Targeting colon cancer stem cells using a new curcumin analogue, GO-Y030.

BACKGROUND: Persistent activation of signal transducers and activators of transcription 3 (STAT3) is commonly detected in many types of cancer, including colon cancer. To date, whether STAT3 is activated and the effects of STAT3 inhibition by a newly developed curcumin analogue, GO-Y030, in colon cancer stem cells are still unknown. METHODS: Flow cytometry was used to isolate colon cancer stem cells, which are characterised by both aldehyde dehydrogenase (ALDH)-positive and CD133-positive subpopulations (ALDH(+)/CD133(+)). The levels of STAT3 phosphorylation and the effects of STAT3 inhibition by a newly developed curcumin analogue, GO-Y030, that targets STAT3 in colon cancer stem cells were examined. RESULTS: Our results observed that ALDH(+)/CD133(+) colon cancer cells expressed higher levels of phosphorylated STAT3 than ALDH-negative/CD133-negative colon cancer cells, suggesting that STAT3 is activated in colon cancer stem cells. GO-Y030 and curcumin inhibited STAT3 phosphorylation, cell viability, tumoursphere formation in colon cancer stem cells. GO-Y030 also reduced STAT3 downstream target gene expression and induced apoptosis in colon cancer stem cells. Furthermore, GO-Y030 suppressed tumour growth of cancer stem cells from both SW480 and HCT-116 colon cancer cell lines in the mouse model. CONCLUSION: Our results indicate that STAT3 is a novel therapeutic target in colon cancer stem cells, and inhibition of activated STAT3 in cancer stem cells by GO-Y030 may offer an effective treatment for colorectal cancer.

Activation of phosphatidylinositol 3-kinase/protein kinase B pathway by a vanadyl compound mediates its neuroprotective effect in mouse brain ischemia.

We previously reported that orthovanadate composed of vanadate (V(5+)) activates phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling through inhibition of protein tyrosine phosphatases, thereby eliciting neuroprotection in brain ischemia/reperfusion injury. However, therapeutic doses of orthovanadate are associated with diarrhea due to inhibition of ATPase. By contrast, vanadyl (V(4+)) organic compounds show low cytotoxicity. Since both vanadate and vanadyl inhibit protein tyrosine phosphatases, we tested whether bis(1-oxy-2-pyridinethiolato)oxovanadium(IV) [VO(OPT)] in a vanadyl form elicits a neuroprotection in brain ischemia. In a mouse transient middle cerebral artery occlusion (MCAO) model, pre- and post-treatments with VO(OPT) significantly reduced infarct volume in a dose-dependent manner. Like orthovanadate, activation of the PI3K/Akt pathway mediated neuroprotective action. VO(OPT) treatment inhibited reduced Akt phosphorylation at Ser-473 following brain ischemia and restored decreased phosphorylation of forkhead box class O (FOXO) family members such as FKHR, FKHRL1, and AFX. Consistent with inhibition of FOXO dephosphorylation, VO(OPT) treatment blocked elevated expression of Fas-ligand, Bim and active caspase-3 24 h after ischemia/reperfusion. Taken together, a vanadyl compound, VO(OPT) elicits neuroprotective effects on brain ischemia/reperfusion injury without apparent side effects.

Clinical features and alterations in the inferior horn sizes in lateral ventricle in Alzheimer's patients with different ApoE genotype in Japanese population.

E4 allele of Apolipoprotein E (ApoE) is considered to be not only a risk factor for Alzheimer's disease (AD) but also a determinant of clinical features in AD. However, it is still controversial whether ApoE e4 allele is related to age at onset, severity of memory impairment or brain morphological changes in AD patients. The present study examined the issue in Japanese population: 1) ApoE genotype on in 38 normal controls and 32 AD patients; 2) association between e4 allele of ApoE and clinical features including Wechsler Memory Scale-Revised in 32 AD patients; and 3) association between e4 allele of ApoE and change in size of inferior horn in lateral ventricle (LV) in 13 out of 32 AD patients. The e4 allele of ApoE frequency was higher in AD patients than in normal controls. There was no significant difference in age at onset or neuropsychological results between AD with and without e4 allele of ApoE. Alteration per month of the inferior horn sizes in LV measured by MRI was similar in the AD patients with and without e4 allele of ApoE. These results suggest that e4 allele of ApoE is a risk factor but not a determinant of clinical features for AD in Japanese population.

A concise enantioselective synthesis of a key A-ring synthon for 1 alpha-hydroxyvitamin D3 compounds.

[figure: see text] This report describes a concise enantioselective synthesis of the A-ring synthon for the synthesis of 1 alpha-hydroxyvitamin D3 compounds. The synthesis involves two notable transformations: (I) stereoselective construction of the enol triflate from the vinyl ketone by Michael addition of Ph2P(O)Li followed by in situ triflation of the resulting enolate and (II) palladium-catalyzed Heck type cyclization of the enol triflate.

Synthesis and evaluation of A-ring diastereomers of 1alpha,25-dihydroxy-22-oxavitamin D3 (OCT).

A-ring diastereomers of 1alpha,25-dihydroxy-22-oxavitamin D3 (OCT) (2), 3-epi-1alpha,25-dihydroxy-22-oxavitamin D3 (3-epiOCT) (3) and 1,3-diepi-1alpha,25-dihydroxy-22-oxavitamin D3 (1,3-diepiOCT) (4) were synthesized by the convergent method. In vitro binding affinity for rat vitamin D binding protein and calf-thymus vitamin D receptor, differentiation-inducing activity on HL-60 cells, and transcriptional activity of 3-epiOCT (3) and 1,3-diepiOCT (4) were evaluated in comparison with OCT (2), 1-epi-1alpha,25-dihydroxy-22-oxavitamin D3 (1-epiOCT) (5) and 1alpha,25-dihydroxyvitamin D3 (1).

A concise enantioselective synthesis of antimalarial febrifugine alkaloids.

Reaction of (S)-2-(tert-butyldiphenylsilyloxy)-5-(mesyloxy)pentanal with hydroxylamine in allyl alcohol brought about simultaneous 1,3-dipolar cycloaddition of the resulting nitrone to allyl alcohol to give three diastereoisomeric adducts, from which (+)-febrifugine and (+)-isofebrifugine, potent antimalarial alkaloids, were synthesized.

Synthesis and biological characterization of 1alpha,24,25-trihydroxy-2beta-(3-hydroxypropoxy)vitamin D(3) (24-hydroxylated ED-71).

24-Hydroxylated derivatives were synthesized in 24(R) and 24(S) forms by the convergent method as analogs related to 1alpha,25-dihydroxy-2beta-(3-hydroxypropoxy)vitamin D(3). In the convergent synthesis, the A-ring fragment, synthesized from diethyl D-tartarate, and the C/D-ring fragments in 24(R) and 24(S) forms (vitamin D numbering), obtained from vitamin D(2) via the Inhoffen-Lythgoe diol, were coupled in moderate yields to give 1alpha,24(R),25-trihydroxy-2beta-(3-hydroxypropoxy)vitamin D(3) and 1alpha,24(S),25-trihydroxy-2beta-(3-hydroxypropoxy)vitamin D(3). In preliminary biological evaluations, 24-hydroxylation of 1alpha,25-dihydroxy-2beta-(3-hydroxypropoxy)vitamin D(3) caused weakened affinity to vitamin D binding protein in vitro and less calcemic activity in vivo compared to the parent compound. While the affinity to vitamin D receptor in 24(R) epimer was sustained, the affinity in 24(S) epimer was less than that of the parent compound.

An enantio- and stereocontrolled synthesis of (-)-mycestericin E via cinchona alkaloid-catalyzed asymmetric Baylis-Hillman reaction.

A new enantiocontrolled synthesis of a potent immunosuppressant(-)-mycestericin E has been accomplished by using cinchona alkaloid-catalyzed asymmetric Baylis-Hillman reaction of an aldehyde with 1,1,1,3,3,3-hexafluoroisopropyl acrylate and Lewis acid-promoted cyclisation of an epoxytrichloroacetimidate as the key steps.

Characterization of new conjugated metabolites in bile of rats administered 24,25-dihydroxyvitamin D(3) and 25-hydroxyvitamin D(3).

The characterization of new conjugated vitamin D metabolites in rat bile was performed using HPLC, liquid chromatography/tandem mass spectrometry combined derivatization, and GC-MS. After the administration of 24,25-dihydroxyvitamin D(3) to rats, 23, 25-dihydroxy-24-oxovitamin D(3) 23-glucuronide, 3-epi-24, 25-dihydroxyvitamin D(3) 24-glucuronide, and 24,25-dihydroxyvitamin D(3) 3-sulfate were obtained as new biliary metabolites together with 24,25-dihydroxyvitamin D(3) 3- and 24-glucuronides. The above metabolites, except 24,25-dihydroxyvitamin D(3) 3-glucuronide, were obtained from rats dosed with 25-hydroxyvitamin D(3). 23, 25-Dihydroxyvitamin D(3) 23-glucuronide was also obtained from the bile of rats administered 25-hydroxyvitamin D(3) in addition to its 3-glucuronide, 25-glucuronide, and 3-sulfate. Thus, it was found that 24,25-dihydroxyvitamin D(3) and 25-hydroxyvitamin D(3) were directly conjugated as glucuronide and sulfate, whereas at the C-23 position, they were hydroxylated and then conjugated. Furthermore, we found that the C-3 epimerization acts as one of the important pathways in vitamin D metabolism.

Lack of topographical specificity in peripheral nerve regeneration in rats.

In a previous study we found that sensory regeneration was neurotropically selective regardless of the end organ, but motor regeneration was not, which made us doubt the existence of topographic specificity. The purpose of the present study was to confirm the existence of topographic specificity in rats. The proximal stump of either the peroneal or tibial nerve was inserted into the proximal limb of a silicone Y-chamber. Both distal stumps of peroneal and tibial nerve were inserted into the distal limbs. The gap between the stumps was set at either 4 mm (n = 8, on each subgroup) or 8 mm (n = 8, on each subgroup). Six weeks later the number of regenerated axons in the distal two limbs were counted and compared. The number of regenerated axons towards the distal tibial nerve side was significantly larger in every model. Regenerated axons from the proximal peroneal stump did not preferentially choose the distal peroneal stump. The existence of topographic specificity is unlikely.

New metabolic pathway of (24R)-24,25-dihydroxyvitamin D3: epimerization of the 3-hydroxy group.

A new metabolic pathway of (24R)-24,25-dihydroxyvitamin D3 [24,25(OH)2D3] was clarified in the in vivo experiments. After the administration of 24,25(OH)2D3 to rats, a new monoglucuronide of a vitamin D metabolite was obtained from the bile together with 24,25(OH)2D3 3- and 24-glucuronides. The genin of the metabolite was identified as 3-epi-24,25(OH)2D3 in comparison with the synthetic sample based on the data from 1H-NMR, GC/MS, and LC/atmospheric pressure chemical ionization-MS. The conjugation position was determined to be the 24-hydroxy group by the LC/electrospray ionization-MS and -MS/MS/MS combined with derivatization. To our knowledge, this is the first reported instance of the epimerization of the 3-hydroxy group of vitamin D compound with no hydroxy group at the 1alpha-position.

Hic-5, a paxillin homologue, binds to the protein-tyrosine phosphatase PEST (PTP-PEST) through its LIM 3 domain.

The Hic-5 protein is encoded by a transforming growth factor-beta1- and hydrogen peroxide-inducible gene, hic-5, and has striking similarity to paxillin, especially in their C-terminal LIM domains. Like paxillin, Hic-5 is localized in focal adhesion plaques in association with focal adhesion kinase in cultured fibroblasts. We carried out yeast two-hybrid screening to identify cellular factors that form a complex with Hic-5 using its LIM domains as a bait, and we identified a cytoplasmic tyrosine phosphatase (PTP-PEST) as one of the partners of Hic-5. These two proteins are associated in mammalian cells. From in vitro binding experiments using deletion and point mutations, it was demonstrated that the essential domain in Hic-5 for the binding was LIM 3. As for PTP-PEST, one of the five proline-rich sequences found on PTP-PEST, Pro-2, was identified as the binding site for Hic-5 in in vitro binding assays. Paxillin also binds to the Pro-2 domain of PTP-PEST. In conclusion, Hic-5 may participate in the regulation of signaling cascade through its interaction with distinct tyrosine kinases and phosphatases.

Interlaboratory validation of the in vitro eye irritation tests for cosmetic ingredients. (3) Evaluation of the haemolysis test.

The haemolysis test using sheep red blood cells (RBC) was evaluated as an alternative method to the Draize rabbit eye irritation test (Draize test) by six to nine laboratories. The participating laboratories performed the test according to the standard operating procedure (SOP). Thirty-eight cosmetic ingredients and isotonic sodium chloride solution were used as test substances in this validation study. The concentrations of the test substances that induced 50% haemolysis (HC(50) value) was obtained to serve as a toxicological index and compared with in vivo Draize scores. HC(50) values were not obtained for coloured or water-insoluble (turbid) substances. Three acids caused denaturation of haemoglobin leaked from RBC and consequently interfered with the determination of the HC(50) value. Interlaboratory reproducibility was relatively good except in the case of water-insoluble substances. The average values of coefficient of variation (CV) was 37%. The correlation coefficient and Spearman's rank correlation between the HC(50) value and maximum average Draize total score (MAS) were -0.631 and 0.641, respectively. The equivalence ratio between the haemolysis test and MAS was 70.0% when MAS 15 was set as the in vivo cut-off point. On the other hand, strong irritants (MAS50) could be correctly classified by this method. These results suggest that the haemolysis test might be applied to cosmetic ingredients as a screening method to distinguish strong irritants that directly affect the cell membrane permeability and do not disturb spectrophotometrical determination of haemoglobin. In order to evaluate the potential for eye irritation of cosmetic ingredients, a combination of haemolysis with other methods based on different mechanism should be employed to improve the predictability.

Interlaboratory Validation of the in vitro Eye Irritation Tests for Cosmetic Ingredients. (4) Haemoglobin Denaturation Test.

Interlaboratory validation of the haemoglobin denaturation (HD) test on 38 cosmetic ingredients was conducted by five to eight participating laboratories. The HD test was evaluated as an alternative method to the Draize eye irritation test (Draize test) based on three indices of protein denaturation: the test substance concentration that induces 50% HD of the positive control (RDC(50)), a relative HD rate at 1% of the test substance (1%RDR) and a relative change in maximum absorption wavelength (1% lambdamax). The coefficients of variation associated with a positive HD test among the participating laboratories were within an acceptable range for practical application. The in vitro test results were in relatively good agreement with the Draize test. The correlation coefficient (r) between the in vivo maximal average Draize total score (MAS) and log (RDC(50)), 1%RDR and 1% lambdamax were -0.91, 0.67 and 0.79, respectively. The results revealed several limitations associated with the HD test: (1) the HD test cannot be applied to coloured test substances with a strong absorption, around 418nm; (2) water-insoluble test substances cannot be evaluated by RDC(50) or 1%RDR; (3) the HD test cannot be applied to strong acids that exceed the buffering capacity of a phosphate buffer solution; (4) the HD test cannot be used to determine the potential for eye irritation caused by factors other than protein denaturation, for example, polyoxyethylene octylphenylether (10 E.O.). Thus, the HD test alone is not appropriate for predicting eye irritation potential. Nevertheless, the good agreement between the HD test results and in vivo irritation scores as well as the ease of application suggest that this test may play an important role in a test system to determine eye irritation potential.

Malignant melanoma extending along the ulnar, median, and musculocutaneous nerves: a case report.

We analyzed a case of malignant melanoma that resembled malignant peripheral nerve sheath tumor with marked neurotropism. The subungual tumor in the right ring finger extended along the ulnar nerve for a distance of 30 cm, as well as along the median and musculocutaneous nerves, with lymph nodal metastases. The tumor consisted of interlacing spindle-shaped cells with large nuclei and distinct nucleolei. Immunohistochemically, the tumor cells were diffusely positive for S-100 protein. Five years after forequarter amputation, the patient is alive without disease. Malignant melanoma has the potential of invading several major peripheral nerves and must be distinguished from malignant peripheral nerve sheath tumor, which rarely metastasizes to regional lymph nodes.

Histological and ultrastructural studies of the effects of tachykinins on protein secretion from the lingual epithelium and the lingual gland of the Tokyo daruma pond frog (Rana porosa porosa).

Four tachykinins were each administered at 20 micrograms/kg body wt. All the tachykinins had a positive effect on granular secretion from the lingual epithelium, and the loss of cytoplasm from cells of the lingual epithelium was greatest with physalaemin and eledoisin, moderate with neurokinin A, and smallest with substance P. These reactions were very different from those in mammals, in which tachykinins induce only watery secretions. Only physalaemin had a positive effect on protein secretion from the lingual gland. Transmission electron microscopy revealed that electron-dense granules in cells of the lingual epithelium were discharged during stimulation with physalaemin and eledoisin by typical exocytosis. Discharge of these granules was indistinct after the administration of substance P and of neurokinin A. Exocytosis of electron-dense granules from cells of the lingual gland was clearly detectable by electron microscopy after the administration of physalaemin, reflecting observations made by light microscopy. However, mucous granules in the lingual gland were secreted by an exocrine process only after administration of physalaemin.

Evolving catalytic antibodies in a phage-displayed combinatorial library.

In vitro affinity maturation for evolving catalytic antibodies has been demonstrated by generating a diverse repertoire of the appropriate complementarity-determining regions on a phage surface. Phage display is followed by a selection based on binding to an altered antigen that was not used at the time of immunization, and provides variants with new catalytic activity and substrate specificity. This library format reduces the time needed to isolate the desired catalytic antibody fragments to under 2 weeks.

Effects of calcitonin and calcitonin gene-related peptide on the substance P-mediated secretion of fluid from the rat submandibular gland.

1. The effects of rat calcitonin gene-related peptide (rCGRP), rat calcitonin (rCT) and salmon calcitonin (sCT) on the substance P-mediated (SP-mediated) secretion of fluid from the rat submandibular gland were investigated. 2. Rat CGRP potentiated and prolonged the SP-mediated secretion of fluid from the rat submandibular gland in a dose-dependent manner. CGRP also enhanced methacholine- and phenylephrine-mediated secretion of fluid. 3. The potentiating effect of the combination of rCGRP and SP was somewhat reduced by pretreatment with spantide, human CGRP8-37 and atropine but not by pretreatment with phentolamine or with propranolol. 4. Salmon CT at a low dose mimicked the effect of rCGRP on the SP-mediated secretion of fluid, but its potency was lower than that of rCGRP. However, rCT had no effect on the SP-mediated secretion of fluid. 5. These results suggest that the potentiating effects of rCGRP and SP might involve cholinergic receptors, as well as CGRP and tachykinin receptors, and that sCT, but not rCT, is able to mimic rCGRP in potentiating the secretion of fluid induced by SP.

Interaction between substance P and beta-adrenergic agonists in the modulation of the secretion of fluid and protein by the rat submandibular gland.

The interactions between substance P and beta-adrenergic agonists such as isoprenaline, dobutamine and terbutaline in the control of the secretion of fluid and protein from the rat submandibular gland have been examined. Substance P elicited large volumes of saliva whereas isoprenaline, dobutamine and terbutaline elicited small volumes only. The secretion of fluid in response to substance P was markedly enhanced when substance P was administered in combination with isoprenaline or dobutamine but not when it was administered in combination with terbutaline. Isoprenaline elicited large amounts of protein, whereas substance P elicited small amounts. The secretion of protein in response to isoprenaline did not change when isoprenaline was administered in combination with substance P. The secretion of fluid and protein induced by substance P in combination with isoprenaline was antagonized by metoprolol and by spantide, but it was unaffected by pretreatment with ICI118551. These results suggest that in the rat submandibular gland stimulation of beta1-adrenoceptors but not of beta2-adrenoceptors potentiates the secretion of fluid that is induced by stimulation of tachykinin receptors, whereas stimulation of tachykinin receptors does not enhance the secretion of protein that is induced by stimulation of beta1-adrenoceptors.