Cinnamaldehyde induces apoptosis and downregulates HPVE6/E7 protein expression in human cervical cancer Siha cells
Cervical cancer is one of the most common malignant tumors in the female reproductive system, with over 99% of cervical cancer patients being associated with high-risk human papillomavirus (HPV) infection, with HPV16 and HPV18 being common high-risk phenotypes. In recent years, its incidence rate has increased significantly. Although drug radiotherapy/chemotherapy and surgical treatment have certain effects on early cervical cancer, the treatment effect on middle and late cervical cancer is not good, and the recurrence rate is high, with obvious side effects. E6/E7, as the main oncogenes in cervical cancer, are the cause of initial changes in epithelial cells and can promote host cell proliferation and viral amplification. Research has shown that HPV E6/E7 oncogenes have become potential drug targets for the treatment of cervical cancer. Therefore, natural compounds that induce E6/E7 degradation may be an important source of anti cervical cancer drugs.
Cinnamon, as one of the commonly used traditional Chinese medicines in clinical practice, has the title of “the longest of all medicines”. Cinnamaldehyde (CA), as the main component of cinnamon volatile oil, is a natural small molecule compound. Research has shown that it has various pharmacological activities such as antipyretic, anti-inflammatory, and hypoglycemic effects, and can inhibit the proliferation of various tumor cells such as non-small cell lung cancer, liver cancer, gastric cancer, and melanoma, exhibiting certain anti-tumor effects. However, the molecular mechanism of inducing tumor cell apoptosis has not been fully elucidated, and there are few reports on its anti cervical cancer effects. This study utilized human cervical cancer Siha cells to investigate the anticancer effect and mechanism of CA in vitro, providing further theoretical and experimental basis for anti cervical cancer research.

 

Cervical cancer is one of the most common gynecological malignancies, often accompanied by high recurrence and metastasis rates, posing a serious threat to women’s health. In recent years, natural compounds have been widely explored as potential treatments for cervical cancer by inhibiting E6/E7. For example, tanshinone IIA inhibits E6/E7 at the transcriptional level, leading to p53 dependent growth inhibition in cervical cancer cells; Anisolic acid downregulates E6/E7 expression at the protein level in cervical cancer cells through proteasomal degradation, and induces p53 independent mitochondrial apoptosis; N-benzylcinnamamide promotes apoptosis in human cervical cancer cells by inhibiting E6/E7 mRNA levels. In this study, we reported for the first time that CA can induce apoptosis in Siha cells and downregulate the expression levels of HPV E6/E7 proteins.

Abnormal cell cycle is one of the main causes of tumor formation, and its regulatory mechanisms include cell cycle drive and monitoring. When the driving mechanism is damaged, it can lead to uncontrolled growth of normal cells and their transformation into tumor cells; When the monitoring mechanism is damaged, it can induce functional disorders in DNA repair and cell death, worsening the entire regulatory mechanism. We found through flow cytometry that CA can induce a significant increase in the proportion of G2/M phase cells in Siha cells, indicating that CA is blocked in the G2/M phase. Apoptosis is a form of programmed cell death, which mainly involves the death receptor signaling pathway, mitochondrial signaling pathway, and endoplasmic reticulum signaling pathway. The representative proteins in the “mitochondrial signaling pathway” are Bcl-2 and Bax, which are activated upon receiving intracellular death signals and interact with each other in the outer membrane or cytoplasm of mitochondria, causing changes in mitochondrial membrane permeability, loss of transmembrane potential, and release of cytochrome C and other proteins. In the preliminary experiment, we first confirmed through Hoechst 33342 staining that CA induces apoptosis in Siha cells; Subsequently, through JC-1 fluorescent probe detection, it was found that CA significantly reduced the transmembrane potential of mitochondria in Siha cells; Further Western blot verification revealed that CA reduced the anti apoptotic proteins Bcl-2 and Survivin in Siha cells, while increasing the pro apoptotic protein Bax, further indicating the occurrence of cell apoptosis. E6/E7 proteins help maintain the malignant phenotype of cervical cancer cells with HPV infection and can regulate many molecules related to cell survival and apoptosis pathways. This article demonstrates that CA can downregulate the expression levels of E6/E7, thereby inducing apoptosis in Siha cells infected with HPV. Subsequently, we investigated the specific mechanism of CA induced degradation of E6/E7. The results showed that the pre action of proteasome inhibitor MG132 significantly reduced CA induced E6/E7 degradation, indicating that these two proteins were previously degraded by the proteasome.

The effects of small molecule drugs on gene function are concentration dependent. If a small molecule drug can exert anti-tumor effects, it can use sublethal concentrations to screen for drugs that can synergistically kill it. This method is called chemical genomics screening strategy. Due to the selectivity of chemotherapy drugs for treating cervical cancer in clinical practice and the potential for drug resistance and other side effects in long-term use, existing clinical combination therapy regimens are only effective for some patients. Therefore, finding and developing new therapeutic drugs and combination therapy regimens is the research work we need to carry out. In this study, we utilized a chemical genomics screening strategy to detect the synergistic effect of CA with commonly used cervical cancer drugs TAX, Pt, and 5-FU in Siha cells. The results confirmed that the combination of CA and the above-mentioned chemotherapy drugs can exhibit synergistic effects at a certain concentration, and the effect of the combination therapy group is better than that of the monotherapy group, with statistical significance. According to literature reports, the above-mentioned chemotherapy drugs can cause DNA damage and block the cell cycle during the anti-tumor process; Among them, TAX can block the G2M phase; Pt can block the G2M phase; 5-FU can block G0/G1 phase. According to the research results of this article, it suggests that CA may exert a synergistic effect with the above-mentioned chemotherapy drugs by jointly blocking the cell cycle, thereby inducing cell apoptosis and enhancing its anti cervical cancer effect.

In summary, this study confirms that CA induces apoptosis in Siha cells through the mitochondrial pathway and downregulates the expression of HPV E6/E7 proteins through the proteasome pathway; Simultaneously, it can exhibit synergistic effects with various commonly used chemotherapy drugs for cervical cancer at the cellular level. The above research provides new research ideas for better treatment of cervical cancer, and also proposes new combination therapy regimens, providing theoretical basis for reducing patients’ drug resistance in anti cervical cancer treatment and improving the effectiveness for patients.