Study on the Killing Effect of γδT Cells Activated by Rukangyin on Breast Cancer MDA-MB-231 Cells.

OBJECTIVE: To investigate the killing effect of rukangyin (RKY) activated γδT cells on breast cancer cells MDA-MB-231 and to provide a basis for Chinese medicine combined with immunotherapy for breast cancer. METHODS: Thus, study isolates peripheral blood mononuclear cells (PBMC) and uses CCK8 to select the optimal concentration of Rukang drink, ZOL (zoledronic acid), and PHA (phytoagglutinin) to activate γδT cells. There are 8 groups including the ① PBMC control group, ② RKY group, ③ ZOL group, ④ PHA group, ⑤RKY+ZOL group, ⑥RKY+PHA group, ⑦ZOL+PHA group, and ⑧ RKY+ZOL+PHA group. At 0 and 14 days of culture, cell viability and γδT cell expansion were detected by flow cytometry. The 8 groups of amplified γδT were cocultured with breast cancer MDA-MB-231 cells labeled with fluorescent dye CFSE at a ratio of 10 : 1 to determine the lethality of γδT cells on breast cancer MDA-MB-231 cells. RESULTS: The optimal concentrations of RKY, ZOL, and PHA to activate γδT cell proliferation were 4.5 mg/l, 3 µM, and 60 µg/ml, respectively. On day 0 of culture, the values ( x ¯ ± s , %) of γδT cells in groups ① to ⑧ were 3.50 ± 0.72, 3.97 ± 0.45, 3.99 ± 0.15, 4.37 ± 0.24, 4.47 ± 0.97, 4.59 ± 1.35, 3.45 ± 0.40, and 3.89 ± 0.48, while when a comparison between groups was made, F = 1.093 and p = 0.412; there is no significant difference between groups. Besides, when being cultured for 14 days, the values ( x ¯ ± s , %) of γδT cells in groups ① to ⑧ were 4.77 ± 0.78, 23.22 ± 2.73, 26.4 ± 0.92, 28.66 ± 1.43, 27.99 ± 1.10, 30.21 ± 1.91, 32.51 ± 0.74, and 33.21 ± 0.42. Then, based on the comparison between groups, F = 119.917 and p < 0.001, there are obvious statistical differences between groups. Furthermore, the expansion values of γδT cells were compared before and after culture for 0 and 14 days. The t values of group ① to group ⑧ were 2.072, 12.051, 41.641, 29.015, 27.777, 18.972, 59.836, and 79.622. Except for the PBMC control group (p = 0.107), there are significant statistical differences (p < 0.001). The number of γδT cell expansion at 14 days was the RKY+ZOL+PHA group>ZOL+PHA group>RKY+PHA group>PHA group>RKY+ZOL group>ZOL group>RKY group>PBMC control group. From group ① to group ⑧, the γδT cell expansion multiples were 1.14 ± 0.44, 5.25 ± 0.77, 5.70 ± 0.89, 6.05 ± 1.03, 6.21 ± 0.09, 6.76 ± 1.46, 7.52 ± 1.05, and 7.97 ± 1.55, respectively, while the comparison between groups was F = 17.772 and p < 0.001. As for the amplification factor, there was RKY+ZOL+PHA group>ZOL+PHA group>RKY+PHA group>RKY+ZOL group>PHA group>ZOL group>RKY group>PBMC control group. In the killing experiment, the killing rate ( x ¯ ± s , %) of group ① to group ⑧ was 1.08 ± 0.03, 1.89 ± 0.14, 1.22 ± 0.11, 1.31 ± 0.09, 1.48 ± 0.10, 2.02 ± 0.21, 2.18 ± 0.27, and 2.37 ± 0.35, whereas the comparison between groups was F = 20.498 and p < 0.001. In terms of killing rate, there was RKY+ZOL+PHA group>ZOL+PHA group>RKY+PHA group>RKY group>RKY+ZOL group>PHA group>ZOL group>PBMC control group. CONCLUSION: Rukangyin can increase the lethality of γδT cells against MDA-MB-231 cells by activating the proliferation of γδT cells, which provides a basis for Chinese medicine combined with immunotherapy for breast cancer.

Systems pharmacology in combination with proteomics reveals underlying mechanisms of Xihuang pill against triple-negative breast cancer.

Xihuang pill (XHP), a traditional Chinese herbal formula, has been clinically used as an adjuvant therapy against triple-negative breast cancer (TNBC) via inhibiting cancer cell invasion and proliferation, as well as promoting cancer cell apoptosis. However, its anti-TNBC bio-active ingredients and possible mechanisms are still unclear. Herein, the hub bio-active compounds and underlying mechanisms of XHP against TNBC were systematically elucidated by integrating systems pharmacology approach and in vitro proteomics analysis. Using systems pharmacology analysis and molecular docking evaluation, 28 bio-active compounds and 10 potential therapeutic targets of XHP were identified. Functional analysis showed that the core therapeutic targets against TNBC were mainly involved in epidermal growth factor receptor (EGFR)-phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway to prevent cancer cell proliferation and angiogenesis, as well as to enhance cancer cell apoptosis. The in vitro proteomics analysis identified 153 differentially expressed proteins (DEPs), including HASP90AA1, AKT1, and EGFR, which were also identified as therapeutic targets against TNBC through systems pharmacology analysis. Protein function analysis showed that the DEPs were mainly involved in PI3K-AKT signaling pathway, which was consistent with the result of systems pharmacology, suggesting the reliability of systems pharmacology analysis. Taken together, these findings uncover the underlying mechanism of XHP against TNBC, and provide a scientific method for the rational development of traditional Chinese medicine.

Multi-targeting chemoprevention of Chinese herb formula Yanghe Huayan decoction on experimentally induced mammary tumorigenesis.

BACKGROUND: Development of safe and effective chemopreventive agents is a winning strategy in reducing the morbidity and mortality of breast cancer. The current study was to investigate the mechanism-based chemopreventive potential of a Chinese herb formula Yanghe Huayan (YHHY) Decoction on the classical 7,12-dimethylbenz(a)anthracene (DMBA) induced rat mammary carcinogenesis model. METHODS: Female Sprague-Dawley rats at 42 days of age were orally administered with a human equivalent dose of YHHY Decoction at 0.02 ml/g (10 mg/ml) once daily, starting 1 wk. before and 4 wks following DMBA treatment. Mammary tumor occurrence was monitored every day. The length of time before palpable tumor is examined is defined as tumor-free survival time. High performance liquid chromatography (HPLC) analyses were adopted to identify major chemical compositions of the decoction. Following bioinformatics data mining and experimental analyses were performed to demonstrate the underlying mechanism of action. RESULTS: DMBA animals receiving YHHY Decoction exhibited a significant delay (P = 0.014) and in some animals prevention (P = 0.046) of tumor occurrence without obvious toxicity. Oncogenic myc activation was significantly suppressed in the DMBA-induced rats by the YHHY treatment. Eight major chemical compositions of the decoction were identified and were shown to interfere with multiple tumorigenic pathways simultaneously in the mammary tumors, including inducing tumor apoptosis and up-regulating pro-apoptotic protein Bax and down-regulating anti-apoptotic protein Bcl-2; suppressing abnormal cell proliferation and the MAPK/ERK, PI3K/AKT signalings; blocking neo-angiogenesis and the VEGF/KDR signaling, and inhibiting oxidative stress in the mammary tumors. CONCLUSION: The multi-components and multi-targeting properties of the YHHY Decoction support its use as a potent chemopreventive drug in breast cancer.

Yanghe Huayan decoction inhibits the capability of trans-endothelium and angiogenesis of HER2+ breast cancer via pAkt signaling.

Background: Yanghe Huayan Decoction (YHD), a traditional Chinese medicine, is one of the most common complementary medicine currently used in the treatment of breast cancer (BC). It has been recently linked to suppress precancerous lesion and tumor development. The current study sought to explore the role of YHD on trans-endothelium and angiogenesis of BC. Methods: HER2+ BC cells were treated with YHD, Trastuzumab, or the combination in vitro and in vivo to compare the effects of them on trans-endothelium and angiogenesis features. The present study also investigated the potential molecular mechanism of YHD in inhibiting angiogenesis of BC. Results: YHD significantly suppressed the invasion and angiogenesis of BC cells via elevated pAkt signaling. Administration of YHD in vivo also strikingly repressed angiogenesis in tumor grafts. Conclusion: YHD could partially inhibit and reverse tumorigenesis of BC. It also could inhibit Akt activation and angiogenesis in vitro and in vivo Its effect was superior to trastuzumab. Thus it was suitable for prevention and treatment of BC.

[Intervention of rukangyin on the lymphangiogenesis in breast cancer metastasis nude spontaneous mouse model].

OBJECTIVE: To observe the effects of rukangyin (RKY) on the lymphangiogenesis and lymph node metastasis of breast cancer transplantation tumor mice, thus exploring its anti-tumor metastasis mechanisms. METHODS: Human breast cancer cell line MDA-MB-435S were in situ implanted into the mammary fat pad of 30 female nude mice to establish breast cancer transplantation tumor spontaneous metastasis model. They were randomly divided into six groups, i.e., the model control group, the 5-FU control group, the small, medium, large dose RKY groups, and the medium dose RKY +5-FU group, 5 in each. Normal saline was given to mice in the model control group at the daily dose of 0.4 mL/kg by gastrogavage. 5-FU was given to mice in the 5-FU control group at the daily dose of 30 mg/kg by peritoneal injection. RKY was given to mice in the small, medium, large dose RKY groups at the daily dose of 18, 45, and 90 g/kg by gastrogavage. 5-FU 30 mg/kg (by peritoneal injection) + RKY 45 g/( kg x d) (by gastrogavage) was given to mice in the medium dose RKY +5-FU group. All medication was carried out once daily for 6 successive weeks. The tumor volume, the tumor inhibition ratio, and the inhibition ratio of axillary lymph node metastasis were detected after medication. The lymphatic microvessel density (LMVD) and expressions of vascular endothelial growth factor-C (VEGF-C) and vascular endothelial growth factor receptor-3 (VEGFR-3) of the breast cancer tissues were detected using immunohistochemical assay. RESULTS: Compared with the model control group, the tumor volume was markedly reduced in the small, medium, large dose RKY groups, and the medium dose RKY +5-FU group, the expressions of VEGF-C and VEGFR-3 were significantly down-regulated and LMVD were obviously lowered, showing statistical difference (P < 0.05, P < 0.01). The inhibition rates of tumor and axillary lymph node metastasis were highest and the LMVD was the lowest in the medium dose RKY +5-FU group, showing statistical difference when compared with other medication groups (P < 0.05, P < 0.01). CONCLUSION: RKY might inhibit the lymph node metastasis of breast cancer possibly through intervening the expressions of VEGF-C and VEGFR-3, and suppressing lymphangiogenesis.

Heme oxygenase type 2 participates in the development of chronic inflammatory and neuropathic pain.

Heme oxygenase (HO) catalyzes the conversion of heme to biliverdin, iron, and carbon monoxide. Recent studies have demonstrated that inhibitors of HO are analgesic in a number of different pain models. In these studies we attempted to define the role of HO type 2 (HO-2) in the development of chronic inflammatory and neuropathic pain. To do this both wild type and C57Bl/6 HO-2 null mutant mice were either injected with complete Freund[apos ]s adjuvant in 1 hind paw or underwent unilateral partial sciatic nerve ligation. The resulting thermal hyperalgesia and mechanical allodynia were monitored for up to 14 days afterward. In both models of chronic pain it was observed that the extent of hyperalgesia and allodynia was significantly less for the HO-2 null mutants than the wild type mice. Additional studies quantified spinal cord dorsal horn Fos expression after brushing of the affected hind paw for both complete Freund[apos ]s adjuvant and partial sciatic nerve ligation treated mice. These studies showed that HO-2 null mutants had less Fos expression after stimulation by brushing than did their wild type counterparts. Our results indicate that HO-2 participates in the thermal hyperalgesia and mechanical allodynia that occur in 2 commonly used models of chronic inflammatory and neuropathic pain.