Assessment of Proliferation, Clonogenic Assay, and Osteogenic Differentiation of Human Periodontal Ligament Stem Cells Following Application of Orthodontic Forces

Context: The proliferation and differentiation of human periodontal ligament stem cells (hPDLSC) into other cell types are also mediated by mechanical stresses; they might offer therapeutic benefits in tissue regeneration and angiogenesis. Objectives: The study was planned to assess the proliferation, clonogenic potential, and osteogenic differentiation of human periodontal ligament stem cells (PDLSC) following the application of light and heavy orthodontic forces. Materials and Methods: A couple forces of 50 gm (light force) were applied on the 1st premolar on the one side and 250 gm (heavy force) on the contralateral side in the upper arch of patients requiring orthodontic treatment with extraction of all 1st premolars. After 30 days, periodontal tissues were scrapped from extracted teeth for the establishment of PDLSC in vitro. PDLC from the lower premolar teeth where no orthodontic force was applied acted as the control group. Morphology, viability, proliferating rate and population doubling time, clonogenicity, and alkaline phosphatase activity were analysed. Result: The osteogenic potential was confirmed by Alizarin red staining and the expression of the osteogenic markers by qRT?PCR. The morphology, growth kinetics, potency, and osteogenic lineage characteristics inferred the application of high force reduced the proliferative ability and osteogenesis of PDLSC, though the difference was not significant. Conclusion: The established PDLSCs demonstrated their MSC?like properties based on morphology, growth kinetics, colony forming ability, and AP activity. The culture?expanded PDLSCs showed their differentiation potential into osteocytes. The application of high force reduced the proliferative ability and osteogenesis of PDLSCs, variations were not significant.differentiation

Actividad citotóxica y antiproliferativa in vitro del extracto acuoso de Phyllanthus comosus

RESUMEN Introducción: Desde los inicios de la medicina antigua, las plantas han sido utilizadas como tratamiento en diversas enfermedades incluyendo las de naturaleza infecto-contagiosa y el cáncer. Son numerosos los informes sobre las propiedades biológicas del género Phyllanthus. Objetivo: Evaluar la actividad citotóxica y antiproliferativa de un extracto acuoso de Phyllanthus comosus en tres líneas celulares, dos de origen tumoral (SiHa y HeLa) y una no tumoral (Vero). Métodos: La actividad citotóxica se evaluó mediante el método del MTT y la capacidad antiproliferativa mediante el ensayo de detección de inhibición de colonias o clonogénico. Se tuvieron en cuenta valores como la concentración citotóxica media (CC50), índice selectivo y porcentaje de disminución de la proliferación celular. Resultados: En el ensayo de citotoxicidad se obtuvieron CC50 similares para ambas líneas tumorales; mientras que el valor para la línea Vero resultó tres veces menos tóxico, con valores de índice de selectividad mayor que tres. El ensayo clonogénico demostró inhibición de la proliferación en las líneas tumorales, mientras que en células Vero no se observó inhibición de la capacidad de formación de colonias. Conclusiones: El extracto de P. comosus es más citotóxico para las líneas tumorales SiHa y HeLa que para las células Vero, no tumorales. Además, la inhibición de la formación de clonos celulares en ambas líneas tumorales evidencia su acción antiproliferativa y selectiva, lo que argumenta su potencialidad antitumoral in vitro

ABSTRACT Introduction: Ever since the onset of ancient medical practice, plants have been used to treat a variety of conditions, including infectious communicable diseases and cancer. A large number of reports are available about the biological properties of the genus Phyllantus. Objective: Evaluate the cytotoxic and antiproliferative activity of an aqueous extract of Phyllanthus comosus on three cell lines: two of tumoral origin (SiHa and HeLa) and one of non-tumoral origin (Vero). Methods: Cytotoxic activity was evaluated by the MTT method, and antiproliferative capacity by colony inhibition detection or clonogenic assay. Mean cytotoxic concentration (CC50), selective index and cell proliferation reduction percentage were some of the values taken into account. Results: The cytotoxicity assay obtained similar CC50 values for both tumor cell lines, whereas the value for the Vero line was three times less toxic, with a selectivity index above three. The clonogenic assay revealed proliferation inhibition in the tumor cell lines, whereas no inhibition of colony forming capacity was observed in Vero cells. Conclusions: The P. comosus extract is more cytotoxic for tumoral cell lines SiHa and HeLa than for non-tumor Vero cells. Additionally, inhibition of the formation of cell clones in both tumor cell lines is evidence of its antiproliferative and selective action, substantiating its in vitro antitumor potential.

Anti-proliferative potential of sodium thiosulfate against HT 29 human colon cancer cells with augmented effect in the presence of mitochondrial electron transport chain inhibitors

Objective: To compare the anti-proliferative effect of sodium thiosulfate on human colorectal cancer cells (HT-29) and normal small intestine cells (IEC6). Methods: Cells (HT-29 and IEC6) were treated with different concentrations of sodium thiosulfate ranging from 0.5 mM to 80 mM for 24 h. Cell viability was measured via crystal violet and MTT assays. HT-29 cells were further treated in the presence and absence of mitochondrial electron transport chain (ETC) inhibitors, K

Anti-proliferative potential of sodium thiosulfate against HT 29 human colon cancer cells with augmented effect in the presence of mitochondrial electron transport chain inhibitors

Objective: To compare the anti-proliferative effect of sodium thiosulfate on human colorectal cancer cells (HT-29) and normal small intestine cells (IEC6). Methods: Cells (HT-29 and IEC6) were treated with different concentrations of sodium thiosulfate ranging from 0.5 mM to 80 mM for 24 h. Cell viability was measured via crystal violet and MTT assays. HT-29 cells were further treated in the presence and absence of mitochondrial electron transport chain (ETC) inhibitors, KATP channel opener and closer and H2S inhibitors for 24 h followed by sodium thiosulfate in order to study their respective roles in the anti-proliferative activity of sodium thiosulfate. Results: The IC50 values of sodium thiosulfate on HT-29 cells were 40.93 mM and 42.45 mM by crystal violet and MTT assay whereas, in the case of IEC6 cells, the values were 45.17 mM and 47.22 mM. The inhibition of endogenous H2S enzymes and KATP channel induced no change in the anti-proliferative capacity of sodium thiosulfate. However, the anti-proliferative activity of sodium thiosulfate was enhanced in the presence of mitochondrial ETC inhibitors. Conclusions: HT-29 cell growth is effectively attenuated by sodium thiosulfate and the anti-proliferative activity of sodium thiosulfate is enhanced in the presence of mitochondrial ETC inhibitors.

Lawaly Maman Manzo1,3, Yijuan Cheng2,3, Ling yang1,3, Jiping liu1 , Ya-Hui Deng1 , Li-Ping Sun2 and Yu liu1 . Small-scale screening program for the identification of cytotoxic Oxazolo[5,4-d]pyrimidine derivatives based on Whole Cell Viability Assay. Indian Journal of Pharmaceutical & Biological Research. 2015 Apr-Jun; 3(2): 58-65.

For over last couple of decades, there has been a robust activity aimed towards the discovery of novel anti-cancer therapeutics. An approach to identify starting points for new drug candidates is high throughput screening of compound library collection. In this work, we describe the application of a Tetrazolium-based, 96-well small scale screening assay to screen a mini library of 19 compounds bearing Oxazolo[5,4-d]pyrimidine structures against human umbilical vein endothelial cells. Primary actives identified against HUVEC were retested and the IC50 value compounds were estimated for HUVEC. The screening program (Primary screening) identified 4 compounds with inhibition rate percentage ≥ 70% each. Retest screening of these compounds, taking into account criteria required for high cytotoxic compounds, afforded a panel of 1 compound for further biological analysis. This compound had IC50 value of 12.19µM, 12.16µM, 10.24µM, 20.43µM for HUVECs, SGC7901, MCF7, and HeLa respectively. Furthermore, a clonogenic assay was performed in order to confirm the cytotoxic activity of the selected compound on the survival and proliferation of MCF7. This compound was found to significantly effect the survival and proliferation of MCF7. Taken together, the selected compound, namely SCYJ32, was found to be highly cytotoxic against the numerous cell lines. Further studies are ongoing in order to unravel various mechanisms of action of this novel small compound.

Radiosensitization of histone deacetylases inhibitor panobinostat in prostate cancer cells / 中华放射肿瘤学杂志

Objective To study the radiosensitization of histone deaeetylases inhibitor(HDACI) panobinistat in prostate cancer cells in vitro,as well as the possible mechanisms.Methods IC20 of two prostate cancer cell lines(LNCaP and PC-3)was determined using MTI assay.Cells received a single dose irradiation of 0,2,4,6,or 8 Gy using 6 MV X-ray for radiosensitivity experiment,but only 2 Gy for western blot and flow cytometry.Radiosensitization of panobinostat was investigated with clonogenic assay,and sensitizing enhancement ratio(SER)was calculated with single-hit multi-target model.Western blot was used to compare γH2AX expression.Flowcytomctry was used to detect the cell cycle distribution.Results IC20 of LNCaP and PC-3 was 2.5 and 10.0 μmol/L,respectively.SER of panobinostat at IC20 was 1.37(D0 ratio)and 1.11(Dq ratio)for LNCaP cells,and 1.78(D0 ratio)and 1.17(Dq ratio)for PC-3 cells.Expression of γH2AX gradually decreased in the 2 Gy irradiation-alone cells standing for the DSB repair,while γH2AX expression was persistent in the combination group.Irradiation triggered a G2/M arrest 6-12 hours after irradiation in LNCaP and PC-3 cells.G2/M arrest was observed when cells were treated with panobinostat for 24 hours,however,no significant change concerning cell cycle distribution was showed when cells received further irradiation.Conclusions Panobinostat Call radiosensitize prostate cancer cells,which may be related with increased DNA DSB,inhibition of DSB repair and attenuation of cell cycle modulation after irradiation.

Immunorestorative in immunosuppressed Balb/c mice and cytotoxic activity of water extract from Trichilia hirta root / Actividad inmunorestauradora en ratones Balb/c inmunodeprimidos y citotóxica del extracto acuoso de raíz de Trichilia hirta

Patients receiving chemotherapy treatment in Santiago de Cuba traditionally use water extracts from Trichilia hirta roots. The study aim was to evaluate the immunorestorative and cytotoxic activity of water extracts from Trichilia hirta root. Administration of root water extract increased the total and differential leukocyte counts in inmunosupressed Balb/c mice. Thymus weight recovered significantly as well as bone marrow cellularity. Moreover, water extract (125 ug/mL) showed selective cytotoxicity against cancer cells T-47D and SK-mel-3 in comparison with non-cancer cells (Vero). The results indicate that Trichilia hirta has significant immunorestorative effects in vivo and selective cytotoxicity in vitro. Therefore, it might be a promising alternative for cancer therapy.

Pacientes bajo tratamiento quimioterapéutico tradicionalmente usan extractos acuosos de raíz de Trichilia hirta en Santiago de Cuba. El objetivo de este estudio fue evaluar la actividad inmunorestauradora y citotóxica de extractos acuosos de raíz de Trichilia hirta. La administración del extracto acuoso de raíz incrementó los conteos globales y diferenciales de leucocitos en ratones inmunodeprimidos. El peso del timo, así como, la celularidad de la médula ósea se recuperaron significativamente. Además, el extracto acuoso (125 ug/mL) mostró citotoxicidad selectiva contra las células tumorales T-47D y SK-mel-3 en comparación con la línea no tumoral (Vero). Los resultados indican que Trichilia hirta posee significativos efectos inmunorestauradores in vivo y citotoxicidad selectiva, por lo cual podría ser una promisoria alternativa para la terapia del cáncer.

Radiosensitizing effect of gemcitabine on human lung cancer cells : A preliminary in vitro study / 中华放射肿瘤学杂志

Objective To investigate whether gemcitabine (GEM) could enhance radiosensitivity of human non-small cell lung cancer cells and its related mechanism.Methods Clonogenic assay was used to analyze radiosensitivity enhancement by GEM on p53 mutant human lung adenocarcinoma cell line 973.Alterations of cell cycle distribution and apoptosis were measured by flow cytometry.Results Mild radiosesitizing effect was observed when 10 nmol/L GEM was administrated before or after irradiation.Marked radiosesitizing effect was demonstrated when 100 nmol/L GEM was administrated before or after irradiation, with much stronger effect of pre-irradiation GEM treatment.Mutation of p53 gene affected cell cycle redistribution and cell apoptosis, but had no relationship with radiosensitivity enhancement of GEM.Conclusions 100 nmol/L GEM could significantly enhance radiosensitivity of human lung cancer cells.However, this effect may not be associated with p53 gene mutation, cell cycle redistribution or cell apoptosis.

The Use of MTT Assay, In Vitro and Ex Vivo, to Predict the adiosensitivity of Colorectal Cancer / 대한방사선종양학회지

PURPOSE: The measurement of radiosensitivity of individuals is useful in radiation therapy. Unfortunately, the measurement of radiation survival using a clonogenic assay, which is the established standard, can be difficult and time consuming. The aim of this study is to compare radiosensitivity results obtained from the MTT and clonogenic assays, and to evaluate whether the MTT assay can be used on clinical specimens. MATERIALS AND METHODS: HCT-8, LoVo, CT-26, and WiDr were the colon cancer cell lines used for this study. The clonogenic assay was performed to obtain the cell survival curves and surviving fractions at a dose of 2 Gy (SF2) as the standard technique for radiosensitivity. Also, the MTT assay was performed for each of the cell lines (in vitro). To simulate clinical specimens, the cell lines were inoculated into nude mice, removed when the tumors reached 1 cm in diameter, and chopped. Next, the tumors were subjected to the same process involved with the MTT assay in vitro. The inhibition rates (IR) of 10 Gy or 20 Gy of irradiation for in vitro and ex vivo were calculated based on the optical density of the MTT assay, respectively. RESULTS: According to SF2 and the cell survival curve, the HCT-8 and WiDr cell lines were more resistant to radiation than LoVo and CT-26 (p<0.05). The IR was measured by in vitro. The MTT assay IR was 17.3%, 21%, 30% and 56.5% for the WiDr, HCT-8, LoVo and CT-26 cell lines, respectively. In addition, the IR measured ex vivo by the MTT assay was 23.5%, 26%, 38% and 53% in the HCT-8, WiDr, LoVo and CT-26 tumors, respectively. CONCLUSION: The radiosensitivity measured by the MTT assay was correlated with the measures obtained from the clonogenic assay. This result highlights the possibility that the MTT assay could be used in clinical specimens for individual radiosensitivity assays.

The Optimal Condition of Performing MTT Assay for the Determination of Radiation Sensitivity / 대한방사선종양학회지

PURPOSE: The measurement of radiation survival using a clonogenic assay, the established standard, can be difficult and time consuming. In this study, We have used the MTT assay, based on the reduction of a tetrazolium salt to a purple formazan precipitate by living cells, as a substitution for clonogenic assay and have examined the optimal condition for performing this assay in determination of radiation sensitivity. MATERIALS AND METHODS: Four human cancer cell lines-PCI-1, SNU-1066, NCI-H630 and RKO cells have been used. For each cell line, a clonogenic assay and a MTT assay using Premix WST-1 solution, which is one of the tetrazolium salts and does not require washing or solubilization of the precipitate were carried out after irradiation of 0, 2, 4, 6, 8, 10 Gy. For clonogenic assay, cells in 25 cm2 flasks were irradiated after overnight incubation and the resultant colonies containing more than 50 cells were scored after culturing the cells for 10~14 days. For MTT assay, the relationship between absorbance and cell number, optimal seeding cell number, and optimal timing of assay was determined. Then, MTT assay was performed when the irradiated cells had regained exponential growth or when the non-irradiated cells had undergone four or more doubling times. RESULTS: There was minimal variation in the values gained from these two methods with the standard deviation generally less than 5%, and there were no statistically significant differences between two methods according to t-test in low radiation dose (below 6 Gy). The regression analyses showed high linear correlation with the R2 value of 0.975~0.992 between data from the two different methods. The optimal cell numbers for MTT assay were found to be dependent on plating efficiency of used cell line. Less than 300 cells/well were appropriate for cells with high plating efficiency (more than 30%). For cells with low plating efficiency (less than 30%), 500 cells/well or more were appropriate for assay. The optimal time for MTT assay was after 6 doubling times for the results compatible with those of clonogenic assay, at least after 4 doubling times was required for valid results. In consideration of practical limits of assay (12 days, in this study) cells with doubling time more than 3 days were inappropriate for application. CONCLUSION: In conclusion, it is found that MTT assay can successfully replace clonogenic assay of tested cancer cell lines after irradiation only if MTT assay was undertaken with optimal assay conditions that included plating efficiency of each cell line and doubling time at least.