Evaluating bio-physicochemical properties of raw powder prepared from whole larvae containing liquid silk of the domestic silkworm.

The domestic silkworm, Bombyx mori, has been widely used in silk production for centuries. It is also used as a bioreactor by the textile and pharmaceutical industries to mass produce recombinant bioactive proteins containing silk-based materials. Furthermore, silkworms are well-known as a source of food and have also been orally administered to prevent and treat several human disorders. In this study, we aimed to investigate the inherent bio-physicochemical properties of edible silkworms to accurately evaluate their clinical and nutritional potential. We prepared raw powder from whole larvae of silkworm. The yield rate of the powder derived from dried larvae was almost 100% (98.1-99.1% in replicates). As "percentage yield" translates to "Budomari" in Japanese, this raw powder was named "B100rw." We further prepared B100dn that was denatured through autoclaving. Thereafter, we examined whether B100rw sustained the original bio-physicochemical properties by comparing it with B100dn. There was no significant difference in nutritional content between B100rw and B100dn. B100rw contained proteins derived from silkworm larvae and mulberry leaves, whereas the proteins of B100dn were mostly degraded. On measuring the enzymatic activity of both powders using trehalase as an indicator enzyme, B100rw was found to maintain trehalase activity. B100rw also maintained a random coil conformation, similar to that of liquid silk. This suggested that B100rw sustained the unique bio-physicochemical properties of living larvae. These findings may facilitate the development of novel food products or orally administered vaccines.

Semisynthesis of Antitrypanosomal p-Quinone Analog Possesing the Komaroviquinone Pharmacophore.

A p-quinone analog having the komaroviquinone pharmacophore fused with a more conformationally flexible cycloheptane ring, was semisynthesized from natural demethlsalvicanol isolated from Perovskia abrotanoides via four steps in 26% overall yield. The IC50 for the antitrypanosomal activity of the analog was 0.55 µM.

Acyl flavonoids, biflavones, and flavonoids from Cephalotaxus harringtonia var. nana.

A methanol extract of the leaves of Cephalotaxus harringtonia var. nana and its ethyl acetate (EtOAc)-soluble fraction demonstrated strong antitumor activity against A549 and HT-29 cell lines. The EtOAc-soluble fraction was purified by column chromatography and high-performance liquid chromatography (HPLC) using a reverse-phase column to yield three novel acyl flavonoids and a biflavonoid, along with 15 other known compounds that included flavonoids, biflavonoids, and other phenolics. The structures of the new compounds were elucidated using spectral data from HR-MS and NMR, including two-dimensional NMR studies, as (2R,3R)-3-O-eicosanoyltaxifolin (1), (2R,3R)-3-O-docosanoyltaxifolin (2), (2R,3R)-3-O-tetracosanoyltaxifolin (3), and 6-methyl-4',7,7″-tri-O-methylamentoflavone (4). The isolated compounds, including the known compounds, were tested for possible antitumor activity; some of the biflavones were found to be active. The potent antitumor activity of the extract was attributed to Cephalotaxus alkaloids, such as homoharringtonine (20).

Antitumor activity of IHL-305, a novel pegylated liposome containing irinotecan, in human xenograft models.

The antitumor effect of IHL-305, a novel pegylated liposome containing irinotecan, was investigated in human xenograft models. After subcutaneous transplantation of several human cancer cell lines (colorectal, non-small cell lung, small cell lung, prostate, ovarian and gastric cancer cells) to nude mice, IHL-305 or CPT-11 was administered intravenously 3 times at 4-day intervals. In all xenograft models tested, IHL-305 showed superior antitumor activity to that of CPT­11 and a comparable tumor-growth-inhibitory effect at one-eighth or less of the dose of CPT-11, even against HT-29 colorectal and NCI-H460 non-small cell lung cancer cell lines, which show intrinsic resistance to CPT-11. A single injection or 2 injections of IHL-305 on several dosing schedules also resulted in a significant antitumor effect compared to that of vehicle control in a dose-dependent manner and showed comparable antitumor activity at about one-fifth the dose of the maximum tolerated dose of CPT-11. The analysis of the concentrations of irinotecan and SN-38, an active metabolite of CPT-11, in plasma and tumors revealed that irinotecan was maintained at high concentrations, and the prolonged presence of SN-38 in plasma and tumors in IHL-305 treated mice compared with CPT-11-treated mice. Therefore, the stronger tumor inhibitory effect of IHL-305, as compared to CPT-11, was associated with the difference in the concentration of irinotecan in plasma or tumors after each agent was administered and with the maintainance of a higher concentration of SN-38. These results indicate that IHL-305 demonstrated superior antitumor activity against a wide range of tumors at lower doses than CPT-11.

Semisynthesis of triptolide analogues: effect of γ-lactone and C-14 substituents on cytotoxic activities.

Triptolide γ-lactone and C-14 analogues were prepared and evaluated cytotoxity against human lung adenocarcinoma epithelial A549 cells and human colon adenocarcinoma HT-29 cells. γ-Lactone substructure and C-14 substituents affected the biological activities significantly.

Efficacy of combination treatment and influence of schedule with irinotecan and amrubicin in human lung carcinoma cells in vivo and in vitro.

The aim of this study was to elucidate the efficacy of combination therapy with irinotecan and amrubicin for lung cancer and the influence of administration schedule in a xenograft mouse model and human cancer cell culture. We investigated the antitumor activity of irinotecan and amrubicin on human small cell lung cancer cell line LX-1 inoculated in mice in vivo and the cytotoxic effect of SN-38 and amrubicinol on human lung cancer cell lines A549 and PC-6 in vitro. Combined administration of irinotecan and amrubicin in divided doses inhibited tumor growth by approximately 90%, with complete recovery observed in one case. Furthermore, combined administration in divided doses induced little loss of body weight. Combination index analysis revealed that the cell growth inhibitory effect of SN-38 combined with amrubicinol was additive, regardless of schedule or cell line. The effect of combination treatment with SN-38 and amrubicinol on cell cycle was investigated. Cell cycle showed arrest at both the S and G2/M phases. The results indicate that combination therapy with irinotecan and amrubicin can be expected to yield improved outcomes, including less toxicity, especially with divided administration.

Rhazinilam and quebrachamine derivatives from Yunnan Kopsia arborea.

Three new rhazinilam-derived alkaloids, kopsiyunnanines C1, C2, and C3, and a new quebrachamine-type alkaloid, kopsiyunnanine D, which possess an unusual methoxymethyl or ethoxymethyl function, were isolated from the aerial parts of Yunnan Kopsia arborea. This is the first report of the presence of these functions in natural alkaloids. The structures and absolute configurations of the alkaloids were determined by spectroscopic methods and confirmed by semisynthesis.

Fluorination of triptolide and its analogues and their cytotoxicity.

The reaction of triptolide and its analogues with a fluorinating agent, that is, bis(2-methoxyethyl)aminosulfur trifluoride (Deoxo-Fluor) or (diethylamino)sulfur trifluoride (DAST), was studied. One of the fluorinated products, 14beta-dehydroxy-14beta-fluoro triptolide, was found to be more cytotoxic than the parent natural triptolide.

Semisynthesis of isetexane diterpenoid analogues and their cytotoxic activity.

Isetexane diterpene analogues were semisynthesized from demethylsalvicanol isolated from Perovskia abrotanoides (Labiatae). The structure and cytotoxic activity relationships (SAR) of the natural parent diterpene, demethylsalvicanol, and its semisynthetic analogues were studied by using P388 murine leukemia cells.

Low-dose oxaliplatin enhances the antitumor efficacy of paclitaxel in human gastric cancer cell lines.

BACKGROUND: The enhanced antitumor effect of paclitaxel when used with oxaliplatin in gastric cancer is reported, however the underlying biological mechanism is unknown. METHODS: We tested the cytotoxic activity, apoptosis, and mitotic catastrophe of paclitaxel and oxaliplatin in MKN-28 and MKN-45 gastric cancer cell lines. The modulation of survivin expression was determined by Western blotting. RESULTS: WST-1 assay indicated that paclitaxel plus oxaliplatin showed better cytotoxicity than paclitaxel alone, even when low concentrations of oxaliplatin were used. Flow cytometry analysis revealed significantly greater increases in apoptotic cells after treatment with paclitaxel followed by low-dose oxaliplatin (1 microM) than after any single-reagent regimen in the MKN-45 cell line. In MKN-28, a difference existed only between combination treatment and oxaliplatin treatment. Morphologic examination showed that the cells undergoing mitotic catastrophe were highest in the combination groups in the both cell lines. Downregulation of survivin expression was found by Western blotting with treatment by paclitaxel, oxaliplatin, or their combination. CONCLUSION: Our findings suggest that the mechanism of enhanced cytotoxicity might be through enhanced mitotic catastrophe and apoptosis, which is possibly due to chemotherapy-induced downregulation of surviving. The combination of paclitaxel and low-dose oxaliplatin should be incorporated into the design of a clinical trial.

Cytotoxicity of abietane diterpenoids from Perovskia abrotanoides and of their semisynthetic analogues.

Seven known abietane diterpenoids and 11-O- and 12-O-acetylcarnosic acids were isolated from a methanol extract of Perovskia abrotanoides (Labiatae). Structure and cytotoxic activity relationships (SAR) of the natural and semisynthetic analogues of the presently isolated abietane diterpenoids were studied by using P388 murine leukemia cells.

Semisynthesis of C-ring modified triptolide analogues and their cytotoxic activities.

Several C-ring modified analogues of a potent antileukemic diterpene, triptolide (1), were synthesized and their structure-activity relationships were studied.

Alleviation of side effects induced by irinotecan hydrochloride (CPT-11) in rats by intravenous infusion.

PURPOSE: Irinotecan hydrochloride (CPT-11) is a potent topoisomerase I inhibitor and is established and used widely as an antitumor agent. However, it sometimes causes severe side effects such as myelosuppression and diarrhea. These dose-limiting toxicities prevent the adoption of CPT-11 in aggressive chemotherapy. Thus we sought to determine in a rat model whether extending the period of infusion of CPT-11 would ameliorate the adverse reactions. METHODS: CPT-11 was administered intravenously (i.v.) to rats at a dose of 60 mg/kg per day for four consecutive days as a bolus injection or as 3-, 8- or 24-h infusions, and then blood cell counts and the incidence of acute and delayed-onset diarrhea were monitored. RESULTS: Serious acute and delayed-onset diarrhea and marked decreases in the number of neutrophils and lymphocytes were observed in the bolus injection group. These symptoms were alleviated in the infusion groups with the degree of alleviation dependent on infusion time. In the bolus injection group, mucosal impairment of the cecal epithelium including wall thickening, edema, a decrease in the number and size of crypts, and the formation of a pseudomembrane-like substance was observed, whereas these changes were less severe in the infusion groups. Areas under the plasma concentration-time curves (AUCpla) of CPT-11 and its metabolite, 7-ethyl-10-hydroxycamptothecin (SN-38), differed little among the bolus injection group, and the 3-h and 8-h infusion groups. However, the AUCpla values of CPT-11 and SN-38 were significantly decreased and increased, respectively, in the 24-h infusion group. The maximum plasma concentrations (Cmax) of CPT-11 decreased with increasing infusion time, but those of SN-38 did not. CONCLUSIONS: It was confirmed that the side effects of CPT-11 were alleviated by extending the infusion time. The pharmacokinetic parameters suggested that the Cmax of CPT-11 is closely related to the incidence and severity of adverse reactions such as myelosuppression and acute and delayed-onset diarrhea.

Preventive effects of Cladosiphon fucoidan against Helicobacter pylori infection in Mongolian gerbils.

BACKGROUND: Recently, the acquisition by Helicobacter pylori of resistance to antibiotics has become a serious problem. Therefore, nonantibiotic substances are required to diminish H. pylori-induced gastric lesions. In the present study, the effects of Cladosiphon fucoidan were examined in terms of H. pylori attachment to porcine gastric mucin in vitro and Helicobacter pylori-induced gastritis in vivo. METHODS: The inhibitory effect of Cladosiphon fucoidan and other polysaccharides on H. pylori attachment to porcine gastric mucin was assayed in vitro with mucin-coated microtiter plates. The effect of Cladosiphon fucoidan on H. pylori-induced gastritis was examined in vivo using Mongolian gerbils. H. pylori-inoculated gerbils were given fucoidan in drinking water. Six weeks after H. pylori-inoculation, gerbils were sacrificed for macroscopic and microscopic examination of gastric lesions and counting of viable H. pylori in the gastric mucosa. RESULTS: Cladosiphon fucoidan inhibited the H. pylori attachment to porcine gastric mucin at pH 2.0 and 4.0. Two other sulfated polysaccharides, Fucus fucoidan and dextran sulfate sodium, also inhibited the attachment but only at pH 2.0. Inhibitory effects of these three sulfated polysaccharides were not observed at pH 7.2 and nonsulfated polysaccharides, such as mannan and dextran, exerted no influence at any pH. In the in vivo experiment, the H. pylori-induced gastritis and the prevalence of H. pylori infected animals were markedly reduced by fucoidan in a dose-dependent manner, at doses of 0.05 and 0.5% in the drinking water. CONCLUSION: Cladosiphon fucoidan may deserve particular attention as a safe agent that can prevent H. pylori infection and reduce the risk of associated gastric cancer.

Selective cyclooxygenase-2 inhibitors show a differential ability to inhibit proliferation and induce apoptosis of colon adenocarcinoma cells.

Although the influence of selective cyclooxygenase (COX)-2 inhibitors on the proliferation of colon adenocarcinoma cells have been the subject of much investigation, relatively little research has compared the effects of different COX-2 inhibitors. Celecoxib strongly suppressed the proliferation of COX-2 expressing HT-29 cells at 10-40 microM. NS-398 and nimesulide also inhibited cell proliferation, whereas rofecoxib, meloxicam, and etodolac did not. Only celecoxib induced apoptosis of HT-29 cells, as detected on the basis of DNA fragmentation, TUNEL positivity, and caspase-3/7 activation. DNA fragmentation was also increasd in COX-2 non-expressing cell lines (SW-480 and HCT-116) by exposure to celecoxib for 6-24 h. All six COX-2 inhibitors suppressed the production of prostaglandin E(2) by HT-29 cells, suggesting that the pro-apoptotic effect of celecoxib was unrelated to inhibition of COX-2. Inactivation of Akt might explain the differential pro-apoptotic effect of these selective COX-2 inhibitors on colon adenocarcinoma cells.

Nonsteroidal anti-inflammatory drugs induce apoptosis in association with activation of peroxisome proliferator-activated receptor gamma in rheumatoid synovial cells.

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been reported to induce apoptosis in a variety of cell lines. In this study, we examined the effect of NSAIDs on the growth and apoptosis of synovial cells from patients with rheumatoid arthritis and analyzed the activation of peroxisome proliferator-activated receptor gamma (PPARgamma) as a possible mechanism of action of NSAIDs. Cell proliferation and viability were assessed from 5-bromo-2'-deoxyuridine incorporation and by 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) assay, respectively. The apoptosis of synovial cells was identified by DNA fragmentation assay and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. Indometacin, diclofenac, oxaprozin, and zaltoprofen reduced cell proliferation and induced apoptotic cell death in synovial cells, whereas ketoprofen and acetaminophen did not. N-[2-(cyclohexyloxyl)-4-nitrophenyl]-methanesulfonamide (NS-398), a selective cyclooxygenase-2 inhibitor, also inhibited cell proliferation, whereas it did not cause apoptosis. Rheumatoid synovial cells expressed PPARgamma mRNA, and the PPARgamma ligands 15-deoxy-Delta(12,14)-prostaglandin J(2) and troglitazone reduced the proliferation and induced apoptosis in synovial cells. Luciferase reporter assay demonstrated that not only PPARgamma ligands but also NSAIDs, which could induce apoptosis, increased the activation of PPARgamma in synovial cells. Furthermore, the ability of NSAIDs and PPARgamma ligands to stimulate the activation of PPARgamma correlated with their ability to decrease cell viability(r = 0.92, p < 0.01) and ability to induce DNA fragmentation (r = 0.97, p < 0.001) in synovial cells. These results suggest that PPARgamma is an attractive target for induction of apoptosis in rheumatoid synovial cells and that the activation of the PPARgamma pathway is associated with the apoptotic action of NSAIDs.

Aspirin and sodium salicylate inhibit proliferation and induce apoptosis in rheumatoid synovial cells.

Aspirin has been reported to induce apoptosis in a variety of cell lines. In this study, we examined whether aspirin and sodium salicylate inhibit cell growth and induce apoptosis in rheumatoid synovial cells. Synovial cells were obtained from patients with rheumatoid arthritis, and the cells were treated with aspirin or sodium salicylate (0.1-10 mM) for 24 h. Cell proliferation and viability were assessed by 5-bromo-2'-deoxyuridine incorporation and by 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) assay, respectively. The apoptosis of synovial cells was identified by DNA fragmentation assay and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay. Aspirin and sodium salicylate suppressed the proliferation (IC50 (concentration causing 50% inhibition of cell proliferation): 2.1 and 1.2 mM, respectively) and reduced the viability (IC50: 2.0 and 1.4 mM, respectively) of synovial cells in a concentration-dependent manner at 0.3-10 mM. Furthermore, they induced DNA fragmentation and increased the number of TUNEL-positive synovial cells. These results suggest that aspirin and sodium salicylate can inhibit the proliferation of rheumatoid synovial cells through induction of apoptosis.