Exploring the effects and molecular mechanisms of Xinjiang swollen fruit licorice chalcone A on the proliferation and apoptosis of cervical cancer cells based on CADD
Cervical cancer is one of the malignant tumors that seriously threaten women’s health, and its incidence rate has increased in recent years. Research has found that the mortality rate of cervical cancer patients in China is increasing at a rate of 3.35% per year, with a 10-year growth rate of 29.9%, and the incidence trend among young people is becoming increasingly evident. According to a screening of nearly a thousand women in Xinjiang for human papillomavirus (HPV), the infection rate was 17.46%, indicating that the prevention and treatment of cervical cancer face severe challenges. At present, the conventional treatment measures for cervical cancer are radiotherapy, chemotherapy, and surgical treatment, which face challenges such as high toxicity, susceptibility to tumor drug resistance, and easy recurrence. Cancer stem cells (CSCs) are a type of self-renewal ability that can initiate and drive the growth and metastasis of tumor cells, playing an important role in the process of tumor recurrence and metastasis. CSCs are a small population present in the tumor microenvironment, and research has reported that cervical cancer stem cells (CCSCs) can be identified and isolated from cervical cancer cells. Cancer stem cells are insensitive to most chemotherapy drugs and are the main cause of cancer recurrence in patients during the recovery stage, seriously affecting the 2-5 year survival rate of patients after radiotherapy and chemotherapy. Therefore, searching and screening for novel structures, high anti-cancer activity, low toxicity, and tumor stem cell inhibitory activity of lead compounds against cervical cancer from natural active ingredients has significant research significance in innovative drug research.

Licorice, as a medicinal and edible resource, has a long history of medicinal use in China. Although there are various varieties of licorice, the Chinese Pharmacopoeia (2020 edition, Part 1) includes two original plant species, namely Glycyrrhiza inflata Bat. and Glycyrrhiza glabra L., among which Glycyrrhiza inflata L. is widely distributed and has abundant resources in southern Xinjiang, Gansu and other northwest regions. The chemical structure of chalcones contained in different varieties of licorice varies. Research has confirmed that licochalcone A (LicoA) has anti-tumor, antibacterial, anti-inflammatory, hepatoprotective, and immunomodulatory effects, but there are few reports on its regulation of cervical cancer-related tumor stem cell markers. On the basis of preliminary basic research, our research group has improved a traditional hospital preparation for treating cervical erosion by adding LicoA containing licorice extract. We are currently developing vaginal effervescent tablets that have the potential to prevent cervical cancer and can be used for vaginal antibacterial, anti-inflammatory, and cervical erosion treatment. In the screening of anticancer activity of natural active ingredients, it was found that LicoA, a component of licorice chalcone, has significant activity in the proliferation, apoptosis, and cell cycle of cervical cancer cells. In addition, it has significant regulatory effects on cervical cancer tumor stem cell markers (Bcl-2, ALDH1A1, OCT-4, UHRF1, BIRC7, BIRC5) genes and cyclin dependent kinase 4 (CDK4) genes. By using computer-aided drug design (CADD) and other methods to predict its possible targets, the effect of LicoA on the mRNA expression levels of the above genes was studied, providing a more comprehensive reference for further elucidating the anticancer activity of LicoA on cervical cancer cells and exploring its possible molecular mechanisms.

At present, surgical resection is still the main treatment for cervical cancer in clinical practice, combined with chemotherapy and radiotherapy as adjuvant therapy. The proposal of the tumor stem cell theory has provided a new interpretation of the occurrence, development, and treatment of cervical tumors. Research has confirmed that 11 tumor stem cells are not only the root cause of tumor cell chemoradiotherapy tolerance, but also one of the reasons for tumor cell metastasis and recurrence. Therefore, using natural active ingredients to safely and effectively intervene in tumor stem cells, thereby reducing the possibility of cervical cancer and preventing the recurrence of cervical cancer after treatment, is a new research direction.
Current research has confirmed that licorice has anti-tumor, antibacterial, anti-inflammatory, hepatoprotective, and immunomodulatory effects. The Xinjiang region is rich in licorice resources, with licorice chalcone A mainly distributed in its roots and stems. Our research group developed and established a detection and identification system for chalcone A in swollen licorice during the preliminary research, and applied for a utility model patent related to instrument equipment (patent number: ZL201721685479.2). Based on this, LicoA components were extracted and prepared. The mechanism of LicoA’s anti-tumor effect may be through inducing tumor cell apoptosis, but there is relatively little research on its regulatory effect on cervical cancer tumor stem cell markers. The results of this study showed that Xinjiang swollen fruit licorice chalcone A has a significant inhibitory effect on the proliferation of cervical cancer cells, and exhibits concentration and time-dependent effects. It can induce apoptosis in SiHa cells and may block the cell cycle in the S and G2/M phases, and has a significant downregulation effect on cervical cancer tumor stem cell markers.

Under normal circumstances, retinoblastoma (Rb) forms a complex with E2F transcription factors in the G1 phase of the cell cycle and is inhibited. CDK4 can phosphorylate Rb complex, leading to the release of E2F to transcribe the necessary genes for entering the S phase of the cell cycle from G1 phase. Without this phosphorylation activity, cells are blocked in G1 phase. In this study, CDK4 gene was significantly inhibited at concentrations of 25 and 50 μ g/mL of glycyrrhizic acid chalcone A. The expression of stem cell markers ALDH1A1 and OCT4 is upregulated in cervical cancer and its precursor lesions, especially in peripheral blood, suggesting that ALDH1A1 and OCT4 can be used as biomarkers for early detection of cervical cancer or monitoring of patient treatment. ALDH1A1 is involved in the oxidation of retinol to retinoic acid in the cytoplasm, which is then transferred into the nucleus and initiates transcription of genes involved in early stem cell differentiation, promoting tumor stem cell proliferation. Octomer binding protein 4 (OCT-4) is a transcription factor expressed in embryonic and adult stem cells, which is associated with cell proliferation, differentiation, pluripotency, and self-renewal. Knocking out OCT-4 in embryonic stem cells leads to their differentiation and loss of stem cell characteristics. Glycyrrhizic acid chalcone A can also significantly downregulate the mRNA expression levels of the above two markers (ALDH1A1, OCT4). In addition, the selected tumor markers Bcl-2, UHRF1, BIRC7, BIRC5, etc. in this study are all highly expressed in cervical cancer cells, and their mRNA expression levels are also downregulated to varying degrees by glycyrrhizic acid chalcone A. This suggests that the Xinjiang swollen fruit glycyrrhizic acid chalcone A component may have good research potential in preventing cervical cancer recurrence.