Oridonin promotes endoplasmic reticulum stress via TP53-repressed TCF4 transactivation in colorectal cancer.

BACKGROUND: The incidence of colorectal cancer and cancer death rate are increasing every year, and the affected population is becoming younger. Traditional Chinese medicine therapy has a unique effect in prolonging survival time and improving the prognosis of patients with colorectal cancer. Oridonin has been reported to have anti-cancer effects in a variety of tumors, but the exact mechanism remains to be investigated. METHODS: Cell Counting Kit-8 assay (CCK8) and 5-Ethynyl-2'-deoxyuridine (EdU) staining assay, Tranwell, and Wound healing assays were performed to measure cell proliferation, invasion, and migration capacities, respectively. The protein and mRNA expression levels of various molecules were reflected by Western blot and Reverse Transcription quantitative Polymerase Chain Reaction (qRT-PCR). Transcription Factor 4 (TCF4) and its target genes were analyzed by Position Weight Matrices (PWMs) software and the Gene Expression Omnibus (GEO) database. Immunofluorescence (IF) was performed to visualize the expression and position of Endoplasmic Reticulum (ER) stress biomarkers. The morphology of the ER was demonstrated by the ER tracker-red. Reactive Oxygen Species (ROS) levels were measured using a flow cytometer (FCM) or fluorescent staining. Calcium ion (Ca2+) concentration was quantified by Fluo-3 AM staining. Athymic nude mice were modeled with subcutaneous xenografts. RESULTS: Oridonin inhibited the proliferation, invasion, and migration of colorectal cancer, and this effect was weakened in a concentration-dependent manner by ER stress inhibitors. In addition, oridonin-induced colorectal tumor cells showed increased expression of ER stress biomarkers, loose morphology of ER, increased vesicles, and irregular shape. TCF4 was identified as a regulator of ER stress by PWMs software and GEO survival analysis. In vitro and in vivo experiments confirmed that TCF4 inhibited ER stress, reduced ROS production, and maintained Ca2+ homeostasis. In addition, oridonin also activated TP53 and inhibited TCF4 transactivation, further exacerbating the elevated ROS levels and calcium ion release in tumor cells and inhibiting tumorigenesis in colorectal cancer cells in vivo. CONCLUSIONS: Oridonin upregulated TP53, inhibited TCF4 transactivation, and induced ER stress dysregulation in tumor cells, promoting colorectal cancer cell death. Therefore, TCF4 may be one of the important nodes for tumor cells to regulate ER stress and maintain protein synthesis homeostasis. And the inhibition of the TP53/TCF4 axis plays a key role in the anti-cancer effects of oridonin.

Identification of bioactive compounds in Shaoyao-Gancao decoction using ß2-adrenoceptor affinity chromatography.

Shaoyao-Gancao decoction, a Chinese herbal formula, is composed of Paeoniae Radix alba and Glycyrrhiza Radix et rhizoma. It has been widely used to treat muscle spasms and asthma. However, little is known about the bioactive components of Shaoyao-Gancao decoction. In the present study, the bioactive compounds in water-extract of Shaoyao-Gancao decoction were separated by the immobilized ß2 -adrenoceptor affinity column and identified using quadrupole time-of-flight mass spectrometry. The affinity constants of the separated compounds that bind to ß2 -adrenoceptor were determined by frontal analysis. Compound bioactivity was tested in a rat tracheal smooth muscle relaxation assay. We identified the bioactive compounds in the water extract of Shaoyao-Gancao decoction that bound to the ß2 -adrenoceptor as paeoniflorin and liquiritin. Paeoniflorin and liquiritin had only one binding site on the immobilized ß2 -adrenoceptor, and the affinity constants were (2.16 ± 0.10) × 104  M-1 and (2.95 ± 0.15) × 104  M-1 , respectively. Both compounds induced a concentration-dependent relaxation of tracheal smooth muscle following K+ -stimulated contraction, and the relaxation effects were abrogated by the ß2 -adrenoceptor antagonist, ICI 118551. Therefore, paeoniflorin and liquiritin are bioactive compounds in Shaoyao-Gancao decoction and the ß2 -adrenoceptor affinity chromatography is a useful tool for identifying potential ß2 -adrenoceptor ligands in natural products used in traditional Chinese medicine.