The Effect of 4-(Dimethylamino)phenyl-5-oxopyrrolidines on Breast and Pancreatic Cancer Cell Colony Formation, Migration, and Growth of Tumor Spheroids.

A series of hydrazones, azoles, and azines bearing a 4-dimethylaminophenyl-5-oxopyrrolidine scaffold was synthesized. Their cytotoxic effect against human pancreatic carcinoma Panc-1 and triple-negative breast cancer MDA-MB-231 cell lines was established by MTT assay. Pyrrolidinone derivatives 3c and 3d, with incorporated 5-chloro and 5-methylbenzimidazole fragments; hydrazone 5k bearing a 5-nitrothien-2-yl substitution; and hydrazone 5l with a naphth-1-yl fragment in the structure significantly decreased the viability of both cancer cell lines. Compounds 3c and 5k showed the highest selectivity, especially against the MDA-MB-231 cancer cell line. The EC50 values of the most active compound 5k against the MDA-MB231 cell line was 7.3 ± 0.4 µM, which were slightly higher against the Panc-1 cell line (10.2 ± 2.6 µM). Four selected pyrrolidone derivatives showed relatively high activity in a clonogenic assay. Compound 5k was the most active in both cell cultures, and it completely disturbed MDA-MB-231 cell colony growth at 1 and 2 µM and showed a strong effect on Panc-1 cell colony formation, especially at 2 µM. The compounds did not show an inhibitory effect on cell line migration by the 'wound-healing' assay. Compound 3d most efficiently inhibited the growth of Panc-1 spheroids and reduced cell viability in MDA-MB-231 spheroids. Considering these different activities in biological assays, the selected pyrrolidinone derivatives could be further tested to better understand the structure-activity relationship and their mechanism of action.

Assessment of Toxicity and Wound Healing Activity of Selaginella Bryopteris Extract.

AIM: The aim of the study was to assess the toxicity profile of Selaginella bryopteris extract and evaluate its wound healing activity. METHODS: In vitro wound healing activity of S. bryopteris extract (5% and 10%) was performed using Clonogenic and Scratch assays. The toxicity profile of S. bryopteris extract ointment was evaluated on animals using acute toxicity and dermal toxicity tests. In vivo wound healing activity of S. bryopteris extract ointment (5% and 10%) was used to determine tensile strength in the incision wound healing model. RESULTS: Results exhibited that the extract was safe up to 2000 mg/kg per oral dose and non-reactive while applied topically. In vitro results showed that S. bryopteris extract closed the wound gap created by 97.13% in 48 h. The clonogenic assay revealed that the surviving factor for HaCaT cells and MEF cells was 0.78 and 0.85 after treated with 10% concentrations of S. bryopteris. The tensile strength exhibited by S. bryopteris 5% and 10% groups was 395.4 g and 558.5 g in comparison to the control group. CONCLUSION: Thus, S. bryopteris extract can be used as an alternative safe drug therapy against topical wounds.

Toward a Transportable Cell Culture Platform for Evaluating Radiotherapy Dose Modifying Factors.

The current tools for validating dose delivery and optimizing new radiotherapy technologies in radiation therapy do not account for important dose modifying factors (DMFs), such as variations in cellular repair capability, tumor oxygenation, ultra-high dose rates and the type of ionizing radiation used. These factors play a crucial role in tumor control and normal tissue complications. To address this need, we explored the feasibility of developing a transportable cell culture platform (TCCP) to assess the relative biological effectiveness (RBE) of ionizing radiation. We measured cell recovery, clonogenic viability and metabolic viability of MDA-MB-231 cells over several days at room temperature in a range of concentrations of fetal bovine serum (FBS) in medium-supplemented gelatin, under both normoxic and hypoxic oxygen environments. Additionally, we measured the clonogenic viability of the cells to characterize how the duration of the TCCP at room temperature affected their radiosensitivity at doses up to 16 Gy. We found that (78±2)% of MDA-MB-231 cells were successfully recovered after being kept at room temperature for three days in 50% FBS in medium-supplemented gelatin at hypoxia (0.4±0.1)% pO2, while metabolic and clonogenic viabilities as measured by ATP luminescence and colony formation were found to be (58±5)% and (57±4)%, respectively. Additionally, irradiating a TCCP under normoxic and hypoxic conditions yielded a clonogenic oxygen enhancement ratio (OER) of 1.4±0.6 and a metabolic OER of 1.9±0.4. Our results demonstrate that the TCCP can be used to assess the RBE of a DMF and provides a feasible platform for assessing DMFs in radiation therapy applications.

Design of the New Closo-Dodecarborate-Containing Gemcitabine Analogue for the Albumin-Based Theranostics Composition.

Combination therapy is becoming an increasingly important treatment strategy because multi-drugs can maximize therapeutic effect and overcome potential mechanisms of drug resistance. A new albumin-based theranostic containing gemcitabine closo-dodecaborate analogue has been developed for combining boron neutron capture therapy (BNCT) and chemotheraphy. An exo-heterocyclic amino group of gemcitabine was used to introduce closo-dodecaborate, and a 5'-hydroxy group was used to tether maleimide moiety through an acid-labile phosphamide linker. The N-trifluoroacylated homocysteine thiolactone was used to attach the gemcitabine analogue to human serum albumin (HSA) bearing Cy5 or Cy7 fluorescent dyes. The half-maximal inhibitory concentration (IC50) of the designed theranostic relative to T98G cells was 0.47 mM with the correlation coefficient R = 0.82. BNCT experiments resulted in a decrease in the viability of T98G cells, and the survival fraction was ≈ 0.4.

Clonogenic assays improve determination of variant allele frequency of driver mutations in myeloproliferative neoplasms.

Molecular diagnostics moves more into focus as technology advances. In patients with myeloproliferative neoplasms (MPN), identification and monitoring of the driver mutations have become an integral part of diagnosis and monitoring of the disease. In some patients, none of the known driver mutations (JAK2V617F, CALR, MPL) is found, and they are termed "triple negative" (TN). Also, whole-blood variant allele frequency (VAF) of driver mutations may not adequately reflect the VAF in the stem cells driving the disease. We reasoned that colony forming unit (CFU) assay-derived clonogenic cells may be better suited than next-generation sequencing (NGS) of whole blood to detect driver mutations in TN patients and to provide a VAF of disease-driving cells. We have included 59 patients carrying the most common driver mutations in the establishment or our model. Interestingly, cloning efficiency correlated with whole blood VAF (p = 0.0048), suggesting that the number of disease-driving cells correlated with VAF. Furthermore, the clonogenic VAF correlated significantly with the NGS VAF (p < 0.0001). This correlation was lost in patients with an NGS VAF <15%. Further analysis showed that in patients with a VAF <15% by NGS, clonogenic VAF was higher than NGS VAF (p = 0.003), suggesting an enrichment of low numbers of disease-driving cells in CFU assays. However, our approach did not enhance the identification of driver mutations in 5 TN patients. A significant correlation of lactate dehydrogenase (LDH) serum levels with both CFU- and NGS-derived VAF was found. Our results demonstrate that enrichment for clonogenic cells can improve the detection of MPN driver mutations in patients with low VAF and that LDH levels correlate with VAF.

Anti-proliferative and pro-apoptotic activity of glycosidic derivatives of lawsone in melanoma cancer cell.

BACKGROUND: Melanoma is a malignant cancer that affects melanocytes and is considered the most aggressive skin-type cancer. The prevalence for melanoma cancer for the last five year is about one million cases. The impact caused of this and other types of cancer, revel the importance of research into potential active compounds. The natural products are an important source of compounds with biological activity and research with natural products may enable the discovery of compounds with potential activity in tumor cells. METHODS: The Sulforhodamine B was used to determine cell density after treatment with lawsone derivatives. Apoptosis and necrosis were analyzed by flow cytometer. Morphological changes were observed by fluorescence using the Phalloidin/FITC and DAPI stains. The clonogenic and wound healing assays were used to analyze reduction of colonies formation and migratory capacity of melanoma cells, respectability. RESULTS: In pharmacological screening, seven compounds derived from lawsone were considered to have high cytotoxic activity (GI > 75%). Three compounds were selected to assess the inhibitory concentration for 50% of cells (IC50), and the compound 9, that has IC50 5.3 µM in melanoma cells, was selected for further analyses in this cell line. The clonogenic assay showed that the compound is capable of reducing the formation of melanoma colonies at 10.6 µM concentration. The compound induced apoptotic morphological changes in melanoma cells and increased by 50% the cells dying from apoptosis. Also, this compound reduced the migratory capacity of melanoma cells. CONCLUSIONS: The results of this study showed that the evaluated lawsone derivatives have potential activity on tumor cells. The compound 9 is capable of inducing cell death by apoptosis in melanoma cells (B16F10).

Verification of electron beam parameters in an intraoperative linear accelerator using dosimetric and radiobiological response methods.

BACKGROUND: The availability of linear accelerators (linac) for research purposes is often limited and therefore alternative radiation sources are needed to conduct radiobiological research. The National Centre for Radiation Research in Poland recently developed an intraoperative mobile linac that enables electron irradiation at energies ranging from 4 to 12 MeV and dose rates of 5 or 10 Gy/min. The present study was conducted to evaluate the electron beam parameters of this intraoperative linac and to verify the set-up to evaluate out-of-field doses in a water phantom, which were determined through dosimetric and biological response measurements. MATERIALS AND METHODS: The distribution of radiation doses along and across the radiation beam were measured in a water phantom using a semiconductor detector and absolute doses using an ionisation chamber. Two luminal breast cancer cell lines (T-47D and HER2 positive SK-BR-3) were placed in the phantom to study radiation response at doses ranging from 2 to 10 Gy. Cell response was measured by clonogenic assays. RESULTS AND CONCLUSION: The electron beam properties, including depth doses and profiles, were within expected range for the stated energies. These results confirm the viability of this device and set-up as a source of megavoltage electrons to evaluate the radiobiological response of tumour cells.

Exploring the role of oral microorganisms in the pathogenesis of mucositis by assessing their impact on metabolic activity and reproductive capacity of epithelial cells in vitro.

PURPOSE: Clinical and in vitro studies showed selected oral microorganisms to be related to delayed wound healing and ulcerative oral mucositis. However, it is not known whether this effect is due to reduced metabolism and/or the reduced reproductive capacity of epithelial cells. Therefore, we studied the influence of the oral microorganisms Porphyromonas gingivalis, Candida glabrata, and Candida kefyr on cell metabolism and reproductive capacity of oral epithelial cells, aimed to further unravel the pathogenesis of oral mucositis. METHODS: Oral epithelial cells were exposed to different concentrations of P. gingivalis, C. glabrata, and C. kefyr as mono-infections or mixed together. An MTT assay was performed to determine the effect on cell metabolism. A clonogenic assay was used to study the effect on the reproductive capacity of oral epithelial cells. RESULTS: The metabolism of oral epithelial cells was reduced when the microorganisms were present in high concentrations: P. gingivalis at a multiplicity of infection (MOI) of 1000 and the Candida spp. at MOI 100. No statistical difference was observed in the ability of a single epithelial cell to grow into a colony of cells between control and P. gingivalis, C. glabrata, and C. kefyr, independent of the concentrations and combinations used. CONCLUSION: P. gingivalis, C. glabrata, and C. kefyr lowered the metabolic activity of oral epithelial cells in high concentrations, yet they did not influence the reproductive capacity of epithelial cells. Their impact on ulcerative oral mucositis is likely due to an effect on the migration, proliferation, and metabolism of epithelial cells.

In Vitro Inhibition of Hsp90 Protein by Benzothiazoloquinazolinequinones Is Enhanced in The Presence of Ascorbate. A Preliminary In Vivo Antiproliferative Study.

A series of benzo[g]benzothiazolo[2,3-b]quinazoline-7,12-quinones were prepared from 2-acylnaphthohydroquinones and 2-aminobenzothiazoles and were evaluated for their in vitro antiproliferative activity. After screening using the MTT reduction assay, their IC50 values were calculated on a panel of cancer cells (T24, DU-145, MCF-7). Current standard anticancer drugs were included as control, and their calculated IC50 values were 7.8 and 23.5 µM for 5-fluorouracil and tamoxifen, respectively. Non-cancer cells (AG1523) were included to assess cancer cell sensitivity and drug selectivity. Four members of the series, with IC50 values from 0.11 to 2.98 µM, were chosen for further assays. The selected quinones were evaluated regarding their effects on cancer cell proliferation (clonogenic assay) and on Hsp90 and poly(ADPribose)polymerase (PARP) protein integrity. The most active compound (i.e., 15) substantially inhibited colony forming unit (CFU) formation at 0.25 µM. In the presence of ascorbate, it induced an oxidative cleavage of Hsp90 but had no effect on PARP protein integrity. In an in vivo animal model, it discreetly increased the mean survival time (m.s.t.) of tumor-bearing mice. In light of these results, compound 15 represents a potential lead-molecule to be further developed.

Six New Polyhydroxysteroidal Glycosides, Anthenosides S1 - S6, from the Starfish Anthenea sibogae.

Six new polyhydroxysteroidal glycosides, anthenosides S1  - S6 (1 - 6), along with a mixture of two previously known related glycosides, 7 and 8, were isolated from the methanolic extract of the starfish Anthenea sibogae. The structures of 1 - 6 were established by NMR and HR-ESI-MS techniques as well as by chemical transformations. All new compounds have a 5α-cholest-8(14)-ene-3α,6ß,7ß,16α-tetrahydroxysteroidal nucleus and differ from majority of starfish glycosides in positions of carbohydrate moieties at C(7) and C(16) (1 - 4, 6) or only at C(16) (5). The 4-O-methyl-ß-d-glucopyranose residue (2) and Δ24 -cholestane side chain (3) have not been found earlier in the starfish steroidal glycosides. The mixture of 7 and 8 slightly inhibited the proliferation of human breast cancer T-47D cells and decreased the colony size in the colony formation assay.

Miniaturization of the Clonogenic Assay Using Confluence Measurement.

The clonogenic assay is a widely used method to study the ability of cells to 'infinitely' produce progeny and is, therefore, used as a tool in tumor biology to measure tumor-initiating capacity and stem cell status. However, the standard protocol of using 6-well plates has several disadvantages. By miniaturizing the assay to a 96-well microplate format, as well as by utilizing the confluence detection function of a multimode reader, we here describe a new and modified protocol that allows comprehensive experimental setups and a non-endpoint, label-free semi-automatic analysis. Comparison of bright field images with confluence images demonstrated robust and reproducible detection of clones by the confluence detection function. Moreover, time-resolved non-endpoint confluence measurement of the same well showed that semi-automatic analysis was suitable for determining the mean size and colony number. By treating cells with an inhibitor of clonogenic growth (PTC-209), we show that our modified protocol is suitable for comprehensive (broad concentration range, addition of technical replicates) concentration- and time-resolved analysis of the effect of substances or treatments on clonogenic growth. In summary, this protocol represents a time- and cost-effective alternative to the commonly used 6-well protocol (with endpoint staining) and also provides additional information about the kinetics of clonogenic growth.

Comparative evaluation of the clonogenic capacity of periodontal ligament fibroblasts in Hank's balanced salt solution and egg albumen: An in vitro study.

BACKGROUND/AIM: The type of storage media for short-term storage of an avulsed tooth is a critical determinant for the success of tooth replantation. If immediate replantation of an avulsed tooth is not possible, it is advised to store the tooth in a suitable storage medium. The viability and clonogenicity of periodontal ligament fibroblasts (PDLF) determines the success of replantation of an avulsed tooth. The aim of this study was to evaluate the effect on the clonogenic capacity of PDLF's upon storage in Hank's balanced salt solution (HBSS) and egg albumen. METHODS: Fibroblast cell culture was established from a human premolar tooth extracted for orthodontic purposes. The PDLF cells thus obtained were treated with either Dulbecco's modified Eagle's medium (DMEM; as a positive control), HBSS, or egg albumen for different durations at room temperature and then allowed to grow in DMEM medium until visible colonies appeared which were then fixed, stained, and scored manually. RESULTS: With increase in the duration of storage in both egg albumen as well as HBSS, there was a reduction in the clonogenic capacity of the PDLF's as compared to DMEM. However, storage in egg albumen led to a significant reduction in the clonogenic capacity of PDLF's (8%-16% for egg albumen) compared to HBSS (80%-90%). CONCLUSION: Due to its limited ability to support the clonogenicity of PDLF's, egg albumen is a poor storage medium for an avulsed tooth compared to either DMEM or HBSS.

Feasibility of in vivo three-dimensional T 2* mapping using dicarboxy-PROXYL and CW-EPR-based single-point imaging.

OBJECTIVES: The aim of this study was to demonstrate the feasibility of in vivo three-dimensional (3D) relaxation time T 2* mapping of a dicarboxy-PROXYL radical using continuous-wave electron paramagnetic resonance (CW-EPR) imaging. MATERIALS AND METHODS: Isotopically substituted dicarboxy-PROXYL radicals, 3,4-dicarboxy-2,2,5,5-tetra(2H3)methylpyrrolidin-(3,4-2H2)-(1-15N)-1-oxyl (2H,15N-DCP) and 3,4-dicarboxy-2,2,5,5-tetra(2H3)methylpyrrolidin-(3,4-2H2)-1-oxyl (2H-DCP), were used in the study. A clonogenic cell survival assay was performed with the 2H-DCP radical using squamous cell carcinoma (SCC VII) cells. The time course of EPR signal intensities of intravenously injected 2H,15N-DCP and 2H-DCP radicals were determined in tumor-bearing hind legs of mice (C3H/HeJ, male, n = 5). CW-EPR-based single-point imaging (SPI) was performed for 3D T 2* mapping. RESULTS: 2H-DCP radical did not exhibit cytotoxicity at concentrations below 10 mM. The in vivo half-life of 2H,15N-DCP in tumor tissues was 24.7 ± 2.9 min (mean ± standard deviation [SD], n = 5). The in vivo time course of the EPR signal intensity of the 2H,15N-DCP radical showed a plateau of 10.2 ± 1.2 min (mean ± SD) where the EPR signal intensity remained at more than 90% of the maximum intensity. During the plateau, in vivo 3D T 2* maps with 2H,15N-DCP were obtained from tumor-bearing hind legs, with a total acquisition time of 7.5 min. CONCLUSION: EPR signals of 2H,15N-DCP persisted long enough after bolus intravenous injection to conduct in vivo 3D T 2* mapping with CW-EPR-based SPI.

Glutathione-dependent and -independent oxidative stress-control mechanisms distinguish normal human mammary epithelial cell subsets.

Mechanisms that control the levels and activities of reactive oxygen species (ROS) in normal human mammary cells are poorly understood. We show that purified normal human basal mammary epithelial cells maintain low levels of ROS primarily by a glutathione-dependent but inefficient antioxidant mechanism that uses mitochondrial glutathione peroxidase 2. In contrast, the matching purified luminal progenitor cells contain higher levels of ROS, multiple glutathione-independent antioxidants and oxidative nucleotide damage-controlling proteins and consume O2 at a higher rate. The luminal progenitor cells are more resistant to glutathione depletion than the basal cells, including those with in vivo and in vitro proliferation and differentiation activity. The luminal progenitors also are more resistant to H2O2 or ionizing radiation. Importantly, even freshly isolated "steady-state" normal luminal progenitors show elevated levels of unrepaired oxidative DNA damage. Distinct ROS control mechanisms operating in different subsets of normal human mammary cells could have differentiation state-specific functions and long-term consequences.

Preclinical Study of Antineoplastic Sinoporphyrin Sodium-PDT via In Vitro and In Vivo Models.

Photodynamic therapy (PDT) investigations have seen stable increases and the development of new photosensitizers is a heated topic. Sinoporphyrin sodium is a new photosensitizer isolated from Photofrin. This article evaluated its anticancer effects by clonogenic assays, MTT assays and xenograft experiments in comparison to Photofrin. The clonogenicity inhibition rates of sinoporphyrin sodium-PDT towards four human cancer cell lines ranged from 85.5% to 94.2% at 0.5 µg/mL under 630 nm irradiation of 30 mW/cm² for 180 s. For MTT assays, the IC50 ranges of Photofrin-PDT and sinoporphyrin sodium-PDT towards human cancer cells were 0.3 µg/mL to 5.5 µg/mL and 0.1 µg/mL to 0.8 µg/mL under the same irradiation conditions, respectively. The IC50 values of Photofrin-PDT and sinoporphyrin sodium-PDT towards human skin cells, HaCaT, were 10 µg/mL and 1.0 µg/mL, respectively. Esophagus carcinoma and hepatoma xenograft models were established to evaluate the in vivo antineoplastic efficacy. A control group, Photofrin-PDT group (20 mg/kg) and sinoporphyrin sodium group at three doses, 0.5 mg/kg, 1 mg/kg and 2 mg/kg, were set. Mice were injected with photosensitizers 24 h before 60 J 630 nm laser irradiation. The tumor weight inhibition ratio of 2 mg/kg sinoporphyrin sodium-PDT reached approximately 90%. Besides, the tumor growths were significantly slowed down by 2 mg/kg sinoporphyrin sodium-PDT, which was equivalent to 20 mg/kg Photofrin-PDT. In sum, sinoporphyrin sodium-PDT showed great anticancer efficacy and with a smaller dose compared with Photofrin. Further investigations are warranted.

Anticarcinogenic Effect of Spices Due to Phenolic and Flavonoid Compounds-In Vitro Evaluation on Prostate Cells.

This study shows the effects of spices, and their phenolic and flavonoid compounds, on prostate cell lines (PNT1A, 22RV1 and PC3). The results of an MTT assay on extracts from eight spices revealed the strongest inhibitory effects were from black pepper and caraway seed extracts. The strongest inhibitory effect on prostatic cells was observed after the application of extracts of spices in concentration of 12.5 mg·mL-1. An LC/MS analysis identified that the most abundant phenolic and flavonoid compounds in black pepper are 3,4-dihydroxybenzaldehyde and naringenin chalcone, while the most abundant phenolic and flavonoid compounds in caraway seeds are neochlorogenic acid and apigenin. Using an MTT assay for the phenolic and flavonoid compounds from spices, we identified the IC50 value of ~1 mmol·L-1 PNT1A. The scratch test demonstrated that the most potent inhibitory effect on PNT1A, 22RV1 and PC3 cells is from the naringenin chalcone contained in black pepper. From the spectrum of compounds assessed, the naringenin chalcone contained in black pepper was identified as the most potent inhibitor of the growth of prostate cells.

An evaluation of novel real-time technology as a tool for measurement of radiobiological and radiation-induced bystander effects.

The xCELLigence real-time cell impedance system uses a non-invasive and label-free method to create a cell index that is a composite measure of cell proliferation. The aim of this study was to evaluate xCELLigence against clonogenic assay (gold standard) for measuring radiobiological effects and radiation-induced bystander effects (RIBE). A radiobiological study was conducted by irradiating EMT6.5, 4T1.2 and NMUMG cell lines with different radiation doses, while a RIBE study was done using transfer of conditioned media (CM) harvested from donor to the same type of recipient cell (EMT6.5, 4T1.2, NMUMG, HACAT and SW48). CM was harvested using two protocols which differed in the dose chosen and the exposure to the recipient cells. Results showed that xCELLigence measured a radiobiological effect which correlated with the clonogenic assay. For the RIBE study, no statistically significant differences were observed between xCELLigence or clonogenic survival in control or recipient cells incubated with CM in protocol one. However, there was a significant increase in cell index slope using CM from EMT-6.5 cells irradiated at 7.5 Gy compared with the control group under the second protocol. No other evidence of RIBE was detected by either xCELLigence or clonogenic assay. In conclusion, xCELLigence methods can measure radiobiological effects and the results correlate with clonogenic assay. We observed a lack of RIBE in all tested cell lines with the clonogenic assay; however, we observed a RIBE effect in EMT6.5 cells under one particular protocol that showed RIBE is cell type dependent, is not universally observed and can be detected in different assays.

Comparative effects of cocaine and cocaethylene on alveolar epithelial type II cells.

Abuse of cocaine (COC) and alcohol have been among the leading causes of non-prescription drug-related deaths in the USA and are known to cause acute and chronic lung diseases. The co-abuse of COC and alcohol results in the production of an active metabolite, cocaethylene (CE). The effects of COC and its metabolites on the respiratory system have been scarcely studied. This study was aimed at comparing the toxic effects of eqimolar concentration (1 mM) of COC and CE on alveolar epithelial type II cells. This was performed by measuring cell growth, viability, clonogenic activity, cell cycle and reactive oxygen species (ROS) generation. The treatment of CE and COC resulted in a significant inhibition of cell proliferation with the formation of an average of three colonies which measured about 1.74×10(-15) m each and 25 colonies each of about 5.73×10(-15) m, respectively, while untreated cells yielded 31 colonies of 8.75×10(-15) m (p<0.05). The treatments of CE and COC resulted in the reduction of the growth fraction of alveolar epithelial type II cells without significant decrease in viability. In addition, there was an approximately twofold increase in ROS generation as compared to the controls (p<0.05). Therefore, CE-induced inhibition of cellular proliferation may contribute to the pathogenesis of diffuse alveolar damage in co-abusers of COC and alcohol.

Mitotic disruption and reduced clonogenicity of pancreatic cancer cells in vitro and in vivo by tumor treating fields.

OBJECTIVES: Tumor Treating Fields (TTFields) are a non-invasive cancer treatment modality approved for the treatment of patients with recurrent glioblastoma. The present study determined the efficacy and mechanism of action of TTFields in preclinical models of pancreatic cancer. METHODS: The effect of TTFields in vitro was assessed using cell counts, clonogenic assays, cell cycle analysis and analysis of mitotic figures. The effect in vivo effect was studied in the PC1-0 hamster pancreatic cancer model. RESULTS: Application of TTFields in vitro showed a significant decrease in cell count, an increase in cell volume and reduced clonogenicity. Further analysis demonstrated significant increase in the number of abnormal mitotic figures, as well as a decrease in G2-M cell population. In hamsters with orthotopic pancreatic tumors, TTFields significantly reduced tumor volume accompanied by an increase in the frequency of abnormal mitotic events. TTFields efficacy was enhanced both in vitro and in vivo when combined with chemotherapy. CONCLUSIONS: These results provide the first evidence that TTFields serve as an effective antimitotic treatment in preclinical pancreatic cancer models and have a long term negative effect on cancer cell survival. These results make TTFields an attractive candidate for testing in the treatment of patients with pancreatic cancer.

Combining a noble gas with radiotherapy: glutamate receptor antagonist xenon may act as a radiosensitizer in glioblastoma.

BACKGROUND: Ionotropic glutamate receptors α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) and N-methyl-D-aspartate receptor (NMDAR) modulate proliferation, invasion and radioresistance in glioblastoma (GB). Pharmacological targeting is difficult as many in vitro-effective agents are not suitable for in patient applications. We aimed to develop a method to test the well tolerated AMPAR- and NMDAR-antagonist xenon gas as a radiosensitizer in GB. METHODS: We designed a diffusion-based system to perform the colony formation assay (CFA), the radiobiological gold standard, under xenon exposure. Stable and reproducible gas atmosphere was validated with oxygen and carbon dioxide as tracer gases. After checking for AMPAR and NMDAR expression via immunofluorescence staining we performed the CFA with the glioblastoma cell lines U87 and U251 as well as the non-glioblastoma derived cell line HeLa. Xenon was applied after irradiation and additionally tested in combination with NMDAR antagonist memantine. RESULTS: The gas exposure system proved compatible with the CFA and resulted in a stable atmosphere of 50% xenon. Indications for the presence of glutamate receptor subunits were present in glioblastoma-derived and HeLa cells. Significantly reduced clonogenic survival by xenon was shown in U87 and U251 at irradiation doses of 4-8 Gy and 2, 6 and 8 Gy, respectively (p < 0.05). Clonogenic survival was further reduced by the addition of memantine, showing a significant effect at 2-8 Gy for both glioblastoma cell lines (p < 0.05). Xenon did not significantly reduce the surviving fraction of HeLa cells until a radiation dose of 8 Gy. CONCLUSION: The developed system allows for testing of gaseous agents with CFA. As a proof of concept, we have, for the first time, unveiled indications of radiosensitizing properties of xenon gas in glioblastoma.