Analytical characterization and purification of plasma membrane from cultured hepatoma cells (HTC cells)

The plasma membrane of the hepatoma cell line, HTC cells, has been characterized and purified by cell fractionation techniques. In the absence of true 5′-nucleotidase in HTC cells, alkaline phosphodiesterase I has been used as a marker enzyme, following conclusions gained from differential and isopycnic centrifugation studies (Lopez Saura, P., Trouet A. and Tulkens P. (1978) Biochim. Biophys. Acta 543, 430–449). To confirm this localization, HTC cells were exposed to anti-plasma membrane IgG at 4°C and fractionated. Alkaline phosphodiesterase I and IgG showed super imposable distribution patterns in linear sucrose gradients. Alkaline phosphodiesterase I is, however, only poorly resolved from enzyme markers of other organelles, especially NADPH-cytochrome $\text{c}$ reductase (endoplasmic reticulum) and galactosyltransferase (Golgi complex). Maximal purification from the homogenate is only 13-fold, on a protein basis, even when using a microsomal fraction (67 and 13% of alkaline phosphodiesterase I and protein, respectively) as the starting material. Improved resolution can be obtained after the addition of small quantities of digitonin (equimolar with respect to the cholesterol content). Digitonin increases the buoyant density of alkaline phosphodiesterase I by approx. 0.05 g/cm³, whereas the buoyant densities of galactosyltransferase and NADPH-cytochrome $\text{c}$ reductase are increased only by 0.03 and 0.015 g/cm³, respectively. Accordingly, a procedure has been designed which yields a fraction containing 22.8% of alkaline phosphodiesterase I with a purification of 21-fold on a protein basis. The content of NADPH-cytochrome $\text{c}$ reductase and galactosyltransferase is 1.2 and 2.1%, respectively. Electron microscopy shows smooth surface membrane elements and vesicles, with only occasional other recognizable elements.     

Triterpenoids from Meehania fargesii with Cytotoxic Activity1

In the investigation of Meehania fargesii, eighteen triterpenoids were isolated and identified, including a previously unknown compound with an 13,27-cycloursane skeleton, using techniques like 1D and 2D NMR, and HR-MS. Furthermore, the cytotoxicity of these compounds were evaluated against HCT116, MCF-7, and AGS cell lines using the CCK-8 method to examine their structure–activity relationship. Remarkably, compounds 13 and 16 exhibited higher cytotoxicity across all three cell lines compared to the positive drug. Western blot analysis revealed that these compounds activated apoptosis in HCT116 cells by promoting the Bax protein and inhibiting the Bcl-2 protein. This suggests that compounds 13 and 16 have potential as apoptosis-inducing agents in HCT116 cells.     

A novel podophyllotoxin derivative with higher anti-tumor activity produced via 4′-demethylepipodophyllotoxin biotransformation by Penicillium purpurogenum

Biological modification of natural products is an important approach to improve the pharmacological properties. 4′-d-β-Demethylepipodophyllotoxin (4′-d-β-DMEP), a typical natural lignin, is used as the aglycon of topoisomerase inhibitor etoposide (VP-16). In this study, 4′-d-β-DMEP was isomerized into the novel product 4′-d-α-DMEP with 43.09% conversion by P. purpurogenum ACCC 32170. 4′-d-α-DMEP was largely produced extracellularly in the late fermentation stage, which was accompanied with the decrease of dry cell weight and the increase of culture pH. A constitutively expressed cytosolic enzyme was involved in the transformation and functioned when culture pH ranged from 7.0–10.0. The IC₅₀ value of 4′-d-α-DMEP was around 0.80 μM against tumor cells MCF-7, which was significantly reduced by 11.27 and 17.34 times than 4′-d-β-DMEP (i.e., 9.82 μM) and VP-16 (i.e., 14.67 μM). This study provided the novel podophyllotoxin derivative with higher anti-tumor activity and microbial transformation process, which would promote transferring lignans into the clinic trial.     

钌络合物通过诱发高尔基体应激在Walker-256荷瘤大鼠中发挥抗肿瘤作用的机制

摘要 目的：探讨钌络合物通过诱发高尔基体应激在Walker-256荷瘤大鼠中发挥抗肿瘤作用的机制。方法：以30只雄性Wistar大鼠为研究对象,通过Walker-256细胞右骨盆肢体皮下注射建立荷瘤大鼠模型,然后根据实验目的将大鼠分为3组,对照组（正常大鼠,PBS干预）,肿瘤模型组（荷瘤模型大鼠,PBS干预）和钌络合物组[荷瘤模型大鼠,管饲法给予5 mg / kg钌络合物溶液（由含2% Tween的PBS溶解）],各10只。通过测厚仪和电子秤分别计算大鼠肿瘤体积及重量;酶联免疫吸附试验试剂盒检测大鼠刚脏组织匀浆中氧化应激水平;蛋白印迹和荧光探针DCFH-DA试剂盒分析LC3 II/I表达和ROS活性;蛋白印迹分析高尔基应激相关蛋白GOLPH3、GRASP65的表达;实时定量PCR分析Bax、Bcl-2和Caspase-3的mRNA表达。结果：钌络合物组较肿瘤模型组肿瘤重量降低（P<0.05）,肿瘤模型组较对照组体重增加降低（P<0.05）,钌络合物组较肿瘤模型组体重增加（P<0.05）。肿瘤模型组较对照组SOD活性和LPO升高（P<0.05）,CAT、GST和GSH活性降低（P<0.05）,钌络合物组较肿瘤模型组LPO降低（P<0.05）,CAT、GST和GSH活性升高（P<0.05）。肿瘤模型组较对照组LC3 II/I蛋白表达和ROS活性升高（P<0.05）,钌络合物组较肿瘤模型组LC3 II/I蛋白表达和ROS活性降低（P<0.05）。肿瘤模型组较对照组GOLPH3、GRASP65的蛋白表达升高（P<0.05）,钌络合物组较肿瘤模型组GOLPH3、GRASP65的蛋白表达降低（P<0.05）。肿瘤模型组较对照组Bax和Caspase-3的mRNA表达升高（P<0.05）,Bcl-2 mRNA表达降低（P<0.05）,钌络合物组较肿瘤模型组Bax和Caspase-3的mRNA表达降低,Bcl-2 mRNA表达升高（P<0.05）。结论：钌络合物通过调节高尔基应激反应,削弱氧化磷酸化从而促进Walker-256细胞死亡发挥抗肿瘤活性。     

Design,synthesis, and anti-tumor activities of novel triphenylethylene–coumarin hybrids,and their interactions with Ct-DNA

Novel triphenylethylene–coumarin hybrid derivatives containing different amounts of amino side chains were designed and synthesized in good yields under microwave radiation. The derivatives 5b–d which possessed two amino side chains (except morpholinyl) showed a broad-spectrum and good anti-proliferative activity against five tumor cells and low cytotoxicity in osteoblast. UV–vis, fluorescence, and circular dichroism (CD) spectroscopies and thermal denaturation exhibited that compounds 10c, 5c, and 13c bearing amino side chain (except morpholinyl) on 4-phenyl had significant interactions with Ct-DNA by the intercalative mode of binding. Structure–activity relationships (SARs) analysis suggested that the amino alkyl chain would play an important role both in the compounds against tumor cells proliferation and their interactions with DNA.     

Two new cembranic diterpenoids from the flowers of Nicotiana tabacum L.

Two new cembranoids, together with fifteen known ones, were isolated from the flowers of Nicotiana tabacum L. The structures of the new compounds were established as (1βH,2E,4αOH,6αOH,7E,10αH,11αH,12βOH)-10,11-epoxy-2,7-cembradiene-4,6,12-triol (1) and (1βH,2E,4αOH,6αOH,7E,10βH,11βH,12αOH)-10,11-epoxy-2,7-cembradiene-4,6, 12-triol (2) by using spectroscopic analysis, including HRESIMS, IR, one- and two-dimensional NMR. A plausible biogenetic relationship of the isolated cembranoids was proposed. The antitumor activities of selected compounds against a panel of three human cancer HepG2, A549 and HCT-116 cell lines were evaluated by the MTT assay. Compound 5 exhibited moderate activity against Hep-G2 cell lines with an IC₅₀ value of 14.38 μM.     

Structure-antioxidant and anti-tumor activity of Teucrium polium phytochemicals

Chemical characterization as well as antioxidant and anti-tumor activity are reported for isolated metabolites from Teucrium polium L. (Lamiaceae). Structures were identified using standard MS and NMR spectroscopic methods. Sesquiterpene absolute stereochemistry was determined based on a modified Mosher’s reaction. Biological activity was evaluated by a cupric reducing antioxidant capacity (CUPRAC) assay and select compounds screened for anti-tumor activity. (1R,4S,10R) 10,11-dimethyl-dicyclohex-5(6)-en-1,4-diol-7-one and (R)-mandelonitrile-β-laminaribioside, together with ten previously reported compounds were identified. Antioxidant versus tumor-inhibition relationships was examined.     

Structure-activity relationships study of neolamellarin A and its analogues as hypoxia inducible factor-1 (HIF-1) inhibitors

The novel marine pyrrole alkaloid neolamellarin A derived from sponge has been shown to inhibit hypoxia-induced HIF-1 activity. In this work, we designed and synthesized neolamellarin A and its series of derivatives by a convergent synthetic strategy. The HIF-1 inhibitory activity and cytotoxicity of these compounds were evaluated in Hela cells by dual-luciferase reporter gene assay and MTT assay, respectively. The results showed that neolamellarin A 1 (IC₅₀ = 10.8 ± 1.0 μM) and derivative 2b (IC₅₀ = 11.9 ± 3.6 μM) had the best HIF-1 inhibitory activity and low cytotoxicity. Our SAR research focused on the effects of key regions aliphatic carbon chain length, aromatic ring substituents and C-7 substituent on biological activity, providing a basis for the subsequent research on the development of novel pyrrole alkaloids as HIF-1 inhibitors and design of small molecule probes for target protein identification.     

Design and synthesis of novel tellurodibenzoic acid compounds as kidney-type glutaminase (KGA) inhibitors

Organotellurium compounds have been reported as an immune-modulator sensitizing chemotherapeutics. Herein, we report the design and synthesis of a series of novel tellurodibenzoic acids as mimics of diphenylarsenic acid (DPAA) and potential selective KGA inhibitors. Representative compound 3B exhibited potent inhibition of KGA and glutamine-dependent HCT-116 cells. Stability experiments indicated that 3B has excellent stability under acidic (HCOOH), basic (NH₃·H₂O) and oxidative (H₂O₂) conditions, but reacts with β-ME, DTT and lysine which suggested that compound 3B may interact with cysteine or lysine residues. Moreover, molecular docking disclosed that compound 3B binds to the allosteric site of the GAC tetramer containing Arg³¹⁷-Lys³²⁰-Leu³²¹-Phe³²²-Tyr³⁹⁴-Glu³²⁵, which helped to rationalize the SAR and further design and optimization. Taken together, compound 3B could be used as a starting point for the development of new KGA inhibitors.     

Chemical synthesis and functional characterization of a new class of ceramide analogues as anti-cancer agents

Deregulation of ceramide metabolism is a hallmark of human cancer. Ceramide analogues thereby represent a new class of anti-cancer agents. We aimed at developing effective and low toxic ceramide analogues and synthesized a new class of ceramide analogues starting from l-threonine. Several analogues exhibit potent cytotoxicity against human cancer cells in vitro with IC₅₀ as low as 4.8?μM. These ceramide analogues decreased xIAP and Bcl-xL level and exhibited significant sensitization activity to overcome human cancer cell resistance to TRAIL, a cancer-selective agent that are being tested in human clinical trials. Furthermore, we determined that these ceramide analogues effectively suppress human cancer xenograft growth in vivo with no significant toxicity at the efficacious dose. Therefore, we have developed a simple and effective method to synthesize functional ceramide analogues using l-threonine as starting material and these analogues have the great potential to be further developed as anti-cancer agents in human cancer therapy.