The killing effect of Tanshinol on breast cancer cells: insight into the reversion of TGF-𝜷1-mediated suppression of NK cell functions

¹ Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, School of Pharmacy, Nanjing University of Chinese Medicine, 210023 Nanjing, Jiangsu, China

² School of Medicine & Holistic Integrative Medicine, Nanjing University of Chinese Medicine, 210023 Nanjing, Jiangsu, China

³ Shandong co-innovation center of TCM formula, College of Traditional Chinese Medicine, Shandong University of Traditional Chinese Medicine, 250399 Jinan, Shandong, China

⁴ Jiangsu Collaborative Innovation Center of Traditional Chinese Medicine (TCM) Prevention and Treatment of Tumor, Nanjing University of Chinese Medicine, 210023 Nanjing, Jiangsu, China

^*Correspondence: wangaiyun@njucm.edu.cn (Aiyun Wang); y.zhao@njucm.edu.cn (Yang Zhao); luyingreen@126.com (Yin Lu)
^† These authors contributed equally.

Front. Biosci. (Landmark Ed) 2021, 26(11), 1106–1118; https://doi.org/10.52586/5013

Submitted: 14 July 2021 | Revised: 30 September 2021 | Accepted: 19 October 2021 | Published: 30 November 2021

This is an open access article under the CC BY 4.0 license (https://creativecommons.org/licenses/by/4.0/).

Abstract

Background: Natural killer (NK) cells play an indispensable role in anti-tumor immunity. TGF- $\beta$ 1 is the main accomplice of tumor immune escape, inhibiting tumor immunity mediated by NK cells. It is reported that Salvia miltiorrhiza can promote the immune killing effect of NK cells. In this study, Tanshinol, a water-soluble active component of Salvia miltiorrhiza, was used to investigate its effect on the inhibition of NK cell functions mediated by TGF- $\beta$ 1 in breast cancer. Methods: We constructed a mouse model of breast cancer by tail vein injection, H&E staining and ELISA were used to verify the role of TGF- $\beta$ 1 and the effects of Tanshinol on breast cancer and NK cells. In vitro, we used CCK8 and cytotoxicity assays to preliminarily evaluate the effect of Tanshinol on the anti-tumor effect of NK cells intervention by TGF- $\beta$ 1. We explored the killing activity of NK cells and related signal pathways by immunofluorescence imaging technology, RT-PCR, ELISA and flow cytometry. Also, Western blot, RT-PCR and immunofluorescence experiments were applied to investigate the expression level of the natural killer group 2 member D (NKG2D)-NKG2D ligands (NKG2DL) signal axis, and combined with immunoprecipitation, to detect the formation of NKG2D-DNAX-activating protein of 10 kD (DAP10) complex. Results: TGF- $\beta$ 1 played a role in promoting lung metastasis of breast cancer and inhibiting the secretion of cytotoxic mediators from NK cells, but Tanshinol could reverse it. High-dose Tanshinol also significantly optimized the survival rate of tumor-bearing mice. TGF- $\beta$ 1 could destroy the NKG2D-NKG2DL axis, down-regulate the expression and nuclear accumulation of p-smad2/3. Moreover, TGF- $\beta$ 1 inhibited the activation of PI3K-ERK1/2-PLC $\gamma$ 2 signaling pathway that is related to the degranulation of NK cells, and diminished the expression of degranulation marker CD107a and the release of anti-tumor cytotoxic killing medium of NK cells. However, Tanshinol was able to interfere with the negative regulation of TGF- $\beta$ 1 on the functions of NK cells, mainly through promoting the expression of NKG2D and its molecular chaperone DAP10, thereby propelling the formation of NKG2D-DAP10 complex. Conclusions: Collectively, Tanshinol enables NK cells to activate and release multiple killing mediators to carry out immune attacks on tumor cells.

Keywords

Tanshinol

Tumor

TGF-β1

NKG2D

DAP10

NK cell functions

Figures

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Fig. 1.

The effects of Tanshinol on breast cancer metastasis. (A) Body weight of mice. (B) Organ index. (C) H&E staining of lung tissue sections ( $\times$ 50). (D) Serum TGF- $\beta$ 1 content in mice. (E) The levels of NK cell effector molecules. All data are presented as the means $\pm{}$ SD, n = 8. (F) The survival rate of tumor-bearing mice, n = 15. #p $<$ 0.05, ##p $<$ 0.01, ###p $<$ 0.001 (vs. control group). ${*}$ p $<$ 0.05, ${**}$ p $<$ 0.01, ${***}$ p $<$ 0.001 (vs. model group).

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