Cell Cycle Phase Distribution Research Articles

Cetyltrimethylammonium Bromide Attenuates the Mesenchymal Characteristics of Hypopharyngeal Squamous Cell Carcinoma Through Inhibiting the EGFR/PI3K/AKT Signaling Pathway.

Cetyltrimethylammonium bromide (CTAB), a quaternary ammonium surfactant, was shown to have antitumor effects in a cellular mode of head and neck squamous cell carcinoma (HNSCC), modulating apoptotic and cytotoxic processes. However, the mechanisms by which CTAB exerts its effects against the epithelial- mesenchymal transition in HNSCC remain poorly understood. In the present study, we investigated whether CTAB inhibits cellular mobility and invasiveness of hypopharyngeal squamous cell carcinoma (HPSCC) cells. WST-1, cell-cycle phase distribution, and wound healing, as well as transwell assays were conducted. Changes in protein expression patterns and related signaling pathways involved in effects of CTAB on HPSCC cell lines were evaluated by western blotting. Treatment of human HPSCC cell lines with CTAB significantly altered their morphology from spindle-like to cobblestone-like by diminishing mesenchymal-like phenotypic characteristics. CTAB also hindered cell functional properties, including migration and invasion, independently of cell viability. In addition, western blot results demonstrated that treatment with CTAB reduced expression of mesenchymal markers. Further investigation showed that CTAB treatment suppressed the phosphorylation of extracellular-regulated kinase 1/2, mechanistic target of rapamycin kinase and AKT serine/threonine kinase 1. CTAB also repressed the expression and phosphorylation levels of epidermal growth factor receptor (EGFR) and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), and the partial restoration of mesenchymal phenotype by EGF addition confirmed that CTAB inhibited migration and invasion in HPSCC cells by blocking the EGFR signaling pathway. Our results suggest that CTAB is involved in the suppression of EGFR-mediated mesenchymal phenotype and the molecular mechanism by which CTAB obstructs HPSCC cell metastasis may represent a promising strategy for use in HPSCC treatment.

Long Non-Coding Transcript in LEF1 Locus Is a Novel Regulator of Cell Cycle and Apoptotic Pathways in Myeloid Leukemic Cells

Myelodysplastic syndrome and acute myeloid leukemia are hematologic disorders characterized by inefficient differentiation and aberrant control of cell growth and apoptosis. In early MDS, blasts, unable to differentiate, exhibit exacerbated proliferation and apoptosis. However, MDS progression and AML transformation are associated with a progressive resistance to apoptosis. The molecular mechanisms that control these cellular decisions are not fully understood. Long non-coding RNAs (lncRNA) have gained great attention in recent years as they have been implicated in a wide variety of biological functions. Here we focus on a long non-coding transcript encoded in the LEF1 locus as a novel regulator of apoptosis and proliferation signaling in hematologic disease.Our previous work using microarray technology identified a long non-coding RNA, antisense to LEF1 gene, which expression was reduced in myelodysplastic syndrome patients. We mapped the sequence to a previously characterized transcript, LEF1-AS1. This transcript is highly conserved and has been recently associated to B-cell differentiation. We observed a modulation of LEF1-AS1 during the first stages of in-vitro erythroid differentiation from CD34+ HSC to erythroblast (from collection day 6 to day 8 after addition of erythropoietin, n=6, 78% mean reduction, P<0.0001).Due to reported reduction on LEF1-AS1 expression in MDS and low endogenous expression of LEF1-AS1 in myeloid cells lines, we decided to overexpress the transcript in myeloid cell lines HL60 and K562 to study the function of the lncRNA. Stably transfected cells were obtained by DMRIE-C mediated transfection using pcDNA vector containing full length lef1-AS1 or empty pcDNA vector. Selection was carried out by addition of geneticin to the culture medium.Unexpectedly, after stable over-expression of LEF1-AS1, we could not detect any alteration of LEF1 expression in the mRNA or protein level in both myeloid cell lines (HL60 and K562). Despite of the lack of regulatory effect upon LEF1, we observed a significant inhibition in cell proliferation evaluated Carboxyfluorescein succinimidyl ester (CFSE) labelling (p<0.05).To investigate the antiproliferative effects of LEF1-AS1, we analyzed the cell cycle phase distribution of the stably transfected cell HL60 by flow cytometry using propidium iodide DNA staining. LEF1-AS1 over-expression resulted in a significant increase in the proportion of S-phase cells (p=0.01) and no difference in G2/M, suggesting intra S-phase imprisonment.Additionally, we evaluated the effect of LEF1-AS1 over-expression upon apoptosis in stably transfected HL60 using annexin/Propidium Iodide Apoptosis flow cytometry assay. We observed no difference in the apoptosis level between LEF1-AS1-overexpressing cells and control cells in basal culture conditions; however, LEF1-AS1 over-expression inhibited significantly serum deprivation-induced apoptosis (reduction of 50%, p<0.05).We screened several apoptosis and proliferation regulators by RT-PCR and western blotting and detected a strong increase in anti-apoptotic BCL2 and CDKN1A/p21 in HL60 cells over-expressing LEF1-AS1. This increase was observed in cells in exponential growth and starvation. Interestingly, we did not observe this antiapoptotic effect in K562, a cell line that do not express BCL2 endogenously.To confirm that the phenotypical effects of lef1-AS over -expression were not mediated by LEF1 function, we also over-expressed lef1-AS1 in Hela, a cell line lacking endogenous expression of LEF1. Empty vector and lef1-AS1-Hela stably-transfected-cells had no detectable expression of LEF1; however, a similar inhibition of cell proliferation and S-phase accumulation were observed.To further investigate the mechanism behind these phenotypical effects we carried out a RNA-pulldown experiment coupled with mass spectrometry to identify the proteins that bind to LEF1-AS1. Our preliminary results identified an apoptosis regulator protein in association to this transcript and we are currently working to unravel the implications of this interaction.Remarkably, BCL2 is simultaneously antiapoptotic and anti-proliferative and S-phase arrest has been associated to p21 up-regulation. Our results depict LEF1-AS1 as a fine-tuner of proliferation and survival decisions, acting, at least partially, through modulation of BCL2 and CDKN1A/p21. DisclosuresNo relevant conflicts of interest to declare.

Synthesis, X-ray structures and cytotoxic effects of a Cu(II)- and a Zn(II) thiosemicarbazones on human epidermoid carcinoma cell A431

Thiosemicarbazone metal complexes [Cu(L)(2-benzpy)]ClO4 (1) and [Zn(L)2] (2) [L = 2-benzoylpyridine-N(4)-methyl-3-thiosemicarbazone), 2-benzpy = 2-benzolylpyridine] were synthesized and X-ray crystallographically characterized. The metal centre in compound 1 has a distorted square planar geometry while in compound 2, the metal centre has a distorted octahedral geometry. Both the molecules induce cytotoxic effects on A431 skin carcinoma cell line in 24 h incubation and significantly enhances hypoploid cell population in cell cycle phase distribution below IC50 dose. Application of both the two molecules exhibit apoptotic induction and genotoxic activity in 24 h exposure.

Retracted] Antitumor‑ and apoptosis‑inducing effects of pomolic acid against SK‑MEL‑2 human malignant melanoma cells are mediated via inhibition of cell migration and sub‑G1 cell cycle arrest.

Following the publication of the above paper, a concerned reader drew to the Editor's attention that several figures (Figs.3‑8 inclusive) contained apparent anomalies, including repeated patternings of data within the same figure panels. After having conducted an independent investigation in the Editorial Office, the Editor of Molecular Medicine Reports has determined that the above paper should be retracted from the Journal on account of a lack of confidence concerning the originality and the authenticity of the data. The authors were asked for an explanation to account for these concerns, but the Editorial Office never received any reply. The Editor regrets any inconvenience that has been caused to the readership of the Journal. [the original article was published on Molecular Medicine Reports 17: 1035‑1040, 2018; DOI: 10.3892/mmr.2017.7977].

Anticancer effects of 7,8-dihydromethysticin in human leukemia cells are mediated via cell-cycle dysregulation, inhibition of cell migration and invasion and targeting JAK/STAT pathway.

The main focus of this research work was to study the anti-cancer properties of 7,8-dihydromethysticin against HL-60 leukemia cells. Investigations were also performed to check its impact on the phases of the cell cycle, cell migration and invasion, JAK/STAT signalling pathway and intracellular mitochondrial membrane potential (MMP) and reactive oxygen species (ROS). Cell proliferation was assessed through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and effects on colony formation were examined via clonogenic assay. Flow cytometry and Western blott analysis were performed to investigate the distribution of cell cycle phases. Flow cytometric analysis was performed for the examination of MMP and ROS production. The effect on JAK/STAT signalling pathway was examined through Western blot analysis. Results depicted that 7,8-dihydromethysticin induced concentration- as well as time-dependent inhibition of cell proliferation in leukemia HL-60 cells. Clonogenic assay indicated potential suppression in leukemia HL-60 cell colonies. The 7,8-dihydromethysticin molecule also caused cell cycle arrest at G2/M-phase along with concentration-dependent inhibition of cyclin B1, D1 and E. ROS and MMP measurements indicated significant ROS enhancement and MMP suppression with increasing 7,8-dihydromethysticin concentrations. Additionally, 7,8-dihydromethysticin led to remarkable dose-reliant inhibition of cell invasion as well as cell migration. Therefore, 7,8-dihydromethysticin should be considered a valuable candidate for leukemia research and chemoprevention.

Pinocembrin flavanone inhibits cell viability in PC-3 human prostate cancer by inducing cellular apoptosis, ROS production and cell cycle arrest.

The main purpose of the present study was to evaluate the antitumor effects of pinocembrin in human prostate cancer cells (PC-3) along with investigating its effects on cell apoptosis, endogenous ROS production and cell cycle. MTT assay and clonogenic assays were used to study the effects on cell viability and cancer colony formation, respectively. Fluorescence microscopy along with Western blotting was used to study apoptotic effects induced by pinocembrin. Flow cytometry was used to study effects on ROS production and cell cycle phase distribution. Results indicated that pinocembrin promoted inhibition cell proliferation along with reducing cancer colony formation of PC-3 cells in a dose-dependent manner. Pinocembrin induced regulatory effects over expressions of caspase-3, caspase-9, Bax and Bcl-2, thereby promoting apoptotic cell death in PC-3 cells. It also led to the dose-dependent G0/G1 cell cycle arrest. In conclusion, pinocembrin exhibits strong anticancer effects in human prostate cancer cells mediated via apoptosis, endogenous ROS production and G0/G1 cell cycle arrest.

An in vitro Study on the Role of Hepatitis B Virus X Protein C-Terminal Truncation in Liver Disease Development.

Hepatitis B virus X protein C-terminal 127 amino acid truncation is often found expressed in hepatocellular carcinoma (HCC) tissue samples. The present in vitro study tried to determine the role of this truncation mutant in the hepatitis B–related liver diseases such as fibrosis, cirrhosis, HCC, and metastasis. HBx gene and its 127 amino acid truncation mutant were cloned in mammalian expression vectors and transfected in human hepatoma cell line. Changes in cell growth/proliferation, cell cycle phase distribution, expression of cell cycle regulatory genes, mitochondrial depolarization, and intracellular reactive oxygen species (ROS) level were analyzed. Green fluorescent protein (GFP)–tagged version of HBx and the truncation mutant were also created and the effects of truncation on HBx intracellular expression pattern and localization were studied. Effect of time lapse on protein expression pattern was also analyzed. The truncation mutant of HBx is more efficient in inducing cell proliferation, and causes more ROS production and less mitochondrial depolarization as compared with wild type (wt) HBx. In addition, gene expression is altered in favor of carcinogenesis in the presence of the truncation mutant. Furthermore, mitochondrial perinuclear aggregation is achieved earlier in the presence of the truncation mutant. Therefore, HBx C-terminal 127 amino acid truncation might be playing important roles in the development of hepatitis B–related liver diseases by inducing cell proliferation, altering gene expression, altering mitochondrial potential, inducing mitochondrial clustering and oxidative stress, and changing HBx expression pattern.

The growth-inhibitory effects of pawpaw (Asimina triloba [L.] Dunal) roots, twigs, leaves, and fruit against human gastric (AGS) and cervical (HeLa) cancer cells and their anti-inflammatory activities.

The pawpaw tree has several beneficial effects. However, no studies have been conducted to address the mechanisms underlying the cytotoxic effects of pawpaw extracts against cancer cells, and no study has investigated the anti-inflammatory effects. Hence, in this study, the growth-inhibitory effects of pawpaw (Asimina triloba [L.] Dunal) extracts against gastric (AGS) and cervical (HeLa) cancer cells and the inhibitory effects of pawpaw extracts against inflammatory factors (NO, TNF-α, IL-6, iNOS, and COX-2) were investigated. The viability of AGS and HeLa cells, the analysis of cell cycle, and the expression of apoptosis marker protein were determined using MTT assay, FACS, western blotting, and TUNEL assays. The inflammatory factors were determined using Griess method, ELISA assay kit, and RAW 264.7 cells. The IC50 values of twig and unripe fruit extracts for AGS cells were 82.01 and 100.61µg/mL, respectively. For HeLa cells, pawpaw twig extracts exhibited the strongest ability to inhibit cervical cancer cell growth (IC50 = 97.73µg/mL). Analysis of the cell cycle phase distribution and expression of the apoptosis regulatory proteins BCL-2, BAX, caspase-3, and PARP showed that pawpaw twig, root, and unripe fruit extracts induced Sub G1 cell cycle arrest and apoptosis of AGS and HeLa cells. In addition, the twig, root, and unripe fruit extracts of pawpaw effectively inhibited the inflammatory makers NO, TNF-α, IL-6, and iNOS. Particularly, the twig, root, and unripe fruit extracts at concentrations of 50µg/mL exhibited > 50% inhibition of TNF-α. These findings indicate that pawpaw extracts are natural therapeutic agents that may be used for the prevention and treatment of gastric and cervical cancers, and encourage further studies on the anti-inflammatory potential of the pawpaw tree.

Quantitative nuclear DNA content and cell cycle analysis of a mixotrophic dinoflagellate by image cytometry

AbstractThe goal of this work was to develop and demonstrate the utility of microscope‐based image cytometry (ICM) as a method for quantifying nuclear DNA content and cell cycle phase distribution in microalgae both in culture and in natural blooms, as an alternative to flow cytometry (FCM). To do so, aliquots from the same samples of the dinoflagellate Karlodinium veneficum were examined using both ICM and FCM. ICM specimen preparation and data acquisition methods were optimized to improve precision and agreement between the two techniques. Accuracy and precision of DNA measurements by ICM were significantly higher using the DNA fluorophore DAPI compared to SYBR® Green I. Milli‐Q H2O was found to be superior to Tris‐EDTA as a staining and slide preparation solution for ICM analyses. Lower‐powered objective magnification (10x, 20x) in image acquisition for ICM produced higher precision in nuclear DNA measurements. Overall precision of ICM analysis of DAPI‐stained K. veneficum cells was comparable to FCM, with respective 1C DNA peak coefficients of variation as low as 6.2%. Cell cycle distributions of mid‐log culture samples analyzed by both ICM and FCM were in agreement (two‐way ANOVA; p = 0.93); while distributions analyzed in a field sample were similar but not identical (Z‐test; p &lt; 0.001). Overall, the results show the feasibility of ICM as a useful tool for microalgal cell cycle analysis, with the potential for more flexible application to mixotrophic/phagotrophic species and complex field populations.

An Evaluation of the Anti-Carcinogenic Response of Major Isothiocyanates in Non-Metastatic and Metastatic Melanoma Cells.

Malignant melanoma is one of the most deadly types of solid cancers, a property mainly attributed to its highly aggressive metastatic form. On the other hand, different classes of isothiocyanates, a class of phytochemicals, present in cruciferous vegetables have been characterized by considerable anti-cancer activity in both in vitro and in vivo experimental models. In the current study, we investigated the anti-cancer response of five isothiocyanates in an in vitro model of melanoma consisting of non-metastatic (A375, B16F-10) and metastatic (VMM1, Hs294T) malignant melanoma as well as non-melanoma epidermoid carcinoma (A431) and non-tumorigenic melanocyte-neighboring keratinocyte (HaCaT) cells. Our aim was to compare different endpoints of cytotoxicity (e.g., reactive oxygen species, intracellular glutathione content, cell cycle growth arrest, apoptosis and necrosis) descriptive of an anti-cancer response between non-metastatic and metastatic melanoma as well as non-melanoma epidermoid carcinoma and non-tumorigenic cells. Our results showed that exposure to isothiocyanates induced an increase in intracellular reactive oxygen species and glutathione contents between non-metastatic and metastatic melanoma cells. The distribution of cell cycle phases followed a similar pattern in a manner where non-metastatic and metastatic melanoma cells appeared to be growth arrested at the G2/M phase while elevated levels of metastatic melanoma cells were shown to be at sub G1 phase, an indicator of necrotic cell death. Finally, metastatic melanoma cells were more sensitive apoptosis and/or necrosis as higher levels were observed compared to non-melanoma epidermoid carcinoma and non-tumorigenic cells. In general, non-melanoma epidermoid carcinoma and non-tumorigenic cells were more resistant under any experimental exposure condition. Overall, our study provides further evidence for the potential development of isothiocyanates as promising anti-cancer agents against non-metastatic and metastatic melanoma cells, a property specific for these cells and not shared by non-melanoma epidermoid carcinoma or non-tumorigenic melanocyte cells.

Investigation of DNA Damage and Cell-Cycle Distribution in Human Peripheral Blood Lymphocytes under Exposure to High Doses of Proton Radiotherapy.

Simple SummaryRadiotherapy is a cornerstone care therapy for many tumors. Despite permanent advances in ra-diation dose delivery, there are unmet needs for further improvement. In principle, proton therapy offers a substantial clinical advantage over conventional modalities using photons in uniform-dose delivery of radiation to tumors, along with significant reductions in the harmful effects on normal tissue. However, the effect and mechanisms of a single high-dose delivery remain unclear. The study aimed to systematically observe and compare the biological effects of DNA damage and cell-cycle phase distribution in the human peripheral blood lymphocytes ex vivo irradiation model of normal tissue after proton versus conventional radiotherapy (X-rays). The effects induced at a single high-dose radiation exposure at a dose range of 8.00–20.00 Gy were studied. The results in-dicate a different distribution of DNA damage following high doses of irradiation with protons versus photons between donors, types of radiation, and doses. The results illuminate the cellular and molecular mechanisms that underlie differences in the distribution of DNA damage and cell-cycle phases. An understanding of the mechanisms in the distinct pathways induced by radiation can facilitate the development of more efficient radiotherapies with beneficial immunological conse-quences.This study systematically investigates how a single high-dose therapeutic proton beam versus X-rays influences cell-cycle phase distribution and DNA damage in human peripheral blood lymphocytes (HPBLs). Blood samples from ten volunteers (both male and female) were irradiated with doses of 8.00, 13.64, 15.00, and 20.00 Gy of 250 kV X-rays or 60 MeV protons. The dose–effect relations were calculated and distributed by plotting the frequencies of DNA damage of excess Premature Chromosome Condensation (PCC) fragments and rings in the G2/M phase, obtained via chemical induction with calyculin A. The Papworth’s u test was used to evaluate the distribution of DNA damage. The study shows that high doses of protons induce HPBL DNA damage in the G2/M phase differently than X-rays do. The results indicate a different distribution of DNA damage following high doses of irradiation with protons versus photons between donors, types of radiation, and doses. The proliferation index confirms the impact of high doses of mitosis and the influence of radiotherapy type on the different HPBL response. The results illuminate the cellular and molecular mechanisms that underlie differences in the distribution of DNA damage and cell-cycle phases; these findings may yield an improvement in the efficacy of the radiotherapies used.

Cytotoxic Effect of trans-Cinnamaldehyde from Cinnamomum osmophloeum Leaves on Human Cancer Cell Lines

Quantitative determination of trans-cinnamaldehyde (TCA) was conducted by re- versed-phase HPLC from young and mature leaves, and leaf branches of Cinnamomum osmo- phloeum, a Taiwan endemic plant. The results showed that highest yield, 23.79 mg/g of TCA (the tree's age was three years) was obtained in the two year old mature leaves. In addition, cytotoxic and inhibitory effects of TCA was evaluated against selected human cancer cell lines such as Jurkat, U937, and normal cell lines primary purified T cells and macrophages. The results re- vealed that TCA exhibited potent inhibitory activity against Jurkat and U937 cell viability, and found that the IC50 values were 0.057, 0.076 µM, respectively. In parallel, no effect on the viabil- ity of primary purified T cells and macrophages. Moreover, interestingly at 0.095 µM, TCA in- hibited proliferation of both Jurkat and U937 cell lines approximately 2-fold at 0.057 µM, com- pared to controls. In contrast, TCA increases approximately 26% proliferation of mito- gen-stimulated human peripheral blood mononuclear cells (PBMCs) during the concentration range studied. Furthermore, by cell cycle analysis, we found that TCA altered the cell cycle phase distribution of Jurkat and U937 cells in a nonlinear concentration-dependent fashion. Taken together our results suggest that TCA may be a useful chemotherapeutic agent for cancer treatment in human.

A quantitative systems pharmacological approach identified activation of JNK signaling pathway as a promising treatment strategy for refractory HER2 positive breast cancer

HER2-positive breast cancer (BC) is a rapidly growing and aggressiveBC subtype that predominantly affects younger women. Despite improvements in patient outcomes with anti-HER2 therapy, primary and/or acquired resistance remain a major clinical challenge. Here, we sought to use a quantitativesystems pharmacological (QSP) approach to evaluate the efficacy of lapatinib (LAP), abemaciclib (ABE) and 5-fluorouracil (5-FU) mono- and combination therapies in JIMT-1 cells, a HER2+ BC cell line exhibiting intrinsic resistanceto trastuzumab. Concentration-response relationships and temporal profiles of cellular viability were assessed upon exposure to single agents and theircombinations. To quantify the nature and intensity of drug-drug interactions, pharmacodynamic cellular response models were generated, to characterize single agent and combination time course data. Temporal changes in cell-cycle phase distributions, intracellular protein signaling, and JIMT-1 cellular viability were quantified, and a systems-based protein signaling network model was developed, integrating protein dynamics to drive the observed changes in cell viability. Global sensitivity analyses for each treatment arm were performed, to identify the most influential parameters governing cellular responses. Our QSP model was able to adequately characterize protein dynamic and cellular viability trends following single and combination drug exposure. Moreover, the model and subsequent sensitivity analyses suggest that the activation of the stress pathway, through pJNK, has the greatest impact over the observed declines of JIMT-1 cell viability in vitro. These findings suggest that dual HER2 and CDK 4/6 inhibition may be a promising novel treatment strategy for refractory HER2+ BC, however, proof-of-concept in vivo studies are needed to further evaluate the combined use of these therapies.

Exploiting DNA repair pathways for tumor sensitization, mitigation of resistance, and normal tissue protection in radiotherapy.

More than half of cancer patients are treated with radiotherapy, which kills tumor cells by directly and indirectly inducing DNA damage, including cytotoxic DNA double-strand breaks (DSBs). Tumor cells respond to these threats by activating a complex signaling network termed the DNA damage response (DDR). The DDR arrests the cell cycle, upregulates DNA repair, and triggers apoptosis when damage is excessive. The DDR signaling and DNA repair pathways are fertile terrain for therapeutic intervention. This review highlights strategies to improve therapeutic gain by targeting DDR and DNA repair pathways to radiosensitize tumor cells, overcome intrinsic and acquired tumor radioresistance, and protect normal tissue. Many biological and environmental factors determine tumor and normal cell responses to ionizing radiation and genotoxic chemotherapeutics. These include cell type and cell cycle phase distribution; tissue/tumor microenvironment and oxygen levels; DNA damage load and quality; DNA repair capacity; and susceptibility to apoptosis or other active or passive cell death pathways. We provide an overview of radiobiological parameters associated with X-ray, proton, and carbon ion radiotherapy; DNA repair and DNA damage signaling pathways; and other factors that regulate tumor and normal cell responses to radiation. We then focus on recent studies exploiting DSB repair pathways to enhance radiotherapy therapeutic gain.

Biofabricated platinum nanoparticles: therapeutic evaluation as a potential nanodrug against breast cancer cells and drug-resistant bacteria.

Use of plant extracts for the synthesis of various metal nanoparticles has gained much importance recently because it is a simple, less hazardous, conservative and cost-effective method. In this research work, platinum nanoparticles were synthesized by treating platinum ions with the leaf extract of Psidium guajava and their structural properties were studied using various characterization techniques. The formation of platinum nanoparticles was confirmed by the disappearance of the absorbance peak at 261 nm in UV-visible spectra. The results of gas chromatography-mass spectrometry (GC-MS) and Fourier transform infrared spectroscopy (FT-IR) analysis showed functional moieties responsible for bio-reduction of metal ions and stabilization of platinum nanoparticles. The use of dynamic light scattering (DLS) imaging techniques confirmed the formation of stable monodispersed platinum nanoparticles showing a zeta potential of −23.4 mV. The morphological examination using high resolution transmission electron microscopy (HR-TEM) and Scanning electron microscopy (SEM) confirmed the formation of spherical platinum nanoparticles with an average diameter of 113.2 nm. X-ray powder diffraction (XRD) techniques showed the crystalline nature of biosynthesized platinum nanoparticles with a face-centered cubic structure. The results of energy-dispersive X-ray spectroscopy (EDAX) showed 100% platinum content by weight confirming the purity of the sample. The cytotoxic effect of biosynthesized platinum nanoparticles assessed in a breast cancer (MCF-7) cell-line by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, revealed an IC50 of 167.2 μg ml−1. The results of a wound healing assay showed that treatment with platinum nanoparticles induced an anti-migratory effect on MCF-7 cells. In the cell cycle phase distribution, treatment with platinum nanoparticles inhibited cell proliferation as determined by flow cytometry with PI staining. Significant cell cycle arrest was detected at the G0/G1 phase with a notable decrease in the distribution of cells in the S and G2/M phases. The anti-bacterial activity of bio-synthesized platinum nanoparticles was evaluated against four pathogenic bacteria i.e. B. cereus (Gram positive), P. aeruginosa (Gram negative), K. pneumonia (Gram negative) and E. coli (Gram negative). The biosynthesized platinum nanoparticles were found to show dose-dependent inhibition against pathogenic bacteria with a significant effect on Gram-negative bacteria compared to Gram-positive bacteria. This synergistic blend of green and simplistic synthesis coupled with anti-proliferative and anti-bacterial properties makes these biogenic nanoparticles suitable in nanomedicine.

Synthesis, antitumor activity and structure-activity studies of novel pyridoxine-based bioisosteric analogs of estradiol

A new efficient approach to the synthesis of 6-alkenyl substituted pyridoxine derivatives has been developed. A series of 31 novel alkenyl pyridoxine derivatives, stilbene-based bioisosteric analogs of estradiol, were synthesized. In vitro cytotoxicity of the obtained compounds against MCF-7 (ER+) breast cancer tumor cells was studied using the MTT assay. The most active compounds with IC50,MCF-7 < 10 μM were also tested for cytotoxicity in vitro against MDA-MB-231 (ER-) breast adenocarcinoma cells and conditionally normal human skin fibroblasts (HSF). The patterns of structure-antitumor activity relationships of the obtained compounds were analyzed. The most active compounds were found to contain a six-membered ketal ring, a methyl group in position 5, a 3,4-dimethoxystyryl fragment in positions 2 or 6 of the pyridoxine ring, and a trans-configuration of the double bond. Using the most active compound 5a as a representative cytotoxic agent, we have demonstrated that it has high specificity and antiproliferative activity against MCF-7 (ER+) tumor cells (IC50 < 5 μM), and a higher therapeutic index compared to the reference compound raloxifene (48 versus 5.8). Compound 5a decreased the mitochondrial membrane potential and increased the level of reactive oxygen species in MCF-7 cells, but not MDA-MB-231 cells. Compound 5a did not affect the distribution of cell cycle phases and induced apoptosis in MCF-7 cells, but not MDA-MB-231. Unlike compound 5a, raloxifene decreased mitochondrial potential, increased the ROS level, and induced apoptosis in both MCF-7 and MDA-MB-231 cells, which indicated a lack of selectivity for cells with estrogen receptor expression. It was also shown that compound 5a reduced the level of ERα expression in cells to a lesser extent than raloxifene and, unlike the latter, did not activate the PI3K/Akt signaling pathway.

Differential Effects of IGF-1R Small Molecule Tyrosine Kinase Inhibitors BMS-754807 and OSI-906 on Human Cancer Cell Lines.

Simple SummaryWe have tested the effects of IGF-1R tyrosine kinase inhibitors BMS-754807 (BMS) and OSI-906 (OSI) on human colon, pancreatic carcinoma cell, and glioblastoma cell lines and primary cultures. Although OSI and BMS are able to inhibit IGF-1R activity at low doses, the differential effect on cell proliferation and cell-cycle phase distribution shown by both compounds probes that many effects observed are mediated by BMS off-target interactions. Using MAPKs ELISAs and phospho-RTK array analysis, we have identified several BMS regulated putative kinases able to mediate BMS off-target effects. Interestingly, molecular docking assays suggest that BMS could affect these kinases not only by blocking their ATP-binding domain, but also by means of allosteric interactions. Since BMS has an important antineoplastic effect on these poor prognosis types of cancer, these compounds could be taken in consideration for treatment independently of IGF-1R status.We have determined the effects of the IGF-1R tyrosine kinase inhibitors BMS-754807 (BMS) and OSI-906 (OSI) on cell proliferation and cell-cycle phase distribution in human colon, pancreatic carcinoma, and glioblastoma cell lines and primary cultures. IGF-1R signaling was blocked by BMS and OSI at equivalent doses, although both inhibitors exhibited differential antiproliferative effects. In all pancreatic carcinoma cell lines tested, BMS exerted a strong antiproliferative effect, whereas OSI had a minimal effect. Similar results were obtained on glioblastoma primary cultures, where HGUE-GB-15, -16 and -17 displayed resistance to OSI effects, whereas they were inhibited in their proliferation by BMS. Differential effects of BMS and OSI were also observed in colon carcinoma cell lines. Both inhibitors also showed different effects on cell cycle phase distribution, BMS induced G2/M arrest followed by cell death, while OSI induced G1 arrest with no cell death. Both inhibitors also showed different effects on other protein kinases activities. Taken together, our results are indicative that BMS mainly acts through off-target effects exerted on other protein kinases. Given that BMS exhibits a potent antiproliferative effect, we believe that this compound could be useful for the treatment of different types of tumors independently of their IGF-1R activation status.

Tropinone-Derived Alkaloids as Potent Anticancer Agents: Synthesis, Tyrosinase Inhibition, Mechanism of Action, DFT Calculation, and Molecular Docking Studies.

A new series of hybrid compounds with tropinone and thiazole rings in the structure was designed and synthesized as potential anticancer agents. They were tested against human multiple myeloma (RPMI 8226), lung carcinoma (A549), breast adenocarcinoma (MDA-MB-231), and mouse skin melanoma (B16-F10) cell lines. Toxicity was tested on human normal skin fibroblasts (HSF) and normal colon fibroblasts (CCD-18Co). The growth inhibition mechanism of the most active derivative was analyzed through investigation of its effect on the distribution of cell cycle phases and ability to induce apoptosis and necrosis in RPMI 8226 and A549 cancer cells. The tyrosinase inhibitory potential was assessed, followed by molecular docking studies. Compounds 3a–3h show high anticancer activity against MDA-MB-231 and B16-F10 cell lines with IC50 values of 1.51–3.03 µM. Moreover, the cytotoxic activity of the investigated compounds against HSF and CCD-18Co cells was 8–70 times lower than against the cancer cells or no toxicity was shown in our tests, with derivative 3a being particularly successful. The mechanism of action of compound 3a in RPMI 8226 cell was shown to be through induction of cell death through apoptosis. The derivatives show ability to inhibit the tyrosinase activity with a mixed mechanism of inhibition. The final molecular docking results showed for IC50 distinct correlation with experiment.

Oleanolicacid-Chitosan Nanocomplex Induced Apoptotic Cell Death Through Mitochondrial Dysfunction in Human Lung Carcinoma: An Improved Synergetic Drug System for Cancer Therapy

Lung carcinoma is a forceful type of malignancy, having great elevated pace of morbidity and mortality with limited therapeutic options. The goal of current examination was to reveal the anticancer potential embedded in the oleanolic acid conjugated chitosan nanocomplex (OAC) in Human lung cancer cells (A-549). The OAC complex was characterized with DLS, Zeta potential, FTIR and SEM analysis. We found that the cytotoxic efficacy of OAC in Human lung cancer cells (A549) was dose dependent and also shows insignificant cytotoxicity towards to Human normal lung epithelial normal cells IMR-90. The influence of OAC nanocomplex on cell cycle phase distribution and also the mitochondrial membrane potential was assessed using flowcytometry. The results revealed that oleanolic acid nanocomplex system induced apoptotic cell death in a dose dependent manner. Subsequently the acridine orange and ethidium bromide staining (AO/EtBr) treatment was done with different concentrations of OAC nanocomplex had initiated a characteristic morphological alterations associated with apoptosis. The present findings concluded that OAC nanocomplex treatment brought about the loss in integrity of mitochondrial membrane potential, which occurred in treated human lung cancer cells. Thus the oleanolic acid nanocomplex could be utilized as a therapeutic mediator in the treatment of cancers in humans.

Mahonia aquifolium Extracts Promote Doxorubicin Effects against Lung Adenocarcinoma Cells In Vitro.

Mahonia aquifolium and its secondary metabolites have been shown to have anticancer potential. We performed MTT, scratch, and colony formation assays; analyzed cell cycle phase distribution and doxorubicin uptake and retention with flow cytometry; and detected alterations in the expression of genes involved in the formation of cell–cell interactions and migration using quantitative real-time PCR following treatment of lung adenocarcinoma cells with doxorubicin, M. aquifolium extracts, or their combination. MTT assay results suggested strong synergistic effects of the combined treatments, and their application led to an increase in cell numbers in the subG1 phase of the cell cycle. Both extracts were shown to prolong doxorubicin retention time in cancer cells, while the application of doxorubicin/extract combination led to a decrease in MMP9 expression. Furthermore, cells treated with doxorubicin/extract combinations were shown to have lower migratory and colony formation potentials than untreated cells or cells treated with doxorubicin alone. The obtained results suggest that nontoxic M. aquifolium extracts can enhance the activity of doxorubicin, thus potentially allowing the application of lower doxorubicin doses in vivo, which may decrease its toxic effects in normal tissues.