Osthole inhibits proliferation of kainic acid­activated BV­2 cells by modulating the Notch signaling pathway.

Epilepsy is a syndrome involving chronic recurrent transient brain dysfunction. Activation and proliferation of microglia serve important roles in epilepsy pathogenesis and may be targets for treatment. Although osthole, an active constituent isolated from Cnidium monnieri (L.) Cusson, has been demonstrated to improve epilepsy in rats, its underlying mechanism remains to be elucidated. The present study investigated the effect of osthole on proliferation of kainic acid (KA)­activated BV­2 cells and explored the molecular mechanism by which it inhibited their proliferation. Using Cell Counting Kit­8, enzyme­linked immunosorbent assay, reverse transcription­quantitative PCR, western blot analysis and immunofluorescence staining, it was identified that following exposure of KA­activated BV­2 cells to 131.2 µM osthole for 24 h, cell proliferation and release of tumor necrosis factor α, interleukin 6 and nitric oxide synthase/induced nitric oxide synthase were significantly inhibited (P<0.05). Further experiments revealed that osthole significantly downregulated mRNA and protein levels of Notch signaling components in KA­activated BV­2 cells (P<0.05). Therefore, it was hypothesized that osthole inhibited the proliferation of microglia by modulating the Notch signaling pathway, which may be useful for the treatment of epilepsy and other neurodegenerative diseases characterized by Notch upregulation.

Osthole inhibits proliferation and induces apoptosis in BV-2 microglia cells in kainic acid-induced epilepsy via modulating PI3K/AKt/mTOR signalling way.

CONTEXT: Osthole is a natural coumarin compound most frequently extracted from plants of the Apiaceae family such as Cnidium monnieri (L.) Cusson, Angelica pubescens Maxin.f., and Peucedanum ostruthium (L.). Osthole is considered to have potential therapeutic applications for the treatment of diseases including epilepsy. However, the mechanism of osthole induced-apoptosis in BV-2 microglia cells is not yet clear. OBJECTIVE: To investigate the molecular mechanisms underlying the effect of osthole on PI3K/AKt/mTOR expression in kainic acid (KA)-activated BV-2 microglia cells. MATERIALS AND METHODS: Optimal culture concentration and time of osthole were investigated by MTT assay. The concentration of osthole was tested from 10 to 400 µM and the culture time was tested from 2 to 72 h. Ultrastructure difference among control, KA and osthole group was analyzed under transmission electron microscope. The mRNA expression of PI3K/AKt/mTOR was investigated using reverse transcription (RT)-PCR and the protein expression was investigated using western blotting and immunofluorescence assay. Apoptosis rate of BV-2 cells between each group was measured by flow cytometry. RESULTS: IC50 for cell viability of BV-2 cells by osthole was 157.7 µM. Treated with osthole (140 µM) for 24 h significantly increased the inhibition rate. Pretreatment with osthole inhibited the KA-induced PI3K/AKt/mTOR mRNA and protein expression. The results of flow cytometry analysis showed that the apoptotic rate of osthole group was obviously higher than KA group. CONCLUSIONS: Date showed that osthole may be useful in the treatment of epilepsy and other neurodegenerative diseases that are characterized by over expression of PI3K/Akt/mTOR.

HOTAIR/miR-326/FUT6 axis facilitates colorectal cancer progression through regulating fucosylation of CD44 via PI3K/AKT/mTOR pathway.

Metastasis is the main cause of death in colorectal cancer (CRC) patients. Aberrant fucosylation, catalyzed by the specific fucosyltransferases (FUTs), is associated with malignant behaviors. Non-conding RNAs, including long non-coding RNAs (lncRNAs) and microRNAs (miRNAs), emerge as key molecules in cancer malignancy. The aim of this study was to investigate HOTAIR/miR-326/FUT6 axis modified fucosylation on sLeX-CD44 (HCELL), which served as E-selectin ligand during CRC progression. Higher levels of HOTAIR and FUT6 were verified in CRC tissues and cell lines, with a positive correlation. HOTAIR was associated with poor clinical prognosis of CRC. Altered HOTAIR levels influenced proliferation, aggressiveness, apoptosis and tumorigenesis of CRC cells. HOTAIR directly harbored miR-326 binding sites and regulated FUT6 expression. Further results corroborated that HOTAIR/miR-326/FUT6 axis modified α1, 3-fucosylation of CD44, which mediated CRC malignancy. Co-modulation of HOTAIR, miR-326 and FUT6 impacted α1, 3-fucosylated CD44, which further triggered PI3K/AKT/mTOR pathway. HOTAIR also mediated CRC tumorigenesis and liver metastasis in vivo. Thus, our findings indicated that HOTAIR/miR-326/FUT6 axis mediated CRC procession through α1, 3-fucosylated CD44 via PI3K/AKT/mTOR pathway. This work rendered new therapeutic targets for CRC.

Long non-coding RNA-SNHG7 acts as a target of miR-34a to increase GALNT7 level and regulate PI3K/Akt/mTOR pathway in colorectal cancer progression.

BACKGROUND: Colorectal cancer (CRC) arises in a multistep molecular network process, which is from either discrete genetic perturbation or epigenetic dysregulation. The long non-coding RNAs (lncRNAs), emerging as key molecules in human malignancy, has become one of the hot topics in RNA biology. Aberrant O-glycosylation is a well-described hallmark of many cancers. GALNT7 acts as a glycosyltransferase in protein O-glycosylation, involving in the occurrence and development of CRC. METHODS: The microarrays were used to survey the lncRNA and mRNA expression profiles of primary CRC cell line SW480 and metastatic CRC cell line SW620. Cell proliferation, migration, invasion, and apoptosis were assayed. Xenograft mouse models were used to determine the role of lncRNA-SNHG7 in CRC in vivo. In addition, CNC analysis and competing endogenous analysis were used to detect differential SNHG7 and relational miRNAs expression in CRC cell lines. RESULTS: SNHG7 expression showed a high fold (SW620/SW480) in CRC microarrays. The CRC patients with high expression of SNHG7 had a significantly poor prognosis. Furthermore, SNHG7 promoted CRC cell proliferation, metastasis, mediated cell cycle, and inhibited apoptosis. SNHG7 and GALNT7 were observed for co-expression by CNC analysis, and a negative correlation of SNHG7 and miR-34a were found by competing endogenous RNA (ceRNA) analysis. Further results indicated that SNHG7 facilitated the proliferation and metastasis as a competing endogenous RNA to regulate GALNT7 expression by sponging miR-34a in CRC cell lines. SNHG7 also played the oncogenic role in regulating PI3K/Akt/mTOR pathway by competing endogenous miR-34a and GALNT7. CONCLUSION: The CRC-related SNHG7 and miR-34a might be implicated in CRC progression via GALNT7, suggesting the potential usage of SNHG7/miR-34a/GALNT7 axis in CRC treatment.

The Antitumor Effect of Xihuang Pill on Treg Cells Decreased in Tumor Microenvironment of 4T1 Breast Tumor-Bearing Mice by PI3K/AKT~AP-1 Signaling Pathway.

To study the antitumor effect of Xihuang pill (XHP) on the number of Treg cells in the tumor microenvironment of 4T1 breast tumor-bearing mice by PI3K/AKT/AP-1 pathway, a mouse model was established. Flow cytometry (FCM) and immunohistochemistry (IHC) were used to detect the number of Treg cells in the tumor microenvironment; terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was used to detect the apoptosis of Treg cells in tumor microenvironment. Quantitative real-time PCR (RT-qPCR) was used to detect the mRNA expression of PI3K, AKT, and AP-1 in Treg cells in tumor microenvironment; immunofluorescence (IF) and Western Blot (WB) were used to detect the protein expression of PI3K, AKT, and AP-1 in Treg cells in tumor microenvironment. Compared with the naive control group, the tumor weight in XHP groups decreased significantly (P < 0.05); FCM and IHC results showed that the number of Treg cells in the tumor microenvironment decreased with the dose of XHP groups (P < 0.05); TUNEL staining showed that the number of Treg cells in tumor microenvironment increased with the dose of XHP groups (P < 0.05); RT-qPCR results showed that the mRNA expression of PI3K and AKT in Treg cells decreased with the dose of XHP groups, while RNA expression of AP-1 increased with the dose of XHP groups (P < 0.05); IF and WB results showed that the protein expression of PI3K and AKT in Treg cells decreased with the dose of XHP groups and the protein expression of AP-1 increased with the dose of XHP groups (P < 0.05). The results suggested that XHP decreased the number of Treg cells via inhibiting PI3K and AKT expression and upregulating AP-1 expression in Treg cells and then promoting the apoptosis of Treg cells. Thus, XHP could improve the immunosuppressive state of tumor microenvironment and reverse the immune escape to inhibit tumor growth.

Xihuang pill promotes apoptosis of Treg cells in the tumor microenvironment in 4T1 mouse breast cancer by upregulating MEKK1/SEK1/JNK1/AP-1 pathway.

OBJECTIVE: To determine the role of the MEKK1/SEK1/JNK1/AP-1 pathway in the action of Xihuang pill (XHP) in reducing regulatory T (Treg) cell numbers in the tumor microenvironment in a 4T1 mouse breast cancer model, and to clarify the anti-tumor mechanism of XHP in breast cancer. METHODS: We established a mouse 4T1 breast cancer model. Model mice were administered XHP for 2 weeks, and tumor tissues were then removed, weighed, sliced, and homogenized. Treg cells in the tumor microenvironment were isolated by magnetic cell sorting and analyzed by immunohistochemistry and flow cytometry. Treg cell apoptosis was detected by TdT-mediated dUTP nick end labeling. mRNA expression levels of MEKK1, SEK1, JNK1, and AP-1 in Treg cells in the tumor microenvironment were detected by quantitative real-time PCR and their protein expression levels were detected by immunofluorescence staining and western blot. RESULTS: Tumor weights were significantly lower in the XHP groups compared with the untreated control group. The overall number of Treg cells in the tumor microenvironment decreased while the number of apoptotic Treg cells increased with increasing doses of XHP. mRNA and protein expression levels of MEKK1, SEK1, JNK1, and AP-1 in Treg cells in the tumor microenvironment increased with increasing doses of XHP. CONCLUSION: XHP might promote Treg cell apoptosis in the tumor microenvironment and further inhibit the tumor growth of 4T1 mouse breast cancer. The mechanism of XHP may be related to upregulation of gene and protein expression of MEKK1, SEK1, JNK1, and AP-1 in Treg cells in the tumor microenvironment.

Effects of triptolide on the expression of MHC II in microglia in kainic acid­induced epilepsy.

The purpose of the present study was to determine whether triptolide (T10) had any effect on major histocompatibility complex class II (MHC II) expression in kainic acid (KA)­activated microglia, and to investigate the underlying molecular mechanism. BV­2 microglia were pretreated with T10 prior to activation with KA. The expression level of MHC II and class II transactivator (CIITA) mRNA was determined via reverse transcription­polymerase chain reaction. The expression of MHC II, CIITA and the phosphorylation level of c­Jun and proto­oncogene c­Fos (c­Fos) was determined by western blotting. The protein expression level of MHC II was determined by immunocytochemistry. It was observed that the mRNA and protein levels of MHC II and CIITA were increased in KA­activated BV­2 microglia, and that this increase was almost completely eliminated by T10. AP­1 is a family of homodimers or heterodimers, composed of Jun family and Fos family proteins. Sequence analysis revealed an AP­1 DNA binding site in the promoter of CIITA. The phosphorylation of c­Jun and c­Fos was increased in KA­activated microglia, while T10 was able to suppress the phosphorylation of c­Jun and c­Fos in KA­activated microglia. These data suggested that T10 may exert suppressive effects on MHC II expression in KA­activated microglia, and that the mechanism may involve the regulation of AP­1 activity.

[Experimental study on anti-tumor effect of xihuang pill and its immune clearance function].

OBJECTIVE: To discuss the anti-tumor effect of Xihuang pill on tumor-bearing rats and its effect on the immune clearance function of tumor-bearing organisms. METHOD: Walker256 tumor cells were adopted to establish the tumor-bearing rat model. The rats were randomly divided into five groups: the normal control group, the model control group, the lentinan group and Xihuang pill low dose, middle dose and high dose groups, with 10 rats in each group, and continuously treated and given drugs for 14 d after modeling. Blood and tumors were collected from abdominal aorta to calculate the tumor inhibition rate. The content of CD3+, CD4+, CD8+ T cells and adhesion molecule B7-1 (CD80) in peripheral blood were detected by flow cytometry (FCM). The expressions of IL-2 and IFN-gamma in were determined by ELISA. RESULT: The tumor inhibition rate of the Xihuang pill high dose group was 33. 1 percent. Compared with the model group, the Xihuang pill large dose group showed significantly low IL-2, IFN-gamma, CD3+, CD4+, B7-1 in peripheral blood, with statistical significance in their differences (P < 0.05). CONCLUSION: Xihuang pill could show its anti-tumor effect by enhancing the immune clearance function and increasing IL-2, IFN-gamma, CD3+ T, CD4+ T, B7-1 in peripheral blood.