Cytotoxic Effect of Saffron Stigma Aqueous Extract on Human Prostate Cancer and Mouse Fibroblast Cell Lines.

PURPOSE: Several lines of experimental evidence have shown that saffron has anticarcinogenic effects. This study aimed at evaluating the possible anticancer effect of saffron stigma aqueous extract on human prostate cancer (PC3) and mouse fibroblast cells (L929) as non-cancerous control cells. MATERIALS AND METHODS: Saffron stigma aqueous extract at concentrations of 100, 200, 400, 600, 800, 1600 and 3200 µg/mL were prepared. PC3 and L929 cells were incubated with different concentrations of saffron extracts in different time intervals (24, 48, 72, 96 and 144 hours). MTT assay was used for each cell line to investigate the cytotoxic effect of saffron. Morphological alterations were also observed under light inverted microscope. RESULTS: In fibroblast cell line after 24 hours, Saffron extract did not affect significantly the normal cells and they were intact in morphologic view. After 96 hours in the cells with highest concentration (1600 µg/mL), cell death and cellular form changes as well as severe granulation was observed. In prostate cell line after 24 hours, the only changes were observed in cells with the concentration of 1600 µg/mL. The cells were granulated and the form of the cells were spherule. After 72 hours, in group with the concentration of 1600 µg/mL, severe granulation was observed and the cell count decreased and some cells were dead. CONCLUSION: Saffron aqueous extract has an in vitro inhibitory effect on the proliferation of human prostate cell and mouse L929 cells which is dose-dependent.

Relationship Between Polymorphisms of Interleukin-1 Receptor Antagonist (IL-1Ra) Genes and Susceptibility to Systemic Lupus Erythematosus in Iranian Population.

OBJECTIVES: Interleukin (IL)-1 has a major role in cell destruction and inflammation. IL-1 receptor antagonist (IL-1RN or IL-1Ra) is a natural anti-inflammatory molecule that blocks IL-1. We intended to determine whether IL-1RN or IL-1Ra variable number tandem repeat (VNTR) polymorphism is associated with susceptibility to systemic lupus erythematosus (SLE) in a series of patients in the Northeastern part of Iran. METHODS: Genomic DNA was extracted from the whole blood of 104 SLE patients and 209 subjects without SLE as a control group. The control group was matched for age and gender with SLE patients. Then, genomic DNA was genotyped by polymerase chain reaction (PCR) method for a length polymorphism in intron 2 of the IL-1RN gene. RESULTS: Of five alleles, only allele 4 of IL-1RN had a higher frequency in healthy subjects (2.4%) compared to SLE patients (0), with a statistically significant difference (P= 0.03). Eleven kinds of polymorphisms of IL-1RN were found including 1/1, 1/2, 2/2, 3/3, 1/3, 3/5, 2/3, 2/5, 1/5, 4/4 and 1/4 in both groups. In genotype frequency, there was no statistically significant difference regarding gene polymorphism kinds between the two groups (P= 0.29). CONCLUSION: Alleles 4 of IL-1RN may have a protective role against SLE susceptibility. However, SLE was not associated with any of the 11 kinds of genotype IL1-RN gene polymorphisms studied here.

Analysis of CFTR Gene Mutations in Children with Cystic Fibrosis, First Report from North-East of Iran.

OBJECTIVE(S): More than 1500 registered mutations in cystic fibrosis transmembrane regulator (CFTR) gene are responsible for dysfunction of an ion channel protein and a wide spectrum of clinical manifestations in patients with cystic fibrosis (CF). This study was performed to investigate the frequency of a number of well-known CFTR mutations in North Eastern Iranian CF patients. MATERIAL AND METHODS: A total number of 56 documented CF patients participated in this study. Peripheral blood was obtained and DNA extraction was done by the use of routin methods. Three steps were taken for determining the target mutations: ARMS-PCR was performed for common CFTR mutations based on previous reports in Iran and neighboring countries. PCR-RFLP was done for detection of R344W and R347P, and PCR-Sequencing was performed for exon 11 in patients with unidentified mutation throughout previous steps. Samples which remained still unknown for a CFTR mutation were sequenced for exon 12. RESULTS: Among 112 alleles, 24 mutated alleles (21.42%) were detected: ΔF508 (10.71%), 1677delTA (3.57%), S466X (3.57%), N1303K (0.89%), G542X (0.89%), R344W (0.89%), L467F (0.89%). Eight out of 56 individuals analyzed, were confirmed as homozygous and eight samples showed heterozygous status. No mutations were detected in exon 12 of sequenced samples. CONCLUSION: Current findings suggest a selected package of CFTR mutations for prenatal, neonatal and carrier screening along with diagnosis and genetic counseling programs in CF patients of Khorasan.

Gene polymorphisms of TNF-α and IL-1ß are not associated with generalized aggressive periodontitis in an Iranian subpopulation.

Cytokines play a part in pathogenesis of periodontitis via inflammation phenomenon. Aggressive periodontitis (AgP) is a multifactorial disease resulting in rapid tooth loss due to severe destruction of tooth supporting apparatus. Recently, researchers have focused on genetic susceptibility of periodontitis through investigating the gene variations of cytokines and other components of immune response. In this study we analyzed single nucleotide polymorphism (SNP) of two cytokines in association with AgP in an Iranian-khorasanian population; Interleukin-1 beta (IL-1ß) +3954 C/T and Tumor Necrosis Factor alpha (TNF-α) -308 G/A. From arm vein of patients (n=58) and periodontally healthy individuals (n=60) blood sample was obtained and the DNA was extracted. Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) procedure was performed to recognize the SNPs. X2 test was used to determine the statistically significant differences between the two groups. The frequency of genotypes and alleles had no significant differences between patients and control groups. The distributions were as follows. IL-1ß +3954: CT, CC and TT genotypes in patients were 39.6%, 60.4% and 0.0% and in controls were 41.7%, 50% and 8.3%, respectively. TNF-α -308: GA, GG and AA genotypes in patients were 44.8%, 41.4% and 13.8% and in controls were 46.7%, 50% and 3.3%, respectively.This investigation do not substantiates the role of IL-1ß +3954 and TNF-α -308 polymorphisms, separately, as risk determinants for AgP in Iranian population. Further research based on all components of immune response, is needed to corroborate the genetic susceptibility of AgP.

Analysis of osteoprotegerin (OPG) gene polymorphism in Iranian patients with chronic periodontitis and peri-implantitis. A cross-sectional study.

PURPOSE: Receptor activator for nuclear factor kappa B ligand (RANKL)/RANK/OPG pathway plays a redundant role in osteoclastogenesis, osteoclast activation and regulation of bone resorption. Recently, single nucleotide polymorphisms (SNP) have been reported as a co-factor in pathogenesis of inflammatory diseases. The aim of this study was to investigate the relationship between osteoprotegerin (OPG) gene polymorphisms and chronic periodontitis and peri-implantitis in an Iranian population. MATERIALS AND METHODS: 77 patients with chronic periodontitis (CP), 40 patients with peri-implantitis (PI) and 89 periodontally healthy subjects were enrolled in this study. A total of 5 ml of blood was obtained from the arm vein of participants. DNA was extracted based on the salting out method. Two functional SNPs (T950C and G1181C) were analysed by using polymerase chain reaction and restriction fragment length polymorphism (PCR_RFLP). The prevalence differences in three batches were evaluated by chi-square test. RESULTS: An evaluation of the T950C genotype found that there was no statistical difference between groups. A comparison of the G1181C genotype distribution between peri-implantitis and chronic periodontitis groups, and between peri-implantitis and normal groups found that the frequency of the CC genotype was significantly higher in patients with peri-implantitis (P = 0.005). CONCLUSIONS: Our results indicate that a SNP at G1181C is associated with the presence of PI.

Cytotoxicity of orthodontic bonding adhesive resins on human oral fibroblasts.

There is little information concerning the cytotoxic effects of no-mix and flowable adhesives used in orthodontics. The aim of the present study was to evaluate the cytotoxic effects of a no-mix (Unite), a light-cured (Tranbond XT), and a flowable (Denfil Flow) adhesives on human oral fibroblasts. Twelve discs of each adhesive were prepared and aged for 1, 3, 5, and 7 days in Dulbecco's Modified Eagle's Medium (DMEM). Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the difference between the groups was tested by analysis of variance and Tukey tests (α = 0.05). After 1 day of storage, the no-mix adhesive showed moderate cytotoxic effects (P < 0.05), while the light-cured and flowable adhesives were essentially non-cytotoxic. Ageing considerably reduced the cytotoxicity of the no-mix adhesive. On days 5 and 7 of the experiment, the cell viability of three adhesives did not differ significantly (P > 0.05), but cell viability was slightly reduced on day 7. Moderate cytotoxic effects of no-mix adhesive on the first day of the experiment suggest that care should be taken to protect dentists and patients when these adhesives are being handled. Despite higher resin components, the flowable adhesive showed excellent biocompatibility.

Role of caspases and Bax protein in saffron-induced apoptosis in MCF-7 cells.

Saffron (Crocus sativus), widely used as a spice in Middle Eastern cuisine and is known for anti-cancer properties. The mechanism of saffron-induced cytotoxicity, in tumor cells has not been adequately explored. Therefore, we investigated the role of caspases and Bax protein in saffron-induced apoptosis in MCF-7 cells, a commonly used cell culture system for in vitro studies on breast cancer. Cells were incubated with different concentrations of saffron extract. Cell viability was quantitated by MTT assay. Apoptotic cells were determined using PI staining of DNA fragmentation by flow cytometry (sub-G1 peak). Role of caspase were studied using the pan-caspase inhibitor. Bax protein expression was analysed by western blotting. Saffron extract (200-2000 microg/ml) decreased cell viability in MCF-7 cells as a concentration- and time-dependent manner with an IC50 of 400+/-18.5 microg/ml after 48 h. Analysis of DNA fragmentation by flow cytometry showed apoptotic cell death in MCF-7 cell treated with saffron extract. Saffron-induced apoptosis could be inhibited by pan-caspase inhibitors, indicating caspase-dependent pathway was induced by saffron in MCF-7 cells. Bax protein expression was also increased in saffron-treated cells. Thus saffron exerts proapoptotic effects in a breast cancer-derived cell line and could be considered as a potential chemotherapeutic agent in breast cancer.

Direct toxicity of Rose Bengal in MCF-7 cell line: role of apoptosis.

Current therapies for breast cancer are often limited by short-term efficacy due to the emergence of drug resistance. In view of this, there is much interest in the identification of new agents for the treatment of breast cancer. Rose Bengal (RB) has been used as a photosensitiser in photodynamic treatment. In the present study, we investigated the direct cytotoxic and proapoptotic effects of RB, not as a photosensitiser, in MCF-7 cells. Cell viability was quantitated by MTT assay. Apoptotic cells were determined using PI staining of DNA fragmentation by flow cytometry. Bax protein expression was studied by western blotting. ROS was measured using DCF-DA by flow cytometry analysis. The result showed RB decreased cell viability in MCF-7 cells in a concentration- and time-dependent manner. RB induced a sub-G1 peak in flow cytometry histogram of treated cells indicating apoptosis is involved in this toxicity. In Western blot analysis, Bax expression significantly increased in RB-treated cells. RB could also increase ROS production in MCF-7 cells but antioxidant GSH could not decrease the toxicity indicating this toxicity was independent of ROS production. Thus RB exerts proapoptotic effects in a MCF-7 cells and could be considered as a potential chemotherapeutic agent in breast cancer.

Cytotoxic effect of saffron stigma aqueous extract on human transitional cell carcinoma and mouse fibroblast.

INTRODUCTION: Saffron has been suggested to have inhibitory effects on tumoral cells. We evaluated the cytotoxic effect of aqueous extract of saffron on human transitional cell carcinoma (TCC) and mouse non-neoplastic fibroblast cell lines. MATERIALS AND METHODS: Human TCC 5637 cell line and mouse fibroblast cell line (L929) were cultivated and incubated with different concentrations of aqueous extract of saffron stigma (50 microg/mL to 4000 microg/mL). Cytotoxic effect of saffron was evaluated by morphologic observation and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay after 24, 48, 72, and 120 hours in each cell line. RESULTS: After 24 hours, morphological observations showed growth inhibitory effects at saffron extract concentrations higher than 200 microg/mL for L929 cells and at concentrations of 50 microg/mL to 200 microg/mL for the TCC cells. These changes became more prominent after 48 hours. However, significant growth inhibitory effects of the extract were shown at concentrations of 400 microg/mL and 800 microg/mL. Higher concentrations of saffron correlated inversely with cell population of both cell lines. Significant reduction of the survived cells was seen at concentrations of 400 microg/mL and 2000 microg/mL for TCC and L929 cell lines, respectively. After 120 hours, decrease in the percentage of survived cells at higher concentrations of saffron extract was seen in both cell lines. At a concentration of 800 microg/mL, the survived L929 cells plummeted to less than 60% after 120 hours, while no TCC cells survived at this time. No L929 cells survived at 2000 microg/mL. CONCLUSION: Saffron aqueous extract has inhibitory effects on the growth of both TCC 5637 and normal L929 cell lines. This effect is dose dependent.

Study of cytotoxic and apoptogenic properties of saffron extract in human cancer cell lines.

Saffron (dried stigmas of Crocus sativus L.) has been used as a spice, food colorant and medicinal plant for millennia. In this study cytotoxic effect of saffron extract was evaluated in HepG2 and HeLa cell lines. Meanwhile role of apoptosis and ROS were explored. Malignant and non-malignant cells (L929) were cultured in DMEM medium and incubated with different concentrations of ethanolic saffron extract. Cell viability was quantitated by MTT assay. Apoptotic cells were determined using PI staining of DNA fragmentation by flow cytometry (sub-G1 peak). ROS was measured using DCF-DA by flow cytometry analysis. Saffron could decrease cell viability in malignant cells as a concentration and time-dependent manner. The IC50 values against HeLa and HepG2 were determined 800 and 950 microg/ml after 48 h, respectively. Saffron induced a sub-G1 peak in flow cytometry histogram of treated cells compared to control indicating apoptotic cell death is involved in saffron toxicity. This toxicity was also independent of ROS production. It might be concluded that saffron could cause cell death in HeLa and HepG2 cells, in which apoptosis or programmed cell death plays an important role. Saffron could be also considered as a promising chemotherapeutic agent in cancer treatment in future.

Cytotoxic effect of a new endodontic cement and mineral trioxide aggregate on L929 line culture.

INTRODUCTION: The aim of this study was to compare the cytotoxicity of Mineral Trioxide Aggregate (MTA) and a New Endodontic Cement (NEC) on L929 mouse fibroblasts. MATERIALS AND METHODS: Different dilutions (Neat, 1/2, 1/10, 1/100) of fresh and set materials placed adjacent flasks of L929 in DMEM medium. Cellular viability was assessed using MTT assay in three time intervals (24, 48, and 72 h after mixing). Differences in mean cell viability values between materials were assessed by using the One-way ANOVA and Bonferoni post-test. Optical microscopic analysis of morphology of the untreated control and the cement-treated cell cultures were carried out in all experimental periods. RESULTS: It was indicated that there was not a significant difference in cytotoxicity among the materials of test and between them and the control group. However, there was a statistically significant difference between different time intervals within each group (P< 0.05) and between different concentration of test materials (P<0.05). In all samples, set materials showed better viability than fresh ones. CONCLUSION: According to results of this study, NEC and MTA have similar cytotoxic effect on L929 cell culture.

D28G mutation in congenital glucose-galactose malabsorption.

BACKGROUND: Congenital glucose-galactose malabsorption is a rare autosomal recessive disorder of the intestinal transport of glucose and galactose, leading to watery diarrhea, dehydration, failure to thrive, and early death. METHODS: In this study, we analyzed D28G mutation in 16 family members of a patient with typical presentation of congenital glucose-galactose malabsorption with polymerase chain reaction-Restriction Fragment Length Polymorphism method. RESULTS: Nine members of this family were heterozygous for D28G mutation. CONCLUSION: To the best of our knowledge this is the first report of D28G mutation in Iran. Moreover, this simple typical PCR-Restriction Fragment Length Polymorphism method, allows immediate identification of D28G mutation.