Marsdeniae tenacissimae extract(MTE) suppresses cell proliferation by attenuating VEGF/VEGFR2 interactions and promotes apoptosis through regulating PKC pathway in human umbilical vein endothelial cells

Marsdeniae tenacissimae extract(MTE), commonly known as Xiao-Ai-Ping in China, is a traditional Chinese herb medicine capable of inhibiting proliferation and metastasis and boosting apoptosis in various cancer cells. However, little is known about the contribution of MTE towards tumor angiogenesis and the underlying mechanism. The present study aimed to evaluate the effects of MTE on the proliferation and apoptosis of human umbilical vein endothelial cells(HUVECs) and the molecular ism. 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium, inner salt(MTS) and PI-stained flow cytometry assays revealed that MTE dose-dependently reduced the proliferation of HUVECs by arresting cell cycle at S phase(P < 0.05). Annexin V-FITC/PI-stained flow cytometry confirmed that MTE(160 μL·m L-1) enhanced the apoptosis of HUVECs significantly(P < 0.001). Real-time quantitative RT-PCR and Western blot analyses showed an increase in Bax expression and a sharply decline in Bcl-2 expression; caspase-3 was activated simultaneously in a dose-dependent manner(P < 0.05). Further study observed the dose-dependent down-regulation of vascular endothelial growth factor(VEGF) receptor-2(VEGFR-2), P2Y6 receptor(P2Y6R), and chemokine(C-C motif) ligand 2(CCL-2), along with the activation of PKC δ and up-regulation of p53 in a dose-dependent manner in MTE-treated selected cells(P < 0.05). Collectively, the results from the present study suggested that MTE suppressed the proliferation by attenuating CCL-2-mediated VEGF/VEGFR2 interactions and promoted the apoptosis through PKCδ-induced p53-dependent mitochondrial pathway in HUVECs, supporting that MTE may be developed as a potent anti-cancer medicine.

傣药“雅解”:宾蒿、傣百解及竹叶兰的生物活性及其解毒机制

傣药“雅解”是傣族传统医学中最具特色的药物和治疗方法。其中,宾蒿(Clerodendrum chinense var.simplex)、傣百解(Marsdenia tenacissima)和竹叶兰(Arundina graminifolia)作为3种常见的“雅解”被广泛使用。本研究使用石油醚、乙酸乙酯、正丁醇或水依次萃取3种“雅解”的乙醇提取物获得各极性部位,并制备3种“雅解”的水煎剂。经测定3种“雅解”的总黄酮质量分数为22.41~586.39 mg/g,高于总多酚质量分数(2.76~28.66 mg/g)。3种“雅解”的提取物均可清除DPPH、·OH和ABTS自由基,其中对ABTS自由基的清除率高于其他自由基(IC50>380μg/mL)。它们对大肠杆菌、铜绿假单胞菌和金黄色葡萄球菌的抑制作用较弱。此外,3种“雅解”具有一定的抗炎作用,在60μg/mL的质量浓度下即可显著下调脂多糖刺激巨噬细胞产生的NO、肿瘤坏死因子-α、白细胞介素-1β和白细胞介素-6。综上所述,本研究通过现代科学手段初步揭示了3种“雅解”的解毒作用机理,为“雅解”的进一步优化与研究奠定了基础。

通关藤中总酚酸的提取工艺

采用正交试验设计法,以绿原酸为对照,分析物液比、提取温度、提取时间、乙醇体积分数4个因子对通关藤(Marsdenia tenacissima)中总酚酸提取率的影响。结果表明,影响提取率的主次因素为乙醇体积分数、提取时间、料液比、提取温度,最佳提取条件为乙醇体积分数70%,提取时间1.0 h,料液比1∶16(m/V,g∶mL),提取温度50℃。

通关藤的适宜采收期研究

测定不同生长期通关藤(Marsdenia tenacissima)的茎粗和茎长,采用香草醛-高氯酸法显色,在462 nm处测定通关藤中总皂苷的含量,研究通关藤的最适宜采收期。结果表明,通关藤总皂苷含量在两年生时达到最高,以后缓慢下降;产量在三年生时增长最快,之后增长缓慢。通关藤的最佳采收期要兼顾其产量和有效成分总皂苷的含量,两者都在相对较高的时候才是最佳采收期,确定其最佳采收期为两年半生至三年生的藤茎。

Two New C_(21) Steroids from Marsdenia tenacissima

Two new C21 steroids were isolated from the CHCl3 extract of the stem of Marsdenia tenacissima. On the basis of spectroscopic analysis and chemical methods, their structures were elucidated as 17β-tenacigenin B (2) and 3-O-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1→4)-β-D- oleandropyranosyl-tenacigenin C (3). The structure of the known aglycon tenacigenin C was revised as 5α, 9α, 17β-pregnane-3β, 8β, 11α, 12β, 14β-pentanol-20-one. Compound 3 is the first reported glycoside of tenacigenin C.

Polyoxypregnanes from the stems of Marsdenia tenacissima

From the stems ofMarsdenia tenacissima two new polyoxypregnanes were isolated, their structures were elucidated by 1D, 2D-NMR as 11α,12β-di-O-tigloyl-tenacigenin B （1） and tenacigenoside E （2）.

A new C_(21) steroid glycoside from Marsdenia tenacissima

A new C21 steroid glycoside,11α-O-tigloyl-12-β-O-acetyl tenacigenin B 3-O-β-D-glucopyranosyl-（1→4）-β-D-glucopyr- anosyl-（1→4）-3-O-methyl-6-deoxy-β-D-allopyranosyl-（1→4）-β-D-oleandropyranoside（1）,named tenacissoside N was isolated from the stems of Marsdenia tenacissima.The structure of the glycoside was identified by HR-ESI-MS and NMR spectra.

Marsdenia tenacissima injection induces the apoptosis of prostate cancer by regulating the AKT/GSK3β/STAT3 signaling axis

Marsdenia tenacissima injection,a standard Marsdenia tenacissima extract(MTE),has been approved as an adjuvant therapeutic agent for various cancers.Our previous study showed that MTE inhibited the proliferation and metastasis of prostate cancer(PCa)cells.However,the underlying mechanisms and active ingredients of MTE against PCa were not completely understood.This study revealed that MTE induced significant decreases in cell viability and clonal growth in PCa cells.In addition,MTE induced the apoptosis of DU145 cells by reducing the mitochondrial membrane potential and increasing the expression of Cleaved Caspase 3/7,Cyt c,and Bax.In vivo,DU145 xenografted NOD-SCID mice treated with MTE showed significantly decreased tumor size.TUNEL staining and Western blot confirmed the pro-apoptotic effects of MTE.Network pharmacology analysis collected 196 ingredients of MTE linked to 655 potential targets,and 709 PCa-associated targets were retrieved,from which 149 overlapped targets were screened out.Pathway enrichment analysis showed that the HIF-1,PI3K-AKT,and ErbB signaling pathways were closely related to tumor apoptosis.Western blot results confirmed that MTE increased the expression of p-AKT^(Ser473) and p-GSK3β^(Ser9),and decreased the expression of p-STAT3^(Tyr705) in vitro and in vivo.A total of 13 compounds in MTE were identified by HPLC-CAD-QTOF-MS/MS and UPLCQTOF-MS/MS.Molecular docking analysis indicated that six compounds may interact with AKT,GSK3β,and STAT3.In conclusion,MTE induces the endogenous mitochondrial apoptosis of PCa by regulating the AKT/GSK3β/STAT3 signaling axis,resulting in inhibition of PCa growth in vitro and in vivo.

Marsdenia tenacissima extract induces G_0/G_1 cell cycle arrest in human esophageal carcinoma cells by inhibiting mitogen-activated protein kinase(MAPK) signaling pathway

Marsdenia tenacissima extract(MTE, trade name: Xiao-Ai-Ping injection) is an extract of a single Chinese plant medicine. It has been used for the treatment of cancer in China for decades, especially for esophageal cancer and other cancers in the digestive tract. In the present study, the potential mechanism for MTE's activity in esophageal cancer was explored. The effects of MTE on the proliferation of human esophageal cancer cells(KYSE150 and Eca-109) were investigated by the MTT assay, the Brd U(bromodeoxyuridine) incorporation immunofluorescence assay, and flow cytometric analysis. MTE inhibited cell proliferation through inducing G0/G1 cell cycle arrest in KYSE150 and Eca-109. Western blot analysis was employed to determine protein levels in the MTE treated cells. Compared with the control cells, the expression levels of the cell cycle regulatory proteins cyclin D1/D2/D3, cyclin E1, CDK2/4/6(CDK: cyclin dependent kinase), and p-Rb were decreased significantly in the cells treated with MTE at 40 mg·m L-1. In addition, MTE had an inhibitory effect on the MAPK(mitogen-activated protein kinase) signal transduction pathway, including ERK(extracellular signal-regulated kinase), JNK(c-Jun N-terminal kinase), and p38 MAPK. Moreover, MTE showed little additional effects on the regulation of cyclin D1/D3, CDK4/6, and p-Rb when the ERK pathway was already inhibited by the specific ERK inhibitor U0126. In conclusion, these data suggest that MTE inhibits human esophageal cancer cell proliferation through regulation of cell cycle regulatory proteins and the MAPK signaling pathways, which is probably mediated by the inhibition of ERK activation.

Pregnane Glycosides from Stems of Marsdenia tenacissima

Objective To study the chemical constituents from the stems of Marsdenia tenacissima.Methods The chemical constituents were isolated by various column chromatography and their structures were identified by spectral and chemical analysis.Results Two pregnane glycosides were isolated from the stems of M.tenacissima and identified as 3-O-β-D-glucopyranosyl-(1→4)-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1→4)-β-D-oleandropyranosyl-11α-O- tigloyltenacigenin B,named as tenacigenoside I(1)and 3-O-β-D-glucopyranosyl-(1→4)-6-deoxy-3-O-methyl-β-D- allopyranosyl-(1→4)-β-D-oleandropyranosyl-11α,12β-di-O-acetyltenacigenin B,named as tenacigenoside K(2).Conclusion Compound 1 is a new compound,1H-NMR and 13C-NMR data of compound 2 are reported for the first time.

Polyoxypregnanes as safe, potent, and specific ABCB1-inhibitory pro-drugs to overcome multidrug resistance in cancer chemotherapy in vitro and in vivo

Multidrug resistance(MDR)mediated by ATP binding cassette subfamily B member 1(ABCB1)is significantly hindering effective cancer chemotherapy.However,currently,no ABCB1-inhibitory drugs have been approved to treat MDR cancer clinically,mainly due to the inhibitor specificity,toxicity,and drug interactions.Here,we reported that three polyoxypregnanes(POPs)as the most abundant constituents of Marsdenia tenacissima(M.tenacissima)were novel ABCB1-modulatory pro-drugs,which underwent intestinal microbiota-mediated biotransformation in vivo to generate active metabolites.The metabolites at non-toxic concentrations restored chemosensitivity in ABCB1-overexpressing cancer cells via inhibiting ABCB1 efflux activity without changing ABCB1 protein expression,which were further identified as specific non-competitive inhibitors of ABCB1 showing multiple binding sites within ABCB1 drug cavity.These POPs did not exhibit ABCB1/drug metabolizing enzymes interplay,and their repeated administration generated predictable pharmacokinetic interaction with paclitaxel without obvious toxicity in vivo.We further showed that these POPs enhanced the accumulation of paclitaxel in tumors and overcame ABCB1-mediated chemoresistance.The results suggested that these POPs had the potential to be developed as safe,potent,and specific pro-drugs to reverse ABCB1-mediated MDR.Our work also provided scientific evidence for the use of M.tenacissima in combinational chemotherapy.

Effect of Marsdenia tenacissima extract on G2/M cell cycle arrest by upregulating 14-3-3σ and downregulating c-myc in vitro and in vivo

Objective: Marsdenia tenacissima extract(MTE) is a traditional Chinese herbal medicine with anti-cancer activity. In some previous studies, different mechanism actions of the anti-cancer effect of MTE have been revealed. In this study, we first observed that MTE exhibited G2/M cell cycle arrest on two different human breast cancer cell lines, MDA-MB-231 and MCF-7 by mediating 14-3-3σ and c-myc.Methods: The effect of MTE on G2/M cell cycle arrest was evaluated in MDA-MB-231 and MCF-7 cell lines. MTT assay was done for evaluation of cell viability. Flow cytometry was employed for cell cycle analysis. Western blotting analysis and immunohistochemistry were performed to analyze the expression of G2/M cell cycle-related key protein in cells and tissue samples. Animal studies have been conducted to elucidate the anti-tumor effect of MTE.Results: Cell cycle is the backbone for developing cancer. Cell cycle proteins play a major role in the progression of cell cycle and cell proliferation. However, some key protein directly or indirectly modulate the action of cell cycle protein that highly affect cell cycle regulation. In order to investigate cellular proliferation of cancer, we observed that MTE induced the upregulation of 14-3-3σ and downregulation of c-myc,and then reduced the expression of G2/M cell cycle associated key protein, leading to the inhibition of cellular entry into mitosis phase. We also confirmed that MTE exerted a significant antitumor effect on the MDA-MB-231 xenograft model in vivo.Conclusion: G2/M cell cycle arrest occurred by the action of MTE, mediated by the upregulation of 14-3-3σ as well as downregulation of c-myc in MDA-MB-231 and MCF-7 cell lines.