Calycosin suppresses breast cancer cell growth via ERβ-dependent regulation of IGF-1R, p38 MAPK and PI3K/Akt pathways.

We previously reported that calycosin, a natural phytoestrogen structurally similar to estrogen, successfully triggered apoptosis of estrogen receptor (ER)-positive breast cancer cell line, MCF-7. To better understand the antitumor activities of calycosin against breast cancer, besides MCF-7 cells, another ER-positive cell line T-47D was analyzed here, with ER-negative cell lines (MDA-231, MDA-435) as control. Notably, calycosin led to inhibited cell proliferation and apoptosis only in ER-positive cells, particularly in MCF-7 cells, whereas no such effect was observed in ER-negative cells. Then we investigated whether regulation of ERβ, a subtype of ER, contributed to calycosin-induced apoptosis in breast cancer cells. The results showed that incubation of calycosin resulted in enhanced expression ERβ in MCF-7 and T-47D cells, rather than MDA-231 and MDA-435 cells. Moreover, with the upregulation of ERβ, successive changes in downstream signaling pathways were found, including inactivation of insulin-like growth factor 1 receptor (IGF-1R), then stimulation of p38 MAPK and suppression of the serine/threonine kinase (Akt), and finally poly(ADP-ribose) polymerase 1 (PARP-1) cleavage. However, the other two members of the mitogen-activated protein kinase (MAPK) family, extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK), were not consequently regulated by downregulated IGF-1R, indicating ERK 1/2 and JNK pathways were not necessary to allow proliferation inhibition by calycosin. Taken together, our results indicate that calycosin tends to inhibit growth and induce apoptosis in ER-positive breast cancer cells, which is mediated by ERβ-induced inhibition of IGF-1R, along with the selective regulation of MAPK and phosphatidylinositol 3-kinase (PI3K)/Akt pathways

Selective regulation of MAPK and PI3K/Akt pathways by calycosin in MCF-7 cells.

<p>Cells were treated with calycosin (0, 25, 50, 100 µM) for 48 h. Then the activity of ERK1/2 and JNK (<b>A</b>) was determined using western blot assay, along with alteration of p38 and Akt activity (<b>B</b>). The antibodies to total ERK1/2, JNK, p38 and Akt served as loading controls. Results are representative of three independent experiments. ** p<0.05 versus control group (0 µM)</p

Inhibited proliferation of ER-positive breast cancer cells by calycosin.

<p>ER-positive cells (MCF-7, T-47D) and ER-negative cells (MDA-231, MDA-435) were separately incubated with varying concentrations of calycosin (0, 25, 50, 100 µM) for 24 (<b>A</b>), 48 (<b>B</b>) and 72 h (<b>C</b>). Then cell proliferation rate (relative to calycosin-untreated cells) was assessed by MTT assay. Results are representative of three independent experiments.</p

Increased cleavage of PARP-1 by calycosin in MCF-7 cells.

<p>After treatment with 0, 25, 50, 100 µM calycosin for 48 h (<b>A</b>), or IGF-1R inhibitor (PPP) prior to 100 µM calycosin (<b>B</b>), the cleavage of PARP-1 in MCF-7 cells was determined by western blot. The expression of β-actin served as loading controls. Results are representative of three independent experiments. ** p<0.05 versus control group (0 µM) or 100 µM group</p

Downregulation of IGF-1R in ER-positive cells with treatment of calycosin.

<p>Western blot assay was performed in MCF-7 (<b>A</b>) and T-47D cells (<b>B</b>) (treated by calycosin as indicated for 48 h) to determine the expression of IGF-1R. Blots were probed for β-actin as loading controls. Results are representative of three independent experiments. ** p<0.05 versus control group (0 µM)</p

Treatment of Parkinson’s disease using focused ultrasound with GDNF retrovirus-loaded microbubbles to open the blood–brain barrier

This study aims to prepare ultrasound-targeted glial cell-derived neurotrophic factor (GDNF) retrovirus-loaded microbubbles (M pLXSN-GDNF) to verify the properties of the microbubbles and to study the therapeutic effect of the GDNF retrovirus-loaded microbubbles combined with ultrasound (U) to open the blood–brain barrier (BBB) in a Parkinson’s disease (PD) model in rats, allowing the retrovirus to pass through the BBB and transfect neurons in the substantia nigra of the midbrain, thereby increasing the expression of GDNF. The results of western blot analysis revealed significant differences between U + MpLXSN-EGFP, U + M + pLXSN-GDNF, and M pLXSN-GDNF (P < 0.05) groups. After 8 weeks of treatment, the evaluation of the effect of increased GDNF expression on behavioral deficits in PD model rats was conducted. The rotation symptom was significantly improved in the U + MpLXSN-GDNF group, and the difference before and after treatment was significant (P < 0.05). Also, the content of dopamine and the number of tyrosine hydroxylase-positive (dopaminergic) neurons were found to be higher in the brain of PD rats in the U + M pLXSN-GDNF group than in the control groups. Ultrasound combined with GDNF retrovirus-loaded microbubbles can enhance the transfection efficiency of neurons in vivo and highly express the exogenous GDNF gene to play a therapeutic role in PD model rats