Research on Resveratrol’s Inhibition of Cervical Cancer by Inducing Autophagic Cell Death
Resveratrol (RES), a non flavonoid polyphenol compound, is widely found in grapes, peanuts and other plants as well as traditional Chinese medicine such as Polygonum cuspidatum. As a natural drug with low toxicity, it has anti-inflammatory, antioxidant, antiplatelet aggregation, anti atherosclerosis and other effects. In addition, resveratrol is also a promising anti-tumor drug. It has a wide range of targets, such as mTOR JAK、β-amyloid、Adenylyl cyclase、IKKβ、DNA polymerase， It is also a specific SIRT1 activator and an effective inhibitor of pregnane X receptor (PXR). Recent studies have found that RES has in vitro and in vivo inhibitory activity against various tumors, but clinical trials of RES as an anti-cancer drug are still limited. The results of testing RES in tumor animal models indicate that RES mainly plays a preventive role in gastrointestinal tumors. In the animal model of breast cancer, RES may become a cancer therapeutic agent. However, this beneficial effect depends on the animal and cell type. There are relatively few reports on the role of RES in the cervical cancer model of human HPV virus infection. This study mainly utilized HeLa cells to generate tumors subcutaneously in nude mice, and examined the anticancer effect of RES in vivo. By detecting the role of RES in autophagy of cervical cancer cells, the mechanism of RES inhibiting cervical cancer by promoting autophagic cell death was explored from both in vivo and in vitro perspectives.

The treatment of cervical cancer is mainly surgical, with postoperative combination of chemotherapy and radiotherapy. Due to the strong adverse reactions and susceptibility to drug resistance of most chemotherapy drugs, the clinical treatment and prognosis of cervical cancer are limited. Therefore, finding drugs with good anti-tumor effects and low toxicity is an urgent need for clinical treatment. In recent years, a large number of studies have confirmed that RES has good anti-tumor ability and is effective in both tumor occurrence and development. Research has shown that tissue protease L mediates autophagic and apoptotic cell death induced by resveratrol in cervical cancer cells. Resveratrol induces a decrease in mitochondrial membrane potential in cervical cancer cell lines, promoting apoptosis and increasing lysosomal permeability. This study further validated the inhibitory effect of resveratrol on cervical cancer through autophagy pathway through in vitro and in vivo experiments. Firstly, it was confirmed through in vivo experiments that RES significantly inhibits the progression of cervical cancer. Moreover, tissue level detection revealed that the expression of autophagy related proteins is regulated by RES. Therefore, it is speculated that the mechanism of RES inhibition of cervical cancer may be related to the occurrence of autophagic cell death (ACD) induced by RES. ACD is the result of excessive autophagy in cells, and it is also a Caspase independent type II programmed cell death that is different from apoptosis. Its main feature is the presence of a large number of autophagosomes and autolysosomes in the cytoplasm, where the majority of substances in the cytoplasm are degraded, but the nucleus remains intact. Under sustained stress and ongoing autophagy, cells will die due to excessive self depletion. The death of such cells often exhibits autophagic characteristics, mainly manifested by overexpression of Beclin1 and the production of a large number of autophagosomes and autolysosomes in the cells. Vacuole membrane protein-1 (VMP1) is a recognized autophagic protein, and its expression level is negatively correlated with tumor malignancy. Together with Beclin-1, it serves as a molecular switch to activate autophagy. The expression and role of VMP1 in cervical cancer have not been reported yet.
Through RT-PCR, Western blot, and autophagy assays, it was confirmed that RES promotes the occurrence of ACD in cervical cancer cells. The results showed that Beclin-1, VMP1, and LC3B located on the inner membrane of autophagosomes and autolysosomes can be induced to be highly expressed by RES in cervical cancer cells, with a similar effect to Rapa. Rapa is an inhibitor of mammalian TOR (mTOR) kinase, which binds to FKBP12 to inhibit mTORC1 activation of autophagy, thereby exerting tumor suppressive effects. Further examination of autophagosomes and autophagic flux revealed that both RES and Rapa can promote the formation of autophagosomes and autolysosomes. After combined use, autophagosomes and autolysosomes further increased, accompanied by a decrease in P62 and mitochondria, but the nucleus remained intact; At the same time, we observed that BAFA1 can block the autophagic flow of cervical cancer cells caused by RES. The combination of BAFA1 and RES resulted in the accumulation of autophagosomes and P62, accompanied by a significant decrease in mitochondria, and obvious abnormalities in the nucleus compared to other groups. BAFA1 is a macrolide antibiotic isolated from Streptomyces species and is a specific vacuolar type H+ATPase (V-ATPase) inhibitor. BAFA1 can target mitochondria, induce apoptosis inducing factors to transfer from mitochondria to the nucleus, and promote caspase independent apoptosis. In addition, BAFA1 induces the binding of Beclin-1 to Bcl-2, further inhibiting autophagy and promoting cell apoptosis. Our experimental results also confirmed this point. By detecting mitochondrial membrane potential and TUNEL positivity rate, we found that RES can induce a decrease in mitochondrial membrane potential to promote cell apoptosis, and produce different responses to cervical cancer cells under autophagy activation and autophagy inhibition. It has been reported in the literature that RES prevents P62 degradation induced by Rapa, so the combination of Rapa and RES can prevent the up regulation of autophagy and induce apoptosis of breast cancer cells. However, in this study, we found that the combination of RES and Rapa can promote excessive autophagy in cervical cancer cells, and the extensive formation of autophagosomes leads to the occurrence of ACD; When the autophagy flow of cervical cancer cells is blocked, RES can induce endogenous cell apoptosis by reducing mitochondrial membrane potential.

From this, it can be seen that RES has good application prospects in the treatment of gynecological tumors such as cervical cancer, and when used in combination with autophagy activator Rapa or autophagy inhibitor BAFA1, it can play a more positive anti-cancer role by promoting autophagic death or cell apoptosis.