An Antitubulin Agent BCFMT Inhibits Proliferation of Cancer Cells and Induces Cell Death by Inhibiting Microtubule Dynamics

Using cell based screening assay, we identified a novel anti-tubulin agent (Z)-5-((5-(4-bromo-3-chlorophenyl)furan-2-yl)methylene)-2-thioxothiazoli din-4-one (BCFMT) that inhibited proliferation of human cervical carcinoma (HeLa) (IC50, 7.2 +/- 1.8 mu M), human breast adenocarcinoma (MCF-7) (IC50, 10.0 +/- 0.5 mu M), highly metastatic breast adenocarcinoma (MDA-MB-231) (IC50, 6.0 +/- 1 mu M), cisplatin-resistant human ovarian carcinoma (A2780-cis) (IC50, 5.8 +/- 0.3 mu M) and multi-drug resistant mouse mammary tumor (EMT6/AR1) (IC50, 6.5 +/- 1 mu M) cells. Using several complimentary strategies, BCFMT was found to inhibit cancer cell proliferation at G2/M phase of the cell cycle apparently by targeting microtubules. In addition, BCFMT strongly suppressed the dynamics of individual microtubules in live MCF-7 cells. At its half maximal proliferation inhibitory concentration (10 mu M), BCFMT reduced the rates of growing and shortening phases of microtubules in MCF-7 cells by 37 and 40%, respectively. Further, it increased the time microtubules spent in the pause (neither growing nor shortening detectably) state by 135% and reduced the dynamicity (dimer exchange per unit time) of microtubules by 70%. In vitro, BCFMT bound to tubulin with a dissociation constant of 8.3 +/- 1.8 mu M, inhibited tubulin assembly and suppressed GTPase activity of microtubules. BCFMT competitively inhibited the binding of BODIPY FL-vinblastine to tubulin with an inhibitory concentration (K-i) of 5.2 +/- 1.5 mu M suggesting that it binds to tubulin at the vinblastine site. In cultured cells, BCFMT-treatment depolymerized interphase microtubules, perturbed the spindle organization and accumulated checkpoint proteins (BubR1 and Mad2) at the kinetochores. BCFMT-treated MCF-7 cells showed enhanced nuclear accumulation of p53 and its downstream p21, which consequently activated apoptosis in these cells. The results suggested that BCFMT inhibits proliferation of several types of cancer cells including drug resistance cells by suppressing microtubule dynamics and indicated that the compound may have chemotherapeutic potential.

Primary Microcephaly Gene MCPH1 Shows Signatures of Tumor Suppressors and Is Regulated by miR-27a in Oral Squamous Cell Carcinoma

Mutations in the MCPH1 (microcephalin 1) gene, located at chromosome 8p23.1, result in two autosomal recessive disorders: primary microcephaly and premature chromosome condensation syndrome. MCPH1 has also been shown to be downregulated in breast, prostate and ovarian cancers, and mutated in 1/10 breast and 5/41 endometrial tumors, suggesting that it could also function as a tumor suppressor (TS) gene. To test the possibility of MCPH1 as a TS gene, we first performed LOH study in a panel of 81 matched normal oral tissues and oral squamous cell carcinoma (OSCC) samples, and observed that 14/71 (19.72%) informative samples showed LOH, a hallmark of TS genes. Three protein truncating mutations were identified in 1/15 OSCC samples and 2/5 cancer cell lines. MCPH1 was downregulated at both the transcript and protein levels in 21/41 (51.22%) and 19/25 (76%) OSCC samples respectively. A low level of MCPH1 promoter methylation was also observed in 4/40 (10%) tumor samples. We further observed that overexpression of MCPH1 decreased cellular proliferation, anchorage-independent growth in soft agar, cell invasion and tumor size in nude mice, indicating its tumor suppressive function. Using bioinformatic approaches and luciferase assay, we showed that the 3'-UTR of MCPH1 harbors two non-overlapping functional seed regions for miR-27a which negatively regulated its level. The expression level of miR-27a negatively correlated with the MCPH1 protein level in OSCC. Our study indicates for the first time that, in addition to its role in brain development, MCPH1 also functions as a tumor suppressor gene and is regulated by miR-27a.

Effect of Ethanol Extract of Whole Plant of Trichosanthes cucumerina var. cucumerina L. on Gonadotropins, Ovarian Follicular Kinetics and Estrous Cycle for Screening of Antifertility Activity in Albino Rats

Ethanol extract of whole plant of Trichosanthes cucumerina L. var. cucumerina was evaluated for antiovulatory activity in adult rats. The ethanol extract at the doses 200 and 400mg/kg body weight (orally) affected the normal estrous cycle showing a significant increase in estrus and metestrus phases and decrease in diestrus and proestrus phases. The extract also significantly reduced the number of healthy follicles (Class I-Class VI) and corpora lutea and increased the number of regressing follicles (Stage IA, Stage IB, Stage IIA, and Stage IIB). The protein and glycogen content in the ovaries were significantly reduced in treated rats. The cholesterol level was significantly increased, whereas, the enzyme activities like 3b-HSD and 17b-HSD were significantly inhibited in the ovary of treated rats. Serum FSH and LH levels were significantly reduced in the treated groups were measured by RIA. In acute toxicity test, neither mortality nor change in the behavior or any other physiological activities in mice were observed in the treated groups. In chronic toxicity studies, no mortality was recorded and there were no significant differences in the body and organ weights were observed between controls and treated rats. Hematological analysis showed no significant differences in any of the parameters examined (RBC, WBC count and Hemoglobin estimation). These observations showed the antiovulatory activity of ethanol extract of whole plant of Trichosanthes cucumerina L. var. cucumerina in female albino rats.

Studies on Rat Ovarian Receptors for Lutropin (Luteinizing Hormone): Applicability of radioimmunoassay to measure lutropin bound to receptors.

Measurement of receptor-bound unlabelled physiologically active lutropin (luteinizing hormone, LH) was possible by a modified radioimmunoassay. The conventional radioimmunoassayconducted at 4°C was inadequate, whereas the modified assay performed at 37'C could measure receptor-bound lutropin. The radioimmunoassay at 37'C takes only 36h for completion compared with 5-7 days at 4°C. The sensitivity and range of dose-response curves are, however, unaltered. The validity of the technique was established by a number of criteria.

Dynamics of Circulating Concentrations of Gonadotropins and Ovarian Hormones Throughout the Menstrual Cycle in the Bonnet Monkey: Role of Inhibin A in the Regulation of Follicle-Stimulating Hormone Secretion

In higher primates, increased circulating follicle-stimulating hormone (FSH) levels seen during late menstrual cycle and during menstruation has been suggested to be necessary for initiation of follicular growth, recruitment of follicles and eventually culminating in ovulation of a single follicle. With a view to establish the dynamics of circulating FSH secretion with that of inhibin A (INH A) and progesterone (P-4)secretions during the menstrual cycle, blood was collected daily from bonnet monkeys beginning day 1 of the menstrual cycle up to 35 days. Serum INH A levels were low during early follicular phase, increased significantly coinciding with the mid cycle luteinizing hormone (LH) surge to reach maximal levels during the mid luteal phase before declining at the late luteal phase, essentially paralleling the pattern Of P-4 secretion seen throughout the luteal phase. Circulating FSH levels were low during early and mid luteal phases, but progressively increased during the late luteal phase and remained high for few days after the onset of menses. In another experiment, lutectomy performed during the mid luteal phase resulted in significant decrease in INH A concentration within 2 hr (58.3 +/- 2 vs. 27.3 +/- 3 pg/mL), and a 2- to 3-fold rise in circulating FSH levels by 24 hr (0.20 +/- 0.02 vs. 0.53 +/- 0.14 ng/mL) that remained high until 48 hr postlutectomy. Systemic administration of Cetrorelix (150 mu g/kg body weight), a gonadotropin releasing hormone receptor antagonist, at mid luteal phase in monkeys led to suppression of serum INH A and P-4 concentrations 24 hr post treatment, but circulating FSH levels did not change. Administration of exogenous LH, but not FSH, significantly increased INH A concentration. The results taken together suggest a tight coupling between LH and INH A secretion and that INH A is largely responsible for maintenance of low FSH concentration seen during the luteal phase. Am. J. Primatol. 71:817-824, 2009.

Effect Of Human Chorionic Gonadotrophin And Ovine Luteinizing Hormone On Rat Ovarian Macromolecular Metabolism

Administration of human chorionic gonadotrophin (HCG) or ovine LH to immature rats primed with pregnant mare serum gonadotrophin (PMSG) stimulated the rate of synthesis of polyadenylic acid (poly A)-rich RNA in the ovaries. The rate of total RNA synthesis was not affected significantly by hormone treatment, whereas protein synthesis was enhanced. The increase in the rate of synthesis of poly(A)-rich RNA in the ovaries could be inferred as induction of messenger RNA synthesis after the hormone treatment. The poly(A)-rich nature of the isolated RNA was established by oligo(dT)–cellulose chromatography, binding to Millipore filter disks and hydridization with [3H]polyuridylic acid. The level of cyclic AMP in the ovaries of such rats was also raised after administration of LH, the increase coincided with the increase in the rate of synthesis of poly(A)-rich RNA. The implications of these results are discussed in the light of the biochemical basis of luteinization and the action of LH.

Middle aged wasps mate through most of the year, without regard to body size, ovarian development and nestmateship: a laboratory study of the primitively eusocial wasp Ropalidia marginata

We studied the mating behaviour of the primi-tively eusocial wasp Ropalidia marginata and the factors that may influence sperm transfer. By introducing a male and a female R. marginata into ventilated transparent plastic boxes, we were able to observe mating behaviour, and it involved mounting and short or long conjugation of the wasps. Dissection of female wasps after the observation indicated that long conjugation is a good behavioural predictor of sperm transfer. This finding makes it possible to obtain mated females without dissecting them every time. We tested the effect of age, season, relatedness, body size and female's ovarian status on mating. Under laboratory conditions, mating success declined rapidly below and above the ages 5-20 days. Within this age range mating success was significantly low in December compared to other months tested. There was no nestmate discrimination, and there was no influence of male and female body size or of the ovarian state of the female on the probability of sperm transfer.

Heat-shock protein 70-2 (HSP70-2) expression in bladder urothelial carcinoma is associated with tumour progression and promotes migration and invasion

Purpose: Testis specific heat-shock protein 70-2 (HSP70-2), a member of HSP70 chaperone family, is essential for the growth of spermatocytes and cancer cells. We investigated the association of HSP70-2 expression with clinical behaviour and progression of urothelial carcinoma of bladder. Experimental design: We assessed the HSP70-2 expression by RT-PCR and HSP70-2 protein expression by immunofluorescence, flow cytometry, immunohistochemistry and Western blotting in urothelial carcinoma patient specimens and HTB-1, UMUC-3, HTB-9, HTB-2 and normal human urothelial cell lines. Further, to investigate the role of HSP70-2 in bladder tumour development, HSP70-2 was silenced in the high-grade invasive HTB-1 and UMUC-3 cells. The malignant properties of urothelial carcinoma cells were examined using colony formation, migration assay, invasion assay in vitro and tumour growth in vivo. Results: Our RT-PCR analysis and immunohistochemistry analysis revealed that HSP70-2 was expressed in both moderate to well-differentiated and high-grade invasive urothelial carcinoma cell lines studied and not in normal human urothelial cells. In consistence with these results, HSP70-2 expression was also observed in superficially invasive (70%) and muscle-invasive (90%) patient's tumours. Furthermore, HSP70-2 knockdown significantly suppressed cellular motility and invasion ability. An in vivo xenograft study showed that inhibition of HSP70-2 significantly suppressed tumour growth. Conclusions: In conclusion, our data suggest that the HSP70-2 expression is associated with early spread and progression of urothelial carcinoma of bladder cancer and that HSP70-2 can be the potential therapeutic target for bladder urothelial carcinoma. (C) 2009 Elsevier Ltd. All rights reserved.

Identification of genes associated with tumorigenesis of meibomian cell carcinoma by microarray analysis

Meibomian cell carcinoma (MCC) is a malignant tumor of the meibomian glands located in the eyelids. No information exists on the cytogenctic and genetic aspects of MCC. There is no report on the gene expression profile of MCC. Thus there is a need, for both scientific and clinical reasons, to identify genes and pathways that are involved in the development and progression of MCC. We analyzed the gene expression profile of MCC by the microarray technique. Forty-four genes were upregulated and 149 genes were downregulated in MCC. Differential expression data were confirmed for 5 genes by semiquantitative RT-PCR in MCC tumors: GTF2H4, RBM12, UBE2D3, DDX17, and LZTS1. We found dysregulation of two major pathways in MCC: MAPK and JAK/STAT. Clusters of genes on chromosomes 1, 12, and 19 were dysregUlated in MCC. The data presented here will facilitate the identification of specific markers and therapeutic targets for the treatment of MCC patients. (c) 2007 Elsevier Inc. All rights reserved.

Simultaneous effect of lead and cadmium on granulosa cells: A cellular model for ovarian toxicity

Lead (Pb) and cadmium (Cd) are known reproductive toxicants, which accumulate in granulosa cells of the ovary. Female Charles foster rats were treated with sodium acetate (control), lead acetate and cadmium acetate either alone or in combination at a dose 0.05 mg/kg body weight intra-peritoneally for 15 days daily. Animals were killed at proestrous stage and granulosa cells were isolated from the ovaries. Binding of I-125-luteinizing hormone (I-125-LH), I-125-follicle stimulating hormone (I-125-FSH) and 17 beta-hydroxysteroid dehydrogenase activity were measured. As these receptors are localized on the surface of the cell membrane, we also estimated the membrane parameters of these cells. Our results demonstrated that both lead and cadmium caused a significant reduction in gonadotropin binding, which altered steroidogenic enzyme activity of granulosa cells. These changes exhibited a positive correlation with membrane changes of the granulosa cells.

Gonadotropin regulation of rat ovarian lysosomes: Existence of a hormone specific dual control mechanism

Gonadotropic hormones PMSG (15 IU/rat), FSH (3 mgrg/rat), LH (9 mgrg/rat) and hCG (3 mgrg/rat) were shown to decrease the free cytosolic lysosomal enzymes during the acute phase of hormone action in rat ovaries. When isolated cells from such rats were analyzed for the cathepsin-D activity, the granulosa cells of the ovary showed a reduction in the free as well as in the total lysosomal enzyme activities in response to FSH/PMSG; the stromal and thecal compartment of the ovary showed a reduction only in the free activity in response to hCG/PMSG. The results suggest the presence of two distinct, target cell specific, mechanisms by which the lysosmal activity of the ovary is regulated by gonadotropins.

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.

Plasmid DNA mediated transfer of the herpes simplex virus thymidine kinase gene to a new bromodeoxyuridine resistant variant of human primary lung carcinoma cells

A human primary lung carcinoma cell line (HPL-R1) established from the tumor biopsy of a lung cancer patient, lacking in cytochrome P1-450 [aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH)], was cloned and used to obtain variants deficient in the expression of thymidine-kinase via treatment with 5-bromo-2'-deoxyuridine, and selection for drug resistance phenotype. The variant cell line, precharacterized for thymidine kinase negative phenotype, was transfected with the thymidine kinase gene bearing p R-tk and px1-tk plasmids. Transfections from both the plasmids, demonstrated a frequency of 5.5 X 10(-5). The transfectants showed a 76-100% retention of the transferred phenotype. These data suggest that transfection in variant human cells can approach significant levels of stability observed with rodent cell recipients.

Measurement of FSH in the ovarian tissue by radioimmunoassay: Correlation to serum FSH levels and follicular development in the hamster

A method is described for monitoring the concentration of endogenous receptor-bound gonadotropin in the ovarian tissue. This involved development of a radioimmunoassay procedure, the validity of which for measuring all of the tissue-bound hormone has been established. The specificity of the method of measurement was indicated by the fact that high levels of FSH could be measured only in target tissue such as follicles, while non-target organs showed little FSH. Using this method, the amount of FSH in the non-luteal ovarian tissue of the hamster at different stages of the estrous cycle was quantitated and compared with serum FSH levels found at these times. No correlation could be found between serum and tissue FSH levels at all times. On the morning of estrus, for example, when the serum level of FSH was high, the ovarian concentration was low, and on the evening of diestrus-2 the ovary exhibited high concentration of FSH, despite the serum FSH concentration being low at this time. The highest concentration of FSH in the ovary during the cycle was found on the evening of proestrus. Although a large amount of this was found in the Graafian follicles, a considerable amount could still be found in the �growing� follicles. Ovarian FSH concentration could be considered to be a reflection of FSH receptor content, since preventing the development of FSH receptors by blocking initiation of follicular development during the cycle resulted in a decrease in the concentration of FSH in the ovary. The high concentration of FSH in the ovary seen on the evening of diestrus-2 was not influenced either by varying the concentration of estrogen or by neutralization of LH. Neutralization of FSH on diestrus-2, on the other hand, caused a drastic reduction in the ovarian LH concentration on the next day (i.e. at proestrus), thus suggesting the importance of FSH in the induction of LH receptors.

Effect of Neutralization of Endogenous Follicle Stimulating Hormone (FSH) or Luteinizing Hormone (LH) on Ovarian Lipids in the Hamster: A Histochemical and Biochemical Evaluation

The effect of neutralizing FSH or LH on ovarian lipids in the cycling hamster was studied. In the normal cycling hamster on the day of proestrus, histochemical examination revealed the presence of sudanophilic lipids in the granulosa cells of the follicles and in the interstitium. A clear reduction in the intensity of lipid staining was observed on proestrus in the ovary of hamsters treated with FSH antiserum on the previous proestrus. Similar treatment with antiserum to LH, on the other hand, caused an accumulation of lipids in these structures. Estimation of the free and esterified fractions of cholesterol and triglycerides in the nonluteal tissue of the ovary of hamsters on proestrus following treatment with FSH antiserum on the previous proestrus revealed a significant reduction in all 3 lipid components. Even a short term deprivation of FSH caused a similar reduction in these lipids in the ovary. In contrast, treatment with LH antiserum either on the previous proestrus or on the previous day (diestrus-2) resulted in an enhancement in esterified cholesterol and triglycerides, while it caused a reduction in the free cholesterol fraction of the ovary on proestrus.It is suggested that though treatment with antisera to either FSH or LH causes a disruption in follicular maturation, their effect on lipid metabolism is different. A positive role for FSH and LH in maintaining normal sterol and triglyceride levels in the nonluteal ovarian tissue of cycling hamster is indicated.