Design,synthesis, and biological evaluation of the analogues of glaziovianin A,a potent antitumor isoflavone

Various analogues of glaziovianin A, an antitumor isoflavone, were synthesized, and their biological activities were evaluated. O⁷-modified glaziovianin A showed strong cytotoxicity against HeLa S₃ cells. Compared to glaziovianin A, the O⁷-benzyl and O⁷-propargyl analogues were more cytotoxic against HeLa S₃ cells and more potent M-phase inhibitors. Furthermore, O⁷-modified molecular probes of glaziovianin A were synthesized for biological studies.     

Synthesis and antitumor activities of novel dipeptide derivatives derived from dehydroabietic acid

A series of dipeptide derivatives from dehydroabietic acid were designed and synthesized as novel antitumor agents. The antitumor activities screening indicated that many compounds showed moderate to high levels of inhibition activities against NCI-H460, HepG2, SK-OV-3, BEL-7404, HeLa and HCT-116 cancer cell lines and that some displayed more potent inhibitory activities than commercial anticancer drug 5-fluorouracil. The mechanism of representative compound 7b was studied by AO/EB staining, Hoechst 33258 staining, JC-1 mitochondrial membrane potential staining, TUNEL assay, DNA ladder assay and flow cytometry, which exhibited that the compound could induce apoptosis in HeLa cells. Further investigation showed that compound 7b induced apoptosis of HeLa cells through a mitochondrial pathway.     

Inhibition of DNA topoisomerase I by natural and synthetic mono- and dimeric protoberberine alkaloids

A series of natural (i.e., 1-7) and synthetic (i.e., 8-23) protoberberine alkaloids were evaluated for their inhibitory activities towards DNA topoisomerase I. Both the natural, monomeric protoberberine alkaloids and their mono-modified congeners showed only minor activities. In contrast, most of the dimeric protoberberine alkaloids, especially compounds 12-22, were highly active, with a similar cleavage efficiency as camptothecin (CPT), a well-known, potent topoisomerase-I inhibitor. Thus, these dimeric compounds are promising candidates to be further elaborated as anticancer leads. The mechanism of topoisomerase-I inhibition seems to be dependent on drug concentration for the dimeric protoberberines. At low concentration, they exhibit similar characteristics as CPT. At high concentration, this ability is mostly lost, and the dimers inhibit the relaxation activity of topoisomerase I. Thus, we suppose that the active, dimeric protoberberines strongly bind to plasmid DNA at elevated drug concentration. This most likely results in blocking the enzyme's access to plasmid DNA, thus inhibiting its relaxation.     

Antifungal 3,5-disubstituted furanones: From 5-acyloxymethyl to 5-alkylidene derivatives

5-Acetoxymethyl-3-(4-bromophenyl)-2,5-dihydrofuran-2-one previously described as highly antifungally active was found to provide the corresponding 5-methylene derivative via an unusual DMSO-promoted elimination of the ester group at C5 under antifungal assay conditions. Since the latter possessed nearly the same antifungal effect as that originally reported for the former, the 5-acetoxymethyl furanone just served as a precursor of the actual antifungally active species. A few series of compounds with alkyloxy, aryloxy and alkylidene substituents at C5 of the parent furanone structure were therefore prepared and evaluated. In line with the ease of elimination of the substituent from C5, low activities of the 5-alkoxy compounds were observed. On the other hand, their 5-aryloxymethyl congeners were found to be capable of liberating the antifungally active 5-methylene furanone into the testing medium. The antifungal effect of the 5-alkylidene derivatives was highly sensitive to substitution of the alkylidene moiety; a substituent in the allylic position was necessary for a compound to retain high activity. Parallel evaluation of cytostatic activity showed moderate activities of the antifungally active derivatives against HeLa S3 and CCRF-CEM lines. Cell cycle analysis of CCRF-CEM cells following the treatment with 5-methylene-3-(4-bromophenyl)-2,5-dihydrofuran-2-one revealed that this compound is a necrotic agent.     

Metallothionein isoform 2A expression is inducible and protects against ROS-mediated cell death in rotenone-treated HeLa cells

The role of MT (metallothionein) gene expression was investigated in rotenone-treated HeLa cells to induce a deficiency of NADH:ubiquinone oxidoreductase (complex I). Complex I deficiency leads to a diversity of cellular consequences, including production of ROS (reactive oxygen species) and apoptosis. HeLa cells were titrated with rotenone, resulting in dose-dependent decrease in complex I activity and elevated ROS production at activities lower than 33%. Expression of MT2A (MT isoform 2A), but not MT1A or MT1B RNA, was significantly inducible by rotenone (up to 7-fold), t-BHP (t-butyl hydroperoxide; 5-fold) and CdCl2 (50-fold), but not ZnCl2. Myxothiazol treatment did not elevate either ROS or MT2A levels, which supports a ROS-related mechanism for rotenone-induced MT2A expression. To evaluate the role of MT2A expression, MT2A and MT1B were overexpressed in HeLa cells and treated with rotenone. Compared with control and MT1B-overexpressing cells, ROS production was significantly lower and cell viability higher in MT2A-overexpressing HeLa cells when ROS production was enhanced by treatment with t-BHP. Mitochondrial membrane potential was noticeably less reduced in both MT-overexpressing cell lines. MT2A overexpression in rotenone-treated cells also significantly reduced or delayed apoptosis induction, as measured by caspase 3/7 activity and cytosolic nucleosome enrichment. We conclude that MT2A offers significant protection against the main death-causing consequences of rotenone-induced complex I deficiency in HeLa cells. Our results are in support of the protective role against oxidative stress ascribed to MTs and provide evidence that MT2A expression may be a beneficial downstream adaptive response in complex I-deficient cells.     

Synthesis and antiproliferative evaluation of 4-anilino-n-methoxyfuro[2,3-b]quinoline derivatives (n=6, 7). Part 5

A series of 27 differently substituted 4-anilinofuro[2,3-b]quinolines were synthesized and evaluated for their antiproliferative activities against the HeLa, SKHep1, SAS, AGS, A549, and CE81T cell lines, cancers commonly found in Asian countries. Among the compounds tested, 1-{4-[(3-chloro-7-methoxyfuro[2,3-b]quinolin-4-yl)amino]phenyl}ethanone (1) was the most potent, with IC(50) values of 3.1, 3.0, and 4.2 microM, resp., against the growth of HeLa, SKHep, and CE81T cells. Compound 1 was, thus, further evaluated by flow cytometry to evaluate its effect on the cell-cycle distribution of HeLa cells. Our results indicated that 1 readily induces cell-cycle arrest in the G2/M phase, followed by DNA fragmentation and, ultimately, cell death.     

Assessment of the antiproliferative and apoptotic roles of sulfonamide carbonic anhydrase IX inhibitors in HeLa cancer cell line

Carbonic anhydrase IX (CA IX) has recently been validated as an antitumor/antimetastatic drug target. In this study, we examined the underlying molecular mechanisms and the anticancer activity of sulfonamide CA IX inhibitors against cervical cancer cell lines. The effects of several sulfonamides on HeLa, MDA-MB-231, HT-29 cancer cell lines, and normal cell lines (HEK-293, PNT-1A) viability were determined. The compounds showed high cytotoxic and apoptotic activities, mainly against HeLa cells overexpressing CA IX. We were also examined for intracellular reactive oxygen species (ROS) production; intra-/extracellular pH changes, for inhibition of cell proliferation, cellular mitochondrial membrane potential change and for the detection of caspase 3, 8, 9, and CA IX protein levels. Of the investigated sulfonamides, one compound was found to possess high cytotoxic and anti-proliferative effects in HeLa cells. The cytotoxic effect occurred via apoptosis, being accompanied by a return of pHe/pHi towards normal values as for other CA IX inhibitors investigated earlier.     

A cytolytic assay for the measurement of palytoxin based on a cultured monolayer cell line

A cytolytic assay that could detect palytoxin and its congeners has been developed by the use of an established cell line grown as monolayer to replace the current hemolytic method. We used MCF-7 cells and cytolysis was measured by the release of cytosolic lactate dehydrogenase (LDH) in the buffer added to treated cells (culture supernatant). A dose-dependent increase in LDH activity in culture supernatants was detected when MCF-7 cells were exposed to palytoxin and its analogue ostreocin D. The cytolytic response induced by palytoxin and ostreocin D was specific for this group of compounds, acting on Na+/K+-ATPase, as it was prevented when cells were preincubated with ouabain. The specificity of our assay for palytoxin and its congeners was confirmed by the finding that cytolysis was not detected when MCF-7 cells were exposed to unrelated toxins such as maitotoxin, tetrodotoxin, okadaic acid, and yessotoxin, even in the case of compounds that elicit cytotoxic responses under our experimental conditions. Using extracts from biological materials after spiking with the palytoxin standard, we found a good correlation between palytoxin levels measured by our cytolytic assay and the expected values. Our cytolytic assay detected palytoxin in naturally contaminated materials, but estimates were significantly higher than the palytoxin contents determined by LC-MS, indicating that naturally contaminated materials contain biologically active palytoxin congeners. We conclude that our cytolytic assay based on the use of MCF-7 cell monolayers is a viable alternative to animal-based methods for the determination of palytoxin and its congeners in contaminated materials.     

One-pot synthesis and biological evaluation of N-(aminosulfonyl)-4-podophyllotoxin carbamates as potential anticancer agents

A series of N-(aminosulfonyl)-4-podophyllotoxin carbamates were synthesized via the Burgess-type intermediate, and their antiproliferative activities were evaluated. Most of them possessed more potent cytotoxic effects against four human tumor cell lines (HeLa, A-549, HCT-8 and HepG2) and less toxic to normal human fetal lung fibroblast WI-38 cells than etoposide. In particular, N-(morpholinosulfonyl)-4-podophyllotoxin carbamate (9) exhibited the most potent activity towards these four tumor cells with IC₅₀ values in the range of 0.5–16.5 μM. Furthermore, immunofluorescence analysis revealed that 9 induced cell apoptosis by up-regulating the expression of p53 and ROS. Meanwhile, 9 effectively inhibited tubulin polymerization and microtubule assembly at cellular levels in HeLa cells. In addition, 9 could induce cell cycle arrest in the G2/M phase in HeLa cells by up-regulating levels of cyclinB1 and cdc2 and decreasing the expression of p-cdc2. These results indicated that 9 had potential for further development as anticancer agents.     

Design, synthesis, and antitumor activity of bile acid-polyamine-nucleoside conjugates

A series of bile acid-polyamine amides conjugated with 3'-azido-3'-deoxythymidine (AZT) as potential antitumor prodrugs in the form of phosphoramidates were synthesized in good yields and their antitumor activities were assayed against two human cancer cells in vitro: cervix cancer HeLa cells and renal cancer 7860 cells. The improved antitumor activity probably derived from the enhanced delivery efficiency of AZT due to bile acid-polyamine conjugates.     

Design and synthesis of piperazine acetate podophyllotoxin ester derivatives targeting tubulin depolymerization as new anticancer agents

In this paper, a series of podophyllotoxin piperazine acetate ester derivatives were synthesized and investigated due to their antiproliferation activity on different human cancer cell lines. Among the congeners, C5 manifested prominent cytotoxicity towards the cancer cells, without causing damage on the non-cancer cells through inhibiting tubulin assembly and having high selectively causing damage on the human breast (MCF-7) cell line (IC₅₀ = 2.78 ± 0.15 μM). Treatments of MCF-7 cells with C5 resulted in cell cycle arrest in G2/M phase and microtubule network disruption. Moreover, regarding the expression of cell cycle relative proteins CDK1, a protein required for mitotic initiation was up-regulated. Besides, Cyclin A, Cyclin B1 and Cyclin D1 proteins were down-regulated. Meanwhile, it seems that the effect of C5 on MCF-7 cells apoptosis inducing was observed to be not obvious enough. In addition, docking analysis demonstrated that the congeners occupy the colchicine binding pocket of tubulin.     

肌醇磷脂激酶(PI4-K)单克隆抗体的制备及其对细胞生长的抑制

制备了抗肌醇磷脂激酶 ( PI4- K)单克隆抗体 ( A6D)并测定了抗原 -抗体反应基本特性及功能 .结果表明 ,单克隆抗体与固相及溶液中肌醇磷脂激酶的亲和常数分别为 7.5× 1 0 6和 6× 1 0 8( mol/L) -1.单抗 1 .9× 1 0 -7mol/L可以抑制从细胞提取液的 PI4- K酶活力 50 % .用 FITC标记单抗在蛋白微球引导下进入细胞内 ,主要富集在细胞质膜区 ,并对 He La细胞和小鼠小脑细胞生长有明显抑制作用 .     

Two forms of purified m7G-cap binding protein with different effects on capped mRNA translation in extracts of uninfected and poliovirus-infected HeLa cells

Eukaryotic mRNA cap binding proteins were purified from ribosomal salt wash in the presence of protease inhibitors by sucrose gradient sedimentation and m7GDP-Sepharose affinity chromatography. Rabbit reticulocyte and erythrocyte proteins with sedimentation constants of less than 6 S yielded a approximately 24,000-dalton cap binding protein. It stimulated capped mRNA translation in extracts of uninfected HeLa cells but did not restore capped mRNA function in extracts prepared from poliovirus-infected cells. Restoring and stimulatory activities both were associated with a larger, approximately 8-10 S complex that included the approximately 24,000-dalton polypeptide and several higher molecular mass components. The same two translational activities were also obtained in a slightly smaller approximately 5-7 S complex from uninfected HeLa cells but were absent from poliovirus-infected cell preparations.     

Application of microarrays to identify and characterize genes involved in attachment dependence in HeLa cells

The ability to modify cellular properties such as adhesion is of interest in the design and performance of biotechnology-related processes. The current study was undertaken in order to evaluate the effectiveness of modulating cellular adhesion in HeLa cells from a genomics perspective. Using DNA microarrays, differences in gene expression between two phenotypically distinct, anchorage-dependent and anchorage-independent, HeLa cell lines were identified. With the aid of several statistical methods and an extensive literature search, two genes were selected as potential targets for further study: siat7e and lama4. Subsequently, experiments were carried out to investigate the effects of siat7e and lama4 separately, on adhesion in HeLa cells by altering their expression in vivo. Decreasing the expression of siat7e, a type II membrane glycosylating sialyltransferase, in anchorage-independent HeLa cells using short interfering RNA (siRNA) resulted in greater aggregation (i.e. clumping) and morphological changes as compared to untreated anchorage-independent HeLa cells. Similar effects were seen in anchorage-independent HeLa cells when the expression of lama4 which encodes laminin alpha4, a member of the laminin family of glycoproteins, was enhanced as compared to untreated anchorage-independent HeLa cells. Using a shear flow chamber, an attachment assay was developed; illustrating either increased expression of siat7e or decreased expression of lama4 in anchorage-dependent HeLa cells reduced cellular adhesion. Collectively, the results of this study are consistent with the roles siat7e and lama4 play in adhesion processes in vivo and indicate modifying the expression of either gene can influence adhesion in HeLa cells. The strategy of applying bioinformatics techniques to characterize and manipulate phenotypic behaviors is a powerful tool for altering the properties of various cell lines for desired biotechnology objectives.     

Overexpression of Hp95 induces G1 phase arrest in confluent HeLa cells

Xp95, a protein recently identified in Xenopus laevis, is potentially involved in progesterone-induced Xenopus oocyte maturation. In this study, we cloned a human homologue of Xp95, designated Hp95, and examined the effect of its overexpression on the growth properties of human malignant HeLa cells which have lost the contact inhibition of cell proliferation. We observed that although HeLa cells did not undergo G1 phase arrest at any stage after confluence, they were able to downregulate their G1 phase CDK activities in response to confluence. When Hp95 was overexpressed in HeLa cells by transfection with a constitutive or an inducible expression vector containing a full-length Hp95 transgene, HeLa cells became able to undergo G1 phase arrest and form a monolayer culture after confluence. However, the G1 phase CDK activities in these Hp95 overexpressing cells were not inhibited further as compared to control cells after confluence. These results indicate that the defects in HeLa cells that cause the loss of contact inhibition of cell proliferation are in components downstream of the G1 phase CDKs and that overexpression of Hp95 counteracts some of these defects.     

The mode of action of cytotoxic and antitumor 1-nitroacridines. III. In vivo interstrand cross-linking of DNA of mammalian or bacterial cells by 1-nitroacridines

To define a critical lesion in presumable target DNA cause in vivo by the antitumor and cytotoxic 1-nitroacridines, Ehrlich ascites tumor (Eat) cells implanted into mice, HeLa cells grown in monolayer culture or Bacillus subtilis SB 1058 strain cells were exposed to Ledakrin [Nitracrine; 1-nitro-9-(3'-dimethylamino-n-propylamino)acridine], its non-antitumor congeners, or mitomycin C tested for comparison; total intracellular DNA was isolated from control or treated cells and denatured by heat, alkali or formamide, after which the chemically-induced fraction of interstrand cross-linked DNA molecules was assessed by thermal denaturation-renaturation curve analysis, hydroxylapatite column chromatography, or partitioning in a Dextran T500-polyethylene glycol 6000 biphasic system. Ledakrin, as compared to mitomycin C, was a very effective cross-linking agent, inducing one covalent cross-link per approx. 20 X 10(3) (B. subtilis), 56 X 10(3) (HeLa) or 80 X 10(3) (Eat) DNA base pairs. The first cross-links were introduced in B. subtilis cell genomes at minimal bactericidal concentrations of Ledakrin of mitomycin C. Ledakrin failed to induce discernible cross-linking of bihelical DNA when complexed with in cell-free system. Unlike the other two 1-nitroacridines which cross-linked DNA of cultured HeLa or B. subtilis cells, the non-antitumor 2-, 3- or 4-nitroacridines did not cause such effect and diminished cross-linking by Ledakrin or mitomycin C. Hence, upon metabolic activation in mammalian or bacterial cell Ledakrin and, most probably other 1-nitroacridines, become very effective DNA cross-linking agents and such effects appear to be responsible for the antitumor and potent cytotoxic activities of 1-nitroacridines.     

Deficient expression of bactericidal/permeability-increasing protein in immunocompromised hosts: translational potential of replacement therapy

BPI (bactericidal/permeability-increasing protein) is a 55?kDa anti-infective molecule expressed in neutrophil and eosinophil granules and on some epithelial cells. BPI's high affinity for the lipid A region of endotoxin targets its opsonizing, microbicidal and endotoxin-neutralizing activities towards Gram-negative bacteria. Several immunocompromised patient populations demonstrate BPI deficiency, including newborns, those with anti-neutrophil cytoplasmic antibodies (as in cystic fibrosis and HIV infection) and those exposed to radiochemotherapy. BPI may be replenished by administering agents that induce its expression or by administration of recombinant BPI congeners, potentially shielding BPI-deficient individuals against Gram-negative bacterial infection, endotoxemia and its toxic sequelae.     

A Click Approach to Novel D-Ring-Substituted 16α-Triazolylestrone Derivatives and Characterization of Their Antiproliferative Properties

A simple and efficient synthesis of novel, D-ring substituted estrone derivatives containing a 16α-triazolyl moiety is described. Two epimeric azido alcohols (16α-azido-17α-hydroxy and 16α-azido-17β-hydroxy) of estra-1,3,5(10)-triene-3-methyl ether were prepared, followed by copper(I)-catalyzed azide-alkyne cycloaddition with various terminal alkynes. The steroidal triazoles were obtained in high yields and their activities against three human cancer cell lines (HeLa, MCF7 and A431) were screened. The most effective analogs were submitted to additional experiments in order to characterize their antiproliferative properties. As evidenced by flow cytometry, the selected steroids induced a disturbance in the HeLa cell cycle in a concentration- and exposure-dependent manner, through an increase of the hypodiploid population (subG1) and a cell cycle arrest in the G2/M phase. A noncancerous human fibroblast cell line (MRC5) was used to determine the selectivities of these compounds. Fluorescent microscopy after Hoechst 33258 - propidium iodide (HOPI) double staining revealed nuclear condensation and disturbed cell membrane integrity. The enhanced activities of caspase-3 and caspase-9 without activation of caspase-8 in the treated cells indicated the activation of the intrinsic pathway of apoptosis. The levels of cell cycle regulators (CDK1, cyclin B1/B2 and cdc25B) were decreased and the ratio Bax/Bcl-2 was increased 24 h after the treatment of HeLa cells (determined at an mRNA level by means of an RT-PCR technique). Under the same conditions, two agents elicited substantially increased degrees of phosphorylation of stathmin, as evidenced by Western blotting. The presented results demonstrate that these steroids can be regarded as appropriate structural scaffolds for the design and synthesis of further steroid analogs as innovative drug candidates with good efficacy.     

Eriodictyol mediated selective targeting of the TNFR1/FADD/TRADD axis in cancer cells induce apoptosis and inhibit tumor progression and metastasis

While the anti-inflammatory activities of Eriodictyol, a plant-derived flavonoid is well-known, reports on its anti-cancer efficacy and selective cytotoxicity in cancer cells are still emerging. However, little is known regarding its mechanism of selective anti-cancer activities. Here, we show the mechanism of selective cytotoxicity of Eriodictyol towards cancer cells compared to normal cells. Investigation reveals that Eriodictyol significantly upregulates TNFR1 expression in tumor cells (HeLa and SK-RC-45) while sparing the normal cells (HEK, NKE and WI-38), which display negligible TNFR1 expression, irrespective of the absence or presence of Eriodictyol. Further investigation of the molecular events reveal that Eriodictyol induces apoptosis through expression of the pro-apoptotic DISC components leading to activation of the caspase cascade. In addition, CRISPR-Cas9 mediated knockout of TNFR1 completely blocks apoptosis in HeLa cells in response to Eriodictyol, confirming that Eriodictyol induced cancer cell apoptosis is indeed TNFR1-dependent. Finally, in vivo data demonstrates that Eriodictyol not only impedes tumor growth and progression, but also inhibits metastasis in mice implanted with 4T1 breast cancer cells. Thus, our study has identified Eriodictyol as a compound with high selectivity towards cancer cells through TNFR1 and suggests that it can be further explored for its prospect in cancer therapeutics.