5-aza-2'-deoxycytidine induces apoptosis and inhibits tumour growth in vivo of FaDu cells, a specific HPVnegative HNSCC cell line.

Head and neck cancer squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, resulting in over 600,000 new diagnoses annually. Traditionally, HNCC has been related to tobacco and alcohol exposure; however, over the past decade, a growing number of head and neck cancers are attributed to human papillomavirus (HPV) infection. 5-Aza-2'-deoxycytidine (5-AzaD) was demonstrated as an effective chemotherapeutic agent for acute myelogenous leukaemia. Preclinical data revealed that 5-aza inhibits growth and increases cell death of HPV(+) cancer cells. These effects are associated with reduced expression of HPV genes, stabilization of TP53, and activation of TP53-dependent apoptosis. The aim of the present study is to test the effect of 5-AzaD on growth of human squamous cell carcinoma (FaDu), a HPV(-) and p53 mutated cells, in vitro and in vivo. The effect of 5-AzaD on cell viability, cell cycle progression and induction of apoptosis was tested in vitro. The effect of 5-AzaD on tumour growth in vivo was tested using xenograft mice inoculated with FaDu cells. The results indicated that 5-AzaD reduced cell viability and induced apoptosis in FaDu cells in vitro. In vivo studies revealed that 5-AzaD suppresses the growth of tumours in xenograft mice inoculated with FaDu cells through inhibition of proliferation and induction of apoptosis. These findings may emphasis that 5-AzaD is effective in treatment of HPV(-) HNSCC tumours through TP53 independent pathway. Future studies are needed in order to clarify the molecular mechanism of action of 5-AzaD in HPV(-) cancer cells.

A Novel Follitropin Analog Inhibits Follitropin Activity In Vitro.

Follitropin (FSH) is a heterodimeric protein composed of an α subunit that is shared with the glycoprotein hormone family, including lutropin (LH), thyrotropin (TSH), human choriogonadotropin (hCG), and a unique ß specific subunit. Both α and FSHß subunits contain two sites of N-linked oligosaccharides, which are important for its function. FSH has a crucial function in the reproductive process in mammals. However, there are some clinical conditions, such as menopausal osteoporosis or adiposity, associated with increased FSH activity. Moreover, in some cases, carcinogenesis is evidently associated with activation of FSH receptor. Therefore, developing a follitropin antagonist might be beneficial in the treatment of these conditions. Here, we describe a novel, engineered, non-glycosylated single-chain FSH variant, prepared by site-directed mutagenesis and fusion of the coding genes of the α and ß subunits. The designed variant was expressed in Chinese hamster ovary (CHO) cells and successfully secreted into the culture medium. We found that the non-glycosylated single-chain FSH analog binds with high affinity to FSH receptor and efficiently inhibits FSH activity in vitro. This variant acts at the receptor level and has the potential to serve as a follitropin antagonist for clinical applications in the future.

Cyathus striatus Extract Induces Apoptosis in Human Pancreatic Cancer Cells and Inhibits Xenograft Tumor Growth In Vivo.

Pancreatic cancer is a highly lethal disease with limited options for effective therapy and the lowest survival rate of all cancer forms. Therefore, a new, effective strategy for cancer treatment is in need. Previously, we found that a culture liquid extract of Cyathus striatus (CS) has a potent antitumor activity. In the present study, we aimed to investigate the effects of Cyathus striatus extract (CSE) on the growth of pancreatic cancer cells, both in vitro and in vivo. The proliferation assay (XTT), cell cycle analysis, Annexin/PI staining and TUNEL assay confirmed the inhibition of cell growth and induction of apoptosis by CSE. A Western blot analysis demonstrated the involvement of both the extrinsic and intrinsic apoptosis pathways. In addition, a RNAseq analysis revealed the involvement of the MAPK and P53 signaling pathways and pointed toward endoplasmic reticulum stress induced apoptosis. The anticancer activity of the CSE was also demonstrated in mice harboring pancreatic cancer cell line-derived tumor xenografts when CSE was given for 5 weeks by weekly IV injections. Our findings suggest that CSE could potentially be useful as a new strategy for treating pancreatic cancer.

Inula Viscosa Extract Inhibits Growth of Colorectal Cancer Cells in vitro and in vivo Through Induction of Apoptosis.

Colorectal cancer (CRC) is the second most common cancer in females and the third in males worldwide. Conventional therapy of CRC is limited by severe side effects and by the development of resistance. Therefore, additional therapies are needed in order to combat the problem of selectivity and drug resistance in CRC patients. Inula viscosa (IV) is a well-known medicinal perennial herb in traditional medicine. It is used for different therapeutic purposes, such as; topical anti-inflammatic, diuretic, hemostatic, antiseptic, antiphlogistic, and in the treatment of diabetes. Several studies attempted to reveal the anti-cancer activity of different extracts prepared by different organic solvents from different parts of the IV plant. The aim of the present study is to examine the potential beneficial effects of IV leaf aqueous extract on the growth of colon cancer cells in vitro and in vivo. The results indicated that exposure of colorectal cancer cells to IV extract, significantly reduced cell viability in a dose and time dependent manner. Moreover, treatment of cells with 300 µg/ml of IV extract induced apoptosis, as it was detected by Annexin V/FITC/PI, TUNEL assay, and the activation of caspases. In vivo studies revealed that treatment with 150 or 300 mg/kg IV extract inhibited tumor growth in mice transplanted with MC38 cells. Tumors' weight and volume were significantly (P < 0.001) reduced when compared to untreated-control group. Staining of the paraffin section of tumors revealed that IV treatment inhibited cell proliferation and induced apoptosis. Additionally, no side effects such as; weight loss, behavior changes, ruffled fur or changes in kidney, and liver functions were observed. These results may indicate that active doses of IV extract are not toxic. Further studies are needed in order to identify the structure of the active compounds. Results from this study may contribute to the development of new and efficient strategies for treatment of human colon cancer.

Development of long-acting recombinant glycoprotein hormones by increasing the carbohydrate content.

Therapeutic recombinant glycoproteins are important for both the biotechnological industry and clinical purposes. Given the rapid clearance of these proteins from the circulation, they have to be injected frequently to obtain optimal therapy. Several strategies have been developed to overcome this limitation, aiming to increase the half-life of such proteins in the circulation. These strategies included chemical attachment of polyethylene glycol, nanocapsulation, fusion to immunoglobulins or to albumin as protein carriers, or enrichment of the carbohydrate content. Here, we describe a strategy for increasing the half-life of recombinant proteins using gene fusion to increase the carbohydrate content of the protein backbone.

Estrogen receptor and laminin genetic polymorphism among women with pelvic organ prolapse.

OBJECTIVE: Laminin is a connective tissue component. The LAMC1 gene encodes for gamma-1 chain of laminin, which is associated with familial clustering of POP. The ERα gene which encodes for cellular estrogen receptor has also been associated with POP. The aim of this study was to evaluate a possible correlation between polymorphism in these genes and the risk for developing POP. MATERIALS AND METHODS: Blood samples were drawn from 33 women with advanced POP (study group) and 33 women without POP (control group). DNA was extracted, and the presence of the rs10911193 C/T mutation in LAMC1 and of the rs2228480 G/A mutation in ERα was detected using the PCR technique. RESULTS: 26 samples were available for each group regarding ERα. 33 samples were available for each group, regarding LAMC1. The prevalence of homozygotes for the ERα rs2228480 G/A mutation was 19.2% and 0% among women with and without POP, respectively (OR 39.77, 95% CI 1.93-817.0, P = 0.00046). The prevalence of heterozygotes for this mutation was 83.3% and 11.5%, respectively (OR 19.2, 95% CI 4.15-88.6, P < 0.0001). The prevalence of homozygotes for the LAMC1 gene rs10911193 C/T mutation was 3.6% and 6.1% among women with and without POP (NS), while the respective for heterozygotes for this mutation was 21.4% and 33.3% (NS). CONCLUSIONS: Polymorphism in the ERα gene is associated with an increased risk for advanced POP. However, polymorphism in the LAMC1 gene does not seem to be associated with such risk.

Endothelial nitric oxide synthase polymorphism and prognosis in systolic heart failure patients.

BACKGROUND: The endothelial nitric oxide synthase (eNOS) gene single nucleotide polymorphism G894T is associated with thrombotic vascular diseases. However, its functional significance is controversial and data are scarce concerning its influence in heart failure (HF). METHODS: We studied 215 patients with chronic systolic HF. DNA was analyzed for eNOS gene G894T polymorphism using PCR and DNA sequencing. Evaluation of clinical characteristics and analysis of factors associated with 2-year mortality were performed for the homozygous G-allele G894T variant (GG), relative to the TT and GT variants. RESULTS: The genotype distributions of eNOS G894T alleles were: GG 135 patients (63%) and TT/GT 80 (37%). Two-year mortality was significantly higher in the GG variant (48%) than the combined TT/GT group (32%). The usage of nitrates was associated with increased 2-year mortality (HR 2.0, 95% CI 1.28-3.17; p = 0.003), which was most significant in the GG group treated with nitrates (73.5%) in comparison to the TT/GT group not treated with nitrates (34%); HR 2.75, 95% CI 1.57-4.79, P < 0.001. CONCLUSIONS: Homozygosity for the G allele of the eNOS G894T polymorphism was associated with worse survival in systolic HF patients, especially in those treated with nitrates. ENOS polymorphism may result in different mechanistic interactions in HF than in thrombotic vascular diseases, suggesting that overexpression of NO may be associated with deleterious effects in systolic HF.

Benzene-poly-carboxylic acid complex, a novel anti-cancer agent induces apoptosis in human breast cancer cells.

Some cases of breast cancer are composed of clones of hormonal-independent growing cells, which do not respond to therapy. In the present study, the effect of Benzene-Poly-Carboxylic Acid Complex (BP-C1) on growth of human breast-cancer cells was tested. BP-C1 is a novel anti-cancer complex of benzene-poly-carboxylic acids with a very low concentration of cis-diammineplatinum (II) dichloride. Human breast cancer cells, MCF-7 and T47D, were used. Cell viability was detected by XTT assay and apoptosis was detected by Flow Cytometry and by annexin V/FITC/PI assay. Caspases were detected by western blot analysis and gene expression was measured by using the Applied Biosystems® TaqMan® Array Plates. The results showed that exposure of the cells to BP-C1 for 48 h, significantly (P<0.001) reduced cell viability, induced apoptosis and activated caspase 8 and caspace 9. Moreover, gene expression experiments indicated that BP-C1 increased the expression of pro-apoptotic genes (CASP8AP1, TNFRSF21, NFkB2, FADD, BCL10 and CASP8) and lowered the level of mRNA transcripts of inhibitory apoptotic genes (BCL2L11, BCL2L2 and XIAP. These findings may lead to the development of new therapeutic strategies for treatment of human cancer using BP-C1 analog.

Absence of the α(2c)-adrenoceptor Del322-325 allele is associated with increased mortality in patients with chronic systolic heart failure.

OBJECTIVE: The Del322-325 polymorphism of the α(2c)-adrenoceptor is considered to be a possible risk factor for heart failure (HF). We investigated the possible clinical association between the presence or absence of the deletion allele and mortality. METHODS AND RESULTS: Of 261 chronic systolic HF patients evaluated, 216 (83%) carried no α(2c)-adrenoceptor Del322-325 alleles (designated II); 28 patients (11%) were heterozygous (ID) and 17 patients (6%) homozygous (DD) for the deletion. Similar genetic distribution of α(2c)-adrenoceptor Del322-325 subgroups was found in a control group of 96 healthy individuals. Mortality was significantly higher in HF patients in whom the deletion allele was absent than in HF patients who carried it: 67 (31%) patients in the II subgroup died compared with 7 (15.5%) in the ID/DD subgroup (P = .01). The odds ratio for death in HF patients who carried no α(2c)-adrenoceptor Del322-325 alleles compared with HF patients with ≥1 allele was 2.45 (95% confidence interval 1.04‒5.74). There were no differences in other relevant clinical parameters between the 2 subgroups of HF patients. CONCLUSIONS: The mortality rate of chronic systolic HF patients carrying no α(2c)-adrenoceptor Del322-325 alleles was significantly higher (almost 2.5-fold) than that of HF patients carrying ≥1 allele.

Conversion of TSH heterodimer to a single polypeptide chain increases bioactivity and longevity.

TSH is a dimeric glycoprotein hormone composed of a common α-subunit noncovalently linked to a hormone-specific ß-subunit. Previously, the TSH heterodimer was successfully converted to an active single-chain hormone by genetically fusing α and ß genes with [TSHß- carboxyl-terminal peptide (CTP)-α] or without (TSHß-α) the CTP of human chorionic gonadotropin ß-subunit as a linker. In the present study, TSH variants were expressed in Chinese hamster ovarian cells. The results indicated that TSHß-α single chain has the highest binding affinity to TSH receptor and the highest in vitro bioactivity. With regard to the in vivo bioactivity, all TSH variants increased the levels of T(4) in circulation after 2 and 4 h of treatment. However, the level of T(4) after treatment with TSH-wild type was significantly decreased after 6 and 8 h, compared with the levels after treatment with the other TSH variants. TSHß-α and TSHß-CTP-α single chains exhibited almost the same bioactivity after 8 h of treatment. Evaluating the half-life of TSH variants, TSHß-CTP-α single chain revealed the longest half-life in circulation, whereas TSH-wild type exhibited the shortest serum half-life. These findings indicate that TSH single-chain variants with or without CTP as a linker may display conformational structures that increase binding affinity and serum half-life, thereby, suggesting novel attitudes for engineering and constructing superagonists of TSH, which may be used for treating different conditions of defected thyroid gland activity. Other prominent potential clinical use of these variants is in a diagnostic test for metastasis and recurrence of thyroid cancer.

The 894G Allele of the Endothelial Nitric Oxide Synthase 3 (eNOS) is Associated with Atrial Fibrillation in Chronic Systolic Heart Failure.

BACKGROUND: Atrial fibrillation (AF) in patients with heart failure signals poor prognosis. The endothelial nitric oxide synthase (eNOS) enzyme is a key player in the counterregulation of oxidative stress, which is related in part to AF pathogenesis. The purpose of this study was to investigate a possible clinical association in heart failure patients between the presence of exon 7 G894T eNOS polymorphism, known to result in the Glu298Asp protein variant, and the occurrence of AF. METHODS: We analyzed the DNA of 344 patients with chronic systolic heart failure for exon 7 G894T eNOS polymorphism, using PCR. Odds ratios for AF were calculated for the homo- and heterozygous G-allele G894T variants relative to the TT variant. RESULTS: Of the 344 patients, 204 (59%) were homozygous for the G allele, 122 (36%) were heterozygous (GT), and 18 (5%) were homozygous for the T allele. AF episodes were documented in 73 patients (36%) with the GG genotype, in 35 (29%) with GT, and in 2 (11%) with TT. The odds ratio for AF, based on the presence of at least one G allele in the eNOS 894 gene, was 3.96 (95% confidence interval, 1.17‒13.56, p=0.04). Having two G alleles increased the odds ratio to 4.5 (95% confidence interval, 1.0‒20.0, p=0.02). CONCLUSION: Patients with systolic heart failure demonstrate strong correlation between AF and the presence of a G allele in the exon 7 G894T eNOS genotype. These findings support the importance of eNOS polymorphism in the pathogenesis of AF in heart failure patients.

The potential efficacy of 3,3'-diindolylmethane in prevention of prostate cancer development.

The objective of this study was to examine the efficacy of 3,3'-diindolylmethane (DIM) in prevention of prostate cancer tumor development in an animal model. Mouse prostate cancer cells (TRAMP-C2, 2x10) were injected subcutaneously into three groups of C57BL/6 mice (10 mice in each group). Two groups were treated earlier with DIM; 2 or 10 mg/kg each, and an additional control group was injected with medium. Animals were treated for five more weeks until sacrificed. Tumor sizes were measured biweekly. At the end of the experiment, mice were sacrificed, and tumors were excised, weighed, measured and tested using immunohistochemical studies. In addition blood samples were collected for biochemical analysis. The results indicated that DIM significantly reduced tumor development in treated animals when compared with controls. Tumors developed in 80% of controls and 40% and 60% of animals treated with 10 or 2 mg/kg of DIM, respectively. Moreover, tumors that developed in treated animals were significantly (P<0.001) smaller than in controls. Additionally, our results indicated that DIM has no effect on animal weight or liver and kidney functions. These results indicated that the DIM agent is not toxic and has an in-vivo preventive effect against the development of prostate cancer in a mouse model.

Human thyrotropin (TSH) variants designed by site-directed mutagenesis block TSH activity in vitro and in vivo.

TSH is a heterodimeric glycoprotein hormone synthesized in the pituitary and composed of a specific beta-subunit and a common alpha-subunit shared with FSH, LH, and human chorionic gonadotropin. The heterodimer was previously converted into a biologically active single chain protein by genetic fusion of the genes coding to both subunits in the presence of the carboxy-terminal sequence of human (h) chorionic gonadotropin-beta subunit as a linker [hTSHbeta-carboxyl-terminal peptide (CTP)-alpha]. N-linked carbohydrate-free single-chain TSH variants were constructed by site-directed mutagenesis and overlapping PCR: one devoid of both N-linked oligosaccharide chains on the alpha-subunit (hTSHbeta-CTP-alpha(deg)) and the other lacking also the oligosaccharides on the beta-subunit (hTSHbeta(deg)-CTP-alpha(deg)). These variants were expressed in Chinese hamster ovary cells and secreted into the culture media. We have previously reported that the variants block the activities of hTSH and thyroid-stimulating immunoglobulins in cultured human thyroid follicles. In the present study, binding affinity of hTSH variants to hTSH receptor and the localization of the antagonistic effect were examined. Moreover, the effect of these variants on TSH activity was tested in vivo. The results of the present study indicate that the hTSH variants bind to the hTSH receptor with high affinity. Experiments using forskolin also indicated that the N-linked carbohydrate-free TSH single-chain variants inhibit TSH activity at the receptor-binding site and not at a postreceptor level. Moreover, the variants significantly inhibited (about 50%) TSH activity with respect to thyroid hormone secretion in vivo in mice. These variants may offer a novel therapeutic strategy in treating hyperthyroidism.

Effects of UV-A irradiation on lens morphology and optics.

Epidemiological studies have indicated that ultraviolet radiation (UVR) is one of the main factors leading to senile cataract formation. We investigated morphological changes in the eye lens caused by UVR-A. Twenty three pairs of lenses obtained from 23 one-year-old calves were used for this study. For each pair, one lens was exposed to 44 J/m(2) UVR in the 365 nm wavelength region while the contralateral lens was not exposed and served as a control. The lenses were placed in specially designed organ culture containers for pre-incubation. Lenses were exposed to UVR after one day in culture. After irradiation, lens optical quality was monitored throughout additional 15 days of the culture period and lenses were taken for morphological analysis by scanning electron microscopy. Damage to lens optical quality was evident as early as day 8 after the irradiation and increased with time in culture. We found irregularity of fiber morphology in lenses exposed to UV-A irradiation (but not in control lenses), similar to that reported previously for aged lenses. At the end of the culture period (day 16), lens fiber membranes also showed holes in fiber membranes. We conclude that UVR-A caused damage to cell membranes of the lens and alterations in lens optics, which may subsequently lead to senile cataract formation.

Long-term lens organ culture system to determine age-related effects of UV irradiation on the eye lens.

Aging of the eye lens represents the life-long accumulation of damage. Factors responsible for age-related cataract are unknown because medical evaluations of aged populations demonstrate a wide range of systemic diseases and medical disorders. There are some main suspected factors, which may contribute to accumulated age-related damage in the eye lens. (1) Diseases, such as diabetes, substantially increase the probability of cataract formation in the age group from 40 to 49, and double or triple this probability for ages 50 to 69. (2) Drugs, including systemic medications such as steroids. (3) Environmental factors, such as UV radiation, heat and electromagnetic radiation. Our study represents an effort to determine the effects of suspected cataractogenic factors on the eye lens. The experiments are performed using a unique long-term lens organ culture system of bovine lenses. In our system it is possible to give controlled amounts of insult and monitor changes in lens optical quality throughout the culture period of 8-15 days. The optical properties, monitored in association with biochemical analysis of lens epithelium, cortex and nuclear samples, help in determining the mechanisms of cataract formation. The present study investigates mechanisms by which UV-A radiation at 365 nm causes damage to the lens. It is believed that solar radiation is one of the major environmental factors involved in lens cataractogenesis. Bovine lenses were placed in our special culture cells for pre-incubation of 24 hr followed by irradiation of 29 or 33 J cm(-2). The lenses were maintained in the cells during irradiation. After irradiation, lens optical quality was monitored throughout the culture period and lens epithelium was taken for enzyme analysis. Using the culture system we learned that: (a) young lenses (less than one-year-old) are less sensitive to UV radiation than 3-year-old lenses; (b) the lenses have the ability to recover in organ culture conditions; (c) applying the insult in one step results in less damage than dividing the same insult in 4 steps with 24 hr interval between each one; and (d) the damage from UV is greater if the intervals between each irradiation stage are insufficient to permit full recovery.