Xihuang Pill (XHP) is a famous patented Chinese medicine, having therapeutic effect on tumor, it was first recorded in the “waike zhengzhi quansheng ji” of ancient Chinese Qing Dynasty. XHP is consisted of bezoar, musk, frankincense (vinegar) and myrrh (vinegar). It has the effects of promoting blood circulation and removing blood stasis, clearing heat and detoxicating, reducing phlegm and resolving masses, decreasing swelling and relieving pain. During recent years, with the rapid grow of integrated traditional Chinese/complementary medicine and modern medicine, the function of XHP in breast cancer progression has been most extensively studied. Clinical studies show clearly that XHP combined with chemotherapy, radiotherapy or endocrine therapy could significantly improve the clinical efficacy, KPS, immune function and reduce the toxicity of chemotherapy or radiotherapy in breast cancer patients[14, 16, 17]. Animal experiments have also confirmed that XHP may inhibit breast cancer through the downregulation of estrogen receptor (ER) and progesterone receptor (PR) expression, inhibition of Bc1-2 expression, increastion of p53 gene expression and IL-2, IFN-γ, CD3+, CD4+ and B7–1 levels[17, 18], XHP can inhibit the growth of breast cancer in 4T1 mice through upregulation of MEKK1, SEK1, JNK1, and AP-1 expression in Tregs, promoting Treg cell apoptosis in the tumor immune microenvironment. In vitro experiments, XHP was found to inhibit the proliferation and migration, but advanced the apoptosis of breast cancer cell lines of MCF-7, Hs578T and MDA-MB435 and breast precancerous lesions MCF-10AT cells.
Its mechanisms might be associated with regulating the protein and gene expression of Cyclin A, CDK2, Bcl-2, Bax, mTOR and VEGF[8, 19, 20]. Based on our research[8–10, 17], we hypothesized that XHP pharmacological mechanism for inhibiting the development of breast precancerous lesions could be closely interrelated to the PI3K/Akt/mTOR signaling pathway.
The current study used a model of 7,12-dimethylbenz[a]anthracene (DMBA) combination with estrogen and progesterone-induced breast precancerous lesion in rats. After confirming that the model was successfully established, XHP was administrated for treatment. subsequently, the specific molecular mechanisms of XHP were investigated. The pathomorphological analysis revealed that Xihuang pill could block and reverse the histopathological changes of breast tissue induced by DMBA combined with estrogen and progestogen. Moreover, the immunofluorescence TUNEL staining assay of breast tissue cells of breast precancerous lesions rats showed that the number of TUNEL positive cells in the XHP group increased compared with the normal control group. In addition, we found greater number of apoptotic cells in the XHP high-dose group than in the middle-dose and low-dose group, these findings revealed that XHP amplified apoptosis in a dose-effect manner. The imbalance between the proliferation and apoptosis of cells was closely associated with breast tumorigenesis and development. PI3K/Akt/mTOR pathway is an important intracellular signaling transduction pathway participate in cell multiplication, differentiation, apoptosis, cycle progression, motility, tumorigenesis, tumor growth and angiogenesis, which plays a major part in both the occurrence and progression of breast cancer[21, 22]. Therefore, inhibition of the PI3K/Akt/mTOR pathway is critical for breast cancer therapy. This signaling pathway involves four key molecules, PI3K, PTEN, Akt and mTOR. PI3K is consisted of a regulatory subunit (p85) and a catalytic subunit (p110), and the activation of PI3K depends upon the activation of the p85 subunit. Once PI3K is activated, its catalytic subunit activates AKT through phosphorylation of AKT, and continuously activates mTOR through phosphorylation of mTOR. AKT has two important phosphorylation sites: Thr308 and Ser473. The phosphorylation of Serine 473 is necessary for the complete activation of AKT. mTOR is a key downstream mediator in the PI3K/Akt/mTOR signaling pathway that plays a key role in regulating cell growth and hyperplasia during breast cancer development. PTEN is a negative regulator in the PI3K-Akt-mTOR signaling pathway, could dephosphorylate the PI3K to down-regulate the activation of PI3K-Akt-mTOR pathway. In this study, we found that XHP could significantly reduce the protein expression of P-PI3K, P-AKT (S473) and P-AKT (T308) in breast precancerous lesion cells, and the higher the dose of XHP, the lower the expression of the three proteins, so there was a negative correlation; XHP could increase the protein expression of PTEN, which was positively related with the dose of XHP.
TSC2 is a potent mTOR suppressor, phosphorylation of TSC2 disturbs the formation of the TSC1-TSC2 complex, which negatively regulates MTOR activity. Wen G Jiang et al.  researched the expression of TSC gene in breast cancer cells or tissues, and found that TSC was strongly positive expressed in normal breast epithelial cells. In invasive tumor tissues, the gene expression was decreased, and the transcription level of TSC2 in normal tissues was apparently higher than that in tumor. the activation of mTOR promotes the phosphorylation of its downstream effectors, including phosphorylation of eIF4E binding protein 1 (4EBP1) and activation of p70 ribosomal S6 protein kinase (p70S6K), which phosphorylates S6 ribosomal protein, hus inducing cell proliferation, angiogenesis and metastasis. Studies have shown that when eIF4E increases, the protein expression of VEGF also increases accordingly. Studies have shown that 4EBP1 could up-regulated the translation of VEGF mRNA, and promoted breast cancer angiogenesis. Shao Bo et al. studied the expression of p-S6 in breast tumor tissues, adjacent nontumorous tissues and benign breast lesion tissues by immunohistochemical method, and and discovered that p-S6 expression was up-regulated in breast tumor and related to the happening and progression of breast cancer. Lao Hai li et al.  studied the expression level of mTOR/p70S6K signaling pathway in in breast cancer, the investigation suggest that the mTOR/P70S6K signaling pathway is specifically activated in breast cancer and it might play an important role in the pathogenesis of breast cancer. Elevated expression of VEGF is a feature of invasive breast cancer. In this project, the upstream regulatory factors (P-Tuberin/TSC2, P-Tuberin (p-S939)), downstream regulatory factors (P-4E-BP1, p-S6, p-p70S6K, VEGF) and p-mTOR protein which were related to PI3K/Akt/mTOR were analyzed. Western blot assays showed that compared with the disease model group, the expression of P-Tuberin/TSC2 and P-Tuberin (p-S939) protein was increased in XHP low, middle and high dose groups, while the expression of p-mTOR and P-4E-BP1 protein were decreased. The effect of high-dose group of XHP was better than that of middle and low-dose groups. Immunohistochemistry results displayed that the protein expression of p-S6, p-p70S6K and VEGF was significantly decreased, and the dose of XHP was negatively correlated with the protein expression, compared with the disease model group. This strongly suggests that XHP can up-regulates the upstream negative regulatory factors of mTOR, such as P-Tuberin/TSC2, P-Tuberin (p-S939), and down-regulates the downstream regulatory factors such as P-4E-BP1, p-S6, p-p70S6K, VEGF, etc., which can block PI3K/Akt/mTOR signaling pathway, thereby effectively inhibiting breast precancerous lesions.
Using qRT-PCR to analysis the expression of PTEN mRNA and VEGF mRNA in breast tissue of rats. compared with the disease model group, the expression levels of PTEN mRNA in each dose group of XHP were significantly increased, while the expression of VEGF mRNA were decreased. It is suggested that XHP can prevent the loss of PTEN mRNA or up-regulate the expression of PTEN mRNA in breast tissue of breast precancerous lesions rats. However, the decrease of VEGF mRNA gene expression indicates that XHP can inhibit the transcription of VEGF mRNA or down-regulate the expression level of VEGF mRNA in breast tissue of breast precancerous lesions rats. The above results were consistent with that of PTEN by Western blot and VEGF by immunohistochemistry. Therefore, PTEN and VEGF genes are relatively antagonistic in the process of transcription and protein synthesis. PTEN gene activation can inhibit VEGF by interrupting PI3K/AKT signaling pathway to affect the synthesis of angiogenic factors, and then inhibit neovascularization of breast cancer.
In summary, we have put forward a new mechanism of XHP inhibit the progress of breast precancerous lesions rats induced by DMBA combined estrogen and progesterone. The PI3K/Akt/mTOR signaling pathways were involved in this process. Intervention on breast precancerous lesions rats with XHP inhibits the proliferation and induces apoptosis through inhibiting the PI3K/AKT/mTOR signaling via upregulating the expression of proteins PTEN, P-Tuberin/TSC2, P-Tuberin (p-S939) and PTEN mRNA, and downregulating the expression of proteins P-PI3K, P-AKT (S473), P-AKT (T308), p-mTOR, P-4E-BP1, p-S6, p-p70S6K, VEGF and VEGF mRNA, suggesting XHP may become a promising anti-tumor drug by directly regulating the PI3K/AKT/mTOR pathway in pre-breast cancer.