MTT Cell Proliferation Assay Research Articles

Non-woven bilayered biodegradable chitosan-gelatin-polylactide scaffold for bioengineering of tracheal epithelium.

ObjectivesThe conversion of tissue engineering into a routine clinical tool cannot be achieved without a deep understanding of the interaction between cells and scaffolds during the process of tissue formation in an artificial environment. Here, we have investigated the cultivation conditions and structural features of the biodegradable non‐woven material in order to obtain a well‐differentiated human airway epithelium.Materials and methodsThe bilayered scaffold was fabricated by electrospinning technology. The efficiency of the scaffold has been evaluated using MTT cell proliferation assay, histology, immunofluorescence and electron microscopy.ResultsWith the use of a copolymer of chitosan‐gelatin‐poly‐l‐lactide, a bilayered non‐woven scaffold was generated and characterized. The optimal structural parameters of both layers for cell proliferation and differentiation were determined. The basal airway epithelial cells differentiated into ciliary and goblet cells and formed pseudostratified epithelial layer on the surface of the scaffold. In addition, keratinocytes formed a skin equivalent when seeded on the same scaffold. A comparative analysis of growth and differentiation for both types of epithelium was performed.ConclusionsThe structural parameters of nanofibres should be selected experimentally depending on polymer composition. The major challenges on the way to obtain the well‐differentiated equivalent of respiratory epithelium on non‐woven scaffold include the following: the balance between scaffold permeability and thickness, proper combination of synthetic and natural components, and culture conditions sufficient for co‐culturing of airway epithelial cells and fibroblasts. For generation of skin equivalent, the lack of diffusion is not so critical as for pseudostratified airway epithelium.

Influence of thoracic drainage fluid on proliferation, migration, apoptosis, and drug resistance in lung cancer cell lines.

BackgroundThis study aimed to clarify the effect of thoracic drainage fluid (DF) on lung cancer cells in vitro.MethodsWe assessed the influence of DF on the proliferation and migration of lung cancer cells (LTEP-a-2 and A549) using the MTT cell proliferation assay and scratch wound assay. Cell apoptosis was determined by flow cytometric analysis. We also investigated the effect of DF on drug chemosensitivity, assessing viability of LTEP-a-2 and A549 cells.ResultsThe proliferative rates of cancer cells in the DF-treated group were significantly higher than those of the control group. Similar results were obtained for cell migration of lung cancer cells. Cells in the DF-treated groups showed a lower percentage of apoptosis than those of the control groups. Chemosensitivity of lung cancer cells to doxycycline and cisplatin (DDP) was lowered by DF.ConclusionThese findings suggest that DF affects lung cancer cells by promoting proliferation and migration, inhibiting apoptosis, and increasing drug resistance.

THE EFFECT OF MANGOSTEEN (GARCINIA MANGOSTANA) PERICARP EXTRACT ON RETINOBLASTOMA CELL CULTURE PROLIFERATION

Introduction: To determine the effect of mangosteen (Garcinia mangostana) pericarp extracts on retinoblastoma cell culture proliferation.
 Methods: This study is a true experimental in vitro design with pre and post-test control group design. Research using retinoblastoma cell culture exposed by mangosteen pericarp extract (Garcinia mangostana) at a dose of 10 µg/ml, 20 µg/ml, and 40 µg/ml.
 Result: This study used retinoblastoma cell line cultures were obtained from the American Type Culture Collection (ATCC) 10801 University Boulevard Manassas, VA 20110 USA. The samples were divided into 4 groups: control group and 3 groups treated with doses of 10 µg/ml, 20 µg/ml and 40 µg/ml and then incubated for 48 hours, and then examined using MTT Cell Proliferation Assay. Group 1 (10 µg/ml) obtained a decrease of 151.8%, group 2 (20 µg/ml) of 134.6% and group 3 (40 µg/ml) of 134.36%.
 Conclusion: Mangosteen pericarp extract (Garcinia mangostana) can reduce retinoblastoma culture cell proliferation.

Suppression of connexin 43 expression by miR-106a promotes melanoma cell proliferation.

Connexin 43 (Cx43), a vital gap junction protein is reported to be involved in melanoma progression. The aim of the study is to investigate the regulatory role of Cx43 in melanoma. Western blot assay was used to detect the protein expression of Cx43 in melanoma cells and the human epidermal melanocytes (HEMn). MTT cell proliferation and cell colony formation assays were used to assess cell proliferation. Bioinformatics prediction, luciferase reporter assay, Western blot and qRT-PCR assays were applied to demonstrate that Cx43 was a direct target of miR-106a in melanoma cells. Connexin 43 (Cx43) was lower expressed in melanoma cells compared with human epidermal melanocytes (HEMn). Cx43 overexpression significantly inhibited melanoma cell proliferation and colony formation ability in vitro. However, knockdown of Cx43 had opposite effects on cell proliferation and colony formation. Bioinformatics prediction and luciferase reporter assays demonstrated that miR-106a targeted the 3' untranslated region (3'UTR) of Cx43 and regulated its mRNA and protein expression levels in melanoma cells. MiR-106a was upregulated in melanoma cells, and its overexpression attenuated the effects caused by upregulating Cx43 expression. Thus, our results indicated that Cx43 was downregulated in melanoma cells and may be a potential target of melanoma treatment.

Synthesis, Characterization, Anticancer and Antimicrobial Activity Studies of Novel Isomeric 2,4‐Disubstituted Ureide Derivatives of Pyrimidinopiperidines

AbstractA series of isomeric ureide derivatives of novel 2,4‐disubstituted pyrimidinopiperidines were synthesized starting from 2,4‐dichloropyrimidine. All the final products were purified on silica and characterized by spectral analysis. Both the 2,4‐disubstituted pyrimidinopiperidines were analyzed for their in vitro anticancer activity on the cell lines HCT116, MIA‐PaCa2 and MDA‐MB 231 by using MTT cell proliferation assay. Further, the antimicrobial studies were carried out against different bacterial and fungal strains by employing cup plate method. These compounds were showed significant anticancer activity on tested three cancer cell lines and exhibited potent antimicrobial activity in tested strains of bacteria and fungi.

Molecular Effects of Treatment of Human Colorectal Cancer Cells with Natural and Classical Chemotherapeutic Drugs: Alterations in the Expression of Apoptosis-related BCL2 Family Members, Including BCL2L12.

Current chemotherapy regimens for the treatment of colorectal cancer (CRC) include oxaliplatin, irinotecan, and fluorouracil along with leucovorin. Cytotoxicity involves the induction of programmed cell death. The purpose of this study was to assess the molecular effects of doxorubicin (a 14-OH derivative of the natural product daunorubicin) and common chemotherapeutic drugs (used in the clinical practice to treat CRC) on the expression of the most prominent members of the BCL2 family, namely BCL2, BAX, BCLX, and MCL1. Moreover, we sought to define the role of BCL2L12, another member of the BCL2 family, the apoptotic role of which is ambiguous. The MTT cell proliferation assay was used to determine the IC50 of each chemotherapeutic drug at 72 hours of treatment of Caco-2 and DLD-1 colorectal adenocarcinoma cell lines. Real-time PCR was used to quantify the antiapoptotic BCL2-α, BLCX-L, and MCL1-L transcripts, the proapoptotic BAX, BLCX-S, BLCX-ES, MCL1-S, and MCL1-ES transcripts, and BCL2L12 expression in relation to GAPDH mRNA levels. We constructed growth curves of Caco-2 and DLD-1 cells and determined the IC50 of each drug at 72 hours of treatment. Significant alterations in the expression levels of the studied BCL2 family genes and/or particular transcripts were observed. The intrinsic apoptotic pathway is activated during treatment of CRC cells with common chemotherapeutic drugs. Moreover, BCL2L12 mRNA expression increases progressively during treatment, similarly to the expression of other BCL2 family genes favoring apoptosis and/or particular proapoptotic transcripts, thus suggesting a proapoptotic role for BCL2L12 in chemotherapy-treated CRC cells.

Cell Cycle Arrest and Induction of Apoptosis in Human Breast Cancer Cells (T-47D) by Annona squamosa L. and Thymus vulgaris L. Ethanolic Extract

Annona squamosa L. is belongs to the Annonaceae family. Similarly, Thymus vulgaris L. is a flowering plant in the mint family Lamiaceae. In this study, Annona squamosa L. seeds ethanolic extract (ASEE) and Thymus vulgaris ethanolic extract (TVEE) were prepared and used against the breast cancer cells. The Breast cancer cells (T-47D) death was analysed by Vybrant® MTT Cell Proliferation Assay Kit. The IC50 of the ASEE was found to be 8.72 ± 0.52 μg/ml. The IC50 of the TVEE was found 7.30 ± 0.6 μg/ml. Membrane marker proteins of apoptotic cells were analysed by Attune flow cytometer. The cell cycle assay was quantified in ASEE and TVEE treated and untreated cells. The ASEE or TVEE treated T-47D cells were found green in color due to the binding of annexin v-FITC to phosphatidylserine (PS) on the membranes of apoptotic cells. This was supported by the upregulated p53 and 14-03-03 sigma expression in cells treated with the ASEE or TVEE. This indicates the inhibition of the sequestring CDK1 in the cytoplasm and inhibition of cell cycle at G2/M phase. The Annona squamosa L. ethanolic extract (ASEE) and Thymus vulgaris L. ethanolic extract (TVEE) were found effective against Breast cancer cells (T-47D).

PSMC2 is Up-regulated in Pancreatic Cancer and Promotes Cancer Cell Proliferation and Inhibits Apoptosis.

Objective: The lack of effective therapeutic targets poses a leading challenge to prolong survival and improve the quality of life for pancreatic cancer patients. Proteasome 26S subunit ATPase 2 (PSMC2), a recently discovered gene, has been implicated in certain human carcinogenesis. However, limited data is available concerning the functional significance of PSMC2 in cancer cell growth, and whether it plays a role in pancreatic carcinogenesis remains unknown.Materials and Methods: Quantitative RT-PCR (qRT-PCR) was performed to assess mRNA expression levels of PSMC2 in different pancreatic cancer cell lines. Knockdown of PSMC2 was achieved by using short hairpin RNA (shRNA). The effects of PSMC2 silencing on pancreatic cancer cell proliferation and apoptosis were evaluated by the MTT cell proliferation assay, Celigoassays, Annexin V fluorescence-activated cell sorting (FACS) assay and Caspase-3/7 array.Results: High expression of PSMC2 was detected in three pancreatic cancer cell lines (SW1990, PANC-1, and AsPC-1). Knockdown of PSMC2 in SW1990 cells inhibited proliferation and enhanced apoptosis.Conclusions: Our primary study suggests that PSMC2 might be involved in the progression of pancreatic cancer and may serve as a potential therapeutic target.

Roles of miR-494 in Intervertebral Disk Degeneration and the Related Mechanism

In this study, we focused on the regulatory roles of miR-494 in the pathogenesis of intervertebral disk degeneration (IDD) and the related mechanism. First, rat IDD models were established, and the expression levels of miR-494 in IDDs of the rats were examined. Next, human nucleus pulposus (NP) cells were cultured and transfected with miR-494 mimics and inhibitors, and the roles of miR-494 on the proliferation and apoptosis of cells were determined using MTT cell proliferation assay and flow cytometry methods. Furthermore, the targeting relationship between miR-494 and neuro-oncological ventral antigen 1 (NOVA1) was examined by dual luciferase reporter assay. Finally, the expression of NOVA1, Caspase-3, Bcl-2-associated X protein (BAX), and B-cell lymphoma-2 (BCL-2) was examined using real-time quantitative polymerase chain reaction and western blot methods. The results demonstrated that the expression of miR-494 was significantly upregulated in IDD rats. Moreover, transfection of miR-494 inhibitors induced a significant increase in the proliferation and marked decrease in the apoptosis of the degenerated human NP cells. Transfection of miR-494 mimics has shown the opposite effects. Furthermore, NOVA1 has been confirmed as a target of miR-494, and the expressions of NOVA1 were significantly downregulated in IDD rats. In addition, transfection of miR-494 inhibitors significantly decreased the expression of Caspase-3 and BAX and markedly increased the expression of NOVA1 and BCL-2. Transfection of miR-494 mimics has shown the opposite effects. miR-494 was upregulated in IDD, and miR-494 might regulate the proliferation and apoptosis of NP cells through targeting NOVA1.

Evaluation of silymarin/duck's feet-derived collagen/hydroxyapatite sponges for bone tissue regeneration

Tissue engineered scaffolds, made of natural derived materials, have the potential to be used in bone regeneration fields due to the biocompatible and biodegradable features. In this study, we propose duck's feet-derived collagen (DC) sponges blended with hydroxyapatite (HAp), incorporated with different concentrations of silymarin (Smn), for improved bone regeneration. The morphological and structural properties of DC/HAp and DC/HAp loaded with 25, 50 and 100 μM of Smn sponges were analyzed using scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). In vitro evaluations were carried out on rabbit bone marrow stem cells (rBMSCs) using MTT assay for cell proliferation, ALP assay for osteogenic differentiation and reverse transcription-polymerase chain reaction (RT-PCR) for expression of mRNAs. For the evaluation of new bone formation in vivo, histological analysis and micro computed tomography (μCT) were used. Preliminary results, on Smn/DC/HAp morphology and mechanical properties, showed an interconnected porosity suitable for cells ingrowth and a higher compressive strength with the presence of Smn. Similarly, the cells proliferation and ALP activity modulation were positively influenced by the Smn content. Especially, the 100 μM Smn/DC/HAp sponge efficiently enhances the rBMSCs adhesion, growth and gene expression of osteogenic markers. The enhanced osteoinductive effects of sponges blended with Smn were confirmed using μ-CT and histological evaluations. In conclusion, results suggest that collagen sponges represent an excellent environment for cells growth and proliferation, while Smn plays an important role to improve materials osteogenic properties.

Exploration of the regulation and control mechanisms of miR-145 in trophoblast cell proliferation and invasion.

Preeclampsia (PE) is the leading cause of maternal and fetal mortality and morbidity. Furthermore, recent studies have reported that miR-145 within the preeclamptic trophoblast debris may cause the high blood pressure via interacting with the maternal endothelium. The aim of the present study was to investigate the functions of miR-145 in PE. Reverse transcription-quantitative polymerase chain reaction and western blotting were used to assess the expression of miR-145 and mucin (MUC1), respectively. TargetScan, miRBase and miRWalk were used to predict the targets of miR-145. Constructed miR-145 mimic plasmids were transfected into HTR-8/SVneo cells for further experiments, including an MTT assay for cell proliferation, Transwell assay for cell invasion and flow cytometry for cell apoptosis analysis. Additionally, the luciferase reporter gene system was employed for target verification. The results demonstrated that miR-145 is downregulated and MUC1 is upregulated in PE tissues and cells compared with normal placenta tissues and cells. The correlation analysis suggests that the expression of miR-145 is negatively correlated with MUC1. Meanwhile, increased proliferation, enhanced invasion and decreased apoptosis of HTR-8/SVneo cells was observed in miR-145 mimic groups compared with mimic control group. Also, the decreased luciferase activity in the miR-145 mimic group indicates that MUC1 may be a target of miR-145. In summary, the results of the present study suggest that miR-145 may serve key roles in the regulation of trophoblast cell proliferation and invasion by targeting MUC1.

Antimicrobial activity of NO-releasing compounds against periodontal pathogens.

This study describes the successful synthesis of nitric oxide (NO)-releasing compounds with biodegradable and injectable properties and demonstrates that the kinetics of NO release vary according to the type of NO donor. The antimicrobial activity of NO-releasing compounds against three common periodontal pathogens, i.e., Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Actinomyces israelii, was investigated using a susceptibility assay. Human gingival fibroblasts were treated with NO-releasing compounds at the minimum concentrations required for bacterial growth and cytotoxicity was evaluated using the MTT cell proliferation assay. Our results suggest that NO-releasing compounds can be used topically to treat both gram-negative and gram-positive periodontal pathogens. Comparison of the antimicrobial activity and cytotoxicity assay results between the NO-releasing compounds revealed that an NO donor comprising a macromolecule without surface charge, a lower instantaneous NO concentration, and an adequate supply of NO were associated with a strong bactericidal effect and low cytotoxicity. NO-releasing compounds with these properties may be suitable for treatment of periodontitis.

Nodal regulates bladder cancer cell migration and invasion via the ALK/Smad signaling pathway

BackgroundBladder cancer is the most common malignant tumor of the urinary tract. We aimed to explore the biological role and molecular mechanism of Nodal in bladder cancer.Materials and methodsThe expression of Nodal in bladder cancer tissues and cells was determined by quantitative real-time polymerase chain reaction. The effect of silencing of Nodal on cell proliferation, clone formation, and migration and invasion was evaluated by MTT cell proliferation assay, colony formation, and transwell assays, respectively. Western blot analysis was employed to detect the expression of proliferation- and invasion-related proteins and proteins involved in ALK/Smad signaling.ResultsWe found that the expression of Nodal was significantly increased in bladder cancer tissues and cell lines. Downregulation of Nodal effectively weakened cell proliferation, clone formation, and cell migration and invasion abilities. The protein expression levels of CDC6, E-cadherin, MMP-2, and MMP-9 were also altered by downregulation of Nodal. Knockdown of Nodal also blocked the expression of ALK4, ALK7, Smad2, and Smad4, which are involved in ALK/Smad signaling. Additionally, the ALK4/7 receptor blocker SB431542 reversed the promotive effects of Nodal overexpression on bladder cancer cell proliferation, migration, and invasion.ConclusionOur study indicated that Nodal functions as an oncogene by regulating cell proliferation, migration, and invasion in bladder cancer via the ALK/Smad signaling pathway, thereby providing novel insights into its role in bladder cancer treatment.

The effect of combined treatment with sodium phenylbutyrate and cisplatin, erlotinib, or gefitinib on resistant NSCLC cells.

BackgroundChemotherapy resistance is the main cause of the marginal clinical benefit of platinum-based chemotherapy and tyrosine kinase inhibitors in advanced non-small-cell lung cancer (NSCLC). Thus, the identification of new therapeutic agents that can enhance the sensitivity of these drugs is of clinical importance. Histone deacetylase inhibitors (HDACIs) are emerging as new promising agents with strong antiproliferative effects against different types of cancers. This study investigates the synergistic potential of sodium phenylbutyrate (NaPB) added on top of standard chemotherapy used against NSCLC.ObjectiveThe objective of this study was to evaluate the ability of NaPB to overcome the resistance of NSCLC cell lines to cisplatin, gefitinib, and erlotinib.MethodsMTT cell proliferation assay was used to measure the anticancer effects of cisplatin, erlotinib, or gefitinib alone or combined with various concentrations of NaPB against A549, Calu1, and H1650 NSCLC cell lines. Synergism was estimated by measuring synergy value (R), which is equal to the ratio of IC50 of each primary drug alone divided by combination IC50s. Student’s t-test analysis was used to evaluate the potential differences between IC50 values. ANOVA followed by Tukey’s post hoc was used to evaluate the potential differences among monotherapy and combination treatment groups. Analyses were performed using R 3.3.2 software. P-value <0.05 was considered to be statistically significant.ResultsNaPB was shown to inhibit the growth of A549, Calu1, and H1650 cell lines in a dose-dependent manner (IC50 10, 8.53, and 4.53 mM, respectively). Furthermore, the addition of NaPB along with cisplatin, erlotinib, or gefitinib to A549, Calu1, and H1650 cell lines resulted in a synergistic antiproliferative effect against the three NSCLC cell lines (R>1.6, P-value <0.05), thus suggesting that NaPB can potentiate the effect of cisplatin, erlotinib, and gefitinib on A549, Calu1, and H1650 cell lines.ConclusionCurrent results suggest a potential role of NaPB as a sensitizing agent in NSCLC.

Biological Activities Evaluation of Enantiopure Isoxazolidine Derivatives: In Vitro, In Vivo and In Silico Studies.

A series of enantiopure isoxazolidines (3a-c) were synthesized by 1,3-dipolar cycloaddition between a (-)-menthone-derived nitrone and various terminal alkenes. The screened compounds were evaluated for their antioxidant activity by two in vitro antioxidant assays, including β-carotene/linoleic acid bleaching, and inhibition of lipid peroxidation (thiobarbituric acid reactive species, TBARS). The results revealed that compound 3b (EC50 = 0.55 ± 0.09mM) was the most potent antioxidant as compared to the standard drug (EC50 = 2.73 ± 0.07mM) using the TBARS assay. Furthermore, the antimicrobial activity was assessed using disc diffusion and microdilution methods. Among the synthesized compounds, 3c was found to be the most potent antimicrobial agent as compared to the standard drug. Subsequently, the acute toxicity study has also been carried out for the newly synthesized compounds and the experimental studies revealed that all compounds were safe up to 500mg/kg and no death of animals were recorded. The cytotoxicity of these compounds was assessed by the MTT cell proliferation assay against the continuous human cell lines HeLa and compound 3c (GI50 = 46.2 ± 1.2μM) appeared to be more active than compound 3a (GI50 = 200 ± 2.8μM) and 3b (GI50 = 1400 ± 7.8μM). Interestingly, all tested compounds displayed a good α-amylase inhibitory activity in competitive manner with IC50 values ranging between 23.7 and 64.35μM when compared to the standard drug acarbose (IC50 = 282.12μM). In addition, molecular docking studies were performed to understand the possible binding and the interaction of the most active compounds to the α-amylase pocket.

A natural flavonoid lawsonaringenin induces cell cycle arrest and apoptosis in HT-29 colorectal cancer cells by targeting multiple signalling pathways.

Colorectal cancer is the third most common malignancy in the world having a high mortality rate. Flavonoids possess many biological activities including anti-cancer activity. lawsonaringenin (LSG) is a flavonoid isolated from leaves of Lawsonia alba Lam. The objective of this study was to demonstrate the anti-cancer potential of LSG in colorectal cancer for the first time. The HT-29 cells were treated with LSG or 5-fluoruracil, as a positive control, to determine its effect on cell cytotoxicity by a MTT cell proliferation assay, and cell cycle progression and apoptosis using flowcytometry. We also determined the mechanisms underlying LSG-mediated growth inhibition of HT-29 cells by by investigating the expression of key oncogenes and apoptosis genes using q-RT PCR and immunocytochemical analysis. The cell cytotoxicity data showed that the IC50 value of LSG was significantly less than the IC50 value of 5-FU (50µM). The anti-proliferative effect of LSG was mediated by arresting cells in the S phase of the cell cycle which then led to the induction of apoptosis the q-RT PCR and immunocytochemical analysis showed that LSG reduced the expression of ß-catenin (non-phosphorylated) and its downstream signalling target c-Myc, whereas it increased the phosphorylation of ß-catenin. Furthermore, LSG also downregulated the expression of oncogene K-Ras and anti-apoptotic proteins, Bcl-2, and Bcl-xL. In conclusion, our data demonstrates that LSG exerted its anti-tumor activity by arresting the cell cycle in S phase, and by downregulating the expression of oncogenes including ß-catenin, c-Myc, K-Ras and anti-apoptosis proteins Bcl-2 and Bcl-xL. This study suggests a potential use of natural flavonoid, lawsonaringenin, to attenuate colorectal cancer growth; however, further pre-clinical/clinical studies are required to establish its role as a therapeutic agent.

Synthesis of Novel Derivatives of Quinazoline Schiff base Compound Promotes Epithelial Wound Healing.

Quinazoline is an aromatic bicyclic compound exhibiting several pharmaceutical and biological activities. This study was conducted to investigate the potential wound healing properties of Synthetic Quinazoline Compound (SQC) on experimental rats. The toxicity of SQC was determined by MTT cell proliferation assay. The healing effect of SQC was assessed by in vitro wound healing scratch assay on the skin fibroblast cells (BJ-5ta) and in vivo wound healing experiment of low and high dose of SQC on adult Sprague-Dawley rats compared with negative (gum acacia) and positive control (Intrasite-gel). Hematoxylin and Eosin (H&E), Masson's Trichrome (MT) staining and immunohistochemistry analysis were performed to evaluate the histopathological alterations and proteins expression of Bax and Hsp70 on the wound tissue after 10 days. In addition, levels of antioxidant enzymes (catalase, glutathione peroxidase and superoxide dismutase), and malondialdehyde (MDA) were measured in wound tissue homogenates. The SQC significantly enhanced BJ-5ta cell proliferation and accelerated the percentage of wound closure, with less scarring, increased fibroblast and collagen fibers and less inflammatory cells compared with the negative control. The compound also increases endogenous enzymes and decline lipid peroxidation in wound homogenate.

Effects of 3-Tetrazolyl Methyl-3-Hydroxy-Oxindole Hybrid (THOH) on Cell Proliferation, Apoptosis, and G2/M Cell Cycle Arrest Occurs by Targeting Platelet-Derived Growth Factor D (PDGF-D) and the MEK/ERK Signaling Pathway in Human Lung Cell Lines SK-LU-1, A549, and A-427.

BackgroundThe aim of this study was to evaluate the effects of 3-tetrazolyl methyl-3-hydroxy-oxindole hybrid (THOH) on cell proliferation, apoptosis, and the cell cycle in human lung cancer cell lines SK-LU-1, A549, and A-427, and the normal lung fibroblast cell line, MRC-5, in vitro.Material/MethodsHuman lung adenocarcinoma cells SK-LU-1, A549, and A-427, and the normal lung fibroblast cells, MRC-5 were cultured and treated with increasing concentrations of 10 mM of a stock solution of THOH in dimethyl sulfoxide (DMSO). An MTT cell proliferation assay was used. Cell apoptosis and the cell cycle were studied using fluorescence-activated cell sorting (FACs) with fluorescein isothiocyanate (FITC), Annexin-V, propidium iodide (PI), and nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI). DNA damage was measured using the comet (single-cell gel electrophoresis) assay. Cell migration was evaluated using a wound healing assay, and Western blotting was used to measure protein expression levels.ResultsTreatment of SK-LU-1 cells with THOH inhibited cell migration. Treatment of lung cancer cells, SK-LU-1, A549, and A-427, with THOH inhibited cell proliferation, with the most marked inhibition found in the SK-LU-1 lung cancer cells (IC50, 12 μM). Treatment of lung cancer cells, SK-LU-1, A549, and A-427, with THOH increased cell apoptosis, resulted in G2/M cell cycle arrest, and inhibited both the platelet-derived growth factor D (PDGF-D) and MEK/ERK signaling pathways.ConclusionsTreatment of adenocarcinoma cells, SK-LU-1, A549, and A-427, with THOH inhibited cell proliferation, apoptosis, and resulted in G2/M cell cycle arrest by targeting PDGF-D and the MEK/ERK signaling pathway.

Pyridazine-based heteroleptic copper(II) complexes as potent anticancer drugs by inducing apoptosis and S-phase arrest in breast cancer cell

A new series of heteroleptic copper(II) complexes of the type [Cu(L1−3)(diimine)](ClO4) (1–6) have been synthesized using three pyridazine-based ligands (3-chloro-6-(salicylidenehydrazinyl)pyridazine (HL1), 3-chloro-6-(4-diethylaminosalicylidenehydrazinyl)pyridazine (HL2) and 3-chloro-6-(5-bromosalicylidenehydrazinyl)pyridazine (HL3), and diimine (2,2′-bipyridine (bpy) or 1,10-phenanthroline (phen)) as co-ligands. The ligands and their copper(II) complexes have been characterized by elemental analyses and spectroscopic methods. The copper(II) complexes display ligand-field band in the region 641–661 nm suggesting square pyramidal geometry. The optimized structures of the complexes and their molecular orbital calculations obtained by the density functional theory (DFT) also showed five coordinated distorted square pyramidal geometry around the copper(II) ion. The cyclic voltammetric analyses of copper(II) complexes exhibit one-electron irreversible reduction wave (Epc = −0.596 to −0.641 V) in the cathodic potential region. Anti-proliferative activity of the complexes against breast cancer MDA-MB-231 cell line was evaluated by MTT cell proliferation assay, and the clonogenic assay revealed improved cytotoxicity for the complexes with potency higher than the standard drug cisplatin. Since the complexes 3 and 4 with diethylamino substituent displayed higher anti-proliferative activity than the other complexes, these complexes were chosen for apoptosis and cell cycle analysis. The apoptosis induction was analyzed by AO/EB staining, and the flow cytometry showed the inhibition of cell growth at the S-phase of the cell cycle. Additionally, the interaction of copper(II) complexes with FGFR kinase receptor have been studied by in silico molecular docking studies.

2036 Extracellular matrix as a novel approach to glioma therapy

2036 Extracellular matrix as a novel approach to glioma therapy