcription factor 6 (ATF6, encoded by ATF6), and inositol-requiring enzyme 1 (IRE1α, encoded by ERN1). These three sensors bind to the endoplasmic reticulum, rendering themselves in a monomeric, inactive state [53, 57, 60, 61]. When the endoplasmic reticulum undergoes stress, BIP dissociates from PERK, ATF6, and IRE1α and binds to misfolded or unfolded proteins due to its higher binding affinity. This enhances the folding ability of the endoplasmic reticulum, according to sources [52, 53, 57, 60, 62]. Mild, stimulus-induced endoplasmic reticulum stress can enhance protein folding ability and promote adaptive transformation and malignant development of tumour cells. However, prolonged and severe endoplasmic reticulum stress has a toxic effect on tumour cells and can promote apoptosis, immunogenic death, and other negative outcomes[52, 53, 57, 60, 63]. Recently, it was discovered that PERK self-phosphorylates and forms dimers upon dissociation from BIP. The activated PERK can then activate the eIF2α translation initiation factor and stimulate its phosphorylation, which limits protein translation and contributes to an increase in selective ATF4 translation. This subsequently induces the activation of the CHOP transcription factor. While PERK-mediated phosphorylation of eIF2α is necessary for autophagy to take place and progress, PERK can also stimulate the expression of autophagy-related genes. Additionally, CHOP causes growth arrest by increasing the expression of genes involved in autophagosome formation, and it promotes apoptosis by reducing BCL-2 expression [52, 57, 64]. The expression of endoplasmic reticulum (ER)-related proteins, including PERK, eIF2α, and CHOP, was detected by Western blotting. The results revealed a considerable upregulation in the expression of PERK, eIF2α, and CHOP, signifying that UA treatment induced ER stress in SKOV3 cells. Recent experiments have shown that antitumour treatments can increase eIF2α phosphorylation in tumour cells, resulting in antitumour effects. Additionally, endoplasmic reticulum stress activators can upregulate endoplasmic reticulum stress via the PERK/AKT/mTOR signalling pathway. Induction of autophagy in tumour cells and inhibition of tumour progression are well-known therapeutic strategies [65]. However, the results of this study demonstrate that treatment with UA promotes endoplasmic reticulum stress in SKOV3 cells and inhibits SKOV3 cell autophagy, which contradicts previous research(Fig. 6).