Study on the Activity and Mechanism of RCE-4, an Active Ingredient of Lucky Grass, in Inhibiting the Proliferation, Invasion, and Migration of Ca Ski Cells in Cervical Cancer
Cervical cancer is a common gynecological malignant tumor, and currently, surgery, radiotherapy, and chemotherapy are the main treatments for it in clinical practice. However, due to the fact that cervical cancer is mostly an invasive tumor with the characteristics of easy metastasis and spread, it often leads to poor treatment expectations. Therefore, the search for new anti cervical cancer chemotherapy drugs that can inhibit its invasion and migration has attracted much attention. Natural source drugs have always been highly valued by scholars due to their multi-target, effective and low toxicity advantages. RCE-4 is a spirostane saponin isolated from Reineckia carnea (Andr.) Kunth, a plant of the Liliaceae family, in our laboratory. Its chemical name is (1 β, 3 β, 5 β, 25S) – spirostane-1,3-diol1- [α – L-rhamnose – (1 → 2) – β – D-xylopyranoside. The structure is shown in Figure 1, and it has the highest content (up to 13%) in the total saponins of Reineckia carnea (Andr.) Kunth. At the same time, its pharmacological activity is also the best. RCE-4 has been included as a landmark component of Reineckia carnea in the local Chinese herbal medicine standards of Hubei Province.
Previous research in our laboratory has found that RCE-4 has good selective cytotoxicity against cervical cancer Ca Ski cells and can significantly inhibit the growth of nude mouse Ca Ski cell transplantation tumors. The highest tumor inhibition rate can reach 69.1%. No tumor cell metastasis, infiltration, or pathological changes were observed in important tissues such as the uterus and ovaries of the treated mice. Taking inspiration from previous results, we speculate that RCE-4 may have inhibitory effects on the invasion and migration of cervical cancer cells. Currently, there have been no reports on its related activities and molecular mechanisms of action.
Wnt/β – catenin and Hippo/YAP are two intracellular signaling pathways closely associated with tumor invasion and migration. Both pathways regulate tumor invasion and migration by entering the nucleus through their core signaling molecules (β – catenin and YAP/TAZ) and binding with transcription factors to transcribe downstream target genes. Blocking the Wnt/β – catenin pathway has a significant inhibitory effect on tumor proliferation. Abnormal activation of the Wnt/β – catenin signaling pathway can cause growth and development defects, as well as the occurrence, migration, and invasion of tumors. Downregulation of downstream proteins such as MMP2 and MMP9 in the Wnt/β – catenin pathway can inhibit tumor cell metastasis. Contrary to the biological function of the Wnt/β – catenin pathway, Hippo/YAP pathway activation inhibits cell proliferation and promotes cell apoptosis. In the absence of Hippo related activating factors, non phosphorylated YAP is transferred into the nucleus and interacts with the transcription activating factor TEAD/TEF family to regulate the expression of target genes. Extracellular stimulation signals can activate Mst1/2 through an unclear mechanism, and then phosphorylate other core members through a series of cascade phosphorylation reactions.
Therefore, this study will focus on cervical cancer Ca Ski cells and investigate the effects and molecular mechanisms of RCE-4 on cervical cancer proliferation, invasion, and migration through the Wnt/β – catenin and Hippo/YAP signaling pathways, providing theoretical support for the future clinical application of RCE-4.

Our laboratory has shown through MTT experiments that RCE-4 can inhibit the proliferation of Ca Ski cells in a time-dependent and dose-dependent manner. The results of the clone formation experiment in this study further validate the effectiveness of RCE-4. In addition, the effect of RCE-4 on the invasion and migration of Ca Ski cells was detected through scratch experiments and Transwell chamber in vitro invasion experiments. The results showed that RCE-4 significantly inhibited the migration and invasion ability of Ca Ski cells, and exhibited concentration dependence.
The Wnt/β – catenin signaling pathway plays an important role in tumor migration, and the entry of β – catenin into the nucleus is the key to the activation of this pathway. The results showed that RCE-4 can inhibit the nuclear entry of β – catenin through three pathways: inhibiting the expression of p-GSK3 β, promoting the phosphorylation of β – catenin, and inhibiting the expression of PAK4, thereby inhibiting the transcription of downstream genes such as matrix metalloproteinases MMP2/7/9/14. Interestingly, the latest research has found that inhibiting the activity of PAK4 can synergistically enhance the therapeutic effect of PD-1 immune blockade therapy. PD-1 therapy has little efficacy in the absence of pre infiltration of immune cells in tumor tissue, which is also the main reason for the development of resistance to PD-1 therapy. The use of PAK4 inhibitors can significantly enhance the response of tumor tissues to PD-1 blockade therapy in vivo. Considering the significant inhibitory effect of RCE-4 on PAK4, this provides us with a new research direction.
The relationship between the Hippo/YAP signaling pathway and tumor cell invasion has also been confirmed, but so far, there are relatively few drugs targeting the Hippo/YAP signaling pathway. In 2018, Gill et al. found that there is a special positive feedback loop around the entry of YAP/TAZ molecules into the nucleus, which is composed of five protein molecules: NUAK2, LATS1, p-LATS1, YAP/TAZ in the cytoplasm and nucleus. NUAK2 can inhibit the expression of LATS1 to prevent its phosphorylation, thereby promoting the transfer of YAP/TAZ complex molecules from the cytoplasm to the nucleus. YAP/TAZ molecules entering the nucleus bind to NUAK2 transcripts, and the transcribed NUAK2 enters the cytoplasm, forming a positive feedback loop. The discovery of this cycle makes the Hippo/YAP signaling pathway more complex and explains the difficulty of regulating the Hippo/YAP signaling pathway to inhibit tumor cell invasion. The results of this experiment showed that RCE-4 can inhibit the nuclear entry of YAP/TAZ through multiple pathways, including inhibiting the phosphorylation of MST1, enhancing the phosphorylation of YAP, and reducing the expression of YAP. The reduction of YAP/TAZ in the nucleus leads to a decrease in the expression of NUAK2, thus breaking this positive feedback loop. The disruption of the cycle leads to a decrease in the expression of downstream proteins such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), CYR61, and SOX2, significantly inhibiting the migration ability of Ca Ski cells.
It is worth noting that the Wnt/β – catenin and Hippo/YAP signaling pathways in cells are not completely unrelated, and they interact in many ways. For example, phosphorylated YAP/TAZ can bind to β – catenin, causing it to remain in the cytoplasm and inhibit its transcriptional activity. YAP can induce inactivation of GSK3 β, which in turn stabilizes cytoplasmic β – catenin and promotes nuclear transfer of β – catenin. This study has not yet conducted in-depth research on the mutual feedback regulation of the two signaling pathways. At the same time, this stage of research also has certain limitations. It has not been validated for other cervical cancer cell lines such as Hela and SiHa, and lacks animal experimental results. This is also our next research direction.
In summary, RCE-4 has a good ability to inhibit the invasion and migration of Ca Ski cells. Its molecular mechanism is mainly achieved by blocking the key step of β – catenin and YAP/TAZ entering the nucleus, thereby inhibiting the activation of the Wnt/β – catenin signaling pathway and activating the Hip po/YAP signaling pathway.