Proapoptotic effects of ceranib-2 in combination with radiation therapy on human breast cancer cells.

OBJECTIVE: Strategies for cancer therapy involve radiation therapy (RT), which accounts for about 40% of all cancer treatment types. As to current chemotherapeutics, cancer cells also develop resistance that remains a clinical problem, such as disease recurrence. Recent studies focused on understanding the molecular mechanisms of radiation-induced cell death. Conventional RT aims at treatment with a single fraction per day of 8-30 Gy per fraction. Radiotherapy increases intracellular ceramide levels that trigger cell death. Additionally, increasing intracellular ceramide by radiation may restore therapeutic sensitivity to cancer treatments. Drugs that inhibit ceramide-metabolizing enzymes like ceramidases are expected to be radiotherapy sensitizers. MATERIALS AND METHODS: In this research, we investigated the proapoptotic effects of SRS alone and in combination with ceranib-2, a ceramidase inhibitor in human breast adenocarcinoma cells. The molecular mechanism of action of RT and ceranib-2 was investigated on MCF-7 cells exposed to 13 µM ceranib-2 for 24 hours following 20 Gy radiation using MTT, radiotherapy, and annexin-V analyses. RESULTS: Results indicated that the dose of 20 Gy radiation induces apoptosis on human breast cancer cells with and without co-treatment with ceranib-2 by causing cytotoxicity in the cells. Based on the results of ceranib-2 exposure, it can be concluded that the mechanism of action may rely on an increase of intracellular ceramides, also called apoptotic lipids. CONCLUSIONS: The study results suggest that co-treatment of human breast adenocarcinoma cells with a ceramidase inhibitor, ceranib-2, and a high dose of radiation of 20 Gy exerted cytotoxicity and apoptosis and might be a solid, potent alternative to current therapy strategies.

Investigation of proapoptotic and cytotoxic effects of 2-aminobenzothiazole on human laryngeal carcinoma cells.

OBJECTIVE: In the present study, we investigated the effects of 2-aminobenzothiazole application on human laryngeal carcinoma cells. MATERIALS AND METHODS: Human larynx epidermoid carcinoma (HEp-2) (ATCC® CCL-23™) cells were purchased from American Type Culture Collection (ATCC, USA). Human larynx epidermoid carcinoma HEp-2 cells were cultured in complete Dulbecco's Modified Eagle's Medium (DMEM) supplemented with fetal bovine serum (FBS) (10%) and penicillin/streptomycin (1%) in a CO2 (5%) incubator under standard cell culture conditions. 2-aminobenzothiazole was prepared, and further dilutions ranging from 3.13 to 100 µM were prepared in fresh culture DMEM. HEp-2 cells on 96 well plates were incubated with the prepared dilutions of 2-aminobenzothiazole for 24, 48, and 72 hours. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test performed cytotoxicity evaluation and viability percentages. The annexin-V staining technique detected 2-aminobenzothiazole-triggered apoptosis of HEp-2 cells. The activated caspases 3/7 on HEp-2 cells after 2-aminobenzothiazole exposure were evaluated with flow cytometric analysis. The membrane potential changing of HEp-2 cells was measured following the Muse™ MitoPotential kit manufacturer instructions. RESULTS: MTT cytotoxicity test results showed that the viability of human laryngeal carcinoma cells decreased with an increase in the application of 2-aminobenzothiazole for 24 hours. The highest growth inhibition by 2-aminobenzothiazole for short-term application of 24 hours was detected at the highest concentration of 2-aminobenzothiazole (100 µM). The results underline that the cytotoxic effect of 2-aminobenzothiazole is dose-dependent. Cytotoxicity test results for an application time of 48 hours showed that the cytotoxicity of 2-aminobenzothiazole is dose-dependent on HEp-2 cells. The required dose of 2-aminobenzothiazole to decrease the cell viability to 50 percent has been 9-fold augmented. Annexin-V findings showed that after exposure to IC50 concentration of 2-aminobenzothiazole for 24 hours, HEp-2 cells underwent the early apoptotic stage (25.99%) and late apoptotic (16.69%), whereas 56.93% of the treated cells were alive. Only 0.39% of 2-aminobenzothiazole treated cells were necrotic. All study results showed that 2-aminobenzothiazole triggered apoptosis on HEp-2 cells with a percentage of total apoptotic cells 42.62 compared to untreated HEp-2 cells. Caspase 3/7 activation results showed that only 0.65% of control HEp-2 cells were with activated caspase 3/7, and 99.35% live cells. The analysis data from the Muse cell analyzer revealed that the percentage of cells with intact mitochondrial membranes was 21.30 after 2-aminobenzothiazole application, and 79.9% were cells with depolarized mitochondrial membranes. It has been understood that the depolarization of the inner mitochondrial membrane has been considered a dysfunction in mitochondria as a sign of apoptosis and drug toxicity. CONCLUSIONS: Based on all study findings, 2-aminobenzothiazole has cytotoxicity on human laryngeal carcinoma cells in a dose and time-dependent manner. That means that it decreased viability via inducing caspase-dependent apoptosis. Consequently, it was concluded that 2-aminobenzothiazole has good potential to lead to cytotoxicity and apoptosis on human laryngeal carcinoma cells and, after deeper in vitro and in vivo investigations, can be a good candidate for designing anticancer drugs with high efficiency.

Effects of ceramide C2 application on human laryngeal carcinoma cells: a cell culture study.

OBJECTIVE: In the present study, we investigated the effects of Ceramide C2 application on human laryngeal carcinoma cells. MATERIALS AND METHODS: Human larynx epidermoid carcinoma HEp-2 (ATCC® CCL-23™) cells were purchased from the American Type Culture Collection (ATCC, USA). Human larynx epidermoid carcinoma HEp-2 cells were cultured in complete Dulbecco's Modified Eagle's Medium (DMEM) supplemented with fetal bovine serum (FBS) (10%) and penicillin/streptomycin (1%) in a CO2 (5%) incubator under standard cell culture conditions. Ceramide C2 was prepared, and further dilutions ranging from 3.13 to 100 µM were prepared in a fresh culture medium. Cells on 96 well plates were exposed to the prepared concentrations of ceramide C2 for 24 and 48 hours. Cytotoxicity evaluation was performed by MTT. Apoptosis profiles of HEp-2 cells were detected by annexin-V analysis. The activated caspases 3/7 on HEp-2 cells after ceramide C2 exposure were evaluated with flow cytometric analysis. The morphological changes on HEp-2 cells caused by ceramide C2 were evaluated by staining with phalloidine and acridine orange via confocal microscopy. For the Wound Healing Assay, HEp-2 cells were cultured in 6 well-plates until they became confluent. RESULTS: MTT cytotoxicity test findings revealed that the viability of human laryngeal carcinoma cells decreased with the increased application of ceramide C2 for 24 hours compared to untreated (control) cells. The highest growth inhibition by ceramide C2 for short-term application for 24 hours was detected at the highest concentration of ceramide C2 (100 µM). Annexin-V findings showed that 98.97 of HEp-2 cells were alive, and 1.63% were detected as early apoptosis for the control group. The results showed that ceramide C2 triggered apoptosis on HEp-2 cells with a percentage of total apoptotic cells of 61,40 compared to untreated HEp-2 cells. Cysteine proteases (caspases) 3/7 activation percentages of HEp-2 cells exposed to ceramide C2 for 24 hours were compared to control cells, and the morphology of HEp-2 cells was changed with clear apoptotic signs that underlined the cytotoxicity and pro-apoptotic activity of ceramide C2. Scratch Assay assessed the migration capability of HEp-2 cells before and after the exposure to ceramide C2. It showed that ceramide C2 reduced human laryngeal carcinoma cells' migration capability and proliferation for 24 hours. CONCLUSIONS: Based on all study findings, it can be considered that short-chain ceramide C2 exerted cytotoxicity on human laryngeal carcinoma cells in a dose and time-dependent manner and reduced the viability via inducing caspase-dependent apoptosis. The overall effect might be derived from the elevated intracellular ceramide levels by the exogenous application of ceramide C2. Consequently, it was concluded that ceramide C2 has good potential to cause cytotoxicity and apoptosis in human laryngeal carcinoma cells and, after deeper in vitro and in vivo investigations, can be a good candidate for designing anti-cancer drugs with high efficiency.

Efficacy of ceramidase inhibition on human renal cell carcinoma: a cell culture study.

OBJECTIVE: Cancer-preventative medicines like curcumin, resveratrol, and nonsteroidal anti-inflammatory medications all have their effects modulated by ceramide. According to research, these medications raise ceramide levels in cancer cells, leading to programmed cell death. Recently, cancer research has been involved in sphingolipid metabolism. The critical molecule here is ceramide. We aimed to investigate if the inhibition of ceramidases induces death in the human renal cell carcinoma cell line. MATERIALS AND METHODS: Human kidney carcinoma A-498 (ATCC® HTB-44™) cells were used as test cells. Ceranib-2, fetal bovine serum (FBS), penicillin/streptomycin, dimethyl sulfoxide, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl-2H-tetrazolium bromide and Dulbecco's Modified Eagle Medium High Glucose, caspase 3/7, annexin-V, Bcl-2 activation dual detection, and MitoPotential kits were used. 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl-2H-tetrazolium bromide (MTT) colorimetric assay, annexin-V analysis, caspase 3/7 analysis, Bcl-2 activation analysis, and measurement of mitochondrial membrane potential were performed. RESULTS: MTT colorimetric assay results for 24 hours indicated that the viability of human renal cell carcinoma cells decreased compared to the control group with an increase in the applied concentration of the ceramidase inhibitor-ceranib-2. The growth inhibition by ceranib-2 for 24 hours did not decrease the viability under 50%; thus, it could not be possible to calculate the IC50 value for the short-term application of ceranib-2 for 24 hours to A-498 cells. A statistically significant decrease in cell viability was recorded at doses of 100, 50, 25, and 12.2 µM of ceranib-2, and no significant decrease was detected at the lower doses of ceranib-2. The highest inhibition caused by ceranib-2 on human renal cell carcinoma cells A-498 was detected at an application time of 72 hours. This inhibition was statistically significant for all applied doses of ceranib-2 on A-498 cells compared to untreated cells. Annexin-V technique that detects the translocation of phosphatidylserine to the outer membrane of apoptotic cells indicated that after the application of ceranib-2, apoptosis was triggered on A-498 cells with a total apoptotic profile of 12.12% compared to the untreated cells that were used as controls. Compared to untreated A-498 cells, a rise in percentage to 16.25% of cells with activated caspases 3/7 was recorded after applying IC50 concentration of ceranib-2 on A-498 cells for 48 hours. CONCLUSIONS: The results of our study indicated that the application of ceramidase inhibitor, ceranib-2 on human renal cell carcinoma A-498 cells cause cytotoxicity, antiproliferative, growth inhibitory, and apoptotic efficacies in a dose and time-dependent manner probably via inhibiting the acid ceramidases that hydrolyze ceramides that induce cell death. For further conclusions, more mechanical, pharmacokinetic, and pharmaceutic, as well as in vitro and in vivo anti-cancer activity investigations are required.

Efficacy and toxicity of anise oil as a potential topical wound healer: a cell culture study.

OBJECTIVE: We studied the cytotoxic effects of topical anise oil on NIH/3T3 fibroblast cells using a cell culture assay. MATERIALS AND METHODS: NIH/3T3 fibroblast cells were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with fetal bovine serum (10%) and penicillin/streptomycin under standard cell culture conditions in a humidified incubator containing 5% carbon dioxide. For the MTT cytotoxicity experiment, NIH/3T3 cells were plated in triplicate at a concentration of 3x103 per well in 96-well plates and incubated for 24 hours. The cells were treated with anise oil concentrations ranging from 3.13 to 100 µM, and the plates were cultured for 24, 48, and 72 hours under standard cell culture conditions. For assessment by confocal microscopy, NIH/3T3 cells were seeded on sterilized coverslips in 6-well plates at a concentration of 105 cells per well in triplicate. For 24 hours, cells were treated with 100 µM of anise oil. Three wells that were not treated with anise oil served as the control group. RESULTS: The MTT findings demonstrated that anise oil is not cytotoxic to NIH/3T3 fibroblast cells. Anise oil stimulated cell growth and triggered cell division at all three incubation intervals of 24, 48, and 72 hours. The maximum growth was obtained in the applied highest concentration of 100 µM anise oil. At doses of 25, 50, and 100 µM, there was also a statistically significant improvement in cell viability. At 72 hours of incubation, dosages of 6.25 and 12.5 micro of anise oil were shown to be viability-inducing for NIH/3T3 cells. In the confocal microscopy pictures, it was found that anise oil was not cytotoxic on NIH/3T3 cells at the applied maximal dose. The experimental group of NIH/3T3 cells exhibited the same cell morphology as the untreated control group. In both sets of NIH/3T3 cells, the nucleus was round and undamaged, and the cytoskeleton was determined to be compact. CONCLUSIONS: Anise oil is not cytotoxic on NIH/3T3 fibroblast cells and initiates cell growth. Anise oil could be used topically to enhance wound healing after surgical procedures if clinical trials will confirm experimental data.

Bromelain: a candidate to enhance wound healing after endonasal surgeries.

OBJECTIVE: In the present study, we investigated the topical bromelain's cytotoxic effects on mouse fibroblast NIH/3T3 cells via cell culture study. MATERIALS AND METHODS: In this cell culture study, Dulbecco's Modified Eagle Medium (DMEM) with fetal bovine serum (FBS, 10%) and penicillin/streptomycin (1%) was used as a cell growth medium for NIH/3T3 mouse fibroblast cells. MTT test was performed in 96-well plates seeded with NIH/3T3 cells 5x103/well and under standard cell culture conditions. Bromelain doses of 3.13 to 100 µM were administered to the wells and incubated for 24, 48, and 72 hours in the same cell culture conditions. For Confocal microscopic evaluation, NIH/3T3 cells were plated on cover slips in 6-well plates (105 cells/well) and treated with 100 µM concentration of bromelain for 24 h. Untreated cells were used as controls. RESULTS: MTT results showed that bromelain is not cytotoxic on mouse fibroblast NIH/3T3 cells. All three incubation times of 24, 48, and 72 hours bromelain initiated cell growth. A statistically significant rise in cell growth was detected in the only applied highest dose of 100 µM bromelain for all incubation times except for 24 hours. The nontoxic effect was further investigated by using confocal microscopy by applying the highest bromelain dose of 100 µM to NIH/3T3 mouse fibroblast cells. Confocal micrographs showed that bromelain did not change the morphology of mouse fibroblast cells at the incubation time of 24h. In untreated cells and bromelain-treated cells, the nucleus of NIH/3T3 cells was undamaged and compact, and the cytoskeleton was fusiform and non-fragmented. CONCLUSIONS: Bromelain is not cytotoxic on mouse fibroblast NIH/3T3 cells and enhances cell growth. If clinical trials will confirm this, it is possible that bromelain will be used topically in humans to enhance wound healing, in rhinosinusitis and chronic rhinosinusitis with nasal polyps and endonasal surgeries due to its anti-inflammatory effects.

Evaluation of spiramycin for topical applications: a cell culture study.

OBJECTIVE: Through a cell culture test, we analyzed the cytotoxic effects of topical spiramycin on NIH/3T3 fibroblast cells. MATERIALS AND METHODS: Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin was used for the growth of NIH/3T3 fibroblast cells in a 5% CO2 incubator. Spiramycin's cytotoxicity was measured using the MTT assay. 5,000 NIH/3T3 cells per well of a 96-well plate were seeded in each well, and the cells were treated with spiramycin (3.13-100 µM) for 24, 48 and 72 hours while the plates were incubated at 37°C in a humidified 5% CO2 atmosphere. First, 105 NIH/3T3 cells were seeded onto coverslips in 6-well plates for morphological analysis of both untreated and spiramycin-treated cells. For 24 hours, NIH/3T3 cells were exposed to a 100 µM dosage of spiramycin. The cells in the control group were grown in complete growth media alone. RESULTS: Spiramycin was non-toxic to NIH/3T3 fibroblast cells in a MTT test. The concentration of spiramycin used to stimulate cell growth increased as the concentration was increased. After 24 and 48 hours of treatment with 100 µM NIH/3T3, the cells showed the most significant increase in size. Cell viability was shown to be significantly reduced at spiramycin doses of 50 and 100 µM. All MTT findings revealed that spiramycin enhanced cell viability and was not harmful to the fibroblast cells for short-term application of 24 and 48 hours but lowered the viability of fibroblast cells at the doses of 50 and 100 µM for long-term application duration of 72 hours. Confocal micrographs showed that spiramycin treatment did not affect the cytoskeleton or nucleus of fibroblast cells, in contrast to the control NIH/3T3 cells. Both untreated and treated with spiramycin, fibroblast cells were found to be fusiform and compact, with their nuclei remaining unaltered and unreduced in size. CONCLUSIONS: It was concluded that spiramycin has a beneficial effect on fibroblast cells and is safe for use over short periods. Spiramycin reduced fibroblast cell viability when applied for 72 hours. Confocal micrographs showed that fibroblast cell skeletons and nuclei were unharmed and undamaged, that cell shapes were fusiform and compact, and that nuclei were neither broken nor shrunken. Topical spiramycin could be recommended for septorhinoplasty procedures due to anti-inflammatory effects for short-term usage if clinical trials will confirm experimental data.

An evaluation of ketoprofen as an intranasal anti-inflammatory agent.

OBJECTIVE: The objective of the study was to evaluate the effect of ketoprofen when locally applied to tissue-cultured nasal epithelium. MATERIALS AND METHODS: Healthy primary nasal epithelial cells were grown in a tissue culture medium. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to evaluate cytotoxicity. Markers of cellular injury revealed by the MTT assay include fragmentation of DNA, condensed nuclei, and changes affecting the cellular outer membrane and cytoskeleton. Epithelial cells at body temperature in cell culture were exposed over a 24-hour period to ketoprofen. Following the MTT assay, the confocal microscopic examination was performed. The extent to which epithelial cells remained capable of proliferating was evaluated by inducing a scratch injury, waiting for the repair to occur, and then examining the result with the ordinary light microscope. RESULTS: Topically applied ketoprofen does not affect the viability of tissue-cultured nasal epithelial cells within a 24-hour period. Furthermore, there were no cellular morphological alterations observed which would indicate toxicity from ketoprofen. In the scratch assay, the cells regained a normal confluent appearance within 24 hours. Thus, ketoprofen neither increases nor alters the rate at which nasal epithelial cells proliferate. CONCLUSIONS: Ketoprofen, when applied topically for 24 hours to nasal epithelial cells in cell culture, does not cause any alterations in cellular appearance which would suggest impairment of the ability to proliferate or indicate a cytotoxic effect. Extrapolating from these results, it appears acceptable to use ketoprofen topically within the nose in cases of rhinosinusitis (acute or chronic) or nasal pain since there is minimal risk of local toxic injury.

Is G. cambogia a promising treatment? Effects on cultured nasal epithelial cells.

OBJECTIVE: The purpose of this study is to assess the effects of applying Garcinia cambogia to cultured human nasal epithelial cells. MATERIALS AND METHODS: A cell culture was set up consisting of human primary nasal epithelial cells harvested during septorhinoplasty from volunteers. The cells came from individuals with no history of rhinosinusitis. One assay for assessing cytotoxicity in cell culture utilizes MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). This method allows visualization of fragmented DNA, condensation of nuclei and changes to the external cellular membrane or cytoskeleton. Our study employed this method. Nasal epithelial cells at 37°C were exposed in culture to G. cambogia for a period of 24 hours. Afterwards an MTT assay was used in conjunction with confocal microscopy to assess evidence of toxicity. The proliferative capability of the nasal epithelial cells was also evaluated by inducing a scratch injury to cultured cells followed by light microscopic examination. RESULTS: Testing for cytotoxicity in this manner indicates that G. cambogia does not appear harmful to cultured nasal epithelial cells when applied directly. The cells exposed to this plant extract were still fully viable 24 hours afterwards. There was no increase in viability at the level of statistical significance. It was noted, however, that proliferation did increase slightly within the exposure period. The MTT assay and confocal microscopy confirm these findings. Under confocal microscopic examination, a compact morphology with unaltered nuclear and cytoskeletal appearances was observed. Thus, there is no evidence suggesting viability is impaired or that cytotoxicity occurs. Ordinary light microscopic examination showed the area denuded of cells had become re-covered completely within 24 hours in the cultures where G. cambogia had been applied. The result suggests that exposure to G. cambogia has no significant effect in terms of stimulating or inhibiting cellular proliferation. CONCLUSIONS: G. cambogia may offer clinical benefit as a supplementary topical treatment for inflammation of the nose and sinuses, as seen in chronic and acute rhinosinusitis, or nasal polyps. The plant appears to increase nasal epitheliocytic proliferation slightly, as revealed by the MTT assay. There were no indications of a cytotoxic effect on epithelial cells of the nose.

Evaluation of ciprofloxacin used as an intranasal antibiotic.

OBJECTIVE: The objective of this research was to examine the effects of topical ciprofloxacin on cultured nasal epithelial cells of human origin. MATERIALS AND METHODS: Human nasal epithelial cells were collected from patients who voluntarily donated tissue left over following septorhinoplasty. The samples were from individuals without any indication of rhinosinusitis. An assay that may be employed to investigate toxic effects at the cellular level is MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). This test reveals where DNA becomes fragmented, the nuclei condense, the outer cell membrane is altered, or the cytoskeleton appears disrupted. The present study employed this technique. Nasal epithelium in cell culture was exposed to ciprofloxacin for 24 hours at a temperature of 37°C, following which the MTT assay was undertaken before examining the cells by confocal microscopy to look for alterations indicating cytotoxicity. Another test of toxicity, the artificial scratch technique, was also used. Cells treated in this way were assessed using a light microscope. RESULTS: The nasal epithelial cells in culture that were exposed to topical ciprofloxacin for 24 hours were less viable than controls and this result was statistically significant. For this length of exposure, the IC50 was calculated as 1.565 mg/mL. The peak reductions in cellular viability occurred with exposure at a concentration of 1.25 mg/mL and 0.625 mg/mL. These was only a mild decrease in viability at other concentrations, but these results were of statistical significance. The MTT assay and confocal microscopy confirmed this result. Cultured nasal epithelium not exposed to ciprofloxacin (i.e., controls) exhibited a compact morphological appearance when examined with confocal microscopy. The epithelial cells had a regular fusiform boundary, and the nuclei were intact. By contrast, the cultures with exposure to the antibiotic were of decreased size and their outline changed from fusiform to round. The epithelial cells cultures where scratch injury was induced were examined by light microscopy, the extent of closure of the denuded area being assessed after 24 hours. It was noted that the area opened up by the experimental scratch was not closed completely by 24 hours later. This result shows that ciprofloxacin decreases the viability of nasal epithelial cells. CONCLUSIONS: Topical application of ciprofloxacin to the nasal lining is not recommended, since this resulted in decreased cellular viability, cellular shrinking and alteration in outline from fusiform to round in cultured nasal epithelial cells. These changes indicate that topically applied ciprofloxacin is toxic to nasal epithelial cells. The outcomes of this study should be studied and correlated in vivo models.

Efficacy and toxicity of Anatolian propolis on healthy nasal epithelial cells.

OBJECTIVE: In our study we aimed to evaluate the effects of applying propolis topically to epithelial cells of the nasal cells, to discover whether this causes any toxic effect upon the cells. MATERIALS AND METHODS: Samples of healthy human primary nasal epithelium harvested during septoplasty from volunteers were incubated in cell culture. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays may be utilized when assessing cellular damage (toxicity), as evidenced by DNA fragmentation, nuclear condensation, alteration in the outer plasma membrane and cytoskeletal alteration. This was the method used in the study. Cultured epithelial cells were incubated with propolis (Bee&You) for 24 hours at 37°C. The MTT assay was then performed, and the cell morphology was examined by confocal microscopy. In addition, via wound healing assay, cellular proliferation was assessed by the artificial scratch method followed by light microscopy. RESULTS: MTT assay results showed that the primary nasal cells were not affected by the topical application of propolis for 24 hours. All of the applied doses not changed significantly the viability of the cells. The agent was not found to be cytotoxic to the primary nasal cells in the application time of 24 hours. Our confocal microscopy findings supported the MTT findings. According to the confocal images, control cells that were not treated with test agent were with compact morphology and undamaged fusiform cell shape and nucleus. In test group of nasal cells, Propolis was found not to be cytotoxic on the cellular morphology and not changed the cells. When evaluating the results from the wound healing assay, the clear area of scratch obtained at the start of incubation (0th) was closed totally with the proliferated primary nasal cells after incubation of 24 hours with propolis. These findings are supported by our MTT findings that imply to the slight induce of proliferation of the primary cells by Propolis. CONCLUSIONS: Topically applied propolis did not have a cytotoxic effect on nasal epithelium cells. Considering its antibacterial and antioxidant effects, it has been concluded that topical application in sinonasal inflammatory diseases (e.g., acute and chronic rhinosinusitis) may have an auxiliary effect in treatment. Moreover, there is a slight induce of proliferation of the primary cells by propolis which may help wound healing in septal surgeries and epistaxis.

The superiority of Dexpanthenol or Vaseline as excipient in nasal formulations.

OBJECTIVE: Dexpanthenol is an ingredient in multiple topical pharmaceutical preparations thanks to its high penetration and localized concentration. It is included in many ointments or lotions for dermatological use, assisting in healing and reducing pruritus. Vaseline is a synthetic product obtained by distilling crude oil. It is commercially available in several grades. The study presented here examined how topically applied agents (dexpanthenol or vaseline) affect nasal epithelial cells in culture. In particular, the study aimed to identify any alterations to epithelial cells which might indicate toxicity. MATERIALS AND METHODS: The nasal epithelial cells used were sourced from mucosal tissue fragments left over the following septorhinoplasty on five patients not suffering from rhinosinusitis. The first step was to dissect the mucosal fragments into smaller pieces on a sterilized Petri dish. These fragments were then placed into the DMEM-F12 cell culture medium, which had been freshly prepared. The dexpanthenol and vaseline were diluted in dimethylsulfoxide (DMSO) to a concentration of 5 mg/mL. The cells in the wells were exposed to varying concentrations of dexpanthenol or vaseline. The actual concentration of the test reagent to which the epithelial cells were exposed ranged from 0.15 mg/mL to 5 mg/mL. The exposure period was 24 hours. The cells were finally examined using a Leica SP5II confocal microscope. The features sought were DNA fragmentation, condensation of the nuclei, changes in the outer membrane, or cytoskeletal abnormality. These features, if present, indicate cytotoxicity. RESULTS: The viability of the cultured nasal epithelial cells was unaltered by a 24-hour exposure to dexpanthenol, nor was the cellular proliferation rate affected at the level of statistical significance. There was evidence of a cytotoxic effect from exposing nasal epithelial cells to vaseline in liquid form for 24 hours. There was a reduction in cellular viability in the plates where the highest dose of vaseline (5 mg/mL) was used. Cellular viability was not affected significantly at any of the doses below 5 mg/mL. CONCLUSIONS: The absence of cytotoxic effects from the application of dexpanthenol to the nasal mucosa indicates that this agent may be safely used within the nose. The cytotoxic effects of liquid vaseline observed in this trial (condensed nuclear chromatin, loss of cellular volume) indicate that this agent may be harmful when used intranasally. For patients who require nasal packing due to nose bleeds or following endoscopic sinus surgical procedures, dexpanthenol should be preferred to vaseline from the point of view of maximizing healing of a nasal injury.

Comparison of p57, c-erbB-2, CD117, and Bcl-2 expression in the differential diagnosis of hydatidiform mole and hydropic abortion.

PURPOSE: To explore the utility of p57, c-erbB-2, CD 117, and Bel-2 immunostaining in the differential diagnosis of complete hydatidiform mole (CHM), partial hydatidiform mole (PHM), and hydropic abortion (HA). MATERIALS AND METHODS: Immunohistochemical expression of the p57, c-erbB-2, CD117, and Bel-2 proteins were investigated semi-quantitatively using paraffin-embedded tissue sections from histologically unequivocal cases of CHM (n = 20), PHM (n = 23), and HA (n = 17). RESULTS: All cases of CHM exhibited a striking absence of p57 expression. The percentage of positive p57 staining was similar between PHMs (73.9%) and HAs (76.5%) (p >0.05). The comparison of c-erbB-2 expression revealed a significantly higher percentage of positive c-erbB-2 staining in CHMs (45%) compared with that in PHMs (8.7%) and HAs (5.9%) (p = 0.006 and 0.01, respectively). The CD 117 expression pattern (immunoreactivity score, percentage of positive cells, and staining intensity) was significantly lower in HAs compared with that in PHMs and CHMs (p < 0.05 for all). A significantly increased Bel-2 expression pattern was observed in HAs compared with that in PHMs and CHMs (p < 0.05 for all). CONCLUSION: Immunohistochemical examination of p57, c-erbB-2, CD 117, and Bel-2 expression represents a relatively simple, reliable, and cost-efficient procedure to definitively distinguish among CHM, PHM, and HA.

Serum level and placental expression of resistin in pregnancies complicated by preeclampsia: relationship with disease severity.

The authors aimed to compare the maternal serum level and placental expression of resistin in pregnancies complicated by preeclampsia and clarify their relationship with disease severity. This cross-sectional study included 50 healthy pregnant women, 50 women with mild preeclampsia, and 48 women with severe preeclampsia. Serum resistin levels were measured by enzyme immunoassay and placental resistin expression was determined by immunohistochemistry. Resistin levels were significantly higher in women with mild and severe preeclampsia than in the healthy controls (p = 0.012 andp < 0.001, respectively). Placental resistin expression was significantly higher in women with severe preeclampsia compared to women with mild preeclampsia (p = 0.003) and healthy controls (p < 0.001). Serum resistin levels were positively correlated with gestational age and umbilical and uterine artery Doppler indices, as well as systolic and diastolic blood pressure, but negatively correlated with birth weight (p < 0.05). On the other hand, placental resistin expression was positively correlated with systolic blood pressure and uterine artery indices, but negatively correlated with birth weight (p < 0.05). In conclusion, increased cir- culating levels and placental expression of resistin in pregnancies complicated by preeclampsia were correlated with disease severity.

AKAP12/Gravin gene expression in colorectal cancer: clinical importance and review of the literature.

PURPOSE: : Colorectal cancer (CRC) is a common and potentially lethal disease. A number of genetic aberrations is known to take place in colorectal carcinogenesis, which leads to progressive alteration of normal mechanisms controlling cell growth. A-kinase-anchoring protein 12 (AKAP12) plays a role in cell proliferation, angiogenesis and cytoskeletal remodeling. The purpose of this study was to demonstrate the role of the AKAP12 gene expression in CRC patients and to determine its relationship (if any) with prognosis. METHODS: AKAP12 gene expression was investigated by immunohistochemistry. RESULTS: A total of 55 patients (63.6% males, 36.4% females) with histologically confirmed CRC were studied. Normal intestinal epithelium showed weak basal staining, dysplastic areas were stained mildly, whereas all of the cancer cells were stained completely with AKAP12. CONCLUSION: AKAP12 gene seems to play a role in colorectal carcinogenesis.

Diagnostic value of thiols, paraoxonase 1, arylesterase and oxidative balance in colorectal cancer in human.

Oxidative stress has been shown to play an important role in carcinogenesis. We hypothesize that serum oxidative and anti-oxidative factors work together to influence colon and rectal cancer through an oxidative balance mechanism. Total thiol (Ttl) is considered a plasma antioxidant and high density lipoprotein (HDL)- dependent paraoxonase l (PON1) is known as a free radical scavenger. This study was undertaken to determine the activity of PON1, arylesterase (ARE) and Ttl levels and oxidative balance in colorectal cancer (CRC) patients and healthy subjects.Paraoxonase and arylesterase activities of the HDL-dependent PON1 enzyme and the level of Ttl, total oxidant status (TOS) ,total antioxidant status (TAS) in forty patients with CRC (19 female, 21 male; mean age, 56.5 ± 2.1) and thirty-nine age and sex matched healthy persons (22 female, 17 male; mean age 56 ± 1.7) were studied.PON1 and ARE activities were significantly lower in patients with CRC compared to controls (p<0.001 for each), whereas oxidant parameters (TOS and OSI) were significantly higher in CRC patients (p<0.001 for each). However, TAS and Ttl significantly decreased in patients with CRC (p=0.03 and p<0.0001, respectively). According to the ROC curve analysis, Ttl was superior to other parameters in terms of diagnostic sensitivity and specificity which were 97.5% and 92.3%, respectively, and the AUC 0.97 in CRC patients. Decreased Ttl and HDL-dependent PON 1 enzyme activities and high TOS levels imply an imbalance of the free radical system which enhances the support the hypothesis that CRC is associated with excess reactive oxygen species.These findings show that of serum oxidative imbalance was associated with an increased risk of CRC. To our knowledge, this is the first report demonstrating lower serum PON1, ARE activities in CRC patients. The mechanism behind this association needs further elucidation.

Prognostic significance of biological apoptosis factors in gastric cancer.

PURPOSE: Gastric cancer is a biologically heterogeneous disease containing many genetic and epigenetic alterations. In our study, the expression status of apoptosis-inducing p53 and apoptosis-inhibiting Bcl-2 in gastric cancer and their relation with prognosis, if any, was investigated. METHODS: Patients that were being followed in our clinic and had histopathologically diagnosed gastric adenocarcinoma were included in this study. The p53 and bcl-2 expressions were investigated immunohistochemically and patients were grouped according to p53 and Bcl-2 expression as follows: group A: both p53 and Bcl-2 negative; group B: p53 positive and Bcl-2 negative; group C: p53 negative and Bcl-2 positive; group D: both p53 and Bcl-2 positive. RESULTS: In 19 (51.4%) patients positive immunostaining with p53 was observed, while negative in 18 (48.6%). A significant relationship between the metastatic ability of the tumor and p53 expression was determined (p=0.004). In 78.6% of the metastatic tumors no p53 expression was observed, while in 69.6% of the non-metastatic tumors p53 expression was positive. No significant relationship was detected between p53 expression and survival. Positive immunostaining with Bcl-2 was observed in 9 (16.7%) patients, and negative in 45 (83.3%). No significant relationship was determined between the Bcl-2 expression and the depth of invasion, dissemination to lymph nodes and metastatic ability of the tumor. A borderline statistically significant relationship was determined between the Bcl-2 expression and survival (p=0.051). Group B patients showed a statistically significant survival difference compared with the other groups (p=0.022). CONCLUSION: The results of this study suggest that concurrent evaluation of p53 and Bcl-2 in patients with gastric adenocarcinoma may have prognostic importance.

The predictive role of Bcl-2 expression in operable locally advanced or metastatic gastric carcinoma.

PURPOSE: Gastric carcinoma is an aggressive disease with different epidemiologic and clinical profiles. Combination chemotherapy containing docetaxel, cisplatin and 5-fluorouracil/5-FU (DCF) is a frequently used regimen in metastatic gastric cancer. We studied the role of B-cell lymphoma 2 (Bcl-2) expression in predicting the response to DCF combination chemotherapy in metastatic gastric carcinoma. METHODS: This study included patients with pathologically confirmed locally advanced, surgically inoperable gastric carcinoma, or with metastatic disease. For immunohistochemical staining of Bcl-2 oncoprotein, lyophilized mouse monoclonal antibody (clone100/D5, 1:50, Thermo Scientific, Fremont, ABD) was used. Bcl-2 expression was evaluated with respect to the nuclear and cytoplasmic staining of the cells. Staining > 10% was accepted as positive and ≤ 10% as negative. RESULTS: Bcl-2 expression was positive in 5 (23.8%) patients and negative in 16 (76.2%), while partial response was achieved in 12 (57%) patients. No complete response was seen in any patient. The effect of positive Bcl-2 expression on survival was statistically significant by log-rank test (p=0.035). CONCLUSION: The patient group that expressed Bcl-2 survived longer confirming that Bcl-2 expression is a good prognostic factor in advanced-stage patients. We believe that Bcl-2 expression has an additional contribution in predicting response to this chemotherapy combination.

Effect of absorbent pads containing oregano essential oil on the shelf life extension of overwrap packed chicken drumsticks stored at four degrees Celsius.

The addition of sachets or pads containing volatile antimicrobial agents into packages has been the most successful commercial application of antimicrobials to packaging. In this study, the effect of oregano (Origanum onites) essential oil on the extension of shelf life of overwrap packed fresh chicken drumsticks was investigated. Meat exudate absorbent pads were sprayed with 5 mL of oregano essential oil at a concentration of 1.5% in distillate water. Sampling was carried out at 0, 3, 5, and 7 d of the refrigerated storage. Total viable count, psychrotrophs, pseudomonads, members of the family Enterobacteriaceae, yeasts, and lactic acid bacteria were enumerated. Physicochemical analysis and sensorial evaluation were also conducted. The shelf life of fresh chicken drumsticks was approximately 3 d. Oregano essential oil extended product shelf life by approximately 2 d. Thus, incorporation of essential oils to absorbent pads may have supplementary applications in food packaging.

Omentum prevents intestinal adhesions to mesh graft in abdominal infections and serosal defects.

BACKGROUND: Many studies have evaluated the use of grafts in the reconstruction of abdominal wall defects. In this study, the effects resulting from the presence or absence of the omentum were evaluated in the setting of infection or serosal defects in the formation of adhesions in abdominal closures using mesh grafts. METHODS: For this study, 60 Wistar albino rats were divided into six groups. A circular 3.79-cm(2) fascioperitoneal defect was created. After group-specific procedures, defects were reconstructed using polypropylene mesh grafts. In group C (control group), only a mesh graft recontruction was performed, whereas group O (O for omentectomy) underwent an omentectomy plus mesh closure. In group SD (serosal defect group), the cecum was abrased with a brush before mesh closure. Group SDO underwent cecal abrasion plus an omentectomy. In group I (infection group), the intraabdominal space was filled with 1 ml of solution containing 100,000 colony-forming units (CFUs) of Escherichia coli per milliliter. Group IO received the same same amount of E. coli solution plus an omentectomy before mesh closure. After 28 days, the groups were evaluated by intraabdominal and blood cultures, grading of intraabdominal adhesions, graft-organ adhesions, proportion of adhesions to graft size, and histopathologic studies. The results were statistically evaluated using one-way variant analysis and Scheffe's and Fisher's definite chi-square tests. RESULTS: For the groups in which the greater omentum was preserved, intestinal adhesions to the graft surface were less frequently observed, especially in cases with intraabdominal infections and serosal defects (p < 0.05). CONCLUSIONS: Preservation of the greater omentum reduces the formation of intestinal adhesions, especially in cases with underlying infections and serosal defects in abdominal closures using mesh grafts. This could be beneficial in related clinical situations in lowering the rate of intestinal fistulas, erosions, and obstructions that can be attributed to the formation of adhesions.