Paracrine action of sFLT-1 secreted by stably-transfected Ehrlich ascites tumor cells and therapy using sFLT-1 inhibits ascites tumor growth in vivo

Background Vascular endothelial growth factor (VEGF) is known to play a major role in angiogenesis. A soluble form of Flt-1, a VEGF receptor, is potentially useful as an antagonist of VEGF, and accumulating evidence suggests the applicability of sFlt-1 in tumor suppression. In the present study, we have developed and tested strategies targeted specifically to VEGF for the treatment of ascites formation.Methods As an initial strategy, we produced recombinant sFLT-1 in the baculovirus expression system and used it as a trap to sequester VEGF in the murine ascites carcinoma model. The effect of the treatment on the weight of the animal, cell number, ascites volume and proliferating endothelial cells was studied. The second strategy involved, producing Ehrlich ascites tumor (EAT) cells stably transfected with vectors carrying cDNA encoding truncated form of Flt-1 and using these cells to inhibit ascites tumors in a nude mouse model. Results The sFLT-1 produced by the baculovirus system showed potent antiangiogenic activity as assessed by rat cornea and tube formation assay. sFLT-1 treatment resulted in reduced peritoneal angiogenesis with a concomitant decrease in tumor cell number, volume of ascites, amount of free VEGF and the number of invasive tumor cells as assayed by CD31 staining. EAT cells stably transfected with truncated form of Flt-1 also effectively reduced the tumor burden in nude mice transplanted with these cells, and demonstrated a reduction in ascites formation and peritoneal angiogenesis. Conclusions The inhibition of peritoneal angiogenesis and tumor growth by sequestering VEGF with either sFlt-1 gene expression by recombinant EAT cells or by direct sFLT-1 protein therapy is shown to comprise a potential therapy. Copyright (C) 2009 John Wiley & Sons, Ltd.

Estrogen modulation of retinol-binding protein in immature chicks: Comparison with riboflavin carrier protein

The kinetics of estrogen (E) modulation of retinol-binding protein (RBP) production in the liver of immature chicks were compared with those governing de novo induction of riboflavin carrier protein (RCP) in the same tissue. A single dose of E markedly enhanced the plasma levels of RBP without any detectable lag period to reach peak value by 24 h and this was followed by a decline to attain the baseline by 4 days. There was no amplification of the response during secondary stimulation unlike the case with RCP induction. With multiple E administration, the 4-fold increased plasma RBP concentrations were sustained at a steady state during both primary and secondary stimulations, whereas concomitant RCP concentration progressively increased with each hormone administration and this response was further amplified during secondary stimulation. Unlike RCP induction, enhanced RBP accumulation was not strictly E dose dependent although a minimal threshold level of the steroid was required to elicit measurable response. Progesterone (P) could neither modulate nor substitute for E in enhancing plasma levels of either of the 2 proteins while the anti-estrogens, en- and zuclomifene citrate severely suppressed the production of both the proteins. RCP induction was completely inhibited by both α-amanitin and cycloheximide for prolonged periods while E-stimulated RBP production was affected only partially by α-amanitin. Likewise, cycloheximide inhibition of RBP accumulation followed a pattern similar to that of hepatic general protein synthesis.

Estrogen Induction Of Biotin-Binding Protein In Immature Chicks - Kinetics, Hormonal Specificity And Modulation

Employing a specific radioimmunoassay for quantification, the kinetics of estrogen-induced elevation in the plasma concentration of biotin-binding protein (BBP) in immature male chicks was investigated. A single injection of the steroid hormone enhanced the plasma BBP content several-fold at 6 h, reaching peak levels around 48 h and declining thereafter. A 2-fold amplification of the response was evident during secondary stimulation with the hormone. The magnitude of the response was hormonal dose-dependent while the initial lag phase and the time of peak protein accumulation were unaltered within the hormonal doses tested. The circulatory half-life of the specific protein in normal and estrogenized birds was 10 h. Hyperthyroidism markedly decreased the hormonal response while the opposite effect was seen during hypothyroidism. The antiestrogens E- and Z-clomiphene citrate effectively blocked the protein induction whereas progesterone, either alone or in combination with estrogen, was ineffective in modulating the induction. Cycloheximide administration drastically inhibited the inductive response. The above observations clearly suggest that the genes corresponding to the two isofunctional proteins of chicken egg, viz. BBP and avidin, are differentially regulated.

Estrogen modulation of riboflavin carrier protein in the bonnet monkey (Macaca radiata)

Circulatory concentrations of riboflavin carrier protein (RCP) were quantitated in bonnet macaques by employing a heterologous radioimmunoassay involving 125I-labelled chicken RCP and its antiserum. The levels of monkey RCP in the serum seem to be governed by the estrogenic status of the animals. An increase in concentration of serum estradiol in the adult females during the menstrual cycle and early pregnancy could be correlated with enhanced serum RCP levels. Estadiol-17β administered to both immature female and male monkeys, specifically brought about elevated levels of RCP with a slower time course of response in males than in females. These results could be a reflection of a more rapid decline of both circulatory estrogen and RCP concentrations in male serum. Repeated administration of estradiol-17β to male animals led to prolonged elevated levels of RCP following estrogen administration. Thus, it would appear that the evolutionary conservation of RCPs from the aves to the primates encompasses not only their physicochemical similarities but also extends to the estrogenic modulation of their elaboration.

Induction of riboflavin-carrier protein in the immature male rat by estrogen: kinetic and hormonal specificity

The kinetics of estrogen-induced accumulation of riboflavin-carrier protein in the plasma was investigated in immature male rats using a specific and sensitive homologous radio-immunoassay procedure developed for this purpose. Following a single injection of the steroid hormone, plasma riboflavin-carrier protein levels increased markedly after an initial lag period of approximately 24 h, reaching peak levels around 96 h and declining thereafter. A 1.5 fold amplification of the inductive response was evident on secondary stimulation with the hormone. The magnitude of the response was dependent on hormonal dose, whereas the initial lag phase and the time of peak riboflavin-carrier protein induction were unaltered within the range of the steroid doses (0.1–10 mg/ kg body wt.) tested. Simultaneous administration of progesterone did not affect either the kinetics or the maximum level of the protein induced. The hormonal specificity of this induction was further adduced by the effect of administration of antiestrogens viz., En and Zu chlomiphene citrates, which effectively curtailed hormonal induction of the protein. That the induction involvedde novo-protein synthesis was evident from the complete inhibition obtained upon administration of cycloheximide. Passive immunoneutralization of endogenous riboflavin-carrier protein with antiserum to the homologous protein terminated pregnancy in rats confirming the earlier results with antiserum to chicken riboflavin-carrier protein.

Comparative mparative Effects of Antiserum to Luteinizing Hormone on Pseudopregnancy and Pregnancy Induced in the Same Rat

The comparative role of luteinizing hormone (LH) in maintaining pregnancy and histamine-induced decidualization in the rat was studied with the help of a new system, wherein the above two states could be brought about simultaneously in the same animal, but in different uterine horns. Specific and well-characterized LH antiserum, administered daily, both during the pre-trauma (days 1-4) and post-trauma (days 5-8) periods, resulted in the termination of pregnancy and inhibition of decidualization. This antiserum effect could be reversed by suitable steroid therapy. Results suggest that the antiserum blockade of ovarian steroidogenesis continued even after cessation of its treatment. Early pregnancy and decidualization seem directly comparable in that both are dependent upon LH to stimulate the ovarian synthesis of much-needed progesterone and estrogen.

Estrogen-induced synthesis of riboflavin-binding protein in immature chicks kinetics and hormonal specificity

The kinetics of estrogen-induced elevation in the plasma concentration of riboflavin-binding protein, a minor yolk constituent, was investigated in immature male chicks, using a specific and sensitive radioimmunoassay proceudre. Following a single injection of the hormone, the plasma riboflavin-binding protein content was enhanced several-fold at 6 h. reaching peak levels around 48 h and declining thereafter. A two-fold amplication of the response was evident on secondary stimulation with the hormone. A 4-h lag phase prior to onset of induction was noticed during both primary and secondary stimulat ions with the steroid hormone. The magnitude of the response was dependent on the hormonal dose whereas the initial lag phase and the time of peak riboflavin-binding protein accumulation were unaltered within the range of hormonal doses tested. The half-life of riboflavin-binding protein in the circulation was 10 h, as calculated from measurement of the rate of disappearance of exogenously administered 125I-labelled protein. Simultaneous administration of progestrone did bot affect the kinetics of riboflavin-binding protein production. On the other hand, the antiestrogens, cis- and trans-clomiphene citrates, given 30 min prior to estrogen and cycloheximide, effectively countered the hormone-induced riboflavin-binding protein elaboration. Both progesterone and the anti-esterogens per se were completely ineffective in substituting for estrogen in the inductive ptrocess.

Gene therapy: Principles, practice, problems and prospects

The remarkable advances made in recombinant DNA technology over the last two decades have paved way for the use of gene transfer to treat human diseases. Several protocols have been developed for the introduction and expression of genes in humans, but the clinical efficacy has not been conclusively demonstrated in any of them. The eventual success of gene therapy for genetic and acquired disorders depends on the development of better gene transfer vectors for sustained, long term expression of foreign genes as well as a better understanding of the pathophysiology of human diseases, it is heartening to note that some of the gene therapy protocols have found other applications such as the genetic immunization or DNA vaccines, which is being heralded as the third vaccine revolution, Gene therapy is yet to become a dream come true, but the light is seen at the end of the tunnel.

Blockade of estrogen synthesis with an aromatase inhibitor affects luteal function of the pseudopregnant rat

The luteotropic action of estrogen (E) was investigated using immature pseudopregnant rat as the model and CGS 16949A (Fadrozole hydrochloride), a potent aromatase inhibitor (AI), to block E synthesis. Aromatase activity could be inhibited by administering CGS 16949A (50 mu g/day/rat) via a mini osmotic Alzet pump (model 2002) for 3 days during pseudopregnancy. This resulted in significant reduction of serum (40%, P < 0.05) and intraovarian (70.6%, P < 0.001) estradiol-17 beta (E(2)) levels. The serum and intraovarian progesterone (P-4) levels as analyzed on day 4 of pseudopregnancy were also reduced by greater than or equal to 50% (for both, P < 0.01). Simultaneous administration of estradiol-3-benzoate (E(2)B) via an Alzet pump during the Al: treatment period at a dose of 1 mu g/day could completely reverse the Al induced reduction in P-4 secretion. The luteal cells of experimental rats depleted of E in vivo showed a significantly reduced response upon incubation with hCG or dbcAMP in vitro (P < 0.05 and 0.001, respectively). Addition of E(2) (500 pg/tube) at the time of in vitro incubation was able to partially increase the responsiveness to hCG. The luteal cell LH/hCG receptor content and the affinity of hCG binding to the receptor remained unchanged following AI treatment in vivo. Both esterified and total cholesterol content of luteal cells of rats treated with Al in vivo was significantly high (P < 0.05) suggesting that E lack results in an impairment in cholesterol utilization for steroidogenesis. The results clearly show that E regulates luteal function in the pseudopregnant rat by acting at a non-cAMP mediated event and this perhaps involves facilitation of cholesterol utilization at the mitochondrial level for P-4 synthesis.

HDAC inhibitor valproic acid enhances tumor cell kill in adenovirus-HSVtk mediated suicide gene therapy in HNSCC xenograft mouse model

Safety, efficacy and enhanced transgene expression are the primary concerns while using any vector for gene therapy. One of the widely used vectors in clinical. trials is adenovirus which provides a safe way to deliver the therapeutic gene. However, adenovirus has poor transduction efficiency in vivo since most tumor cells express low coxsackie and adenovirus receptors. Similarly transgene expression remains low, possibly because of the chromatization of adenoviral genome upon infection in eukaryotic cells, an effect mediated by histone deacetylases (HDACs). Using a recombinant adenovirus (Ad-HSVtk) carrying the herpes simplex thymidine kinase (HSVtk) and GFP genes we demonstrate that HDAC inhibitor valproic acid can bring about an increase in CAR expression on host cells and thereby enhanced Ad-HSVtk infectivity. It also resulted in an increase in transgene (HSVtk and GFP) expression. This, in turn, resulted in increased cell kill of HNSCC cells, following ganciclovir treatment in vitro as well as in vivo in a xenograft nude mouse model.

Ternary Iron(III) complex showing photocleavage of DNA in the photodynamic therapy window

A new ternary iron(III) complex [FeL(dpq)] containing dipyridoquinoxaline (dpq) and 2,2-bis(3,5-di-tert-butyl-2-hydroxybenzyl)aminoacetic acid (H3L) is prepared and structurally characterized by X-ray crystallography. The high-spin complex with a FeN3O3 core shows a quasi-reversible iron(III)/iron(II) redox couple at -0.62 V (vs SCE) in DMF/0.1 M TBAP and a broad visible band at 470 nm in DMF/Tris buffer. Laser photoexcitation of this phenolate (L)-to-iron(III) charge-transfer band at visible wavelengths including red light of >= 630 nm leads to cleavage of supercoiled pUC19 DNA to its nicked circular form via a photoredox pathway forming hydroxyl radicals.

Examination of the role of follicle stimulating hormone in estrogen biosynthesis Image Image and Image Image in the ovary of cyclic hamster

The effect of neutralizing endogenous follicle stimulating hormone (FSH) or luteinizing hormone (LH) with specific antisera on the Image Image and Image Image synthesis of estrogen in the ovary of cycling hamster was studied. Neutralization of FSH or LH on proestrus resulted in a reduction in the estradiol concentration of the ovary on diestrus-2 and next proestrus, suggesting an impairment in follicular development.Injection of FSH antiserum at 0900 h of diestrus-2 significantly reduced the ovarian estradiol concentration within 6–7 h. Further, these ovaries on incubation with testosterone(T) Image Image at 1600 h of the same day or the next day synthesized significantly lower amounts of estradiol, compared to corresponding control ovaries. Although testosterone itself, in the absence of endogenous FSH, could stimulate estrogen synthesis to some extent, FSH had to be supplemented with T to restore estrogen synthesis to the level seen in control ovaries incubated with T. Lack of FSH thus appeared to affect the aromatization step in the estrogen biosynthetic pathway in the ovary of hamster on diestrus-2. In contrast to this, FSH antiserum given on the morning of proestrus had no effect on the Image Image and Image Image synthesis of estrogen, when examined 6–7 h later. The results suggest that there could be a difference in the need for FSH at different times of the cycle.Neutralization of LH either on diestrus-2 or proestrus resulted in a drastic reduction in estradiol concentration of the ovary. This block was at the level of androgen synthesis, since supplementing testosterone alone Image Image could stimulate estrogen synthesis to a more or less similar extent as in the ovaries of control hamsters.

Studies on follicular growth in the immature rat and hamster: Effect of a single injection of gonadotropin or estrogen on the rate of3H-thymidine incorporation into ovarian DNAin vitro

Initiation of follicular growth by specific hormonal stimuli in ovaries of immature rats and hamsters was studied by determining the rate of incorporation of3H-thymidine into ovarian DNAin vitro. Incorporation was considered as an index of DNA synthesis and cell multiplication. A single injection of pregnant mare serum gonadotropin could thus maximally stimulate by 18 hr3H-thymidine incorporation into DNA of the ovary of immature hamsters. Neutralization of pregnant mare serum gonadotropin by an antiserum to ovine follicle stimulating hormone only during the initial 8–10 hr and not later could inhibit the increase in3H-thymidine incorporationin vitro observed at 18 hr, suggesting that the continued presence of gonadotropin stimulus was not necessary for this response. The other indices of follicular growth monitored such as ovarian weight, serum estradiol and uterine weight showed discernible increase at periods only after the above initial event. A single injection of estrogen (diethyl stilbesterol or estradiol-l7β) could similarly cause 18 hr later, a stimulation in the rate of incorporation of3H-thymidine into DNAin vitro in ovaries of immature rats. The presence of endogenous gonadotropins, however, was obligatory for observing this response to estrogen. Evidence in support of the above was two-fold: (i) administration of antiserum to follicle stimulating hormone or luteinizing hormone along with estrogen completely inhibited the increase in3H-thymidine incorporation into ovarian DNAin vitro; (ii) a radioimmunological measurement revealed following estrogen treatment, the presence of a higher concentration of endogenous follicle stimulating hormone in the ovary. Finally, administration of varying doses of ovine follicle stimulating hormone along with a constant dose of estrogen to immature rats produced a dose-dependent increment in the incorporation of3H-thymidine into ovarian DNAin vitro. These observations suggested the potentiality of this system for developing a sensitive bioassay for follicle stimulating hormone.