Analysis of the cellular composition of the native auto-transplantation mixture used for plastics of bone tissue defects

The cell composition of native transplant autosmes (NTA) used for bone plastics was studied. The histological examination showed the fragments of bone beams with preserved osteoblasts, the foci of myeloid and lymphoid hematopoiesis and the fibrin deposits, which suggested the presence of MMSCs. Immunophenotyping of the NTA cell population revealed a high level of expression of the surface markers CD105, CD73, and CD90 characteristic for MMSC. DNA-flow cytometry of the bone dust confirmed almost complete preservation of graft viability on the 3rd day of culturing (97.7 % of live cells). The data of this study confirm the presence of the osteogenic, osteoinductive, and osteoconductive properties of the bone dust and emphasize the importance of a further study of this-type bone graft for use in surgical interventions.

Myrtus Communis Leaf Extract - A Source of Secondary Metabolites Exhibiting Anticancer and Antimycobacterial Activities

Background: Plant-derived products or extracts are widely used in folk/traditional medicine to treat several, infections, ailments, or disorders. A notable therapeutic herb Myrtus communis, worldwide utilized in the traditional medication for centuries, is an evergreen aromatic and medicinal plant of the Mediterranean region. Materials and methods: The SulphoRhodamine-B assay and DNA flow cytometry were used to investigate the proliferation and subsequent distribution of cells among different phases of the cell cycle. Annexin V-FITC/PI staining coupled with flow cytometry was used to analyze apoptosis and necrosis of the cancer cells. Western blotting detected the expression of pro- and anti-apoptotic proteins. Zone of inhibition and MIC were determined by well diffusion method and microplate alamar blue assay, respectively. Biofilm formation was studied by crystal violet method. For statistical analysis, a two-tailed Student’s t-test of GraphPad Prism 6.0 was used.Results: In this study, the secondary metabolites of M. communis leaves extracted in ethanol showed the highest cytotoxicity and thus the greatest anticancer effects against diverse cancer cell lines of the breast (MCF-7), liver (HepG2), cervix (HeLa), and colon (HCT116) (IC50; ranging from 33 to 83 mg/ml). The cancer cells arrested in the G1 phase of the cell cycle undergo apoptosis. The induction of the latter is mediated by activation of the intrinsic mitochondrial pathway. Furthermore, the extract showed a strong growth inhibitory effect (zone of inhibition; 20.3±1.1 - 26.3±2.5 mm, MIC; 4.88 - 312.5 µg/ml, and MBC; 39.07 - 1250 μg/ml) against several rapid and slow-growing mycobacterial strains that cause tuberculosis and several other mycobacterial infections. The biofilm formation in BSL2 microorganisms, M. smegmatis and S. aureus, is strongly inhibited by the extract. Conclusion: These results suggest that M. communis leaf extract is a potential source of secondary metabolites, which could be developed further as potential anti-cancer and anti-mycobacterial agents to treat diverse types of cancers and mycobacterial infections.

Design and Synthesis of Newly Synthesized Acrylamide Derivatives as Potential Chemotherapeutic Agents against MCF-7 Breast Cancer Cell Line Lodged on PEGylated Bilosomal Nano-Vesicles for Improving Cytotoxic Activity

Cancer is a multifaceted disease. With the development of multi drug resistance, the need for the arousal of novel targets in order to avoid these drawbacks increased. A new series of acrylamide derivatives was synthesized from starting material 4-(furan-2-ylmethylene)-2-(3,4,5-trimethoxyphenyl)oxazol-5(4H)–one (1), and they are evaluated for their inhibitory activity against β-tubulin polymerization. The target molecules 2–5 d were screened for their cytotoxic activity against breast cancer MCF-7 cell line. The results of cytotoxicity screening revealed that compounds 4e and 5d showed good cytotoxic profile against MCF-7 cells. Compounds 4e produced significant reduction in cellular tubulin with excellent β-tubulin polymerization inhibition activity. In addition, compound 4e exhibited cytotoxic activity against MCF-7 cells by cell cycle arrest at pre-G1 and G2/M phases, as shown by DNA flow cytometry assay. Aiming to enhance the limited aqueous solubility and, hence, poor oral bioavailability of the prepared lead acrylamide molecule, 4e-charged PEGylated bilosomes were successfully fabricated via thin film hydration techniques as an attempt to improve these pitfalls. Twenty-three full factorial designs were manipulated to examine the influence of formulation variables: types of bile salt including either sodium deoxy cholate (SDC) or sodium tauro cholate (STC), amount of bile salt (15 mg or 30 mg) and amount of DSPE–mPEG-2000 amount (25 mg or 50 mg) on the characteristics of the nanosystem. The F7 formula of entrapment efficiency (E.E% = 100 ± 5.6%), particle size (PS = 280.3 ± 15.4 nm) and zeta potential (ZP = −22.5 ± 3.4 mv) was picked as an optimum formula with a desirability value of 0.868. Moreover, prominent enhancement was observed at the compound’s cytotoxic activity (IC50 = 0.75 ± 0.03 µM) instead of (IC50 = 2.11 ± 0.19 µM) for the unformulated 4e after being included in the nano-PEGylated bilosomal system.

Design, Synthesis and Anticancer Evaluation of Substituted Cinnamic Acid Bearing 2-Quinolone Hybrid Derivatives

A new series of hybrid molecules containing cinnamic acid and 2-quinolinone derivatives were designed and synthesized. Their structures were confirmed by 1H-NMR, 13C-NMR and mass analyses. All the synthesized hybrid molecules were assessed for their in vitro antiproliferative activity against more than one cancer cell lines. Compound 3-(3,5-dibromo-7,8-dihydroxy-4-methyl-2-oxoquinolin-1(2H)-ylamino)-3-phenylacrylic acid (5a) with IC50 = 1.89 μM against HCT-116 was proved to the most potent compound in this study, as compared to standard drug staurosporin. DNA flow cytometry assay of compound 5a revealed G2/M phase arrest and pre-G1 apoptosis. Annexin V-FITC showed that the percentage of early and late apoptosis was increased. The results of topoisomerase enzyme inhibition activity showed that the hybrid molecule 5a displays potent inhibitory activity compared with control.

Testicular fixation and its effect on ipsilateral and contralateral testis in prepubertal rat model

Background While performing orchidopexy, various suture materials or fibrin glues are used to achieve testicular fixation. This study was designed to assess the histological changes in testis after orchidopexy using fibrin glue and suture material. Methods Male Wistar rats (n = 80) were divided randomly into four groups. Group I, (n = 20): sham operation, Group II (n = 20): Dartos Pouch (DP), Group III (n = 20): Transtunical fixation (TF), Group IV (n = 20): Tissue Adhesive (TA). Ipsilateral and contralateral testicular histology was assessed at 70 and 120 days of life after sacrificing animals by using thiopental sodium intraperitoneally at a dose of 100 mg/kg. Results Morphologically, at day 70, contralateral testis in Group III had a significant (p value 0.046) decrease testicular width (0.92 ± 0.01 vs 1.24 ± 0.39 cm). At 120 of life, Group I, II, III, and IV had a significant (p value < 0.001 each) decrease testicular width and weight in ipsilateral and decrease testicular length (p value 0.002) in contralateral testis. Histologically, mean seminiferous tubular diameter and DNA flow cytometry had a significant (p value < 0.001) decrease in size in Group I, II, III, and IV both ipsilateral as well as contralateral testis. Intergroup comparison at 70 and 120 days of life showed a significant decrease in seminiferous tubular diameter in Group II, III and IV and in Johnsen maturation score, seminiferous tubular diameter, DNA flow cytometry in Group I, II, III, and IV. Conclusions Dartos Pouch is most suitable procedure for treatment of orchidopexy. Suture fixation must be avoided and if the need arises then instead of suture materials, fibrin glue should be used for testicular fixation.

Persistent or recurrent Barrett’s neoplasia after an endoscopic therapy session is associated with DNA content abnormality and can be detected by DNA flow cytometric analysis of paraffin-embedded tissue

Endoscopic therapy is currently the standard of care for the treatment of high-grade dysplasia (HGD) or intramucosal adenocarcinoma (IMC) in patients with Barrett’s esophagus (BE). Visible lesions are treated with endoscopic mucosal resection (EMR), which is often coupled with radiofrequency ablation (RFA). However, endoscopic therapy may require multiple sessions (one session every 2-3 months) and does not always assure complete eradication of neoplasia. Furthermore, despite complete eradication, recurrences are not uncommon. This study assesses which potential risk factors can predict a poor response after endoscopic sessions. Forty-five BE patients who underwent at least one endoscopic session (EMR alone or ablation with or without preceding EMR) for the treatment of HGD/IMC, low-grade dysplasia (LGD), or indefinite for dysplasia (IND) were analyzed. DNA flow cytometry was performed on 82 formalin-fixed paraffin-embedded samples from the 45 patients, including 78 HGD/IMC, 2 LGD, and 2 IND. Eight non-dysplastic BE samples were used as controls. Three to four 60-micron thick sections were cut from each tissue block, and the area of HGD/IMC, LGD, or IND was manually dissected. Potential associations between clinicopathologic risk factors and persistent/recurrent HGD/IMC following each endoscopic session were examined using univariate and multivariate Cox models with frailty terms. Sixty (73%) of the 82 specimens showed abnormal DNA content (aneuploidy or elevated 4N fraction). These were all specimens with HGD/IMC (representing 77% of that group). Of these 60 HGD/IMC samples with abnormal DNA content, 42 (70%) were associated with subsequent development of persistent/recurrent HGD/IMC (n = 41) or esophageal adenocarcinoma (EAC; n = 1) within a mean follow-up time of 16 months (range: 1 month to 9.4 years). In contrast, only 6 (27%, all HGD/IMC) of the 22 remaining samples (all with normal DNA content) were associated with persistent/recurrent HGD/IMC. For outcome analysis per patient, 11 (24%) of the 45 patients developed persistent/recurrent HGD/IMC or EAC, despite multiple endoscopic sessions (mean: 3.6, range: 1–11). In a univariate Cox model, the presence of abnormal DNA content (hazard ratio [HR] = 3.8, p = 0.007), long BE segment ≥ 3 cm (HR = 3.4, p = 0.002), endoscopic nodularity (HR = 2.5, p = 0.042), and treatment with EMR alone (HR = 2.9, p = 0.006) were significantly associated with an increased risk for persistent/recurrent HGD/IMC or EAC. However, only abnormal DNA content (HR = 6.0, p = 0.003) and treatment with EMR alone (HR = 2.7, p = 0.047) remained as significant risk factors in a multivariate analysis. Age ≥ 60 years, gender, ethnicity, body mass index (BMI) ≥ 30 kg/m2, presence of hiatal hernia, and positive EMR lateral margin for neoplasia were not significant risk factors for persistent/recurrent HGD/IMC or EAC (p > 0.05). Three-month, 6-month, 1-year, 3-year, and 6-year adjusted probabilities of persistent/recurrent HGD/IMC or EAC in the setting of abnormal DNA content were 31%, 56%, 67%, 79%, and 83%, respectively. The corresponding probabilities in the setting of normal DNA content were 10%, 21%, 28%, 38%, and 43%, respectively. In conclusion, in BE patients with baseline HGD/IMC, both DNA content abnormality and treatment with EMR alone were significantly associated with persistent/recurrent HGD/IMC or EAC following each endoscopic session. DNA content abnormality as detected by DNA flow cytometry identifies HGD/IMC patients at highest risk for persistent/recurrent HGD/IMC or EAC, and it also serves as a diagnostic marker of HGD/IMC with an estimated sensitivity of 77%. The diagnosis of HGD/IMC in the setting of abnormal DNA content may warrant alternative treatment strategies as well as long-term follow-up with shorter surveillance intervals.

Vandetanib versus Cabozantinib in Medullary Thyroid Carcinoma: A Focus on Anti-Angiogenic Effects in Zebrafish Model

Medullary thyroid carcinoma (MTC) is a tumor deriving from the thyroid C cells. Vandetanib (VAN) and cabozantinib (CAB) are two tyrosine kinase inhibitors targeting REarranged during Transfection (RET) and other kinase receptors and are approved for the treatment of advanced MTC. We aim to compare the in vitro and in vivo anti-tumor activity of VAN and CAB in MTC. The effects of VAN and CAB on viability, cell cycle, and apoptosis of TT and MZ-CRC-1 cells are evaluated in vitro using an MTT assay, DNA flow cytometry with propidium iodide, and Annexin V-FITC/propidium iodide staining, respectively. In vivo, the anti-angiogenic potential of VAN and CAB is evaluated in Tg(fli1a:EGFP)y1 transgenic fluorescent zebrafish embryos by analyzing the effects on the physiological development of the sub-intestinal vein plexus and the tumor-induced angiogenesis after TT and MZ-CRC-1 xenotransplantation. VAN and CAB exert comparable effects on TT and MZ-CRC-1 viability inhibition and cell cycle perturbation, and stimulated apoptosis with a prominent effect by VAN in MZ-CRC-1 and CAB in TT cells. Regarding zebrafish, both drugs inhibit angiogenesis in a dose-dependent manner, in particular CAB shows a more potent anti-angiogenic activity than VAN. To conclude, although VAN and CAB show comparable antiproliferative effects in MTC, the anti-angiogenic activity of CAB appears to be more relevant.

Synthesis, Characterization, Anti-proliferative Evaluation, and DNA Flow Cytometry Analysis of Some 2-Thiohydantoin Derivatives

Background and Objective: Due to the well-documented anti-proliferative activity of 2-thiohydantoin incorporated with pyrazole, oxadiazole, quinazoline, urea, β-naphthyl carbamate and Schiff bases, they are noteworthy in pharmaceutical chemistry. Methods: An efficient approach for the synthesis of a novel series of 2-thiohydantoin derivatives incorporated with pyrazole and oxadiazole has proceeded via the reaction of the acyl hydrazide with chalcones and/or triethyl orthoformate. Schiff bases were synthesized by the reaction of the acyl hydrazide with different aromatic aldehydes. Moreover, Curtius rearrangement was applied to the acyl azide to obtain the urea derivative, quinazoline derivative, and carbamate derivative. Results: The synthesized compounds structures were discussed and confirmed depending on their spectral data. The anticancer activity of these heterocyclic compounds was evaluated against the breast cancer cell line (MCF-7), where they showed variable activity. Compound 5d found to have a superior anticancer activity, where it has (IC50 = 2.07 ± 0.13 μg/mL) in comparison with the reference drug doxorubicin that has (IC50 = 2.79 ± 0.07 μg / mL). Then compound 5d subjected to further studies such as cell cycle analysis and apoptosis. Apoptosis was confirmed by the upregulation of Bax, downregulation of Bcl-2, and the increase of the caspase 3/7percentage. Conclusion: Insertion of pyrazole, oxadiazole and, quinazoline moieties with 2-thiohydantoin moiety led to the enhancement of its anti-proliferative activity. Hence they can be used as anticancer agents.

Comparative DNA Flow Cytometric Study of Primary Intraocular and Central Nervous System Lymphomas

Primary intraocular lymphoma is generally considered as a subset of primary CNS lymphoma. This study attempts to show that they may in fact represent distinct entities by comparing their respective proliferation rates using DNA flow cytometry. Four samples of primary intraocular lymphoma and seven samples of primary CNS lymphoma were analyzed, all from paraffin-embedded tissue. All tumors were of the large B-cell type. A normal human tonsil sample was used as a control. Tissue samples were analyzed by DNA flow cytometry, which is a precise and objective method to measure DNA content and cell proliferation of a tumor. S-phase fraction (SPF) and DNA content were measured for each sample. The average SPF for primary intraocular lymphoma was significantly higher than that of primary CNS lymphoma, 23.8 (range: 18.9 to 29.6) versus 15.1 (range: 1.1 to 25.1) respectively. Of the 11 tumors analyzed, 2 brain tumors were aneuploid and 1 eye tumor was peridiploid. All other tumors were diploid. Thus, no significant pattern was detected in the DNA content of the tumors. This lack of clinical significance of tumor aneuploidy is consistent with data reported in the literature. The results of this study indicate that primary intraocular lymphoma is more aggressive and of higher grade than primary CNS lymphoma. The different proliferation rates of intraocular and CNS lymphomas may be explained by either their different spatial location or a distinct genetic composition, the latter reinforcing the hypothesis that the two are fundamentally different entities

In Vitro Anticancer Evaluation of Some Synthesized 2H-Quinolinone and Halogenated 2H-Quinolinone Derivatives as Therapeutic Agents

Background: Searching for new cytotoxic agents with apoptosis induction may represent a viable strategy for cancer treatment to overcome the increased resistance to available anticancer agents. Objective: The purpose of the current study was aimed at preparation and anticancer evaluation of two new series of 2H-quinolinone and halogenated 2H-quinolinone derivatives against two cancer cell lines. Methods: Two new series of 2H-quinolinone and halogenated 2H-quinolinone derivatives were prepared and screened for their cytotoxicity against breast MCF-7 and liver HepG-2 cancer cell lines as well as normal breast MCF-10a. Results: The tested molecules revealed good cytotoxicity and selectivity toward cancer cell lines relative to normal cells. These compounds were analyzed by DNA flow cytometry on MCF-7 cells. They were found to cause G2/M phase arrest and induced apoptosis at the pre-G1 phase. In addition, increased caspase 3/7 activity and decreased osteopontin expression verified the apoptotic activity. Conclusion: The potent compounds discovered in this study can be a hit for the discovery of new cytotoxic agents and are worthy of further investigation.