The inhibitory effects of capillarisin on cell proliferation and invasion of prostate carcinoma cells

Objectives

Capillarisin (Cap), an active component of Artemisia capillaris root extracts, is characterized by its anti‐inflammatory, anti‐oxidant and anti‐cancer properties. Nevertheless, the functions of Cap in prostate cancer have not been fully explored. We evaluated the potential actions of Cap on the cell proliferation, migration and invasion of prostate carcinoma cells.

Materials and methods

Cell proliferation and cell cycle distribution were measured by water‐soluble tetrazolium‐1 and flow cytometry assays. The expression of cyclins, p21, p27, survivin, matrix metallopeptidase (MMP2 and MMP9) were assessed by immunoblotting assays. Effects of Cap on invasion and migration were determined by wound closure and matrigel transmigration assays. The constitutive and interlukin‐6 (IL‐6)‐inducible STAT3 activation of prostate carcinoma cells were determined by immunoblotting and reporter assays.

Results

Capillarisin inhibited androgen‐independent DU145 and androgen‐dependent LNCaP cell growth through the induction of cell cycle arrest at the G0/G1 phase by upregulating p21 and p27 while downregulating expression of cyclin D1, cyclin A and cyclin B. Cap decreased protein expression of survivin, MMP‐2, and MMP‐9 and therefore blocked the migration and invasion of DU145 cells. Cap suppressed constitutive and IL‐6‐inducible STAT3 activation in DU145 and LNCaP cells.

Conclusions

Our data indicate that Cap blocked cell growth by modulation of p21, p27 and cyclins. The inhibitory effects of Cap on survivin, MMP‐2, MMP‐9 and STAT3 activation may account for the suppression of invasion in prostate carcinoma cells. Our data suggest that Cap might be a therapeutic agent in treating advanced prostate cancer with constitutive STAT3 or IL‐6‐inducible STAT3 activation.

     

Cytogenetic rearrangements involving the loss of the Sonic Hedgehog gene at 7q36 cause holoprosencephaly

Holoprosencephaly (HPE) is a genetically heterogeneous disorder that affects the midline development of the forebrain and midface in humans. As a step toward identifying one of the HPE genes, we have set out to refine the HPE3 critical region on human chromosome 7q36 by analyzing 34 cell lines from families with cytogenetic abnormalities involving 7q, 24 of which are associated with HPE. Genomic clones surrounding the DNA marker D7S104, which has previously been shown to be in the HPE3 critical region, have been examined by fluorescent in situ hybridization and microsatellite analysis of our panel of patient cell lines. We report the analysis of a cluster of four translocation breakpoints within a 300-kb region of 7q36 that serves to define the minimal critical region for HPE3 and that has directed the search for candidate genes. The human Sonic Hedgehog (hSHH) gene maps to this region and has been shown to be HPE3 on the basis of mutations within the coding region of the gene. We present evidence that cytogenetic deletions and/or rearrangements of this region of chromosome 7q containing Sonic Hedgehog, and translocations that may suppress Sonic Hedgehog gene expression through a position effect are common mechanisms leading to HPE. Received: 23 December 1996 / Accepted: 17 March 1997     

Effect of different salinities on the coniform chloride cells of mayfly larvae

The larvae of Callibaetis coloradensis can tolerate a fairly wide range of salinities at hypotonic concentrations. However, they are more sensitive to increasing than to decreasing salt concentrations. Exposure to isotonic concentration results in profound degenerations of the chloride cells within 1 day. Long-term adaptation to diluted fresh water causes a significant increase in the number of chloride cells, whereas the gradual concentration of fresh water to finally 120 mM sodium chloride within a period of 15 days leads to approximately 50 per cent mortality and significantly reduces the number of chloride cells in the survivors. The same reciprocal relation between the number of chloride cells and the external salinity was found in larvae of C. floridans collected from fresh- and brackish-water habitats. These results suggest that the adaptive behaviour of the chloride cells is correlated with the osmoregulatory situation and enables these animals to live in habitats of different salinities.     

The Relationship between the Effects of Cytokinin on the Expension and Metabolism of Excised Cucumber Cotyledons and Water Stress

Excised cucumber (Cucumis sativus, var. Jin-ian No. 4) cotyledons were incubated with 10 ppm of BA (benzyladenine) or water for 1 h, then thouroughly rinsed with water and grown in darkness on filter paper saturated with different concentrations of mannitol solution. Up to 24 h, the fresh weight, carotenoid, RNA, DNA and lipid contents of cotyledons were determined. Although mannitol solution reduced the effectiveness of BA treatment, in the same condition of osmotic potential, the increases of fresh weight, carotenoid, RNA and DNA contents, as well as the decrease of lipid per cotyledon were always much higher in BA treated tissues. BA enhanced the rate of water uptake by the cotyledons. The fresh weight of BA and 0.2 M mannitol treated cotyledons was equal to that of water control, but the increases of carotenoid and nucleic acid contents and the decrease of lipid were much higher in tho former than the latter. 0.3 M and 0.5 M mannitol solu- tions almost interrupted the water uptake of water and BA treated cotyledons respectively. However, the increases of carotenoid and nucleic acid contents as well as decrease of lipid were still occurred in these conditions. The different osmotic potential did nearly not affect the ratio of the increases of carotenoid and nucleic acid contents between BA treatment and control. It means the effectiveness of BA was almost the same under different osmotic potential It is evident that BA stimulated simultaneously the water uptake and metabolism of the cotyledons. They are probably different processes but closely related to each other.     

The Effects of Cytokinins on Unfolding of Cotyledons and Opening of Hypocotylar Hooks of Cucumber

Benzyladenine (BA), zeatin and kinetin caused the unfolding of cucumber (Cucumis sativus L. Var. Jing-ye No. 4) cotyledons. But the other plant hormones e.g. gibberellic acid (GA3), indoleacetic acid(IAA), ethylene (ethrel), abscisic acid (ABA) and other related compound e.g. KCl, adenine were not so. This response was caused by even lower concentration (0.01 ppm). Therefore, it could be used as a bioassay for cytokinins. KCl affaeted synergistically on cytokinin-induced unfolding. Basally applied BA (10 ppm) stimulated the opening of the intact hypocotylar hook. The light affacted synergistically on BA-induced opening. However, basally applied BA prevented the opening of decapitated hooks, especially in the light. The cotyledons were found to play a part of effects in maintaining the hook.     

The Effect of Actinomycin D and Cycloheximide on the Dedifferentiation of Cotyledon in Phaseolus radiatus L. and Its Nucleic Acid and Protein

Calli were induced from cotyledon segment of mung bean (Phaseolus radiatus L.) in Miller medium supplemented with NAA 4 mg/l, kinetin 10 mg/L. The callus formation was completely prevented by the addition of actinomycin D 15 μg/mL or cyclo- heximidc 0.5 μg/mL at 0 hour. The inhibitory effect of actinomycin D or cycloheximide was increased with the increment of concentration but decreased when the inhibitory agents were added a few hours later. If actinomycin D or cycloheximide was added at 24 hour culture it inhibits neither the induction of callus formation nor the proliferation. The content of RNA, DNA and protein were determined. RNA in each segment increased obviously in the early stage of callus formation, but DNA and protein increased slightly afterward. It is suggested that a large increase of RNA is the characteristic of dedifferentiation of cotyledon in P. radiatus. In addition, it has also been shown that an actinomycin D or cycloheximide-sensitive process in the early stage of dedifferentiation is crucial for the callus formation. Both RNA and protein synthesis are required for the initiation of dedifferentiation.     

Suppression and synthetic‐lethal genetic relationships of ΔgpsB mutations indicate that GpsB mediates protein phosphorylation and penicillin‐binding protein interactions in Streptococcus pneumoniae D39

GpsB regulatory protein and StkP protein kinase have been proposed as molecular switches that balance septal and peripheral (side‐wall like) peptidoglycan (PG) synthesis in Streptococcus pneumoniae (pneumococcus); yet, mechanisms of this switching remain unknown. We report that ΔdivIVA mutations are not epistatic to ΔgpsB division‐protein mutations in progenitor D39 and related genetic backgrounds; nor is GpsB required for StkP localization or FDAA labeling at septal division rings. However, we confirm that reduction of GpsB amount leads to decreased protein phosphorylation by StkP and report that the essentiality of ΔgpsB mutations is suppressed by inactivation of PhpP protein phosphatase, which concomitantly restores protein phosphorylation levels. ΔgpsB mutations are also suppressed by other classes of mutations, including one that eliminates protein phosphorylation and may alter division. Moreover, ΔgpsB mutations are synthetically lethal with Δpbp1a, but not Δpbp2a or Δpbp1b mutations, suggesting GpsB activation of PBP2a activity. Consistent with this result, co‐IP experiments showed that GpsB complexes with EzrA, StkP, PBP2a, PBP2b and MreC in pneumococcal cells. Furthermore, depletion of GpsB prevents PBP2x migration to septal centers. These results support a model in which GpsB negatively regulates peripheral PG synthesis by PBP2b and positively regulates septal ring closure through its interactions with StkP‐PBP2x.     

The TGF-β inhibitory activity of antibody 37E1B5 depends on its H-CDR2 glycan

Excessive transforming growth factor (TGF)-β is associated with pro-fibrotic responses in lung disease, yet it also plays essential roles in tissue homeostasis and autoimmunity. Therefore, selective inhibition of excessive and aberrant integrin-mediated TGF-β activation via targeting the α-v family of integrins is being pursued as a therapeutic strategy for chronic lung diseases, to mitigate any potential safety concerns with global TGF-β inhibition. In this work, we reveal a novel mechanism of inhibiting TGF-β activation utilized by an αvβ8 targeting antibody, 37E1B5. This antibody blocks TGF-β activation while not inhibiting cell adhesion. We show that an N-linked complex-type Fab glycan in H-CDR2 of 37E1B5 is directly involved in the inhibition of latent TGF-β activation. Removal of the Fab N-glycosylation site by single amino acid substitution, or removal of N-linked glycans by enzymatic digestion, drastically reduced the antibody's ability to inhibit latency-associated peptide (LAP) and αvβ8 association, and TGF-β activation in an αvβ8-mediated TGF-β signaling reporter assay. Our results indicate a non-competitive, allosteric inhibition of 37E1B5 on αvβ8-mediated TGF-β activation. This unique, H-CDR2 glycan-mediated mechanism may account for the potent but tolerable TGF-b activation inhibition and lack of an effect on cellular adhesion by the antibody.     

Assessment of Phytotoxicity of Chromium in Flooded Soils using Embedded Selective Ion Exchange Resin Method

Chromium present in the forms of Cr(VI) or Cr(III) in soils. Since the toxicity and mobility of Cr(VI) are higher than those of Cr(III), it would be important to estimate soil Cr(VI) accurately in order to assess the phytotoxicity of Cr. Soil redox potential can influence the distribution of Cr between Cr(VI) and Cr(III) forms, and thus an in situ method which is not affected by the soil redox condition is needed for determining Cr(VI) availability in paddy fields. In this study, the Cu-saturated selective ion exchange resin (DOWEX M4159), serving as an infinite sink, was embedded in soils to extract available Cr(VI) from three representative saturated soils with different amounts of Cr(VI). The results suggested that Cr(VI) reduction occurred in the flooded soils, and the acid environment favored the adsorption and reduction of Cr(VI). There was a significant dose-response relationship between the soil resin-extractable Cr(VI) and the plant height of rice seedlings for test soils. The experimental results suggested that the embedded selective ion exchange resin method could be a suitable in situ method for assessing the phytotoxicity of Cr in flooded soils.