Salidroside regulates tumor microenvironment of non-small cell lung cancer via Hsp70/Stub1/Foxp3 pathway in Tregs.

BACKGROUND: The treatment of non-small cell lung cancer (NSCLC) is challenging due to immune tolerance and evasion. Salidroside (SAL) is an extract in traditional Chinese medicine and has a potential antitumor effect. However, the mechanism of SAL in regulating the immunological microenvironment of NSCLC is yet to be clarified. METHODS: The mouse model with Lewis lung cancer cell line (3LL) in C57BL/6 mice was established. And then, the percentage of tumor-infiltrating T cell subsets including Treg was detected in tumor-bearing mice with or without SAL treatment. In vitro, the effect of SAL on the expression of IL-10, Foxp3 and Stub1 and the function of Treg were detected by flow cytometry. Network pharmacology prediction and molecular docking software were used to predict the target of SAL and intermolecular interaction. Furthermore, the effect of SAL on the expression of Hsp70 and the co-localization of Stub1-Foxp3 in Treg was confirmed by flow cytometry and confocal laser microscopy. Finally, Hsp70 inhibitor was used to verify the above molecular expression. RESULTS: We discovered that SAL treatment inhibits the growth of tumor cells by decreasing the percentage of tumor-infiltrated CD4+Foxp3+T cells. SAL treatment downregulates the expression of Foxp3 in Tregs, but increases the expression of Stub1, an E3 ubiquitination ligase upstream of Foxp3, and the expression of Hsp70. Inhibiting the expression of Hsp70 reverses the inhibition of SAL on Foxp3 and disrupts the colocalization of Stub1 and Foxp3 in the nucleus of Tregs. CONCLUSIONS: SAL inhibits tumor growth by regulating the Hsp70/stub1/Foxp3 pathway in Treg to suppress the function of Treg. It is a new mechanism of SAL for antitumor therapy.

Structural and functional analysis of an asymmetric bidirectional promoter in Arabidopsis thaliana.

Bidirectional promoters are relatively abundant in eukaryotic genomes, suggesting that they have an important biological significance. As yet, few of these promoters have been characterized in detail. Here, using a promoter::GUS transgene approach has revealed that the intergenic region of Arabidopsis thaliana divergent genes At1g71850 and At1g71860 is an asymmetric bidirectional promoter, which exhibits an orientation-dependent expression profile. The strength of the forward promoter was greater than that of the reverse promoter, and their tissue specificities were not identical. Deletion analyses revealed that this bidirectional promoter could be divided into three functional regions. The basal level and tissue specificity of the promoter in the reverse orientation were regulated positively by region II and negatively by region III, whereas promoter activity in the forward orientation was regulated negatively by region II and positively by region I. Thus the 52-bp stretch of region II had a dual function, enhancing expression in the reverse orientation and suppressing it in the forward orientation. These results demonstrated that the activity of the At1g71850-At1g71860 bidirectional promoter was modulated by complex interactions between both positive and negative cis-acting elements. These findings will enhance our understanding of the regulatory mechanisms of plant bidirectional promoters.

[Ethanol extract of Rhodiola rosea L. regulates the number of tumor infiltrating T cells to enhance antitumor effect in Lewis lung cancer-bearing mice].

Objective To explore the anti-tumor immune function of the Tibetan medicine Rhodiola rosea L. (RRL). Methods Lewis lung cancer-bearing mice were randomly divided into normal saline group, 500 mg/kg RRL ethanol extract treatment group, and 10 mg/kg cyclophosphamide (CTX) treatment group. All the groups underwent the treatment for 10 days. The mouse survival rate and tumor inhibitory rate were calculated. Additionally, the numbers of CD4+T and CD8+ T cells of tumor infiltrating lymphocytes as well as the proportion of FOXP3+ regulatory T cells (Tregs) in the CD4+CD25+Tregs were detected by flow cytometry. Besides, the serum levels of interleukin 2 (IL-2) and γ-interferon (IFN-γ) in the tumor-bearing mice were examined through ELISA, and the spleen cytotoxic T lymphocytes (CTL) activity was detected by the lactic dehydrogenase (LDH) release assay. Results Lewis tumor-bearing mice treated with the ethanol extract of RRL showed remarkably enhanced survival rate and inhibited tumor growth. Furthermore, the number of tumor infiltrating CD4+T and CD8+ T cells increased, while the proportion of FOXP3+ Tregs in the CD4+CD25+Tregs showed a declined tendency. Meanwhile, the serum IFN-γ and IL-2 levels in Lewis tumor-bearing mice increased, and the killing capacity of spleen CTL was enhanced. Conclusion The ethanol extract of RRL has a positive role in enhancing the anti-tumor immunity by regulating the number and function of immunocytes.

[Salidroside induces anti-tumor effect in dendritic cells via ERK pathway].

Objective To investigate the role of mitogen activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway in the salidroside (SAL)-regulated antitumor immunity of dendritic cells (DCs). Methods Lewis lung cancer-bearing mouse model was established and treated with normal saline (NS) (0.2 mL/d), SAL [500 mg/(kg.d)] and cyclophosphamide (CTX) [10 mg/(kg.d)] in corresponding groups. Then their survival and tumor quality were recorded. Meanwhile, the bone marrow-derived DCs from the Lewis lung cancer-bearing mice were isolated in vitro and cultured for 7 days before collection. After sorted with magnetic beads, cells were co-cultured with PBS, SAL (0.05 mg/mL), LPS (200 ng/mL) or SAL combined with ERK inhibitor U0126 separately for 48 hours, followed by detection of the expression of phosphorylated ERK (p-ERK) by flow cytometry. In addition, after the stimulation of DCs by SAL, the expression of p38 MAPK and phosphorylated c-jun N-terminal kinase (p-JNK) was detected by flow cytometry. Moreover, after sorted with magneticbeads, DCs were co-cultured with PBS, SAL (0.05 mg/mL), LPS (200 ng/mL) or SAL combined with U0126. Then, the supernatant was collected, and IL-12p70 secretion ability of DCs was detected by ELISA. Finally, sensitized bone marrow-derived DCs from Lewis lung cancer-bearing mice were stimulated with SAL and co-cultured with spleen T lymphocytes purified by nylon fiber column from C57BL/6 mice as effector cells. 3LL target cells were then added at the target ratio of 10:1, 25:1, 50:1, followed by the detection of cell viability of cytotoxic T lymphocytes (CTLs) using CCK-8 assay. Results SAL could significantly inhibit tumor growth and improve the survival rate. Compared with PBS group, the phosphorylation of ERK protein on DC was enhanced significantly, and then reduced significantly after U0126 treatment. The phosphorylation of p38MAPK and c-jun N-terminal kinase (JNK) protein was not statistically affected by SAL. The level of IL-12p70 significantly increased in SAL group, and became remarkably higher after U0126 treatment. Finally, the killing ability of CTL was significantly enhanced by SAL. Conclusion SAL can inhibit the tumor growth of Lewis lung cancer-bearing mice and activate the ERK signaling pathway, thereby promoting IL-12p70 secretion in DCs and enhancing the cytotoxicity of CTL.