In vitro Culture and Morphometry of Porcine Adipose Derived Mesenchymal Stem Cells (pAD-MSCs)

Background: Mesenchymal stem cells are well known for their self-renewal capacity and ability to differentiate into multiple cell lineages. The aim of the study was to develop a simple technique for isolation of mesenchymal stem cells from porcine adipose tissue and to study the morphometric characteristics of porcine mesenchymal stem cells. Methods: Porcine adipose derived mesenchymal stem cells were isolated in vitro by using collagenase type II enzyme. Cell yield and viability of the cells were calculated by using trypan blue exclusion method using Neubauer’s chamber. Characterization of MSCs were done by using specific cell markers. The morphological changes, morphometry were analysed in culture using Leishman’s stain. The cell doubling (CD) and Population doubling time (PDT) were also calculated. Result: The isolated adherent cells start forming colony and demonstrated an elongated, round and spindle like fibroblastic morphology by day 1. Almost 80-90 per cent confluency was attained on day 8-9 after the initial seeding and was reduced to day 3-4 in the subsequent passages. RT-PCR reactions revealed positive expression of mesenchymal stem cell markers CD44, CD73 and negative expression of CD34, a hematopoietic cell surface marker. Immunocytochemistry also revealed positive expression for CD44 and negative for CD34. In morphometric studies, the cell length, nucleus length, cell width and nucleus width were increased between 24 and 48 hours in both P2 and P3.

Highly diluted compounds effects on B16-F10 melanogenesis, reactive oxygen species (ROS) production and tumorigenesis.

Background: Cutaneous melanoma is a highly malignant tumor derived from pigment-producing (melanin) melanocytes of skin epidermis. Cutaneous pigmentation is described as the major physiologic defense against UV radiation. During melanin biosynthesis and other tumorigenic process, reactive oxygen species (ROS) are produced and might be critically involved in several melanomagenesis stages. ROS play key roles on regulation of many types cell proliferation, including melanoma cells. Aims: In this work we evaluated the effects of highly diluted compounds on melanogenesis and changes in reactive oxygen species after 96 hours of treatment and possible involvement in tumorigenesis. Methodology: Melanin content was measured in B16-F10 cells after 96 hours of treatment with highly diluted compounds, as well as the superoxide anion, hydrogen peroxide and nitric oxide. Furthermore, the effects of highly diluted compounds on cell proliferation were investigated by trypan blue exclusion method after 48 hours of treatment. Results: Treatment led to an increase in B16-F10 melanin content and a decrease in nitrite concentration, an intermediate product of nitric oxide. We also observed a decrease in cell proliferation after treatment. It is well recognized that nitric oxide (NO) is involved in tumor progression, including melanoma. Several articles show that NO treated B16-F10 cells exhibited higher metastatic capacity. Thereby, reduction in cell proliferation can be due to low NO levels. It is speculated that melanocytes are programmed to survive in order to preserve their photoprotective role, thus in a compensatory manner the cell may be synthesizing melanin in response to cell proliferation reduction. Conclusions: These results suggest that treatment may be reducing tumorigenic capacity via ROS reduction. However further studies are need to better understand highly diluted compounds mechanisms of action.

Confocal Microscopy in Conjunction With Haemocytometry to Evaluate the Imperative Physical Characteristics of Multicellular Tumor Spheroids

Background: Tumor tissues resist penetration of therapeutic molecules. Multicellular tumor spheroids (MCTSs) were used as an in vitro tumor model. The aim of this study was to determine the growth of MCTSs with the age of spheroids, which could be applied and compared with in vivo drug uptake and penetration. Method: Spheroids were generated by liquid overlay techniques, and their diameter was measured by confocal microscopy for up to two weeks. The trypan blue exclusion method was used to count dead and live cells separately via a hemocytometer. Results: The pentaphysical characteristics of spheroids, including diameter, cell number, volume per cell, viability status, and estimated shell of viable and core of dead cells, were determined. The growth of spheroids was linear over the first week but declined in the 2nd week, which may be due to an overconcentration of dead cells and degraded products inside the spheroids, hence lowering the ratio of live cells in spheroids. Compaction of spheroids occurs from day 3 to day 7, with the mature spheroids having a low amount of extracellular space compared to intracellular volume. Conclusion: Age-oriented growth of MCTSs provides a rationale to predict less rapid penetration as spheroids get older and could be correlated with in vivo tumors to predict pharmaceutical and therapeutic intervention.

Ethanolic extract of gabirobeira (Campomanesia adamantium) leaves reduces proliferation of osteosarcoma cells in vitro

Osteosarcoma is the most commonly diagnosed malignant bone tumor in humans, with a higher incidence in children and young people. It is highly aggressive and has a high metastatic potential. Its treatment is based on both chemotherapy and surgical intervention. However, currently used chemotherapeutic agents, such as doxorubicin, have several adverse effects on the patient. Therefore, there is a growing demand for new chemotherapeutic agents that stimulate new researches, such as those involving compounds extracted from plants, such as the gabirobeira. In this study, we aimed to evaluate the cytotoxic effects of ethanolic extract, both crude and ethyl acetate, of gabirobeira leaves on osteosarcoma cells in vitro. Cytotoxicity was evaluated using the Trypan blue exclusion method and the IC50 values were calculated using the tetrazolium reduction method. The ethanolic extract of gabirobeira leaves showed a cytotoxic effect on osteosarcoma cells in vitro. The group treated with the crude extract at 1. 0μL mL-1 concentration for 48 hours showed higher cytotoxicity and the lowest IC50 value for this extract was found in the 24 to 48 hours interval. The ethanolic extract of gabirobeira leaves is cytotoxic for osteosarcoma cells.

Cytotoxic metabolites from Thysanolaena maxima Roxb. available in Bangladesh

Background Thysanolaena maxima (Roxb.) Kuntze, a perennial grass plant, is usually distributed in hilly regions of the Indian Subcontinent. Different parts of T. maxima have been used as herbal medicine by traditional healers of this region. In this present study, T. maxima plant extract has been screened for examination of its secondary metabolite content with their probable cytotoxic activity. Methods Secondary metabolites of the crude T. maxima plant extract were isolated by different chromatographic methods. The structures were elucidated by spectroscopic data (1H NMR, 13C NMR) as well as comparison with available literature sources. Antiradical activity by DPPH radical scavenging assay and antimicrobial activity by disc diffusion method of the fractions and cytotoxic activity by trypan blue exclusion method of the isolated compounds were also evaluated. Results Three phenolic compounds 4-hydroxybenzaldehyde (1), 4-hydroxycinnamic acid (2), 4-hydroxybenzoic acid (3) and two steroids stigmast-4-en-3-one (4) and β-stigmasterol (5) were isolated from the aerial part of T. maxima. Among the compounds 4-hydroxycinnamic acid, 4-hydroxybenzoic acid and stigmast-4-en-3-one exhibited notable cytotoxic activity against African Green Monkey Kidney Cell line (Vero cell). Conclusion Bioassay investigation of the isolated compounds and fractions suggested that T. maxima could be a potential source of bioactive secondary metabolites. Graphical abstract

Proanthocyanidins reduce cellular function in the most globally diagnosed cancers in vitro

Background Growing evidence indicates that proanthocyanidins (PACs) may be effective in treating and preventing various cancers. The fundamental mechanism of PACs inhibiting the proliferation at cellular and molecular levels in most of the cancer types remains unclear. Objective The anticancer efficacy of PACs was investigated in vitro using three human cancer cell lines: human colorectal adenocarcinoma (HT-29), human breast carcinoma (MCF-7), and human prostatic adenocarcinoma (PC-3). Methods Cytotoxicity was evaluated by MTT assay, while cell proliferation was measured by trypan blue exclusion method. Cell migration was measured by wound healing assay, and DAPI staining was used to evaluate apoptotic nucleus morphology. RT-PCR was used to analyze the expression of Bax and Bcl-2, and caspase enzyme activity assay was measured by caspase colorimetric assay. Results PACs could inhibit both cellular viability and proliferation in a concentration- and time-dependent fashion in all investigated cells. Further, all tested cells showed similarly decreased migration after 24- and 48-h PAC treatment. We observed increased apoptotic nucleus morphology in treated cells (p ≤ 0.01). BAX expression significantly increased in HT-29 (p < 0.01), PC-3(p < 0.01), and MCF-7 (p < 0.05) cells, while BCL-2 expression significantly declined (p < 0.05). Caspase activities were significantly increased in all tested cancer cell lines after 24-h PAC treatment. Conclusion PACs may have potential therapeutic properties against colorectal, breast, and prostate cancer.

Molecular Response of Pulp Fibroblasts after Stimulation with Pulp Capping Materials

This in vitro study evaluated cell viability and metabolism, nitric oxide release and production of two chemokines and one cytokine by cultured human dental pulp fibroblasts (HDPF) in contact with two glass ionomer cements (Ketac Molar-KM and Vitrebond-VB), Single Bond (SB) and calcium hydroxide (Dycal-DY). Cultures of HDPF were established by means of an explant technique. The specimens were prepared under sterile conditions and in disks measuring 5 mm x 2 mm obtained from a prefabricated mold and placed on a permeable membrane to avoid direct contact with the cells. Cytotoxicity was assessed by Trypan Blue exclusion method and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Nitric oxide release in cell supernatant was detected by the Griess Method whereas stromal derived factor-1 alpha (SDF-1α or CXCL12), chemokine (C-X-C motif) ligand 8 [Interleukin 8 (IL-8 or CXCL8)] and interleukin-6 (IL-6) were detected by ELISA. RT-qPCR was employed for gene expression analysis. Statistical analyses were performed by One-way ANOVA followed by Tukey’s post hoc test for materials independent of the time, and Two-way ANOVA followed by Bonferroni correction test for the comparisons between materials and experimental time (p<0.05). Cytotoxic tests showed significant differences only for DY. Protein levels and mRNA expression were significantly increased for IL-8 for both periods of time. IL-6 production increased when fibroblasts were stimulated by KM. SDF-1α protein production and mRNA expression were not affected by any of the materials. There was a decrease in nitrate/nitrite levels only for KM. Although DY caused intense cell death and did not stimulate the production of the inflammatory mediators evaluated in this work, it is known that this event seems to be fundamental for the process of repair of the pulp tissue and formation of mineralized barrier. KM and VB increased production of proteins related to the inflammatory process, thus favoring tissue repair. Therefore, although these glass ionomer cements did not lead to large cell death, they should be used with caution.

Antileukemic Cell Proliferation of Active Compounds from Kaffir Lime (Citrus hystrix) Leaves

Kaffir lime (Citrus hystrix) is a plant member of family Rutaceae, and its leaves are commonly used in folk medicine. The present study explores antileukemic effects of the extracts and purified active compounds from the leaves. The antileukemic activity was investigated via inhibition of Wilms’ tumor 1 (WT1), which is a protein that involves in leukemic cell proliferation. In addition, the compounds were investigated for their effects on WT1 gene expression using real time RT-PCR and Western blotting. Cell cycle arrest and total cell number were investigated using flow cytometry and trypan blue exclusion method, respectively. The results demonstrated that the hexane fractionated extract had the greatest inhibitory effect on WT1 gene expression of many leukemic cell lines and significantly decreased WT1 protein levels of K562 cells (representative of the leukemic cells), in a dose- and time-dependent manner. Subfraction No. 9 (F9) after partial purification of hexane fractioned extract showed the highest suppression on WT1 protein and suppressed cell cycle at G2/M. The organic compounds were isolated from F9 and identified as phytol and lupeol. The bioassays confirmed antiproliferative activities of natural products phytol and lupeol. The results demonstrated anticancer activity of the isolated phytol and lupeol to decrease leukemic cell proliferation.

Evaluation of in vitro Cytotoxic Effects of Especifico Pessoa Phytotherapic Tincture on Ehrlich Tumor Cells and Mice Spleen Cells

Especifico Pessoa (EP) is traditionally used for the treatment of snakebite envenoming. The traditional use of EP and its properties have been reported. In this study, we evaluated the in vitro cytotoxic effects of EP on Ehrlich tumor and mice spleen cells. Cytotoxicity assay was carried out by using Trypan blue exclusion method. Spleen cell suspension was prepared (n=2) with RPMI medium and tumor cell suspension was prepared from ascitic fluid of Ehrlich tumor-bearing mice (n=1); both the suspensions contained 4 x 106 cells mL-1. Pure EP or EP diluted in RPMI (1:2; 1:4) was used. The results were expressed as percentage of cell viability and demonstrate that EP is toxic to Ehrlich cells at all concentrations (Control: 96.42 ± 3.40; Pure: 1.55 ± 2.91; 1:2: 4.85 ± 5.04; 1:4: 13.39 ± 5.08), but nontoxic to spleen cells in at the lowest dilution (Control: 72.86 ± 13.79; Pure: 13.52 ± 6,36; 1:2: 41.36 ± 13.51; 1:4: 56.59 ± 8.62). Therefore, the results demonstrate that EP has cytotoxic effects, depending on the dose and the cell line evaluated.

ANTIOXIDATIVE ACTIVITY OF ALPHA-MANGOSTIN IN ACETALDEHYDE-INDUCED HEPATIC STELLATE CELLS: AN IN VITRO STUDY

Objective: Alcohol accumulation in the liver can cause pathological disorders such as liver fibrosis that can develop into hepatocellular carcinoma,one of the main causes of mortality associated with liver disease. The previous studies have shown that a plant compound, alpha-mangostin, has anantioxidant effect in the inhibition of pancreatic tumor growth in vitro. This study aimed to analyze the antioxidative properties of alpha-mangostin inacetaldehyde-induced liver fibrosis in vitro.Methods: Immortalized hepatic stellate cells (HSCs), of the LX-2 cell line, were incubated with acetaldehyde in the presence or absence of alphamangostin(10 and 20 μM). The cells were then counted and lysed, and LX-2 cell viability was determined with the trypan blue exclusion method. Themalondialdehyde levels, superoxide dismutase activity, and glutathione (GSH) levels were also determined using the cell lysates.Results: Acetaldehyde treatment resulted in a significant increase in HSC cell viability and decreased the production of GSH. Alpha-mangostintreatment resulted in reduced cell viability of the HSCs and prevention of the loss of intracellular GSH.Conclusion: Alpha-mangostin reduced acetaldehyde-induced cell proliferation, and this was affected at least in part by its antioxidative properties