The role of calcium, calcium-activated K+ channels, and tyrosine/kinase in psoralen-evoked responses in human melanoma cells

8-Methoxy psoralen (8-MOP) exerts a short-term (24 h) mitogenic action, and a long-term (48-72 h) anti-proliferative and melanogenic action on two human melanoma cell lines, SK-Mel 28 and C32TG. An increase of intracellular calcium concentration was observed by spectrofluorometry immediately after the addition of 0.1 mM 8-MOP to both cell lines, previously incubated with calcium probe fluo-3 AM (5 µM). The intracellular Ca2+ chelator BAPTA/AM (1 µM) blocked both early (mitogenic) and late (anti-proliferative and melanogenic) 8-MOP effects on both cell lines, thus revealing the importance of the calcium signal in both short- and long-term 8-MOP-evoked responses. Long-term biological assays with 5 and 10 mM tetraethylammonium chloride (TEA, an inhibitor of Ca2+-dependent K+ channels) did not affect the responses to psoralen; however, in 24-h assays 10 mM TEA blocked the proliferative peak, indicating a modulation of Ca2+-dependent K+ channels by 8-MOP. No alteration of cAMP basal levels or forskolin-stimulated cAMP levels was promoted by 8-MOP in SK-Mel 28 cells, as determined by radioimmunoassay. However, in C32TG cells forskolin-stimulated cAMP levels were further increased in the presence of 8-MOP. In addition, assays with 1 µM protein kinase C and calcium/calmodulin-dependent kinase inhibitors, Ro 31-8220 and KN-93, respectively, excluded the participation of these kinases in the responses evoked by 8-MOP. Western blot with antibodies anti-phosphotyrosine indicated a 92% increase of the phosphorylated state of a 43-kDa band, suggesting that the phosphorylation of this protein is a component of the cascade that leads to the increase of tyrosinase activity.

Anti-inflammatory and antinociceptive activities of an acid fraction of the seeds of Carpotroche brasiliensis (Raddi) (Flacourtiaceae)

Carpotroche brasiliensis is a native Brazilian tree belonging to the Oncobeae tribe of Flacourtiaceae. The oil extracted from its seeds contains as major constituents the same cyclopentenyl fatty acids hydnocarpic (40.5%), chaulmoogric (14.0%) and gorlic (16.1%) acids found in the better known chaulmoogra oil prepared from the seeds of various species of Hydnocarpus (Flacourtiaceae). These acids are known to be related to the pharmacological activities of these plants and to their use as anti-leprotic agents. Although C. brasiliensis oil has been used in the treatment of leprosy, a disease that elicits inflammatory responses, the anti-inflammatory and analgesic activities of the oil and its constituents have never been characterized. We describe the anti-inflammatory and antinociceptive activities of C. brasiliensis seed oil in acute and chronic models of inflammation and in peripheral and central nociception. The mixture of acids from C. brasiliensis administered orally by gavage showed dose-dependent (10-500 mg/kg) anti-inflammatory activity in carrageenan-induced rat paw edema, inhibiting both the edema by 30-40% and the associated hyperalgesia. The acid fraction (200 mg/kg) also showed significant antinociceptive activity in acetic acid-induced constrictions (57% inhibition) and formalin-induced pain (55% inhibition of the second phase) in Swiss mice. No effects were observed in the hot-plate (100 mg/kg; N = 10), rota-road (200 mg/kg; N = 9) or adjuvant-induced arthritis (50 mg/kg daily for 7 days; N = 5) tests, the latter a chronic model of inflammation. The acid fraction of the seeds of C. brasiliensis which contains cyclopentenyl fatty acids is now shown to have significant oral anti-inflammatory and peripheral antinociceptive effects.

Anti-tumor properties of blackseed (Nigella sativa L.) extracts

The objective of the present study was to evaluate the in vitro and in vivo anti-cancer effect of Nigella sativa L. seed extracts. The essential oil (IC50 = 0.6%, v/v) and ethyl acetate (IC50 = 0.75%) extracts were more cytotoxic against the P815 cell line than the butanol extract (IC50 = 2%). Similar results were obtained with the Vero cell line. Although all extracts had a comparable cytotoxic effect against the ICO1 cell line, with IC50 values ranging from 0.2 to 0.26% (v/v), tests on the BSR cell line revealed a high cytotoxic effect of the ethyl acetate extract (IC50 = 0.2%) compared to the essential oil (IC50 = 1.2%). These data show that the cytotoxicity of each extract depends on the tumor cell type. In vivo, using the DBA2/P815 (H2d) mouse model, our results clearly showed that the injection of the essential oil into the tumor site significantly inhibited solid tumor development. Indeed, on the 30th day of treatment, the tumor volume of the control animals was 2.5 ± 0.6 cm³, whereas the tumor volumes of the essential oil-treated animals were 0.22 ± 0.1 and 0.16 ± 0.1 cm³ when the animals were injected with 30 µL (28.5 mg)/mouse and 50 µL (47.5 mg)/mouse per 48 h (six times), respectively. Interestingly, the administration of the essential oil into the tumor site inhibited the incidence of liver metastasis development and improved mouse survival.

Response of the growth plate of uremic rats to human growth hormone and corticosteroids

Children with chronic renal failure in general present growth retardation that is aggravated by corticosteroids. We describe here the effects of methylprednisolone (MP) and recombinant human growth hormone (rhGH) on the growth plate (GP) of uremic rats. Uremia was induced by subtotal nephrectomy in 30-day-old rats, followed by 20 IU kg-1 day-1 rhGH (N = 7) or 3 mg kg-1 day-1 MP (N = 7) or 20 IU kg-1 day-1 rhGH + 3 mg kg-1 day-1 MP (N = 7) treatment for 10 days. Control rats with intact renal function were sham-operated and treated with 3 mg kg-1 day-1 MP (N = 7) or vehicle (N = 7). Uremic rats (N = 7) were used as untreated control animals. Structural alterations in the GP and the expression of anti-proliferating cell nuclear antigen (PCNA) and anti-insulin-like growth factor I (IGF-I) by epiphyseal chondrocytes were evaluated. Uremic MP rats displayed a reduction in the proliferative zone height (59.08 ± 4.54 vs 68.07 ± 7.5 µm, P < 0.05) and modifications in the microarchitecture of the GP. MP and uremia had an additive inhibitory effect on the proliferative activity of GP chondrocytes, lowering the expression of PCNA (19.48 ± 11.13 vs 68.64 ± 7.9% in control, P < 0.0005) and IGF-I (58.53 ± 0.96 vs 84.78 ± 2.93% in control, P < 0.0001), that was counteracted by rhGH. These findings suggest that in uremic rats rhGH therapy improves longitudinal growth by increasing IGF-I synthesis in the GP and by stimulating chondrocyte proliferation.

Inhibition of cyclooxygenase activity by standardized hydroalcoholic extracts of four Asteraceae species from the Argentine Puna

We determined the anti-inflammatory activity of standardized extracts of four medicinal plant species (Baccharis incarum, B. boliviensis, Chuquiraga atacamensis, Parastrephia lucida) that grow in the Argentine Puna (3800 m above sea level) and that are used to reduce oxidative stress and alleviate gout and arthritic pain. The extracts of plant aerial parts were standardized in terms of total phenolic compounds and flavone/flavanone content and free radical scavenging activity. All extracts showed high phenolic compound concentration (0.5-1.6 mg/mL), mainly flavones and flavonols (0.1-0.8 mg/mL). The extracts showed hydrogen donating ability (DPPH and ABTS) and reactive oxygen species scavenging activity (O2●-, OH-, H2O2). The ability of the extracts to inhibit cyclooxygenase enzymes (COX-1 and COX-2) was determined by calculating percent inhibition of PGE2 production measured by enzyme immunoassay. All extracts inhibited both enzymes with IC50 values of 2.0 to 16.7 µg/mL. The anti-inflammatory activity of B. incarum and C. atacamensis extracts was higher than that of B. boliviensis and P. lucida. The IC50 values obtained for indomethacin were 0.11 and 0.78 µM for COX-1 and COX-2, respectively. The present results are consistent with the anecdotal use of these species in phytotherapic preparations.

In vivo and in vitro anti-inflammatory and anti-nociceptive activities of lovastatin in rodents

Statins are among the most prescribed drugs in recent clinical practice. They are also known for their pleiotropic actions, which are independent of their lipid-lowering properties. The effect of lovastatin was investigated against carrageenan-induced paw edema in male Wistar rats (200-250 g) and on leukocyte migration, as measured by carrageenan-induced peritonitis in male Swiss mice (20-25 g), which are models of acute inflammation. Lovastatin (administered 1 h prior to carrageenan), at oral doses of 2, 5, and 10 mg/kg, markedly attenuated paw edema formation in rats at the 4th hour after carrageenan injection (25, 43, and 37% inhibition, respectively). Inhibitions of 20, 45 and 80% were observed in the leukocyte migration, as evaluated by carrageenan-induced peritonitis in mice with lovastatin doses of 0.5, 1 and 5 mg/kg, as compared to controls. Furthermore, lovastatin (administered 1 h before initiation) reduced the nociceptive effect of the formalin test in mice, at both phases, at doses of 2, 5, and 10 mg/kg: first phase (51, 65, and 70%, respectively) and second phase (73, 57, and 66% inhibition of licking time, respectively). The anti-nociceptive activity of lovastatin was inhibited by naloxone (3 mg/kg, sc). Lovastatin (0.01, 0.1, and 1 µg/mL) inhibited by 23, 79, and 86%, respectively, the release of myeloperoxidase from human neutrophils. Leukocyte (predominantly neutrophils) infiltration was almost completely reduced by lovastatin treatment, as observed in the model of acute paw edema with hematoxylin and eosin staining. In addition, lovastatin decreased the number of cells expressing tumor necrosis factor-α (TNF-α) and the inducible form of nitric oxide synthase (iNOS) activity. Therefore, the alterations in leukocyte activity and cytokine release could contribute to the anti-inflammatory activity of lovastatin.

In vitro anti-inflammatory, cytotoxic and antioxidant activities of boesenbergin A, a chalcone isolated from Boesenbergia rotunda (L.) (fingerroot)

The current in vitro study was designed to investigate the anti-inflammatory, cytotoxic and antioxidant activities of boesenbergin A (BA), a chalcone derivative of known structure isolated from Boesenbergia rotunda. Human hepatocellular carcinoma (HepG2), colon adenocarcinoma (HT-29), non-small cell lung cancer (A549), prostate adenocarcinoma (PC3), and normal hepatic cells (WRL-68) were used to evaluate the cytotoxicity of BA using the MTT assay. The antioxidant activity of BA was assessed by the ORAC assay and compared to quercetin as a standard reference antioxidant. ORAC results are reported as the equivalent concentration of Trolox that produces the same level of antioxidant activity as the sample tested at 20 µg/mL. The toxic effect of BA on different cell types, reported as IC50, yielded 20.22 ± 3.15, 10.69 ± 2.64, 20.31 ± 1.34, 94.10 ± 1.19, and 9.324 ± 0.24 µg/mL for A549, PC3, HepG2, HT-29, and WRL-68, respectively. BA displayed considerable antioxidant activity, when the results of ORAC assay were reported as Trolox equivalents. BA (20 µg/mL) and quercetin (5 µg/mL) were equivalent to a Trolox concentration of 11.91 ± 0.23 and 160.32 ± 2.75 µM, respectively. Moreover, the anti-inflammatory activity of BA was significant at 12.5 to 50 µM and without any significant cytotoxicity for the murine macrophage cell line RAW 264.7 at 50 µM. The significant biological activities observed in this study indicated that BA may be one of the agents responsible for the reported biological activities of B. rotunda crude extract.

Kaurene diterpene induces apoptosis in U87 human malignant glioblastoma cells by suppression of anti-apoptotic signals and activation of cysteine proteases

Gliomas are the most common and malignant primary brain tumors in humans. Studies have shown that classes of kaurene diterpene have anti-tumor activity related to their ability to induce apoptosis. We investigated the response of the human glioblastoma cell line U87 to treatment with ent-kaur-16-en-19-oic acid (kaurenoic acid, KA). We analyzed cell survival and the induction of apoptosis using flow cytometry and annexin V staining. Additionally, the expression of anti-apoptotic (c-FLIP and miR-21) and apoptotic (Fas, caspase-3 and caspase-8) genes was analyzed by relative quantification (real-time PCR) of mRNA levels in U87 cells that were either untreated or treated with KA (30, 50, or 70 µM) for 24, 48, and 72 h. U87 cells treated with KA demonstrated reduced viability, and an increase in annexin V- and annexin V/PI-positive cells was observed. The percentage of apoptotic cells was 9% for control cells, 26% for cells submitted to 48 h of treatment with 50 µM KA, and 31% for cells submitted to 48 h of treatment with 70 µM KA. Similarly, in U87 cells treated with KA for 48 h, we observed an increase in the expression of apoptotic genes (caspase-8, -3) and a decrease in the expression of anti-apoptotic genes (miR-21 and c-FLIP). KA possesses several interesting properties and induces apoptosis through a unique mechanism. Further experiments will be necessary to determine if KA may be used as a lead compound for the development of new chemotherapeutic drugs for the treatment of primary brain tumors.

Antibacterial and anti-inflammatory activities of an extract, fractions, and compounds isolated from Gochnatia pulchra aerial parts

This paper reports on the in vitro antibacterial and in vivo anti-inflammatory properties of a hydroethanolic extract of the aerial parts of Gochnatia pulchra (HEGP). It also describes the antibacterial activity of HEGP fractions and of the isolated compounds genkwanin, scutellarin, apigenin, and 3,5-O-dicaffeoylquinic acid, as evaluated by a broth microdilution method. While HEGP and its fractions did not provide promising results, the isolated compounds exhibited pronounced antibacterial activity. The most sensitive microorganism was Streptococcus pyogenes, with minimum inhibitory concentration (MIC) values of 100, 50 and 25 µg/mL for genkwanin and the flavonoids apigenin and scutellarin, respectively. Genkwanin produced an MIC value of 25 µg/mL against Enterococcus faecalis. A paw edema model in rats and a pleurisy inflammation model in mice aided investigation of the anti-inflammatory effects of HEGP. This study also evaluated the ability of HEGP to modulate carrageenan-induced interleukin-1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), and monocyte chemoattractant protein-1 (MCP-1) production. Orally administered HEGP (250 and 500 mg/kg) inhibited carrageenan-induced paw edema. Regarding carrageenan-induced pleurisy, HEGP at 50, 100, and 250 mg/kg diminished leukocyte migration by 71.43%, 69.24%, and 73.34% (P<0.05), respectively. HEGP suppressed IL-1β and MCP-1 production by 55% and 50% at 50 mg/kg (P<0.05) and 60% and 25% at 100 mg/kg (P<0.05), respectively. HEGP abated TNF-α production by macrophages by 6.6%, 33.3%, and 53.3% at 100, 250, and 500 mg/kg (P<0.05), respectively. HEGP probably exerts anti-inflammatory effects by inhibiting production of the pro-inflammatory cytokines TNF-α, IL-1β, and MCP-1.

Effect of Cysticercus cellulosae fractions on the respiratory burst of pig neutrophils

Neutrophils, eosinophils and macrophages are cells that interact with invading parasites and naive hosts have been shown to have anti-parasitic activity. The initial reaction of these leukocytes is the generation of reactive oxygen species (ROS) to play in parasite expulsion. The present work was carried out to study the effect of total extract, scolex and membrane fractions from Cysticercus cellulosae on respiratory burst by pig neutrophils. Hydrogen peroxide (H2O2) production by neutrophils incubated with metacestode fractions from C. cellulosae showed an increase of: 190% (total extract), 120% (scolex) and 44% (membrane). High antioxidant catalatic activity (33%, 28%, 28% by total extract, scolex and membrane, respectively) was observed in neutrophils incubated with metacestode fractions, which could be an attempt at self-protection. Scolex and membrane fractions increased the phagocytic capacity of neutrophils (44% and 28%, respectively). On the other hand, total cysticerci did not alter the phagocytosis, possibly due to modifications in membrane function, caused by high ROS production from neutrophils in the presence of total cysticerci. Total fraction from C. cellulosae is toxic for neutrophils as shown by the decrease in phagocytic capacity, probably caused by high levels of ROS formation. The difference in toxicity of total extract, scolex and membrane fractions on neutrophils can be explained by the presence of an antigenic effect of the vesicular fluid in the total extract of C. cellulosae.

Analysis of the use of fenthion via epicutaneous in dogs for Rhipicephalus sanguineus control

The action of fenthion was studied in a 15% epicutaneous formulation upon Rhipicephalus sanguineus, which may transmit pathogens to men and other animals, such as Ehrlichia, Babesia and Ricketsia. Dogs were artificially infected for the trial. The fenthion bioassays were begun four months after artificial infestation. The test group, having a mean of 186 ticks per dog, received the formulation dosage according to body weight on the neck region. Tick counts were performed, considering diameters > or = 2mm, during 11 days of treatment, in the most affected body areas: back, ears and paws. Before the application of fenthion in the dogs, it were observed an average 43.3% ticks in the ears, 38.1% in the back area and 17.6% in the paws. The number of ticks in dogs decreased by 36.2%, 63.8%, 82.7%, 67%, 40% and 4.9%, respectively on days 1, 2, 3, 5, 7, 9 and 11 after treatment. R. sanguineus anti-tick activity, lower than that officially recommended, was verified. The number of ticks increased progressively after the 5th day, demonstrating residual insecticide inefficacy. The results obtained did not indicate the use of this formulation, at the tested dosage, as an elective measure for R. sanguineus control.

In vitro action of antiparasitic drugs, especially artesunate, against Toxoplasma gondii

INTRODUCTION: Toxoplasmosis is usually a benign infection, except in the event of ocular, central nervous system (CNS), or congenital disease and particularly when the patient is immunocompromised. Treatment consists of drugs that frequently cause adverse effects; thus, newer, more effective drugs are needed. In this study, the possible activity of artesunate, a drug successfully being used for the treatment of malaria, on Toxoplasma gondii growth in cell culture is evaluated and compared with the action of drugs that are already being used against this parasite. METHODS: LLC-MK2 cells were cultivated in RPMI medium, kept in disposable plastic bottles, and incubated at 36ºC with 5% CO2. Tachyzoites of the RH strain were used. The following drugs were tested: artesunate, cotrimoxazole, pentamidine, pyrimethamine, quinine, and trimethoprim. The effects of these drugs on tachyzoites and LLC-MK2 cells were analyzed using nonlinear regression analysis with Prism 3.0 software. RESULTS: Artesunate showed a mean tachyzoite inhibitory concentration (IC50) of 0.075µM and an LLC MK2 toxicity of 2.003µM. Pyrimethamine was effective at an IC50 of 0.482µM and a toxicity of 11.178µM. Trimethoprim alone was effective against the in vitro parasite. Cotrimoxazole also was effective against the parasite but at higher concentrations than those observed for artesunate and pyrimethamine. Pentamidine and quinine had no inhibitory effect over tachyzoites. CONCLUSIONS: Artesunate is proven in vitro to be a useful alternative for the treatment of toxoplasmosis, implying a subsequent in vivo effect and suggesting the mechanism of this drug against the parasite.

Müllerian adenosarcoma of the uterus with sarcomatous overgrowth following tamoxifen treatment for breast cancer

Müllerian adenosarcoma with sarcomatous overgrowth presented by a 52-year-old female patient after adjuvant tamoxifen treatment for breast carcinoma is described. The diagnosis was made on histological basis after curettage and complementary total hysterectomy with bilateral salpingo-oophorectomy. The immunohistochemical study showed high expression of estrogen receptors in the epithelial component of the lesion and irregularly positive findings in the stroma. The proliferative activity evaluated by Ki-67 immunoexpression was higher in the stroma than the epithelium. Some of the stromal cells showed rhabdomyoblastic differentiation. The association of tamoxifen use and development of mesenchymal neoplasms is discussed.

Pentoxifylline Attenuates Cardiac Remodeling Induced by Tobacco Smoke Exposure

Abstract Background: Tobacco smoke exposure is an important risk factor for cardiac remodeling. Under this condition, inflammation, oxidative stress, energy metabolism abnormalities, apoptosis, and hypertrophy are present. Pentoxifylline has anti‑inflammatory, anti-apoptotic, anti-thrombotic and anti-proliferative properties. Objective: The present study tested the hypothesis that pentoxifylline would attenuate cardiac remodeling induced by smoking. Methods: Wistar rats were distributed in four groups: Control (C), Pentoxifylline (PX), Tobacco Smoke (TS), and PX-TS. After two months, echocardiography, invasive blood pressure measurement, biochemical, and histological studies were performed. The groups were compared by two-way ANOVA with a significance level of 5%. Results: TS increased left atrium diameter and area, which was attenuated by PX. In the isolated heart study, TS lowered the positive derivate (+dp/dt), and this was attenuated by PX. The antioxidants enzyme superoxide dismutase and glutathione peroxidase were decreased in the TS group; PX recovered these activities. TS increased lactate dehydrogenase (LDH) and decreased 3-hydroxyacyl Coenzyme A dehydrogenases (OH-DHA) and citrate synthase (CS). PX attenuated LDH, 3-OH-DHA and CS alterations in TS-PX group. TS increased IL-10, ICAM-1, and caspase-3. PX did not influence these variables. Conclusion: TS induced cardiac remodeling, associated with increased inflammation, oxidative stress, apoptosis, and changed energy metabolism. PX attenuated cardiac remodeling by reducing oxidative stress and improving cardiac bioenergetics, but did not act upon cardiac cytokines and apoptosis.

Trypanosoma cruzi: effect of phenothiazines on the parasite and its interaction with host cells

Phenothiazines were observed to have a direct effect on Trypanosoma cruzi and on its in vitro interaction with host cells. They caused lysis of trypomastigotes (50 uM/24 h) and,to a lesser extent, epimastigote proliferation. Treatment of infected peritoneal macrophages with 12.5 uM chlorpromazine or triflupromazine inhibited the infection; this effect was found to be partially reversible if the drugs were removed after 24 h of treatment. At 60 uM, the drugs caused damage to amastigotes interiorized in heart muscle cells. However, the narrow margin of toxity between anti-trypanossomal activity and damage to host cells mitigates against in vivo investigation at the present time. Possible hypothesis for the mechanism of action of phenothiazines are discussed.