A versatile transposon-based technology to generate loss- and gain-of-function phenotypes in the mouse liver.

BACKGROUND: Understanding the contribution of gene function in distinct organ systems to the pathogenesis of human diseases in biomedical research requires modifying gene expression through the generation of gain- and loss-of-function phenotypes in model organisms, for instance, the mouse. However, methods to modify both germline and somatic genomes have important limitations that prevent easy, strong, and stable expression of transgenes. For instance, while the liver is remarkably easy to target, nucleic acids introduced to modify the genome of hepatocytes are rapidly lost, or the transgene expression they mediate becomes inhibited due to the action of effector pathways for the elimination of exogenous DNA. Novel methods are required to overcome these challenges, and here we develop a somatic gene delivery technology enabling long-lasting high-level transgene expression in the entire hepatocyte population of mice. RESULTS: We exploit the fumarylacetoacetate hydrolase (Fah) gene correction-induced regeneration in Fah-deficient livers, to demonstrate that such approach stabilizes luciferase expression more than 5000-fold above the level detected in WT animals, following plasmid DNA introduction complemented by transposon-mediated chromosomal gene transfer. Building on this advancement, we created a versatile technology platform for performing gene function analysis in vivo in the mouse liver. Our technology allows the tag-free expression of proteins of interest and silencing of any arbitrary gene in the mouse genome. This was achieved by applying the HADHA/B endogenous bidirectional promoter capable of driving well-balanced bidirectional expression and by optimizing in vivo intronic artificial microRNA-based gene silencing. We demonstrated the particular usefulness of the technology in cancer research by creating a p53-silenced and hRas G12V-overexpressing tumor model. CONCLUSIONS: We developed a versatile technology platform for in vivo somatic genome editing in the mouse liver, which meets multiple requirements for long-lasting high-level transgene expression. We believe that this technology will contribute to the development of a more accurate new generation of tools for gene function analysis in mice.

Calcium Influx Kinetics and the Characteristics of Potassium Channels in Peripheral T Lymphocytes in Systemic Sclerosis.

BACKGROUND: Systemic sclerosis (SSc) is a chronic, immune-mediated, connective tissue disease causing microvascular abnormalities and fibrosis. The cytoplasmic calcium influx kinetics in T lymphocytes governs lymphocyte activation in this inflammatory process. The inhibition of Kv1.3 and IKCa1 potassium channels reduces calcium influx. METHODS: This study aimed to analyze cytoplasmic calcium influx kinetics following activation in Th1, Th2, and CD8 cells in peripheral blood of 12 healthy individuals and 16 patients with systemic sclerosis using flow cytometry. We also evaluated the effect of the specific inhibition of the Kv1.3 and IKCa1 potassium channels. RESULTS: We observed higher levels of activation in CD8 compared with Th1 cells in SSc. However, the activation of CD8 cells was lower in SSc compared to healthy controls. Moreover, activation of Th1 lymphocytes was slower in SSc than in healthy controls. The inhibition of IKCa1 channels decreased the activation of Th1 cells, while the inhibition of Kv1.3 channels modified the dynamics of activation of Th1 and Th2 lymphocytes in SSc. CONCLUSION: Th1 and CD8 cells demonstrate specific activation dynamics and sensitivity to potassium channel inhibition in SSc, distinguishing this condition both from healthy controls and other autoimmune diseases.

Synthesis and In Vitro Antitumor Activity of Naringenin Oxime and Oxime Ether Derivatives.

Naringenin is one of the most abundant dietary flavonoids exerting several beneficial biological activities. Synthetic modification of naringenin is of continuous interest. During this study our aim was to synthesize a compound library of oxime and oxime ether derivatives of naringenin, and to investigate their biological activities. Two oximes and five oxime ether derivatives were prepared; their structure has been elucidated by NMR and high-resolution mass spectroscopy. The antiproliferative activity of the prepared compounds was evaluated by MTT assay against human leukemia (HL-60) and gynecological cancer cell lines isolated from cervical (HeLa, Siha) and breast (MCF-7, MDA-MB-231) cancers. Tert-butyl oxime ether derivative exerted the most potent cell growth inhibitory activity. Moreover, cell cycle analysis suggested that this derivative caused a significant increase in the hypodiploid (subG1) phase and induced apoptosis in Hela and Siha cells, and induced cell cycle arrest at G2/M phase in MCF-7 cells. The proapoptotic potential of the selected compound was confirmed by the activation of caspase-3. Antioxidant activities of the prepared molecules were also evaluated with xanthine oxidase, DPPH and ORAC assays, and the methyl substituted oxime ether exerted the most promising activity.

Investigation of the Antiproliferative Properties of Natural Sesquiterpenes from Artemisia asiatica and Onopordum acanthium on HL-60 Cells in Vitro.

Plants and plant extracts play a crucial role in the research into novel antineoplastic agents. Four sesquiterpene lactones, artecanin (1), 3ß-chloro-4α,10α-dihydroxy-1α,2α-epoxy-5α,7αH-guaia-11(13)-en-12,6α-olide (2), iso-seco-tanapartholide 3-O-methyl ether (3) and 4ß,15-dihydro-3-dehydrozaluzanin C (4), were isolated from two traditionally used Asteraceae species (Onopordum acanthium and Artemisia asiatica). When tested for antiproliferative action on HL-60 leukemia cells, these compounds exhibited reasonable IC50 values in the range 3.6-13.5 µM. Treatment with the tested compounds resulted in a cell cycle disturbance characterized by increases in the G1 and G2/M populations, while there was a decrease in the S phase. Additionally, 1-3 elicited increases in the hypodiploid (subG1) population. The compounds elicited concentration-dependent chromatin condensation and disruption of the membrane integrity, as revealed by Hoechst 33258-propidium staining. Treatment for 24 h resulted in significant increases in activity of caspases-3 and -9, indicating that the tested sesquiterpenes induced the mitochondrial pathway of apoptosis. The proapoptotic properties of the sesquiterpene lactones were additionally demonstrated withannexin V staining. Compounds 1 and 2 increased the Bax/Bcl-2 expression and decreased the expressions of CDK1 and cyclin B2, as determined at the mRNA level by means of RT-PCR. These experimental results indicate that sesquiterpene lactones may be regarded as potential starting structures for the development of novel anticancer agents.

A molecular understanding of D-homoestrone-induced G2/M cell cycle arrest in HeLa human cervical carcinoma cells.

2-Methoxyestradiol (ME), one of the most widely investigated A-ring-modified metabolites of estrone, exerts significant anticancer activity on numerous cancer cell lines. Its pharmacological actions, including cell cycle arrest, microtubule disruption and pro-apoptotic activity, have already been described in detail. The currently tested D-ring-modified analogue of estrone, D-homoestrone, selectively inhibits cervical cancer cell proliferation and induces a G2/M phase cell cycle blockade, resulting in the development of apoptosis. The question arose of whether the difference in the chemical structures of these analogues can influence the mechanism of anticancer action. The aim of the present study was therefore to elucidate the molecular contributors of intracellular processes induced by D-homoestrone in HeLa cells. Apoptosis triggered by D-homoestrone develops through activation of the intrinsic pathway, as demonstrated by determination of the activities of caspase-8 and -9. It was revealed that D-homoestrone-treated HeLa cells are not able to enter mitosis because the cyclin-dependent kinase 1-cyclin B complex loses its activity, resulting in the decreased inactivation of stathmin and a concomitant disturbance of microtubule formation. However, unlike 2-ME, D-homoestrone does not exert a direct effect on tubulin polymerization. These results led to the conclusion that the D-homoestrone-triggered intracellular processes resulting in a cell cycle arrest and apoptosis in HeLa cells differ from those in the case of 2-ME. This may be regarded as an alternative mechanism of action among steroidal anticancer compounds.

Periosteal microcirculatory reactions in a zoledronate-induced osteonecrosis model of the jaw in rats.

OBJECTIVES: Nitrogen-containing bisphosphonates induce osteonecrosis mostly in the jaw and less frequently in other bones. Because of the crucial role of periosteal perfusion in bone repair, we investigated zoledronate-induced microcirculatory reactions in the mandibular periosteum in comparison with those in the tibia in a clinically relevant model of bisphosphonate-induced medication-related osteonecrosis of the jaw (MRONJ). MATERIALS AND METHODS: Sprague-Dawley rats were treated with zoledronate (ZOL; 80 i.v. µg/kg/week over 8 weeks) or saline vehicle. The first two right mandibular molar teeth were extracted after 3 weeks. Various systemic and local (periosteal) microcirculatory inflammatory parameters were examined by intravital videomicroscopy after 9 weeks. RESULTS: Gingival healing disorders (∼100%) and MRONJ developed in 70% of ZOL-treated cases but not after saline (shown by micro-CT). ZOL induced significantly higher degrees of periosteal leukocyte rolling and adhesion in the mandibular postcapillary venules (at both extraction and intact sites) than at the tibia. Leukocyte NADPH-oxidase activity was reduced; leukocyte CD11b and plasma TNF-alpha levels were unchanged. CONCLUSION: Chronic ZOL treatment causes a distinct microcirculatory inflammatory reaction in the mandibular periosteum but not in the tibia. The local reaction in the absence of augmented systemic leukocyte inflammatory activity suggests that topically different, endothelium-specific changes may play a critical role in the pathogenesis of MRONJ. CLINICAL RELEVANCE: This model permits for the first time to explore the microvascular processes in the mandibular periosteum after chronic ZOL treatment. This approach may contribute to a better understanding of the pathomechanism and the development of strategies to counteract bisphosphonate-induced side effects.

Anticancer and multidrug resistance-reversal effects of solanidine analogs synthetized from pregnadienolone acetate.

A set of solanidine analogs with antiproliferative properties were recently synthetized from pregnadienolone acetate, which occurs in Nature. The aim of the present study was an in vitro characterization of their antiproliferative action and an investigation of their multidrug resistance-reversal activity on cancer cells. Six of the compounds elicited the accumulation of a hypodiploid population of HeLa cells, indicating their apoptosis-inducing character, and another one caused cell cycle arrest at the G2/M phase. The most effective agents inhibited the activity of topoisomerase I, as evidenced by plasmid supercoil relaxation assays. One of the most potent analogs down-regulated the expression of cell-cycle related genes at the mRNA level, including tumor necrosis factor alpha and S-phase kinase-associated protein 2, and induced growth arrest and DNA damage protein 45 alpha. Some of the investigated compounds inhibited the ABCB1 transporter and caused rhodamine-123 accumulation in murine lymphoma cells transfected by human MDR1 gene, expressing the efflux pump (L5178). One of the most active agents in this aspect potentiated the antiproliferative action of doxorubicin without substantial intrinsic cytostatic capacity. The current results indicate that the modified solanidine skeleton is a suitable substrate for the rational design and synthesis of further innovative drug candidates with anticancer activities.

Preparation of dearomatized p-coumaric acid derivatives as DNA damage response inhibitors with potent in vitro antitumor effect.

Our research group previously identified graviquinone (1) as a promising antitumor metabolite that is formed in situ when the antioxidant methyl caffeate scavenges free radicals. Furthermore, it exerted a DNA damaging effect on cancer cells and a DNA protective effect on normal keratinocytes. To expand and explore chemical space around qraviquinone, in the current work we synthesized 9 new alkyl-substituted derivatives and tested their in vitro antitumor potential. All new compounds bypassed ABCB1-mediated multidrug resistance and showed highly different cell line specificity compared with 1. All compounds were more potent in MDA-MB-231 than on MCF-7 cells. The n-butyl-substituted derivatives 2 and 8 modulated the cell cycle and inhibited the ATR-mediated phosphorylation of checkpoint kinase-1 in MCF-7 cells. As a significant expansion of our previous findings, our results highlight the potential antitumor value of alkyl-substituted graviquinone derivatives.

Different administration schedules of darbepoetin alfa affect oxidized and reduced glutathione levels to a similar extent in 5/6 nephrectomized rats.

BACKGROUND: The development of erythropoiesis-stimulating agents (ESAs) with extended serum half-lives has allowed marked prolongation of the administration intervals. The level of oxidative stress is increased in chronic kidney disease, and is reportedly decreased after long-term ESA treatment. However, the effect of different dosing regimens of ESAs on oxidative stress has not been elucidated. METHODS: Five-sixths nephrectomized (NX) rats received either 0.4 µg/kg darbepoetin alfa (DA) weekly or 0.8 µg/kg DA fortnightly between weeks 4 and 10. NX animals receiving saline and a sham-operated (SHAM) group served as controls. The levels of oxidized and reduced glutathione (GSSG, GSH) were followed from blood samples drawn fortnightly. RESULTS: During the follow-up, the ratios GSSG/GSH showed similar trends in both DA groups, levels being significantly lower than those in the SHAM group at weeks 8 and 10. GSSG levels were lower than the baseline throughout the study in all groups except for NX controls. The GSH levels were increased in all three NX groups (weeks 6-10) compared with both the baseline and the SHAM group CONCLUSION: Our results suggest that the extent of oxidative stress is similar in response to different dosing regimens of DA in 5/6 NX rats when comparable hemoglobin levels are maintained. These findings remain to be confirmed in chronic kidney disease patients.

Direct antiproliferative effect of nonsteroidal 17ß-hydroxysteroid dehydrogenase type 1 inhibitors in vitro.

Inhibition of the local formation of estrogens seems to be an attractive strategy for pharmacological intervention in hormone-dependent disorders. The direct antiproliferative properties of ten nonsteroidal 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1) inhibitors were investigated on human cancer cell lines of gynecological origin. The mechanism of the antiproliferative action was approximated by cell cycle analysis, fluorescent microscopy, BrdU assay, determination of caspase-3 activity and quantification of the expression of cell cycle regulators at mRNA level. Treatment of HeLa cells with some of the compounds resulted in a concentration-dependent inhibition of the G1-S transition and an increase in the apoptotic population. The most effective agents increased the expression of tumor suppressors p21 and p53, while CDK2 and Rb were down-regulated. The reported anticancer actions of the tested compounds are independent of the 17ß-HSD1-inhibiting capacity. These results indicate that it is possible to combine direct antiproliferative activity and 17ß-HSD1 inhibition resulting in novel agents with dual mode of action.

Heterocyclic androstane and estrane d-ring modified steroids: Microwave-assisted synthesis, steroid-converting enzyme inhibition, apoptosis induction, and effects on genes encoding estrogen inactivating enzymes.

d-ring-fused and d-homo lactone compounds in estratriene and androstane series were synthesized using microwave-assisted reaction conditions. Microwave-irradiated synthesis methods were convenient and effective, and provided high yields with short reaction times. Their inhibition of C17,20-lyase and 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1) activities were studied in in vitro enzyme assays. d-ring-fused triazolyl estrone analog 24 showed potent inhibition of NADH-complexed 17ß-HSD1, with a binding affinity similar to that of the substrate estrone; its inhibition against NADPH-complexed 17ß-HSD1 was markedly weaker. Compound 24 also significantly and selectively reduced proliferation of cancer cell lines of gynecological origin. This estrane triazole changed the cell cycle and induced apoptosis of HeLa, SiHa, and MDA-MB-231 cancer cells, measured by both increased subG1 fraction of cells and activation of caspase-independent signaling pathways. A third mode of anti-estrogenic action of 24 saw increased mRNA expression of the SULT1E1 gene in HeLa cells; in contrast, its 3-benzyloxy analog 23 increased mRNA expression of the HSD17B2 gene, thus showing pronounced pro-drug anti-estrogenic activity. Estradiol-derived d-ring triazole compound 24 thus acts at the enzyme, gene expression and cellular levels to decrease the production of active estrogen hormones, demonstrating its pharmacological potential.

Cotinus coggygria Scop. induces cell cycle arrest, apoptosis, genotoxic effects, thermodynamic and epigenetic events in MCF7 breast cancer cells.

Current plant-derived anticancer therapeutics aim to reach higher effectiveness, to potentiate chemosensitivity and minimize the toxic side effects compared to conventional chemotherapy. Cotinus coggygria Scop. is a herb with high pharmacological potential, widely applied in traditional phytotherapy. Our previous study revealed that leaf aqueous ethanolic extract from C. coggygria exerts in vitro anticancer activity on human breast, ovarian and cervical cancer cell lines. The objective of the present research was to investigate possible molecular mechanisms and targets of the antitumor activity of the extract in breast cancer MCF7 cells through analysis of cell cycle and apoptosis, clonogenic ability assessment, evaluation of the extract genotoxic capacity, characterization of cells thermodynamic properties, and analysis on the expression of genes involved in cellular epigenetic processes. The obtained results indicated that in MCF7 cells C. coggygria extract causes S phase cell cycle arrest and triggers apoptosis, reduces colony formation, induces DNA damage, affects cellular thermodynamic parameters, and tends to inhibit the relative expression of DNMT1, DNMT3a, MBD3, and p300. Further studies on the targeted molecules and the extract anti-breast cancer potential on animal experimental model system, need to be performed in the future.

2-(3-Aryl-2-propenoyl)-3-methylquinoxaline-1,4-dioxides: a novel cluster of tumor-specific cytotoxins which reverse multidrug resistance.

A series of 2-(3-aryl-2-propenoyl)-3-methylquinoxaline-1,4-dioxides 3a-l were prepared by condensation of various aryl aldehydes with 2-acetyl-3-methylquinoxaline-1,4-dioxide 2. These compounds inhibit the growth of human Molt 4/C8 and CEM T-lymphocytes and the IC(50) values are mainly in the 5-30 microM range. The quinoxaline 1,4-dioxide 3j inhibited the growth of 58 human tumor cell lines, particularly leukemic and breast cancer neoplasms. All of the compounds 3a-l reversed the multidrug resistance (MDR) properties of murine L-5178Y leukemic cells which were transfected with the human MDR1 gene. The MDR-reversing effect may be due to the conjugated pi-electron system forming a weak electron charge transfer complex with the P-glycoprotein-mediated efflux pump. The compounds in series 2 and 3 were assessed against HL-60, HSC-2, HSC-3 and HSC-4 malignant cells as well as HGF, HPC and HPLF normal cell lines which revealed that the majority of the compounds displayed a greater toxicity to neoplastic than normal cells. Various ways in which the project may be expanded are presented.

Perturbation of the lipid phase of a membrane is not involved in the modulation of MRP1 transport activity by flavonoids.

The expression of transmembrane transporter multidrug resistance-associated protein 1 (MRP1) confers the multidrug-resistant phenotype (MDR) on cancer cells. Since the activity of the other MDR transporter, P-glycoprotein, is sensitive to membrane perturbation, we aimed to check whether the changes in lipid bilayer properties induced by flavones (apigenin, acacetin) and flavonols (morin, myricetin) were related to their MRP1 inhibitory activity. All the flavonoids inhibited the efflux of MRP1 fluorescent substrate from human erythrocytes and breast cancer cells. Morin was also found to stimulate the ATPase activity of erythrocyte ghosts. All flavonoids intercalated into phosphatidylcholine bilayers as judged by differential scanning calorimetry and fluorescence spectroscopy with the use of two carbocyanine dyes. The model of an intramembrane localization for flavones and flavonols was proposed. No clear relationship was found between the membrane-perturbing activity of flavonoids and their potency to inhibit MRP1. We concluded that mechanisms other than perturbation of the lipid phase of membranes were responsible for inhibition of MRP1 by the flavonoids.

Differential effect of phenothiazines on MRP1 and P-glycoprotein activity.

BACKGROUND: Overexpression of ATP-binding cassette (ABC) transporters such as P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1) or breast cancer resistance protein (BCRP) accounts for majority of cases of multidrug resistance (MDR) of cancer cells. MATERIALS AND METHODS: In the present work, the interactions of seven commercially available phenothiazine derivatives, known P-glycoprotein inhibitors, with this transporter and MRP1 were compared. By flow cytometry, it was shown that all the drugs increased the accumulation of rhodamine 123 in the P-gp-overexpressing lymphoma cell line L5178 MDR. On the other hand, phenothiazine derivatives stimulated MRP1-mediated efflux of fluorescent probe (BCPCF) out of human erythrocytes. RESULTS: In this way, these phenothiazine derivatives were identified as a group of atypical MDR modulators that differently interact with P-gp (as inhibitors) and MRP1 (as stimulators). CONCLUSION: This observation clearly shows that the activity of all new modulators should be tested for their effects towards different ABC transporters as a standard procedure.

Antitumor activity of alkaloids derived from Amaryllidaceae species.

The aim of the present study was to investigate the anticancer properties of five alkaloids isolated from Amaryllidaceae, including the inhibitory effect on P-glycoprotein (P-gp) and the apoptosis-inducing capacity. The tested alkaloids were evaluated for their multidrug resistance (MDR)-reversing activity on human MDR1-gene-transfected L5178 mouse lymphoma cells, using the rhodamine-123 (Rh-123) assay. Trisphaeridine and pretazettine increased the intracellular Rh-123 concentration 30- and 50-fold, respectively, as compared to the non-treated cells, and 2-O-acetyllycorine and trisphaeridine were demonstrated by means of the checkerboard method to enhance the antiproliferative activity of doxorubicin on L5178 MDR mouse lymphoma cells. The MTT assay revealed that pretazettine, trisphaeridine and 2-O-acetyllycorine displayed excellent antiproliferative effects on both the human and the mouse cell lines. The apoptosis-inducing activities of selected agents (2-O-acetyllycorine and trisphaeridine) were measured via acridine orange and ethidium bromide dual staining and flow cytometry of the subG1 population.

Screening of some saponins and phenolic components of Tribulus terrestris and Smilax excelsa as MDR modulators.

BACKGROUND: Cytotoxic activity of saponins and phenolic compounds have been described in the literature, but no reports were found on their multidrug resistance (MDR)-modulating effects on human mdr1 gene-transfected mouse lymphoma cell line. MATERIALS AND METHODS: Methylprototribestin, structurally related compounds and a mixture of 3 acetylated isomers of methylprotodioscin were investigated for antiproliferative effect and modulation of drug accumulation. RESULTS: The growth inhibitory dose (ID50) of the compounds ranged from 12.64 to 20.62 mug/ml. Methylprototribestin was the most effective resistance modifier. However, methylprotodioscin, pseudoprotodioscin, prosapogenin A of dioscin, tribestin and 5-O-caffeoylshikimic acid showed moderate MDR reversal activity. In a checkerboard method, methyloprototribestin and the mixture of the 3 acetylated isomers enhanced the antiproliferative effects on MDR cells in combination with doxorubicin. CONCLUSION: Based on these results, methylprototribestin and the mixture of the 3 acetylated isomers can be recommended for further in vivo experiments in combination with anthracyclines in human MDR-cancer xenograft transplanted mice.

Microcirculatory consequences of limb ischemia/reperfusion in ovariectomized rats treated with zoledronic acid.

BACKGROUND: Nitrogen-containing bisphosphonates (BIS) are potent therapeutics in osteoporosis, but their use may result in osteonecrotic side-effects in the maxillofacial region. Periosteal microcirculatory reactions may contribute to the development of bone-healing complications, particularly in osteoporotic bones, where ischemia-reperfusion (IR) events often develop during orthopaedic/trauma interventions. The effect of BIS on the inflammatory reactions of appendicular long bones has not yet been evaluated; thus, we aimed to examine the influence of chronic zoledronate (ZOL) administration on the periosteal microcirculatory consequences of hindlimb IR in osteopenic rats. MATERIALS AND METHODS: Twelve-week-old female Sprague-Dawley rats were ovariectomized (OVX) or sham-operated, and ZOL (80 µg/kg iv, weekly) or a vehicle was administered for 8 weeks, 4 weeks after the operation. At the end of the pre-treatment protocols, 60-min limb ischemia was induced, followed by 180-min reperfusion. Leukocyte-endothelial interactions were quantitated in tibial periosteal postcapillary venules by intravital fluorescence videomicroscopy. CD11b expression of circulating polymorphonuclear leukocytes (PMN, flow cytometry) and plasma TNF-alpha levels (ELISA) were also determined. Two-way RM ANOVA followed by the Holm-Sidak and Dunn tests was used to assess differences within and between groups, respectively. RESULTS: Limb IR induced significant increases in PMN rolling and firm adhesion in sham-operated and OVX rats, which were exacerbated temporarily in the first 60 min of reperfusion by a ZOL treatment regimen. Postischemic TNF-alpha values showed a similar level of postischemic elevations in all groups, whereas CD11b expression only increased in rats not treated with ZOL. CONCLUSIONS: The present data do not show substantial postischemic periosteal microcirculatory complications after chronic ZOL treatment either in sham-operated or OVX rats. The unaltered extent of limb IR-induced local periosteal microcirculatory reactions in the presence of reduced CD11b adhesion molecule expression on circulating PMNs, however, may be attributable to local endothelial injury/activation caused by ZOL.

Investigation of natural phenanthrenes and the antiproliferative potential of juncusol in cervical cancer cell lines.

BACKGROUND: Phenanthrenes isolated from Juncus species possess different biological activities, including antiproliferative and antimigratory effects. PURPOSE: In this study, nine phenanthrenes isolated from the roots of Juncus inflexus were investigated for their antiproliferative activity on several gynecological cancer cell lines, using non-cancerous cells as controls. METHODS: Antiproliferative activities of the compounds were determined by means of MTT assay. Flow cytometry was used for cell cycle analysis and determination of mitotic cells. Activities of caspase-3, -8, and -9 were detected by colorimetric kits. Tubulin polymerization was followed by kinetic absorbance determination. Action on tumor cell migration was described using wound healing assay. Western blot assays were used to determine apoptosis-related factors at protein level. RESULTS: Among the compounds tested, juncusol exhibited the most substantial antiproliferative effect against cervical cancer HeLa cells. It was also revealed that juncusol has a distinct growth inhibitory effect in cervical cancer cell lines of various HPV status: it was highly active in HPV type 18-positive HeLa cells, while it was inactive in HPV type 16-positive SiHa and CaSki cells. Cell cycle analysis showed an increase in G2/M and subG1 cell populations after juncusol treatment. Caspase-3, -8, and -9 were detected to be activated by juncusol in HeLa cells, indicating that juncusol induces apoptotic cell death. Moreover, juncusol inhibited tubulin polymerization, as well as EGFR activation, suggesting two possible additional mechanisms that may account for juncusol's inducing a G2/M-phase cell cycle arrest and inhibiting cell migration. CONCLUSION: These results suggest that juncusol is a potent antiproliferative agent against HPV-18 related cervical cancer and may be considered as a lead compound for the development of innovative anticancer agents.

Antitumour properties of acridone alkaloids on a murine lymphoma cell line.

The aim of the present study was to investigate the anticancer properties of a set of furanoacridone alkaloids, arborinine and evoxanthine, including the inhibitory effect of P-glycoprotein (Pgp) and the apoptosis-inducing capacity. The tested alkaloids were evaluated for multidrug resistance (MDR)-reversing activity on human Pgp-transfected L5178 mouse lymphoma cells, using the rhodamine-123 (Rh-123) assay. The antiproliferative effects of natural compounds and their interactions with doxorubicin were determined in MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. Apoptosis-inducing activity was additionally measured by means of dual annexin V and propidium iodide staining. RT-PCR was used to test the expression of Pgp mRNA after acridone treatment. All of the acridones investigated increased the accumulation of Rh-123. Gravacridonetriol and gravacridonediol monomethyl ether increased the antiproliferative effect of doxorubicin on resistant L5178 cells. Treatment with these agents resulted in a decrease in Pgp mRNA levels. Naturally occurring acridone alkaloids exhibit a beneficial combination of anticancer effects and, accordingly, the acridone skeleton can be considered useful in the design of novel antiproliferative agents.