Baicalin Regulates Rho ROCK-MLC Pathway to Inhibit the Growth and Metastasis of breast cancer Cells
Breast cancer is known as “breast rock”, “breast stone carbuncle”, etc. in the category of traditional Chinese medicine. It is one of the most common malignant tumors among women. In recent years, the incidence rate has been rising, and the age of onset is getting younger and younger, posing a serious threat to women’s health. Although breast cancer has made significant progress in early screening, surgery, chemoradiotherapy and other technologies, for the systemic metastasis of advanced breast cancer, the clinical lack of safe and effective drugs is the difficulty of clinical treatment at present, and also one of the main causes of death of breast cancer patients. Traditional Chinese medicine and its active ingredients have been reported to have the effect of inhibiting breast cancer metastasis, and become one of the research hotspots of breast cancer metastasis drugs. Baicalein is one of the main bioactive components of traditional Chinese medicine Scutellaria baicalensis, which has been proven to have anti-tumor, anti-inflammatory, anti cardiovascular disease, and antibacterial activities in both in vitro and in vivo studies. In recent years, baicalein has attracted more and more attention because it can inhibit the proliferation of tumor cells, or induce apoptosis and autophagic cell death of low toxic cancer cells, especially because it can inhibit the metastasis of breast cancer. The abnormally activated RhoA and ROCK in breast cancer tissue cause the phosphorylation of downstream MLC to increase, leading to cell migration and invasion. The purpose of this study was to establish a nude mouse model of human breast cancer xenograft tumor, observe the effect of baicalein on the growth and metastasis of breast cancer in vivo, and explore the mechanism of baicalein participating in ROCK mediated cytoskeleton reorganization and inhibiting the migration of human breast cancer MDA-MB-231 cells.

Scutellaria baicalensis extract, as an effective extract of Scutellaria baicalensis root, has shown its anti-cancer ability in various cancers. Some studies have shown that baicalein can inhibit PI3K/AKT signal transduction to induce apoptosis and autophagy of breast cancer cells, and affect the invasion and migration ability of breast cancer cells by inhibiting the expression of MMP-2 and MMP-9. However, the anti-tumor effect of baicalein has not been confirmed in clinical trials, and the specific mechanism of its inhibition of breast cancer cell migration has not yet been clarified. In this study, on the nude mouse model of human breast cancer xenograft tumor, it was observed that baicalein can inhibit the growth of breast cancer in situ tumor and lung metastasis, and its inhibitory effect is less than that of paclitaxel, but it has no significant impact on the weight, liver and kidney functions of nude mice, indicating that baicalein may be relatively safe for the prevention and treatment of breast cancer, which is consistent with the previous in vivo research results of baicalein inhibiting lung metastasis of breast cancer in nude mice.

The migration ability of tumor cells is one of the main reasons for tumor metastasis, and cell migration is regulated by Rho family genes. They participate in cancer metastasis by regulating cell skeleton reorganization, cell adhesion, and cell movement. The migration of individual or collective tumor cells is a key step in this complex process, involving extracellular matrix (ECM) remodeling and dynamic recombination with adjacent cells and basal connective tissue cells, signaling proteins, and scaffold components related to the cytoskeleton to coordinate the adhesion of cells to ECM. In this study, it was observed that baicalein has an inhibitory effect on the migration of MDA-MB-231 cells. Similar to the experimental results of this study, there are reports that baicalein may inhibit the proliferation and migration of MDA-MB-231 cells by suppressing the expression of MMP-9 protein.

RhoA family proteins play a regulatory role in cell migration and movement. RhoA is one of the small molecular weight G protein members in the Ras superfamily, mainly involved in regulating tumor cell invasion and metastasis by affecting the cytoskeleton. Rac1 is a member of the Rho family Rac subfamily, which can regulate cell growth and cytoskeleton reorganization. It is a multifunctional regulator of many cellular processes, including cell cycle, intercellular adhesion, and regulating cell motility through the actin network. Rho related protein kinase (ROCK) is a kinase belonging to the AGC (PKA/PKG/PKC) serine threonine kinase family. It is a downstream target effector molecule of RhoA and mainly participates in regulating cell shape and movement by acting on the cytoskeleton. It has been found that rat ROCK is the first effector of Rho, which induces stress fiber formation and adhesive plaques by phosphorylating downstream myosin light chain MLC. Currently, two mouse ROCK isoforms, ROCK1 and ROCK2, have been identified. We found in our experiment that baicalein, when used alone or in combination with the specific inhibitor Y-27632 of ROCK, can alter the cytoskeleton reorganization of cells, inhibit cell migration, and suppress the protein expression levels of ROCK1, Rac1, and RhoA, as well as inhibit the phosphorylation of MLC. This suggests that baicalein’s inhibition of cancer cell migration may be related to the Rho ROCK-MLC pathway.

To sum up, baicalein can inhibit the tumor growth and lung metastasis of human breast cancer cell MDA-MB-231 xenograft tumor in nude mice, and has no obvious hepatorenal toxicity. Moreover, baicalein may affect the expression of cytoskeleton proteins by regulating the Rho ROCK-MLC pathway, thereby inhibiting the migration of breast cancer cells.