Dutasteride combined with androgen receptor antagonists inhibit glioblastoma U87 cell metabolism, proliferation, and invasion capacity: Androgen regulation.

Glioblastoma (GB) is the most common and aggressive primary brain tumor in adult humans. Therapeutic resistance and tumor recurrence after surgical resection contributes to a poor prognosis for glioblastoma patients. Men are known to be more likely than women to develop an aggressive form of GB. Although the reasons for this disparity remain poorly understood, differences in sex steroids have emerged as a leading explanation. Studies indicate that GB-derived cells express androgen receptors (ARs) and synthesize androgens, suggesting that androgens may have a role in the tumor pathogenesis. Thus, our objective was to investigate the effects of the 5α-reductase enzyme inhibitor dutasteride, the AR antagonists cyproterone and flutamide, and combinations of these drugs on the metabolism, proliferation, and invasion capacity of GB-derived U87 cells. We also examined the effects of three natural androgens testosterone, androstenedione and dihydrotestosterone (T, A4, and DHT) on these cells. Cell metabolism was investigated by MTT assay, proliferation was assessed by the bromodeoxyuridine (BrdU) incorporation assay, and invasion was assessed by Boyden chamber assay. The results revealed that T and especially DHT, but not A4, increased U87 cell metabolism and proliferation. Following these findings, we examined the effect of adding dutasteride, cyproterone, or flutamide to the culture media and found that they all significantly decreased cell metabolism and proliferation. Dutasteride also significantly reduced cell invasion. Moreover, any combination of these drugs enhanced their inhibitory effects; the combination of dutasteride to flutamide was most effective at decreasing GB cell proliferation. Our results suggest that administering a combination of AR antagonists and enzyme blockers may be a more effective alternative treatment for GB.

Effect of mice Taenia crassiceps WFU cysticerci infection on the ovarian folliculogenesis, enzyme expression, and serum estradiol.

The murine infection with Taenia crassiceps WFU (T. crassiceps WFU) cysticerci has been widely used as an experimental model to better understand human cysticercosis. Several reports have established that the host hormonal environment determines the susceptibility and severity of many parasite infections. Female mice are more susceptible to infection with T. crassiceps cysticerci suggesting that a rich estrogen environment facilitates their reproduction. Ovarian androgens and estrogens are synthesized by key enzymes as P450-aromatase and 17α-hydroxilase/17, 20 lyase (P450C17). The aim of this study was to determine the effect of chronic intraperitoneal infection of T. crassiceps WFU cysticerci on mice ovarian follicular development, ovulation, the expression of ovarian P450-aromatase and P450C17, and serum 17ß-estradiol, key enzymes of the ovarian steroidogenic pathway. To perform this study ovaries and serum were obtained at two, four and six months from T. crassiceps WFU cysticerci infected mice, and compared to those of healthy animals. The ovaries were fixed and processed for histology or lysed in RIPA buffer for Western blot using specific antibodies for P450C17 and P450-aromatase. 17ß-estradiol serum concentration was measured by ELISA. The results showed that the infection with T. crassiceps WFU cysticerci significantly reduced the number of primordial and primary follicles after two months of infection. Through the course of the study, the corpus luteum number began to decrease, whereas atretic follicles increased. The expression of ovarian P450C17 and P450-aromatase as well as serum E2 concentration were significantly increased in the infected group compared to control. These findings show that chronic infection with Taenia crassiceps WFU may alter the reproductive functions of the female mice host.

The synthesis of steroids by Taenia crassiceps WFU cysticerci and tapeworms is related to the developmental stages of the parasites.

Taeniids tapeworms are hermaphroditic helminths that gradually develop testis and ovaries in their reproductive units. The larval stage of the tapeworms named cysticercus is a vesicle that contains the scolex and proliferates asexually in the abdominal cavity of mice. Once in the host, they evaginate, attach to the gut and develop into an adult organism, the tapeworm. We have previously reported reported that T. crassiceps ORF and solium cysticerci transform steroid precursors to androgens and estrogens. Taenia crassiceps WFU cysticerci can also synthesize corticosteroids. The aim of the present work is to investigate the relationship between steroid synthesis ability and the developmental stage of the parasite T. crassiceps WFU. To this purpose, cysticerci were obtained from the abdominal cavity of female mice, manually separated in invaginated (IC) and evaginated parasites (EC) and preincubated for 24 h in DMEM plus antibiotics/antimycotics. Next step consisted in incubation for different periods in the fresh media added with tritiated androstenedione (3H-A4) or progesterone (3H-P4) and incubated for different periods. Taenia crassiceps WFU tapeworms were recovered from the intestine of golden hamsters that had been orally infected with cysticerci. The worms were pre-cultured in DMEM plus FBS and antibiotics, and then incubated without FBS for different time periods, in the presence of 3H-A4 or 3H-P4. At the end of the experiments the media from cysticerci and tapeworms were analyzed by thin layer chromatography. Results showed that testosterone synthesis was significantly higher in the evaginated cysticerci and increased with time in culture. The invaginated and evaginated cysticerci also synthesized small quantities of 17ß-estradiol (E2) and estrone. The evaginated cysticerci synthesized twice more 3H-deoxycorticosterone (3H-DOC) than the invaginated parasites, the production increased significantly with time in culture. Taenia crassiceps WFU tapeworms synthesized significant quantities of 3H-testosterone and small amounts of estrone after only 3 h of culture in the presence of 3H-A4. The tapeworms also transformed 3H-P4 to 3H-DOC and increased its synthesis after 24 h in culture. In summary, our data show the pathways that T. crassiceps WFU cysticerci use to synthesize sexual steroids in both larval developmental stages and reveals the steroidogenic capacity of the tapeworms.

Sphingosine-1-phosphate, regulated by FSH and VEGF, stimulates granulosa cell proliferation.

Sphingosine-1-phosphate (S1P) is a bioactive polar sphingolipid which stimulates proliferation, growth and survival in various cell types. In the ovary S1P has been shown protect the granulosa cells and oocytes from insults such as oxidative stress and radiotherapy, and S1P concentrations are greater in healthy than atretic large follicles. Hence, we postulate that S1P is fundamental in follicle development and that it is activated in ovarian granulosa cells in response to FSH and VEGF. To test this hypothesis we set out: i) to evaluate the effect of FSH and VEGF on S1P synthesis in cultured bovine granulosa cells and ii) to analyse the effect of S1P on proliferation and survival of bovine granulosa cells in vitro. Seventy five thousand bovine granulosa cells from healthy medium-sized (4-7mm) follicles were cultured in 96-well plates in McCoy's 5a medium containing 10ng/mL of insulin and 1ng/mL of LR-IGF-I at 37°C in a 5% CO2/air atmosphere at 37°C. Granulosa cell production of S1P was tested in response to treatment with FSH (0, 0.1, 1 and 10ng/mL) and VEGF (0, 0.01, 0.1, 1, 10 and 100ng/mL) and measured by HPLC. Granulosa cells produced S1P at 48 and 96h, with the maximum production observed with 1ng/mL of FSH. Likewise, 0.01ng/mL of VEGF stimulated S1P production at 48, but not 96h of culture. Further, the granulosa cell expression of sphingosine kinase-1 (SK1), responsible for S1P synthesis, was demonstrated by Western blot after 48h of culture. FSH increased the expression of phosphorylated SK1 (P<0.05) and the addition of a SK1 inhibitor reduced the constitutive and FSH-stimulated S1P synthesis (P<0.05). Sphingosine-1-phosphate had a biphasic effect on granulosa cell number after culture. At low concentration S1P (0.1µM) increased granulosa cell number after 48h of culture (P<0.05) and the proportion of cells in the G2 and M phase of the cell cycle (P<0.05), whereas higher concentrations decreased cell number (10µM; P<0.05) by an increase (P<0.05) in the proportion of cells in apoptosis (hypodiploid cells). In addition, treatment with SK-178 suppressed the FSH- and VEGF-stimulated rise of the granulosa cells number (P<0.05). Interestingly, the effect of 0.1µM S1P on granulosa cell number and their proportion in G2/M phases is similar to that observed with 1ng/mL FSH. The results of this study are the first to demonstrate sphingosine-1-phosphate (S1P) synthesis in granulosa cells under the control of FSH and VEGF. The later achieved through the regulation of sphingosine kinase 1 expression. This S1P augments the proportion of cells in the G2/M phase of the cell cycle that translates in increased granulosa cell proliferation.

Sphingosine-1-phosphate and ceramide are associated with health and atresia of bovine ovarian antral follicles.

The follicle destiny towards ovulation or atresia is multi-factorial in nature and involves outcries, paracrine and endocrine factors that promote cell proliferation and survival (development) or unchain apoptosis as part of the atresia process. In several types of cells, sphingosine-1-phospate (S1P) promotes cellular proliferation and survival, whereas ceramide (CER) triggers cell death, and the S1P/CER ratio may determine the fate of the cell. The aim of present study was to quantify S1P and CER concentrations and their ratio in bovine antral follicles of 8 to 17 mm classified as healthy and atretic antral follicles. Follicles were dissected from cow ovaries collected from a local abattoir. The theca cell layer, the granulosa cells and follicular fluid were separated, and 17ß-estradiol (E2) and progesterone (P4) concentrations were measured in the follicular fluid by radioimmunoassay. Based on the E2/P4 ratio, the follicles were classified as healthy (2.2±0.3) or atretic (0.2±0.3). In both follicular compartments (granulosa and theca cell layer), sphingolipids were extracted and S1P and CER concentrations were quantified by HPLC (XTerra RP18; 5 µm, 3.0×150 mm column). Results showed that in both follicular compartments, S1P concentrations were higher in healthy antral follicles than in atretic antral follicles (P<0.05). The concentration of CER in the granulosa cells was higher in atretic antral follicles than in healthy antral follicles, but no differences were observed in the theca cell layer. The S1P/CER ratio in both follicular compartments was also higher in healthy antral follicles. Interestingly, in these follicles, there was a 45-fold greater concentration of S1P than CER in the granulosa cells (P<0.05), whereas in the theca cell layer, S1P had only a 14-fold greater concentration than CER when compared with atretic antral follicles. These results suggest that S1P plays a role in follicle health, increasing cellular proliferation and survival. In contrast, reduction of S1P and the S1P/CER in the antral follicle could trigger cellular death and atresia.

Taenia solium tapeworms synthesize corticosteroids and sex steroids in vitro.

Cysticercosis is a disease caused by the larval stage of Taenia solium cestodes that belongs to the family Taeniidae that affects a number of hosts including humans. Taeniids tapeworms are hermaphroditic organisms that have reproductive units called proglottids that gradually mature to develop testis and ovaries. Cysticerci, the larval stage of these parasites synthesize steroids. To our knowledge there is no information about the capacity of T. solium tapeworms to metabolize progesterone or other precursors to steroid hormones. Therefore, the aim of this paper was to investigate if T. solium tapeworms were able to transform steroid precursors to corticosteroids and sex steroids. T. solium tapeworms were recovered from the intestine of golden hamsters that had been orally infected with cysticerci. The worms were cultured in the presence of tritiated progesterone or androstenedione. At the end of the experiments the culture media were analyzed by thin layer chromatography. The experiments described here showed that small amounts of testosterone were synthesized from (3)H-progesterone by complete or segmented tapeworms whereas the incubation of segmented tapeworms with (3)H-androstenedione, instead of (3)H-progesterone, improved their capacity to synthesize testosterone. In addition, the incubation of the parasites with (3)H-progesterone yielded corticosteroids, mainly deoxicorticosterone (DOC) and 11-deoxicortisol. In summary, the results described here, demonstrate that T. solium tapeworms synthesize corticosteroid and sex steroid like metabolites. The capacity of T. solium tapeworms to synthesize steroid hormones may contribute to the physiological functions of the parasite and also to their interaction with the host.

Cloning, characterization and functional expression of Taenia solium 17 beta-hydroxysteroid dehydrogenase.

The 17ß-hydroxysteroid dehydrogenases (17ß-HSD) are key enzymes involved in the formation (reduction) and inactivation (oxidation) of sex steroids. Several types have been found in vertebrates including fish, as well as in invertebrates like Caenorhabditis elegans, Ciona intestinalis and Haliotis diversicolor supertexta. To date limited information is available about this enzyme in parasites. We showed previously that Taenia solium cysticerci are able to synthesize sex steroid hormones in vitro when precursors are provided in the culture medium. Here, we identified a T. solium 17ß-HSD through in silico blast searches in the T. solium genome database. This coding sequence was amplified by RT-PCR and cloned into the pcDNA 3.1(+) expression vector. The full length cDNA contains 957bp, corresponding to an open reading frame coding for 319 aa. The highest identity (84%) at the protein level was found with the Echinococcus multilocularis 17ß-HSD although significant similarities were also found with other invertebrate and vertebrate 17ß-HSD sequences. The T. solium Tsol-17ßHSD belongs to the short-chain dehydrogenase/reductase (SDR) protein superfamily. HEK293T cells transiently transfected with Tsol17ß-HSD induced expression of Tsol17ß-HSD that transformed 3H-androstenedione into testosterone. In contrast, 3H-estrone was not significantly transformed into estradiol. In conclusion, T. solium cysticerci express a 17ß-HSD that catalyzes the androgen reduction. The enzyme belongs to the short chain dehydrogenases/reductase family and shares motifs and activity with the type 3 enzyme of some other species.

Steroid synthesis by Taenia crassiceps WFU cysticerci is regulated by enzyme inhibitors.

Cysticerci and tapeworms from Taenia crassiceps WFU, ORF and Taenia solium synthesize sex-steroid hormones in vitro. Corticosteroids increase the 17ß-estradiol synthesis by T. crassiceps cysticerci. T. crassiceps WFU cysticerci synthesize corticosteroids, mainly 11-deoxycorticosterone (DOC). The aim of this work was to investigate whether classical steroidogenic inhibitors modify the capacity of T. crassiceps WFU cysticerci to synthesize corticosteroids and sex steroid hormones. For this purpose, T. crassiceps WFU cysticerci were obtained from the abdominal cavity of mice, pre-cultured for 24h in DMEM+antibiotics/antimycotics and cultured in the presence of tritiated progesterone ((3)H-P4), androstendione ((3)H-A4), or dehydroepiandrosterone ((3)H-DHEA) plus different doses of the corresponding inhibitors, for different periods. Blanks with the culture media adding the tritiated precursors were simultaneously incubated. At the end of the incubation period, parasites were separated and media extracted with ether. The resulting steroids were separated by thin layer chromatography (TLC). Data were expressed as percent transformation of the tritiated precursors. Results showed that after 2h of exposure of the cysticerci to 100 µM formestane, the (3)H-17ß-estradiol synthesis from tritiated androstenedione was significantly inhibited. The incubation of cysticerci in the presence of (3)H-DHEA and danazol (100 nM) resulted in (3)H-androstenediol accumulation and a significant reduction of the 17ß-estradiol synthesis. The cysticerci (3)H-DOC synthesis was significantly inhibited when the parasites were cultured in the presence of different ketoconazole dosis. The drug treatments did not affect parasite's viability. The results of this study showed that corticosteroid and sex steroid synthesis in T. crassiceps WFU cysticerci can be modified by steroidogenic enzyme inhibitors. As was shown previously by our laboratory and others, parasite survival and development depends on sex steroids, therefore the inhibition of their synthesis is a good starting point exploited in situations where the inhibition of steroidogenesis could help to control the infection for the development of new treatments, or replacement of the usual therapy in resistant parasite infections. We raise the possibility that these drug actions may be beneficially.

Taenia crassiceps WFU cysticerci synthesize corticosteroids in vitro: metyrapone regulates the production.

Taenia solium and Taenia crassiceps WFU cysticerci and tapeworms have the ability to synthesize sex steroid hormones and have a functional 3ß-hydroxisteroid dehydrogenase. Corticosteroids (CS) like corticosterone and dexamethasone have been shown to stimulate in vitro estrogen production by Taenia crassiceps WFU cysticerci. The aim of this work was to study the ability of T. crassiceps WFU cysticerci to synthesize corticosteroids, and the effect of the inhibitor metyrapone on the CS synthesis. For this purpose T. crassiceps WFU cysticerci were obtained from the abdominal cavity of mice, thoroughly washed and pre-incubated in multiwells for 24 h in DMEM plus antibiotics/antimycotics. The tritiated CS precursor progesterone ((3)H-P4) was added to the culture media and parasites cultured for different periods. Blanks containing the culture media plus the (3)H-P4 were simultaneously incubated. Blanks and parasite culture media were ether extracted and analyzed by thin layer chromatography (TLC) in two different solvent systems. Corticosterone production was measured in the culture media by RIA. In some experiments metyrapone (0.1-0.5 mM) was added for 24, 48 or 72 h. Results showed that cysticerci mainly synthesized tritiated 11-deoxy corticosterone (DOC) and small amounts of corticosterone that was also detected by RIA. Small amounts of (3)H-11-deoxy cortisol were also found. Corticosteroid synthesis was time dependent. The addition of metyrapone significantly inhibited tritiated DOC, deoxycortisol and corticosterone synthesis. These results show for the first time that parasites have the capacity to synthesize CS that is modulated by metyrapone. Data suggest that DOC is the main corticosteroid in the parasites.

The effect of glucocorticoids on sex steroid synthesis in cultured Taenia crassiceps Wake Forest University (WFU) cysticerci.

We have shown previously that cultured Taenia crassiceps Wake Forest University (WFU) and Taenia solium cysticerci, as well as the adult worms, synthesize sex steroid hormones from [3H]steroid precursors and that androgens and oestrogens influence the in vitro development of the parasites. Glucocorticoids (GCs) are used to control the inflammation caused by T. solium cysticerci in the brain. These steroids stimulate oestrogen synthesis in several tissues. Since there is no information on the effect of GC on the endocrine function of cysticerci, we investigated the effect of natural and synthetic GCs on the synthesis of oestrogens in cultured T. crassiceps WFU cysticerci. The cysticerci were obtained from the peritoneal cavity of infected female BALB/c mice; the cysts were washed extensively and pre-cultured in Dulbecco's Modified Eagle's Medium (DMEM) plus antibiotics for 5 days. The parasites were further cultured with different doses of corticosterone, dexamethasone or the vehicle for 5 days. [3H]Dehydroepiandrosterone (3H-DHEA) was added to the media and the cysticerci were further incubated for 6 or 24 h. Media were then removed and the steroids ether-extracted. Aliquots of the media were seeded on silica gel plates and developed in solvent systems. Parasites incubated in the presence of 3H-DHEA synthesized [3H]androstenediol, [3H]testosterone and [3H]17ß-oestradiol ([3H]17ß-E2). The addition of 100 nm or higher corticosterone doses to the media increased [3H]17ß-E2 synthesis fourfold after 24 h. Dexamethasone also increased [3H]17ß-E2 synthesis. The experiments presented here show for the first time that corticosterone and the synthetic GC dexamethasone modulate the synthesis of oestrogens by cysticerci.

"Il corpo ritrovato": dermocosmetological skin care project for the oncologic patient.

Neoplastic disease and its therapeutic options have a huge impact on the patient's quality of life from both the emotional and the working point of view. The project "Il Corpo Ritrovato" aims at creating an interdisciplinary network of physicians to improve the quality of life of the oncologic patient, focusing on such important aspects as dermocosmetological skin care but also on the evaluation of new therapeutic and diagnostic algorithms in order to make further progress in the field of prevention.

Vascular endothelial growth factor isoforms 120, 164 and 205 are reduced with atresia in ovarian follicles of sheep.

Vascular endothelial growth factor (VEGF) is a potent stimulator of endothelial cell proliferation and neo-vasculogenesis. In the ovary, VEGF mRNA is localised in the follicle, and it is associated with follicular growth and dominance. Alternative splicing of VEGF mRNA produces eight mature forms of mRNA for equal number of VEGF isoforms. In the present study, the VEGF isoforms in granulosa and theca cells of large (4-6mm) and preovulatory (>6mm) sheep follicles were studied during the process of atresia. Follicles were classified as healthy, early atretic and atretic, and the granulosa and theca cells were isolated. The mRNA for three of these isoforms was found in both theca and granulosa cells, and was quantified by image analysis after RT-PCR using primers that amplified VEGF120, VEGF164, VEGF188 and VEGF205 isoforms. The mRNA for these three isoforms was found in both theca and granulosa cells of healthy and atretic follicles. Atresia was accompanied with a reduction in mRNA for VEGF164 and VEGF120 in granulosa and theca cells (P<0.05). Amounts of both isoforms were reduced with the extent of atresia in the granulosa cells, whilst in the theca cells this reduction was only evident in advanced atretic follicles. Furthermore, after the onset of atresia, VEGF205 was not detectable in the granulosa cells. Follicle size did not affect the amount of VEGF mRNA. Hence, the onset of atresia in follicles of sheep is coupled with a reduction in VEGF mRNA. The decrease in VEGF observed with atresia in follicles of sheep was greater in granulosa than in theca cells.

Differential in vitro effects of insulin on Taenia crassiceps and Taenia solium cysticerci.

Hormones play a significant role in murine cysticercosis (Taenia crassiceps), and increase the frequency of porcine cysticercosis caused by Taenia solium. In the present study, we report the in vitro effect of insulin on the larval stages of T. crassiceps (ORF strain) and T. solium. In vitro exposure of T. crassiceps cysticerci to insulin was found to stimulate this parasite's reproduction twofold with respect to control values, while the same treatment had no effect on T. solium cysticerci. Moreover, normal female mice (BALB/cAnN) infected with T. crassiceps cysticerci previously exposed to insulin presented larger parasite loads than mice inoculated with vehicle-treated cysticerci. To determine the possible molecular mechanisms by which insulin affects T. crassiceps, the insulin receptor was amplified by means of reverse transcriptase-polymerase chain reaction (RT-PCR). Interestingly, both T. crassiceps and T. solium expressed the insulin receptor, although insulin had effects only on T. crassiceps. These results demonstrate that insulin has a dichotomistic effect; it acts directly only on T. crassiceps cysticerci reproduction, possibly through its binding to a specific insulin receptor synthesized by the parasite. Thus, insulin may be recognized by T. crassiceps cysticercus cells as a mitogenic factor, and contribute to parasite proliferation inside the host, as well as to the female mouse susceptibility to T.crassiceps. This phenomenon has not been reported for cysticercosis caused by T. solium, which could, in part, be related to the poor effect of insulin upon the human parasite.

Signaling pathways involved in the effect of follicle-stimulating hormone on chick embryo testis cell proliferation.

The aim of the present study was to evaluate the signaling pathways involved in the follicle-stimulating hormone (FSH) regulated mitogenic activity. For this purpose, 18-d-old chick embryo testis cells were dissociated and cultured for 60 h on polycarbonate membranes. The culture medium was Dulbecco modified Eagle's medium with or without high pure human FSH (hFSH), human recombinant FSH, or different regulators of tyrosine kinase activity as herbimycin A and genistein, or serine/threonine kinases [cyclic adenosine monophosphate (cAMP)-dependent protein serine kinase and protein kinase C] as cAMP, phorbol myristate, and forskolin. In some experiments the regulators were added simultaneously with hFSH. The [3H]-thymidine incorporation was used as an indicator of DNA synthesis. In addition, fragments of chick embryo testis were cultured in the presence or absence of FSH or herbimycin A, and 5-bromo-2'-deoxy-uridine was added to identify the proliferating cell subpopulations. The effect of hFSH on [3H]-thymidine incorporation began at 24 h, and the increment was significant at 36 and 60 h of culture. The hFSH as well as human recombinant FSH significantly stimulated [3H]-thymidine incorporation to testicular cells. The 5-bromo-2'-deoxy-uridine technique showed a high signal in pericordonal and interstitial cells of the hFSH-treated groups, confirming the results obtained using [3H]-thymidine uptake. The treatment with the tyrosine kinase inhibitor herbimycin A increased the [3H]-thymidine uptake, but genistein did not. Regulators of PKA such as cAMP and forskolin, as well as PKC regulators and the phorbol ester phorbol myristate, did not influence cell proliferation. In summary, an inhibitor of tyrosine kinase, herbimycin A, induced per se an increment in chick embryo testis cell proliferation, a fact that strongly suggests that tyrosine kinase signaling pathway functions by inhibiting the proliferation of these cells. On the other hand, the cAMP-PKA pathway had no significant role during the embryonic stage of chick embryo testis. Our results also showed that the effect of FSH on chick embryo cell proliferation occurs mainly in pericordonal and interstitial testis cells.

Taenia crassiceps: chloride currents expressed in Xenopus oocytes upon injection of mRNA of cysticerci (WFU strain) isolated from mice.

To study the properties of ion channels of the tapeworm Taenia crassiceps, mRNA was isolated from cysticerci and injected into mature oocytes of the frog Xenopus laevis and ion currents were recorded four days after injection with the two-electrode voltage clamp technique. Oocytes injected with mRNA of T. crassiceps expressed outward currents (I(TC)) that activated instantly after onset of the test pulse, followed by a slow inactivation at potentials over +40 mV, with a reversal potential of -23.2+/-5 mV. They were not affected by changes on monovalent cationic composition of external media, but replacement of external chloride by gluconate shifted significantly the reversal potential, suggesting that I(TC) are anion currents, with a permeability sequence of NO3->Cl(-)>I(-)>>Gluconate. These currents were sensitive to changes of external pH but not to hypotonic challenges. They were significantly inhibited by DIDS, NPPB and Niflumic acid, but not by 9-anthracene. These results suggest that I(TC) are the result of expression of anion channels from the tapeworm T. crassiceps.

Steroidogenic capacity of Trypanosoma cruzi trypomastigotes.

American Trypanosomiasis is caused by the hemoflagellate Trypanosoma cruzi (T. cruzi) and affects millions of persons causing variable degrees of digestive and heart disturbances. As far as we concerned, T. cruzi capacity to synthesize steroid hormones has not been investigated. Therefore, the aim of this work was to investigate the capacity of T. cruzi trypomastigotes to transform tritiated steroid precursors into androgens and estrogens. The T. cruzi Tulahuén strain was obtained from mice blood. The trypomastigotes were cultured for 6 and 24h in Dulbbeco's modified Eagle's medium plus FCS and antibiotics. Tritiated dehydroepiandrosterone or androstendione were added to the culture media and parasites were incubated for 6 or 24h. The cultures were centrifuged and ether extracted. The steroids were analyzed by thin layer chromatography (TLC) in two solvent systems. After incubation with 3H-androstenedione, T. cruzi trypomastigotes synthesized 3H-testosterone (T), 3H-17beta-estradiol (E2) and 3H-estrone (E1). Metabolism of 3H-DHEA by the parasites yielded 3H-androstendione and 3H-androstendiol at 6h of incubation. The recrystallization procedure further demonstrated the 3H-androstendiol and 3H-17beta-estradiol syntheses. Results indicate for the first time that T. cruzi trypomastigotes produce androgens and estrogens when incubated in the presence of steroid precursors and suggest the presence of active parasite steroidogenic enzymes.

Metabolism of steroid hormones by Taenia solium and Taenia crassiceps cysticerci.

Previous in vitro experiments showed that both, Taenia crassiceps and Taenia solium cysticerci have the ability to metabolize exogenous androstenedione to testosterone. Here we evaluate on the capacity of both cysticerci to synthesize several sex steroid hormones, using different hormonal precursors. Experiments using thin layer chromatography (TLC) showed that both cysticerci were able to produce (3)H-hydroxyprogesterone, (3)H-androstenedione and (3)H-testosterone when (3)H-progesterone was used as the precursor. They also synthesized (3)H-androstenediol and (3)H-testosterone when (3)H-dehydroepiandrosterone was the precursor. In addition, both cysticerci interconverted (3)H-estradiol and (3)H-estrone. These results, strongly suggest the presence and activity of the Delta4 and Delta5 steroid pathway enzymes, 3beta-hydroxysteroid dehydrogenase/Delta(5-4) isomerase-like enzyme (3beta-HSD), that converts androstenediol into testosterone; and the 17beta-hydroxysteroid dehydrogenase that interconverts estradiol and estrone, in both types of cysticerci.

Insulin stimulates proliferation but not 17beta-estradiol production in cultured chick embryo ovarian cells.

The development of the chick embryo gonads is influenced by gonadotropins [follicle-stimulating hormone (FSH), luteinizing hormone, human chorionic gonadotropin (hCG)]. We have previously shown that insulin enhanced the production of androgens in the testis of the newly hatched chicken and increased the proliferation of chick embryo testis cells. In the present paper, we have studied the effect of insulin on embryonic chick embryo ovarian cells and compared them with those of human FSH and hCG. The ovaries of 18-d-old chick embryos were dissociated and cultured for different periods in Dulbecco's modified Eagle's medium in the presence and absence of insulin, human FSH, hCG, and combinations of them. 3H-thymidine incorporation was used as an indicator of cell proliferation; steroids were measured by radioimmunoassay. Results showed that insulin enhanced the proliferation of ovarian cells in a dose- and time-dependent manner. Gonadotropins did not affect significantly the ovarian cell proliferation. Insulin did not change 17beta-estradiol production. The combination of insulin and FSH or insulin and hCG decreased the stimulation of estrogen secretion caused by the addition of the gonadotropins. In some experiments, ovarian cells were cultured with or without insulin, and subpopulations were identified. The results showed that insulin but not human FSH or hCG increased the proliferation of germinal cells after 60 h in culture. Insulin and human FSH did stimulate the other 2 subpopulations. In summary, present results suggest that insulin is an important hormone in the development of the chick embryo ovary.

Taenia solium cysticerci synthesize androgens and estrogens in vitro.

Cysticerci from Taenia solium develop in the pig muscle and cause severe diseases in humans. Here we report on the capacity of T. solium cysticerci to synthesize sex steroid hormones. T. solium cysticerci were dissected from infected pork meat. Parasites were incubated for different periods in culture media plus antibiotics and tritiated steroid precursors. Blanks and parasite culture media were extracted and analyzed by thin-layer chromatography (TLC) in two different solvent systems. In some experiments, the scoleces were incubated separately. Results showed that T. solium cysticerci transform [(3)H]androstenedione to [(3)H]testosterone in a time-dependent manner. The production was confirmed in two different solvent systems. The incubation with [(3)H]testosterone yielded only small amounts of [(3)H]androstenedione. The recrystallization procedure further demonstrated that the metabolite identified by TLC was testosterone. The isolated scoleces incubated in the presence of [(3)H]androstenedione yielded [(3)H]testosterone and small quantities of [(3)H]17beta-estradiol. The results reported here demonstrate that T. solium cysticerci have the capacity to synthesize steroid hormones.

Differentiation of normal and cancer cells induced by sulfhydryl reduction: biochemical and molecular mechanisms.

We examined the morphological, biochemical and molecular outcome of a nonspecific sulfhydryl reduction in cells, obtained by supplementation of N-acetyl-L-cysteine (NAC) in a 0.1-10 mM concentration range. In human normal primary keratinocytes and in colon and ovary carcinoma cells we obtained evidences for: (i) a dose-dependent inhibition of proliferation without toxicity or apoptosis; (ii) a transition from a proliferative mesenchymal morphology to cell-specific differentiated structures; (iii) a noticeable increase in cell-cell and cell-substratum junctions; (iv) a relocation of the oncogenic beta-catenin at the cell-cell junctions; (v) inhibition of microtubules aggregation; (vi) upregulation of differentiation-related genes including p53, heat shock protein 27 gene, N-myc downstream-regulated gene 1, E-cadherin, and downregulation of cyclooxygenase-2; (vii) inhibition of c-Src tyrosine kinase. In conclusion, a thiol reduction devoid of toxicity as that operated by NAC apparently leads to terminal differentiation of normal and cancer cells through a pleiade of converging mechanisms, many of which are targets of the recently developed differentiation therapy.