Cepharanthine,a novel selective ANO1 inhibitor with potential for lung adenocarcinoma therapy

Anoctamin-1 (ANO1), also known as transmembrane protein 16A (TMEM16A), is identified as a Ca²⁺-activated Cl⁻ channel that is expressed in many organs and tissues. It is involved in numerous major physiological functions and especially in tumor growth. By screening 530 natural compounds, we identified cepharanthine as a potent blocker of ANO1 channels with an IC₅₀ of 11.2 ± 0.9 μM and E_max of 92.7 ± 1.7%. The Lys³⁸⁴, Arg⁵³⁵, Thr⁵³⁹, and Glu⁶²⁴ in ANO1 are critical for the inhibitory effect of cepharanthine. Similar to its effect on ANO1, cepharanthine inhibits ANO2, the closest analog of TMEM16A. In contrast, up to 30 μM of cepharanthine showed limited inhibitory effects on recombinant ANO6 and bestrophin-1-encoded Ca²⁺-activated Cl⁻ currents, but it showed no effects on endogenous volume-regulated anion currents (VRAC). Cepharanthine could also potently suppress endogenous ANO1 currents, significantly inhibit cell proliferation and migration, and induce apoptosis in LA795 lung adenocarcinoma cells. Moreover, animal experiments have shown that cepharanthine can dramatically inhibit the growth of xenograft tumors in mice. The high specificity provided by cepharanthine could be an important foundation for future studies of the physiological role of ANO1 channels, and these findings may reveal a new mechanism of its anticancer effect.     

Inhibition of neutrophil priming and tyrosyl phosphorylation by cepharanthine, a nonsteroidal antiinflammatory drug.

Receptor-mediated superoxide (O2.-)-generation and tyrosyl phosphorylation of neutrophil proteins, such as 58, 65, 84, 108 and 115 kDa, were enhanced by priming cells with granulocyte colony stimulating factor (G-CSF) [Akimura, K. et al. Arch. Biochem. Biophys. 298: 703-709, 1992]. To elucidate the possible involvement of tyrosyl phosphorylation of neutrophil proteins in the enhancing mechanism of O2.- generation, the effect of cepharanthine, a biscoclaurine alkaloid that inhibits phorbol 12-myristate 13-acetate (PMA)- and receptor-mediated O2.- generation, on the priming of human peripheral neutrophils (HPPMN) was studied. Both enhancement of formyl-methionyl-leucyl- phenylalanine (FMLP)-mediated O2.- generation and tyrosyl phosphorylation of some neutrophil proteins, i.e., 115, 108 and 84 kDa proteins, by HHPMN after treatment with G-CSF were strongly inhibited by cepharanthine in a concentration- and treatment-time-dependent manner. In contrast, inhibition of PMA-mediated O2.- generation by cepharanthine was weak and independent of treatment time. These results suggest that cepharanthine might inhibit the priming step of neutrophil activation concomitantly with its inhibition of the tyrosyl phosphorylation of some neutrophil proteins that might underlie the mechanism for priming of neutrophils with G-CSF.     

Radiation-induced peroxidation of lipid dissolved in organic solvent and its inhibition by alpha-tocopherol and cepharanthine

Effects of cepharanthine and alpha-tocopherol on radiation-induced peroxidation of lipids dissolved in methanol(MeOH)-chloroform (CHCl3)-H2O(v/v, 2/1/0.8) were examined. alpha-Tocopherol strongly inhibited radiation-induced peroxidation of lipids dissolved in MeOH-CHCl3-H2O. However, cepharanthine exhibited a weak inhibitory action in this system. The change in the absorption spectrum of alpha-tocopherol and cepharanthine by X-irradiation was measured. The reagents were dissolved in 95% EtOH acidified with 20 mM HCl and in MeOH-CHCl3-H2O. alpha-Tocopherol exhibited the change in its absorption spectrum in both systems, and seemed to be oxidized at a high rate by free radicals. However, cepharanthine slightly exhibited the change in its spectrum in MeOH-CHCl3-H2O, but not in acidified EtOH.     

Potentiation of harringtonine cytotoxicity by calcium antagonist diltiazem and biscoclaurine alkaloid cepharanthine in adriamycin-resistant P388 murine leukemia and K562 human leukemia cells

Harringtonine showed cross resistance in adriamycin-resistant murine leukemia P388 (P388/ADM) and human leukemia K562 (K562/ADM) cells. The relative resistance of the P388/ADM and K562/ADM cells to harringtonine was about 7 and 40, respectively. Calcium influx blockers, diltiazem and the biscoclaurine alkaloid cepharanthine enhanced the cytotoxicity of harringtonine in P388/ADM and K562/ADM cells. The extent of enhancement was different for the two drugs, and up to a 9- to 10-fold increase in harringtonine cytotoxicity occurred in P388/ADM cells, and 14- to 22-fold enhancement in K562/ADM cells with diltiazem or cepharanthine. Harringtonine resistance of P388/ADM was circumvented completely, and the resistance of K562/ADM was circumvented partially, by diltiazem or cepharanthine. The mechanism of enhanced cytotoxicity by diltiazem and cepharanthine is probably inhibition of active efflux of harringtonine in P388/ADM and K562/ADM cells.     

Bisbenzylisoquinoline alkaloids and P-glycoprotein function: A structure activity relationship study

Conflicts with the notion that specific substrate interactions were required in the control of reaction path in active transport systems, P-glycoprotein showed extraordinarily low specificity. Therefore, overexpression P-glycoprotein excluded a large number of anticancer agents from cancer cells, and multidrug resistance happened. Several kinds of bisbenzylisoqunoline alkaloids were reported to modulate P-glycoprotein function and reverse drug resistance. In order to provide more information for their structure activity relationship on P-glycoprotein function, the effects of tetrandrine, isotetrandrine, fangchinoline, berbamine, dauricine, cepharanthine and armepavine on the P-glycoprotein function were compared by using daunorubicin-resistant leukemia MOLT-4 cells in the present study. Among them, tetrandrine exhibited the strongest P-glycoprotein inhibitory effect, followed with fangchinoline and cepharanthine, and subsequently with berbamine or isotetrandrine. However, dauricine and armepavine showed little influence on the P-glycoprotein function. These data revealed that the 18-membered ring of the bisbenzylisoquinoline alkaloids maintained the P-glycoprotein inhibitory activity, suggesting that double isoquinoline units connected by two oxygen bridges were indispensable. Moreover, stereo-configuration of bisbenzylisoquinoline 3D structures determined their inhibitory activities, which provided a new viewpoint to recognize the specificity of binding pocket in P-glycoprotein. Our data also indicated that 3D chemical structure was more sensitive than 2D to predict the P-glycoprotein inhibitory-potencies of bisbenzylisoqunoline alkaloids.     

Modulators of the multidrug-transporter, P-glycoprotein, exist in the human plasma

P-glycoprotein (P-gp) is thought to mediate the transport of anticancer drugs and to be responsible for the multidrug-resistant (MDR) phenotype. P-gp is also expressed in normal human tissues, such as the adrenal gland, kidney, liver, colon and capillary endothelium of the brain. However, the function and transporting substrates of P-gp in normal tissues are still not understood. This paper explains that some compounds in the human plasma can modulate the transporting activity of P-gp. A partially purified fraction from the human plasma enhanced the accumulation of anti-cancer agents in MDR cells. This fraction inhibited the efflux of vinblastine from MDR cells, and also inhibited the photoaffinity labeling of P-gp with azidopine as effectively as vinblastine, quinidine and cepharanthine. The compounds in this purified fraction may be physiological substrates of P-gp and can probably overcome MDR.     

Screening and identification of HTNVpv entry inhibitors with high-throughput pseudovirus-based chemiluminescence

Hantaviruses, such as Hantaan virus (HTNV) and Seoul virus, are the causative agents of Hantavirus cardiopulmonary syndrome (HCPS) and hemorrhagic fever with renal syndrome (HFRS), and are important zoonotic pathogens. China has the highest incidence of HFRS, which is mainly caused by HTNV and Seoul virus. No approved antiviral drugs are available for these hantaviral diseases. Here, a chemiluminescence-based high-throughput-screening (HTS) assay was developed and used to screen HTNV pseudovirus (HTNV_pv) inhibitors in a library of 1813 approved drugs and 556 small-molecule compounds from traditional Chinese medicine sources. We identified six compounds with in vitro anti-HTNV_pv activities in the low-micromolar range (EC₅₀ values of 0.1–2.2 μmol/L; selectivity index of 40–900). Among the six selected compounds, cepharanthine not only showed good anti-HTNV_pv activity in vitro but also inhibited HTNV_pv-fluc infection in Balb/c mice 5 h after infection by 94% (180 mg/kg/d, P < 0.01), 93% (90 mg/kg/d, P < 0.01), or 92% (45 mg/kg/d, P < 0.01), respectively, in a bioluminescent imaging mouse model. A time-of-addition analysis suggested that the antiviral mechanism of cepharanthine involves the membrane fusion and entry phases. Overall, we have established a HTS method for antiviral drugs screening, and shown that cepharanthine is a candidate for HCPS and HFRS therapy. These findings may offer a starting point for the treatment of patients infected with hantaviruses.     

Cepharanthine inhibited HIV-1 cell–cell transmission and cell-free infection via modification of cell membrane fluidity

The anti-HIV-1 activity of cepharanthine (CEP), a natural product derived from Stephania cepharantha Hayata, was evaluated. CEP stabilized plasma membrane fluidity and inhibited HIV-1 envelope-dependent cell-to-cell fusion of HIV-1-infected cells as well as cell-free infection. It is suggested that CEP inhibited the HIV-1 entry process by reducing plasma membrane fluidity, and the plasma membrane is therefore an identical target to prevent viral infection.     

Selective inhibition of stimulation responses of neutrophils by membrane modulators

Polymorphonuclear leukocytes undergo a series of morphological and biochemical changes in response to various chemical stimuli. Transmembrane potential change is an early event that follows stimulation and membrane depolarization may act as a trigger for superoxide generation. To determine if there is a correlation between membrane depolarization and superoxide generation, we investigated the effects of different membrane modulators on stimulus-dependent depolarization. The membrane modulators mepacrine, chlorpromazine and cepharanthine inhibited the superoxide generation produced by chemotactic peptide, FMLP, and/or digitonin in neutrophils. Inhibitory profiles of the activation parameters, however, demonstrate that membrane depolarization is not associated with superoxide generation: FMLP-induced depolarization was inhibited by the modulators tested and was accompanied by the suppression of superoxide generation, but the depolarization produced by digitonin was stimulated somewhat by these drugs. Our results indicate that receptor-mediated membrane depolarization is not a necessary event for the activation of superoxide generation by digitonin.     

Antioxidant activity of bisbenzylisoquinoline alkaloids from Stephania rotunda: cepharanthine and fangchinoline

In the present study, we determined the antioxidant activity of cepharanthine and fangchinoline from Stephania rotunda by performing different in vitro antioxidant assays, including 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical scavenging, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging, N,N- dimethyl-p-phenylenediamine dihydrochloride (DMPD) radical scavenging, superoxide anion (O₂^?–) radical scavenging, hydrogen peroxide scavenging, total antioxidant activity, reducing power, and ferrous ion (Fe²⁺) chelating activities. Cepharanthine and fangchinoline showed 94.6 and 93.3% inhibition on lipid peroxidation of linoleic acid emulsion at 30 μg/mL concentration, respectively. On the other hand, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), α-tocopherol, and trolox indicated inhibitions of 83.3, 92.2, 72.4, and 81.3% on peroxidation of linoleic acid emulsion at the same concentration (30 μg/mL), respectively. According to the results, cepharanthine and fangchinoline have effective antioxidant and radical scavenging activity.     

药用异喹啉生物碱的研究：Ⅱ.地不容生物碱的研究

从地不容块根中分得四种生物碱A、B、C、D,经UV,IR,~1H-NMR,~(13)C-NMR,HRMS等方法鉴定了它们的结构,分别是cepharanthine (A),1-curine (B),isochondodendrine (C),ushinsunine (D)。C,D系首次从该植物中获得。     

Alkaloids of Stephania sinica

A new hasubanan alkaloid named as runanine was isolated from Stephania sinica. On the basis of spectral analysis, four methoxyl groups could be located at C-2, C-3, C-7 and C-8, respectively. Its structure was determined to be 1. Two other known compounds, cepharanthine and sitosterol, were also obtained from this plant.     

Improvement of the anoxia-induced mitochondrial dysfunction by membrane modulation

The mitochondrial dysfunction induced by anoxia in vitro was improved with chlorpromazine, cepharanthine, bromophenacyl bromide, and mepacrine without affecting phospholipid or adenine nucleotide metabolisms. The drugs inhibited lipid peroxidation by Fe²⁺, mitochondrial disruption by Ca²⁺, and membrane perturbation by lysolecithin, and retained the activity to control H⁺ permeability across mitochondrial membranes. The drugs appeared to preserve the functions by acting to suppress the development of membrane deterioration which may have resided in the deenergization of mitochondria in the absence of oxygen.     

Biscoclaurine alkaloids in callus tissues of Stephania cepharantha

Although callus tissues derived from tubers of Stephania cepharantha cannot synthesize the main alkaloids of the original plant, cepharanthine and isotetrandrine, they are able to synthesize biscoclaurine alkaloids, berbamine and aromorine, the latter not being found in the original plant. These results suggest that enzymes controlling specific methylation and methylenedioxy group formation are absent from the callus. The maximum content of total alkaloid in the callus tissues subcultured for 9 months was more than 3 times that of original plant. Alkaloid content was affected by addition of various auxins, IAA being most effective.     

Simple,sensitive and rapid HPLC–MS/MS method for the determination of cepharanthine in human plasma

A rapid, sensitive and specific method for the determination of cepharanthine in human plasma using high performance liquid chromatography coupled with tandem mass spectrometry (HPLC–MS/MS) was described. Cepharanthine and the internal standard (I.S.), telmisartan, were extracted from human plasma by methanol to precipitate the protein. A centrifuged upper layer was then evaporated and reconstituted with 100 μL methanol. Chromatographic separation was performed on an AGILENT XDB-C₈ column (150 mm × 2.1 mm, 5.0 μm, Agilent, USA) using a gradient mobile phase with 1 mmol/L ammonium acetate in water with 0.05% formic acid and methanol. Detection and quantitation was performed by MS/MS using electrospray ionization (ESI) and multiple reaction monitoring (MRM) in the positive ion mode. The most intense [M+H]⁺ MRM transition of cepharanthine at m/z 607.3 → 365.3 was used for quantitation and the transition at m/z 515.5 → 276.4 was used to monitor telmisartan. The calibration curve was linear within the concentration range of 0.5–200.0 ng/mL (r = 0.9994). The limit of quantification (LOQ) was 0.5 ng/mL. The extraction recovery was above 81.1%. The accuracy was higher than 92.3%. The intra- and inter-day precisions were less than 9.66%. The method was accurate, sensitive and simple and was successfully applied to a pharmacokinetic study after single intravenous administration of 50 mg cepharanthine in 12 healthy Chinese volunteers.     

千金藤属植物生物碱的高效液相色谱定量分析

采用Zorbax CN柱,以MeOH:H_2O:NH_4OH为流动相,在282nm紫外吸收波长检测下,测定了云南和贵州产9种千金藤属(Stephania)植物12个样品中千金藤素、汉防己甲素、轮环藤宁、光千金藤碱、颅痛定、克斑宁、异紫堇定及荷包牡丹碱等8个异喹啉类生物碱的含量,讨论了这些生物碱的分布规律。     

The interaction between cepharanthine and two serum albumins: multiple spectroscopic and chemometric investigations

The binding modes of cepharanthine (CEPT) with bovine serum albumin (BSA) and human serum albumin (HSA) have been established by reproducing physiological conditions, which is very important to understand the pharmacokinetics and toxicity of CEPT. These spectral data were further analyzed by the multivariate curve resolution‐alternating least squares method. Moreover, the concentration profiles and pure spectra of three species (BSA/HSA, CEPT and CEPT–BSA/HSA) and the apparent equilibrium constants K_app were evaluated. The experimental results showed that CEPT could quench the fluorescence intensity of BSA/HSA by a combined quenching (static and dynamic) procedure. The binding constant (K), the thermodynamic parameters (ΔG, ΔH and ΔS) and binding subdomain were measured, and indicated that CEPT could spontaneously bind to BSA/HSA on subdomain IIA through the hydrophobic interactions. The effect of CEPT on the secondary structure of proteins has been analyzed by circular dichroism, 3D fluorescence and Fourier transform infrared spectra. The binding distance between CEPT and tryptophan of BSA/HSA was 2.305/1.749 nm, which is based on the Förster resonance energy transfer theory. Copyright © 2013 John Wiley & Sons, Ltd.     

Polymorphic mimicry in Papilio dardanus: mosaic dominance, big effects, and origins

The mocker swallowtail, Papilio dardanus, has a female-limited polymorphic mimicry. This polymorphism is controlled by allelic variation at a single locus with at least 11 alleles. Many of the alternative morphs are accurate mimics of different species of distasteful butterflies. Geneticists have long been interested in the mechanism by which a single gene can have such diverse and profound effects on the phenotype and in the process by which these complex phenotypic effects could have evolved. Here we present the results of a morphometric analysis of the pleiotropic effects of the mimicry gene on the array of elements that makes up the overall pattern. We show that the patterns controlled by mimicking alleles are more variable and less internally correlated than those controlled by nonmimicking alleles, suggesting the two are subject to different degrees of selection and mutational variance. Analysis of the pleiotropic dominance of the alleles reveals a consistent pattern of dominance within a coevolved genetic background and a mosaic pattern of dominant and recessive effects (including overdominance) in a heterologous genetic background. The alleles of the mimicry gene have big effects on some pattern elements and small effects on others. When the array of big phenotypic effects of the mimicry gene is applied to the presumptive ancestral color pattern, it produces a reasonable resemblance to distasteful models and suggests the initial steps that may have produced the mimicry as well as the polymorphism.     

Gender, sex hormones, and vascular tone

The greater incidence of hypertension and coronary artery disease in men and postmenopausal women compared with premenopausal women has been related, in part, to gender differences in vascular tone and possible vascular protective effects of the female sex hormones estrogen and progesterone. However, vascular effects of the male sex hormone testosterone have also been suggested. Estrogen, progesterone, and testosterone receptors have been identified in blood vessels of human and other mammals and have been localized in the plasmalemma, cytosol, and nuclear compartments of various vascular cells, including the endothelium and the smooth muscle. The interaction of sex hormones with cytosolic/nuclear receptors triggers long-term genomic effects that could stimulate endothelial cell growth while inhibiting smooth muscle proliferation. Activation of plasmalemmal sex hormone receptors may trigger acute nongenomic responses that could stimulate endothelium-dependent mechanisms of vascular relaxation such as the nitric oxide-cGMP, prostacyclin-cAMP, and hyperpolarization pathways. Additional endothelium-independent effects of sex hormones may involve inhibition of the signaling mechanisms of vascular smooth muscle contraction such as intracellular Ca2+ concentration and protein kinase C. The sex hormone-induced stimulation of the endothelium-dependent mechanisms of vascular relaxation and inhibition of the mechanisms of vascular smooth muscle contraction may contribute to the gender differences in vascular tone and may represent potential beneficial vascular effects of hormone replacement therapy during natural and surgically induced deficiencies of gonadal hormones.