Effect of artemisinin on proliferation and apoptosis-related protein expression in vivo and in vitro

Artemisinin is the first-line drugs for the treatment of malaria. In recent years, a large number of reports showed that artemisinin exhibit anti-tumor activity. In this study, we used C6 glioma cells and rat C6 brain-glioma model to study anti-tumor activity of artemisinin in vivo and in vitro. We found that artemisinin inhibited the proliferation in C6 cells and induced cell cycle arrest and a caspase-3-dependent cell apoptosis. It also inhibited the growth of C6 brain-glioma in vivo and enhanced living state of rat brain-glioma model. These results suggested that artemisinin had significant anti-tumor activities on C6 cells both in vitro and in vivo. Artemisinin might be exploited as a promising clinical anti-cancer drug in future.     

Antagonistic interactions between fungal pathogens and phylloplane fungi of guava

Dominant phylloplane fungi of guava (Psidium guajava L.) were screened for their antagonistic activities against the two pathogens,Colletotrichum gloeosporioides andPestalotia psidii, bothin vitro andin vivo. Culture filtrates ofAspergillus niger, Fusarium oxysporum andPenicillium citrinum caused more than 50% growth inhibition ofC. gloeosporioides. Filtrates ofCephalosporium roseo-griseum andF. oxysporum were most effective in reducing the growth ofP. psidii. Volatiles produced from the cultures ofA. niger, F. oxysporum, P. citrinum andP. oxalicum inhibited the growth ofC. gloeosporioides, whereas volatiles fromC. roseo-griseum, F. oxysporum andTrichoderma harzianum inhibited the growth ofP. psidii. The inhibitory effect of volatiles decreased with increase in incubation time. In general, the maximum effect of volatiles was noticed after 48 h incubation. Different grades of colony interactions in dual cultures were recognised between the two pathogens and the phylloplane fungi examined. Maximum inhibition ofC. gloeosporioides was caused byAureobasidium pullulans, Cladosporium cladosporioides, epicoccum purpurascens, F. oxysporum andMyrothecium roridum, whereasAspergillus terreus, C. roseo-griseum andP. oxalicum significantly reduced the growth ofP. psidii. Application of a spore suspension of each test fungus inhibited lesion development of guava leaves caused by the test pathogensin vitro. Inhibition was more pronounced when the spore concentration was increased.A. pullulans, C. cladosporioides, E. purpurascens, F. oxysporum, andT. harzianum were found to be strongly antagonistic toC. gloeosporioides. A. niger, A. terreus, C. roseo-grisem andT. harzianum were strongly antagonistic toP. psidii.     

The use of flavonoid aglycones in in vitro systems to test biological activities: based on bioavailability data,is this a valid approach?

The majority of in vitro assays on biological activities of flavonoids have used the aglycone form as the test compound. This form is readily available from commercial sources and comparable approaches have been used for testing efficacy of drugs. This paper presents the hypothesis that aglycones are only transiently present in vivo at significant concentrations at specific sites. The pathway of metabolism of flavonoids in mammals in vivo, focusing on aglycone formation, is examined to facilitate better design in the future of in vitro cell culture experiments. In vitro experiments using flavonoids and cultured cells require careful consideration of absorption and bioavailability for their appropriate interpretation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Water-soluble ferulic acid derivatives improve amyloid-β-induced neuronal cell death and dysmnesia through inhibition of amyloid-β aggregation

Ferulic acid (FA) has been reported to exhibit protective effects against amyloid-β (Aβ)-induced neurodegeneration in vitro and in vivo. Recently, we developed two water-soluble FA derivatives: 1-feruloyl glycerol and 1-feruloyl diglycerol. In this study, we examined the neuroprotective effects of these water-soluble FA derivatives on Aβ-induced neurodegeneration both in vitro and in vivo. FA and water-soluble FA derivatives inhibited Aβ aggregation and destabilized pre-aggregated Aβ to a similar extent. Furthermore, water-soluble FA derivatives, as well as FA, inhibited Aβ-induced neuronal cell death in cultured neuronal cells. In in vivo experiments, oral administration of water-soluble FA derivatives to mice improved Aβ-induced dysmnesia assessed by contextual fear conditioning test and protected hippocampal neurons against Aβ-induced neurotoxicity. This study provides useful evidence suggesting that water-soluble FA derivatives are expected to be effective neuroprotective agents.     

The pH-dependent yield threshold of the cell wall in a glycerinated hollow cylinder (in vitro system) of cowpea hypocotyl

In order to determine whether the pH-dependent yield threshold of the cell wall still exists in an in vitro system, an extensometer was devised to enable the perfusion of any experimental solution through the hollow cylinder of a hypocotyl segment excised from a cowpea seedling. Stress-strain experiments on glycerinated hollow cylinders revealed the existence of a definite yield threshold (y) of the cell wall in this in vitro system. The y value decreased reversibly with acidification (pH 4) to the same extent as the decrease of the yield threshold obtained in vivo (Y) with auxin-induced growth acceleration of hypocotyl segments. Heat treatment of the glycerinated hollow cylinder completely inhibited the decrease in y with acidification. The increase in the extensibility of the cell wall with acidification was inhibited significantly but not completely by heat treatment. These results support strongly the ‘acid growth’ theory and provide evidence that the acid-induced decrement of the yield threshold is mediated by an enzymatic reaction of a wall-binding protein. The combination of in vitro and in vivo studies presented here provides a basis for the establishment of a molecular theory on the nature of the growth parameters Y and Ф which control the yielding of the cell wall.     

Effects of Interactions BetweenPasteurella haemolyticaandBordetella parapertussisonin vitroPhagocytosis by Lung Macrophages

The aim of the work was to determine the effect of exposing ovine bronchoalveolar macrophages (BAM)in vivotoPasteurella haemolyticaand/orBordetella parapertussison the subsequent uptake and killing ofP. haemolyticaby these cellsin vitro. Exposurein vivotoP. haemolyticadid not affect the uptake ofP. haemolyticaby BAMin vitrobut reduced (P< 0·05) the intracellular killing of bacteria. Exposurein vivotoB. parapertussishad no significant effect on either the uptake of killing ofP. haemolytica in vitro. However, sequential exposurein vivotoB. parapertussisandP. haemolyticareduced both the ingestion (P< 0·05) and killing (P< 0·001) ofP. haemolytica in vitro. These results indicate that exposure toP. haemolyticacompromised the bacterial killing mechanisms of BAM and that synergy betweenB. parapertussisandP. haemolyticareduced the ability of BAM to ingest bacteria.     

In vitro effect of prostaglandins,prostaglandin endoperoxides and thromboxane on rat intestinal alkaline phosphatase and (Ca2+-Mg2+) adenosine triphosphatase

The activity of alkaline phosphate and²⁺-Mg²⁺ adenosine triphosphatase, two of the enzymes involved in limpid and calcium uptake across the intestinal membrane, were increased in experimental atherosclerosis. Administration ofAnnapavala sindhooram, an antiatherosclerotic drug, lowers these enzyme levels to near normal values. Prostaglandin E2 stimulated the enzyme activitiesin vitro, while prostaglandin endoperoxide inhibited the activity. Thromboxane and other prostaglandins had no effect on the enzyme activities. Addition of the antiatherosclerotic drug to thein vitro assay system reversed the effect of both prostaglandin E₂ and prostaglandin endoperoxide.     

Cell culture aging

Summary Cellular research in aging has been stimulated by the observation that human diploid cells have a limited number of cell divisions in culture. This loss of cellular proliferation (in vitro senescence) has been extensively studied by biochemical, clonal, and genetic analysis. Studies of human skin fibroblast cultures have revealed thatin vitro senescense is related toin vivo human cellular aging. Recently differentiated cells have been proposed for aging studies. These cells may provide additional information on aging since alterations ofin vitro cellular functions may be related to thein vivo behavior of specific differented cell types.     

Lack of FasL-mediated killing leads to in vivo tumor promotion in mouse Lewis lung cancer

Lewis lung carcinoma (3LL) cells were constitutively resistant to Fas-mediated apoptosis, but overexpression of Fas on 3LL cells allowed Fas-mediated apoptosis after crosslinking with agonist anti-Fas antibody (Jo2) in vitro. Surprisingly, Fas-overexpressing 3LL cells showed enhanced in vivo tumor progression, whereas no promotion of in vivo tumor growth was observed for dominant negative (DN) Fas-overexpressing 3LL transfectants in which the cytoplasmic death domain was deleted. In addition, the promotion of in vivo tumor growth by Fas-overexpression was reduced in gld (FasL-mutation) mice compared to normal mice. These data indicate that intact Fas/FasL cell signaling is required for the promotion of in vivo tumor growth by Fas overexpression in 3LL cells. In contrast to the efficient Fas-mediated killing induced in vitro by crosslinking with anti-Fas antibody, Fas-overexpressing 3LL cells were resistant in vitro to Fas-mediated apoptosis by activated T cells or transient FasL transfection. These data suggest that agonist anti-Fas antibody and natural FasL can transmit qualitatively different signals, and crosslinking of Fas with natural FasL on 3LL cells does not deliver the expected death signal. Thus, our results demonstrate that in some cases overexpression of Fas can result in a survival advantage for tumor cells in vivo.     

Suppressive effects of swainsonine on C6 glioma cell in vitro and in vivo

Swainsonine, an extract from Astragalus membranaceus, is known for its anti-cancer effects and could prevent metastases. In order to investigate the effects and mechanisms of swainsonine in C6 glioma cells, we carry out correlated experiments in vitro and in vivo. After treatment with swainsonine, the effective dose and IC₅₀ value of swainsonine in the C6 glioma cell were examined using the MTT assay. Cell cycle distribution and apoptotic rates were analyzed using FCM and [Ca²⁺]_i was measured by LSCM. Expressions of p16 and p53 protein were evaluated by immunocytochemical methods. Simultaneously, glioma-bearing rats were administered swainsonine at doses of 2, 4 and 8 mg/kg body wt. The inhibition rate was calculated and pathological sections were observed. The results indicated that the growth of C6 glioma cells is inhibited by swainsonine in vitro, with an IC₅₀ value within 24 h of 0.05 μg/ml. Increases in swainsonine correlate with S phase percentages of 11.3%, 11.6% and 12.4%, respectively. Moreover, the expression of apoptosis inhibiting p53 and p16 protein decreases gradually. Tumor weight in vivo decreased clearly and HE dyeing of tumor tissue showed gray, its texture was soft, with necrosis and hemorrhagic concentrated inward. Swainsonine could inhibit the proliferation of C6 glioma cells in vitro and the growth of C6 glioma in vivo. The mechanisms of swainsonine-induced apoptosis may relate with the expression of apoptosis-related genes and overloading-[Ca²⁺]_i-induced endoplasmic reticulum stress.     

Targeting EP4 downstream c‐Jun through ERK1/2‐mediated reduction of DNMT1 reveals novel mechanism of solamargine‐inhibited growth of lung cancer cells

Lung cancer is the most common cancer and the leading cause of cancer deaths worldwide. We previously showed that solamargine, one natural phytochemicals from traditional plants, inhibited the growth of lung cancer cells through inhibition of prostaglandin E2 (PGE₂) receptor EP4. However, the potential downstream effectors of EP4 involving in the anti‐lung cancer effects of solamargine still remained to be determined. In this study, we further verified that solamargine inhibited growth of non‐small‐cell lung cancer (NSCLC) cells in multiple cell lines. Mechanistically, solamargine increased phosphorylation of ERK1/2. Moreover, solamargine inhibited the protein expression of DNA methyltransferase 1 (DNMT1) and c‐Jun, which were abrogated in cells treated with MEK/ERK1/2 inhibitor (PD98059) and transfected with exogenously expressed DNMT1 gene, respectively. Interestingly, overexpressed DNMT1 gene antagonized the effect of solamargine on c‐Jun protein expression. Intriguingly, overexpressed c‐Jun blocked solamargine‐inhibited lung cancer cell growth, and feedback resisted the solamargine‐induced phosphorylation of ERK1/2. A nude mouse xenograft model implanted with lung cancer cells in vivo confirmed the results in vitro. Collectively, our results show that solamargine inhibits the growth of human lung cancer cells through reduction of EP4 protein expression, followed by increasing ERK1/2 phosphorylation. This results in decrease in DNMT1 and c‐Jun protein expressions. The inter‐correlations between EP4, DNMT1 and c‐Jun and feedback regulation of ERK1/2 by c‐Jun contribute to the overall responses of solamargine in this process. This study uncovers an additional novel mechanism by which solamargine inhibits growth of human lung cancer cells.     

Cell Cycle Defect in Connection with Oxygen and Iron Sensitivity in Fanconi Anemia Lymphoblastoid Cells

Fanconi anemia (FA) is an autosomal recessive disorder involving progressive pancytopenia, skeletal malformations, and a predisposition to leukemia. Thein vitrogrowth of FA fibroblasts is impaired, due to a defective G2 phase traverse of the cell cycle. Analyzing the cell cycle of lymphoid cell lines (LCLs) obtained from peripheral blood of FA patients by transformation with Epstein–Barr virus, we found a similar G2 phase defect, which was dependent upon the oxygen concentration. In addition, FA cells exhibited hypersensitivity towardcis-dichlorodiammineplatinum and mitomycin C, and moderate sensitivity towardtrans-dichlorodiammineplatinum. FA cells, however, showed no elevated sensitivity toward paraquat, an intracellular generator of superoxide radicals, or cumene hydroperoxide, a model organic peroxide. Chelating iron with low concentrations ofo-phenanthrolin improved cell proliferation and G2 phase transit of FA cells at 20% oxygen, but little at 5% oxygen. LCL cultures from healthy subjects were inhibited in their proliferation rate at all concentrations ofo-phenanthrolin. Exposure to excess iron, on the other hand, was very toxic to FA cells at 20%, but less toxic at 5% oxygen. In conclusion, the FA mutation leads to a cell cycle defect, which is expressed in cultures of lymphoid cells from FA patients, and involves hypersensitivity toward bifunctional alkylating agents, oxygen, and iron.     

The effect of conjugation on antitumor activity of vindoline derivatives with octaarginine,a cell‐penetrating peptide

Some Vinca alkaloids (eg, vinblastine, vincristine) have been widely used as antitumor drugs for a long time. Unfortunately, vindoline, a main alkaloid component of Catharanthus roseus (L.) G. Don, itself, has no antitumor activity. In our novel research program, we have prepared and identified new vindoline derivatives with moderate cytostatic activity. Here, we describe the effect of conjugation of vindoline derivative with oligoarginine (tetra‐, hexa‐, or octapeptides) cell‐penetrating peptides on the cytostatic activity in vitro and in vivo. Br‐Vindoline‐(l )‐Trp‐OH attached to the N‐terminus of octaarginine was the most effective compound in vitro on HL‐60 cell line. Analysis of the in vitro activity of two isomer conjugates (Br‐vindoline‐(l )‐Trp‐Arg₈ and Br‐vindoline‐(d )‐Trp‐Arg₈ suggests the covalent attachment of the vindoline derivatives to octaarginine increased the antitumor activity significantly against P388 and C26 tumour cells in vitro. The cytostatic effect was dependent on the presence and configuration of Trp in the conjugate as well as on the cell line studied. The configuration of Trp notably influenced the activity on C26 and P388 cells: conjugate with (l )‐Trp was more active than conjugate with the (d )‐isomer. In contrast, conjugates had very similar effect on both the HL‐60 and MDA‐MB‐231 cells. In preliminary experiments, conjugate Br‐vindoline‐(l )‐Trp‐Arg₈ exhibited some inhibitory effect on the tumor growth in P388 mouse leukemia tumor‐bearing mice. Our results indicate that the conjugation of modified vindoline could result in an effective compound even with in vivo antitumor activity.     

Stimulation of prostaglandin synthetase activity in rat granulosa cells by gonadotropins in vivo

The effect of $\text{in vivo}$ exposure to gonadotropin on prostaglandin synthetase activity in rat granulosa cells was examined in two experimental settings. The first setting was immature rats treated with pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). The second was mature rats on the day of proestrus. In the experiments using immature rats, the administration of hCG (20 I.U.) at noon of the second day after the PMSG (20 I.U.) injection led to large (more than 5 fold) increases in granulosa cell prostaglandin synthetase activity 5 and 10 h later. Follicular fluid PGE levels were also markedly increased at 5 and 10 h after hCG. Similar results were also found in experiments performed with mature proestrus rats. Granulosa cell prostaglandin synthetase activity was elevated at approximately 4 and 8 h after the endogenous LH surge (about 4 p.m. on proestrus), in comparison with the activity at midnight of diestrus, or noon and 4 p.m. on proestrus. In these experiments the changes in prostaglandin synthetase activity (10 fold) also paralleled the increases in follicular fluid PGE concentrations. Thus the exposure to gonadotropin $\text{in vivo}$ produced essentially the same effect as we had reported earlier for isolated granulosa cells incubated with LH $\text{in vitro}$. The stimulation of prostaglandin synthetase activity must therefore be ascribed an important role in the physiological regulation of granulosa cell prostaglandin synthesis by LH.     

Verdinexor,a Selective Inhibitor of Nuclear Exportin 1, Inhibits the Proliferation and Migration of Esophageal Cancer via XPO1/c-Myc/FOSL1 Axis

Esophageal carcinoma (EC) ranks sixth among cancers in mortality worldwide and effective drugs to reduce EC incidence and mortality are lacking. To explore potential anti-esophageal cancer drugs, we conducted drug screening and discovered that verdinexor, a selective inhibitor of nuclear exportin 1 (XPO1/CRM1), has anti-esophageal cancer effects both in vivo and in vitro. However, the mechanism and role of verdinexor in esophageal cancer remain unknown. In the present study, we observed that verdinexor inhibited the proliferation and migration of EC cells in vitro and suppressed tumor growth in vivo. Additionally, we found that verdinexor induced cleavage of PARP and downregulated XPO1, c-Myc, and FOSL1 expression. RNA-sequence analysis and protein-protein interaction (PPI) analysis revealed that verdinexor regulated the XPO1/c-Myc/FOSL1 axis. The results of immunoprecipitation and proximity ligation assays confirmed that verdinexor disrupted the interaction between XPO1 and c-Myc. Overexpression of c-Myc rescued the inhibition of cell proliferation and cell migration caused by verdinexor. Overexpressed FOSL1 restored the inhibited migration by verdinexor. Taken together, verdinexor inhibited cell proliferation and migration of esophageal cancer via XPO1/c-Myc/FOSL1 axis. Our findings provide a new option for the development of anti-esophageal cancer drugs.     

A comparative study of cytotoxic effects of N-ethyl-N-nitrosourea,adriamycin, and mono-(2-ethylhexyl)phthalate on mouse primordial germ cells

Several strategies for the assessment of reproductive toxicity of chemical compounds has have been proposed. In the present work, we devised experimental in vitro assays to test the effect of potential toxicants on proliferating primordial germ cells (PGCs) in vitro using recently developed methods for isolation and culture of mouse PGCs. Primordial germ cells are the embryonic precursors of gametes of the adult that carry the genome from generation to generation. Any damage or mutations caused to these cells by potential toxicants might impair normal reproduction and be transmitted to next generation. Three representative compounds, N-ethyl-N-nitrosourea (ENU), adriamycin (ADR), and mono-(2-ethylhexyl)phthalate (MEHP), toxic to different targets and known to affect germ cell development and impair fertility, were tested on PGCs in culture using three different experimental protocols. Survival and growth of PGCs and their ability to adhere to cell monolayers, were taken as endpoints for drug effects. For each compound, sublethal and acute toxicity doses were determined. In addition, information about the mechanisms of action of these compounds on PGCs was obtained. Whereas the effects of ENU and ADR on PGCs were attributable to growth inhibition and apoptosis induction, MEHP affected PGC adhesion to somatic cells without significantly altering their growth and survival. The results of our in vitro tests were not always exactly predictive of the effects of the tested compounds on PGCs in vivo, determined in parallel experiments in which pregnant mice were exposed to the same compounds. Nevertheless, they can provide information on the sensitivity of PGCs to the direct action of drugs or the mechanisms of action of such agents. This revised version was published online in August 2006 with corrections to the Cover Date.     

Anti-Angiogenic Effects of Conjugated Docosahexaenoic Acid in Vitro and in Vivo

The anti-angiogenic effects of conjugated docosahexaenoic acid (CDHA), which was prepared by an alkaline treatment of docosahexaenoic acid and contained conjugated double bonds, were investigated in vitro and in vivo. CDHA inhibited tube formation by the bovine aortic endothelial cell (BAEC), and also inhibited the proliferation of BAEC at a concentration of CDHA that suppressed tube formation, but did not influence cell migration. The inhibition of BAEC growth caused by CDHA was accompanied by a marked change in cellular morphology. Nuclear condensation and brightness were observed in Hoechst 33342-stained cells treated with CDHA, indicating that CDHA induced apoptosis in BAEC. We also evaluated the angiogenesis inhibition of CDHA in vivo. The vessel formation which was triggered by tumor cells was clearly suppressed in mice orally given CDHA. Our findings suggest that CDHA has potential use as a therapeutic dietary supplement for minimizing tumor angiogenesis.