Confirming therapeutic target of protopine using immobilized β -adrenoceptor coupled with site-directed molecular docking and the target-drug interaction by frontal analysis and injection amount-dependent method.
Liu Guangxin,Wang Pei,Li Chan,Wang Jing,Sun Zhenyu,Zhao Xinfeng,Zheng Xiaohui
Journal of molecular recognition : JMR
Drug-protein interaction analysis is pregnant in designing new leads during drug discovery. We prepared the stationary phase containing immobilized β -adrenoceptor (β -AR) by linkage of the receptor on macroporous silica gel surface through N,N'-carbonyldiimidazole method. The stationary phase was applied in identifying antiasthmatic target of protopine guided by the prediction of site-directed molecular docking. Subsequent application of immobilized β -AR in exploring the binding of protopine to the receptor was realized by frontal analysis and injection amount-dependent method. The association constants of protopine to β -AR by the 2 methods were (1.00 ± 0.06) × 10 M and (1.52 ± 0.14) × 10 M . The numbers of binding sites were (1.23 ± 0.07) × 10 M and (9.09 ± 0.06) × 10 M, respectively. These results indicated that β -AR is the specific target for therapeutic action of protopine in vivo. The target-drug binding occurred on Ser in crystal structure of the receptor. Compared with frontal analysis, injection amount-dependent method is advantageous to drug saving, improvement of sampling efficiency, and performing speed. It has grave potential in high-throughput drug-receptor interaction analysis.
Protopine attenuates inflammation stimulated by carrageenan and LPS via the MAPK/NF-κB pathway.
Alam Md Badrul,Ju Mi-Kyoung,Kwon Yoon-Gyung,Lee Sang Han
Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association
We investigated the anti-inflammatory activity of protopine (PTP) and sought to determine its mechanism of action in LPS-stimulated BV2 cells and a carrageenan (CA)-induced mouse model. Treatment with PTP (5, 10, and 20 μM) significantly suppresses the secretion of NO and PGE in a concentration-dependent manner without affecting cell viability by downregulating iNOS and COX-2 expression in LPS-induced BV2 cells. PTP also attenuates the production of pro-inflammatory chemokines, such as MCP-1, and cytokines, including TNF-α, IL-1β and IL-6, and augments the expression of the anti-inflammatory cytokine IL-10. In addition, PTP suppresses the nuclear translocation of NF-κB by hindering the degradation of IκB and downregulating the expression of mitogen-activated protein kinases (MAPKs), including p38, ERK1/2 and JNK protein. Furthermore, PTP treatment significantly suppresses CA-induced paw oedema in mice compared to that seen in untreated mice. Expression of iNOS and COX-2 proteins is also abrogated by PTP (50 mg/kg) treatment in CA-induced mice. PTP treatment also abolishes IκB phosphorylation, which hinders the activation of NF-κB. Collectively, these results suggest PTP has potential for attenuating CA- and LPS-induced inflammatory symptoms through modulation of MAPKs/NF-κB signaling cascades.
Protopine, a novel microtubule-stabilizing agent, causes mitotic arrest and apoptotic cell death in human hormone-refractory prostate cancer cell lines.
Chen Chun-Han,Liao Cho-Hwa,Chang Ya-Ling,Guh Jih-Hwa,Pan Shiow-Lin,Teng Che-Ming
In this study, we investigated the anticancer effect of protopine on human hormone-refractory prostate cancer (HRPC) cells. Protopine exhibited an anti-proliferative effect by induction of tubulin polymerization and mitotic arrest, which ultimately led to apoptotic cell death. The data suggest that protopine increased the activity of cyclin-dependent kinase 1 (Cdk1)/cyclin B1 complex and that contributed to cell apoptosis by modulating mitochondria-mediated signaling pathways, such as Bcl-2 phosphorylation and Mcl-1 down-regulation. In conclusion, the data suggest that protopine is a novel microtubule stabilizer with anticancer activity in HRPC cells through apoptotic pathway by modulating Cdk1 activity and Bcl-2 family of proteins.
Protopine Protects Mice against LPS-Induced Acute Kidney Injury by Inhibiting Apoptosis and Inflammation via the TLR4 Signaling Pathway.
Zhang Beibei,Zeng Mengnan,Li Meng,Kan Yuxuan,Li Benke,Xu Ruiqi,Wu Yuanyuan,Wang Shengchao,Zheng Xiaoke,Feng Weisheng
Molecules (Basel, Switzerland)
Migo is a traditional Chinese medicine that clears away damp heat, relieves sore. Protopine (PRO) is an alkaloid component isolated from Migo. However, the role of protopine in acute kidney injury (AKI) has not yet been reported. This study aims to investigate the effect and mechanism of protopine isolated from Migo on lipopolysaccharide (LPS)-induced AKI in mice. Inflammation accumulation was assessed by small animal living imaging. The blood urea nitrogen (BUN), and serum creatinine (Scr) were measured to assess the effects of protopine on renal function in LPS-induced AKI. The levels of tumor necrosis factor (TNF), interleukin-2 (IL-2), interferon-γ (IFN-γ), and (interleukin-10) IL-10 in serum were detected by cytometric bead array. Flow cytometry was used to detect the levels of reactive oxygen species (ROS) in primary kidney cells. The proportions of granulocytes, neutrophils, and macrophages in peripheral blood were examined to evaluate the effect of protopine on immune cells in mice with AKI. Toll-like receptor (TLR4) and apoptotic signaling pathway were detected by Western blot analysis. The results showed that protopine markedly improved the renal function, relieve inflammation, reversed inflammatory cytokines, transformed apoptosis markers, and regulated the TLR4 signaling pathway in mice with AKI induced by LPS. The protopine isolated from Migo protected mice against LPS-induced AKI by inhibiting apoptosis and inflammation via the TLR4 signaling pathway, thus providing a molecular basis for a novel medical treatment of AKI.
Protopine isolated from Nandina domestica induces apoptosis and autophagy in colon cancer cells by stabilizing p53.
Son Younglim,An Younju,Jung Jaeyeon,Shin Sora,Park InWha,Gwak Jungsug,Ju Bong Gun,Chung Young-Hwa,Na MinKyun,Oh Sangtaek
Phytotherapy research : PTR
The tumor suppressor p53 plays essential roles in cellular protection mechanisms against a variety of stress stimuli and its activation induces apoptosis or autophagy in certain cancer cells. Here, we identified protopine, an isoquinoline alkaloid isolated from Nandina domestica, as an activator of the p53 pathway from cell-based natural compound screening based on p53-responsive transcription. Protopine increased the p53-mediated transcriptional activity and promoted p53 phosphorylation at the Ser15 residue, resulting in stabilization of p53 protein. Moreover, protopine up-regulated the expression of p21 and BAX, downstream genes of p53, and inhibited the proliferation of HCT116 colon cancer cells. Apoptosis was elicited by protopine as indicated by caspase-3/7 activation, poly ADP ribose polymerase cleavage, and increased population of Annexin V-FITC-positive cells. Furthermore, protopine induced the formation of microtubule-associated protein 1 light chain 3 (LC3) puncta and LC3-II turnover, typical biochemical markers of autophagy, in HCT116 cells. Our findings suggest that protopine exerts its antiproliferative activity by stimulating the p53 pathway and may have potential as a chemopreventive agent for human colon cancer.
Protopine reduces the inflammatory activity of lipopolysaccharide-stimulated murine macrophages.
Bae Deok Sung,Kim Young Hoon,Pan Cheol-Ho,Nho Chu Won,Samdan Javzan,Yansan Jamyansan,Lee Jae Kown
Protopine is an isoquinoline alkaloid contained in plants in northeast Asia. In this study, we investigated whether protopine derived from Hypecoum erectum L could suppress lipopolysaccharide (LPS)-induced inflammatory responses in murine macrophages (Raw 264.7 cells). Protopine was found to reduce nitric oxide (NO), cyclooxygenase-2 (COX-2), and prostaglandin E(2) (PGE(2)) production by LPS-stimulated Raw 264.7 cells, without a cytotoxic effect. Pre-treatment of Raw 264.7 cells with protopine reduced the production of pro-inflammatory cytokines. These inhibitory effects were caused by blocking phosphorylation of mitogen-activated protein kinases (MAP kinases) and also blocking activation of a nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB).