Cellular Metabolic Viability Research Articles

Comparative investigation of exosome extraction from rat bone marrow mesenchymal stem cells using three different methodologies

AbstractExosomes have been identified as crucial mediators in numerous physiological and pathological processes, emerging as a focal point of scientific inquiry. This study aims to compare three methods for isolating exosomes from rat bone marrow mesenchymal stem cells: ultracentrifugation (UC), ultrafast separation system (EXODUS), and commercial precipitation kit (EXO‐kit). First, the investigation compared exosomal morphology, particle size distribution, and expression of marker proteins. Subsequently, the RNA content, protein concentration, and purity of exosomes were evaluated. Finally, the impact of these exosomes on cellular metabolic viability and migration capacity was assessed. Results indicated that exosomes exhibited spherical or elliptical membrane structures, and most of the exosomes extracted by the three methods were in the range of 30to 200 nm. UC‐extracted exosomes demonstrated the least impurities and clearest background, followed by EXODUS‐extracted exosomes, and lastly EXO‐kit‐extracted exosomes. The EXO‐kit‐extracted exosomes yielded the highest RNA and protein content, whereas those isolated through UC exhibited superior purity. Furthermore, exosomes extracted from EXODUS and EXO‐kit methods effectively enhanced the metabolic viability and migratory ability of osteoblast precursor cells compared to UC‐extracted exosomes. In conclusion, each of the three methodologies presents advantages and limitations. Therefore, the selection of an appropriate exosome extraction technique should be based on specific experimental objectives and requirements.

Antimicrobial Activity and Mechanisms of Walnut Green Husk Extract.

Walnut green husks (WGHs), by-products of walnut production, are believed to possess antimicrobial properties, making them a potential alternative to antibiotics. In this study, the antibacterial activities of three extracts, derived from WGH, against Staphylococcus aureus, Bacillus subtilis, and Escherichia coli were investigated, and the antibacterial mechanisms of an anhydrous ethanol extract of WGH (WGHa) were examined. The results showed that WGHa exhibited inhibitory effects on all tested bacteria. The ultrahigh-performance liquid chromatography-tandem mass spectrometry analysis revealed that the major active compounds present in WGHa were terpenoids, phenols, and flavonoids. Treatment with WGHa resulted in the leakage of intracellular ions and alkaline phosphatase; a reduction in intracellular ATP content, ATPase activity, and nucleic acid content; as well as cellular metabolic viability. The transmission electron microscopy images showed varying degrees of cell deformation and membrane damage following WGHa treatment. The transcriptome sequencing and differentially expressed gene enrichment analyses revealed an up-regulation in pathways associated with RNA degradation, translation, protein export, and oxidative phosphorylation. Conversely, pathways involved in cell movement and localization, as well as cell wall organization and carbohydrate transport, were found to be down-regulated. These findings suggest that WGHa alters cell membrane permeability and causes damage to the cell wall. Additionally, WGHa interferes with cellular energy metabolism, compromises RNA integrity, and induces DNA replication stress, consequently inhibiting the normal growth and proliferation of bacteria. These findings unveiled the antimicrobial mechanisms of WGHa, highlighting its potential application as an antibiotic alternative.

Exploiting the biological response of two Serratia fonticola strains to the critical metals, gallium and indium

The use of microorganisms that allows the recovery of critical high-tech elements such as gallium (Ga) and indium (In) has been considered an excellent eco-strategy. In this perspective, it is relevant to understand the strategies of Ga and In resistant strains to cope with these critical metals. This study aimed to explore the effect of these metals on two Ga/In resistant strains and to scrutinize the biological processes behind the oxidative stress in response to exposure to these critical metals. Two strains of Serratia fonticola, A3242 and B2A1Ga1, with high resistance to Ga and In, were submitted to metal stress and their protein profiles showed an overexpressed Superoxide Dismutase (SOD) in presence of In. Results of inhibitor-protein native gel incubations identified the overexpressed enzyme as a Fe-SOD. Both strains exhibited a huge increase of oxidative stress when exposed to indium, visible by an extreme high amount of reactive oxygen species (ROS) production. The toxicity induced by indium triggered biological mechanisms of stress control namely, the decrease in reduced glutathione/total glutathione levels and an increase in the SOD activity. The effect of gallium in cells was not so boisterous, visible only by the decrease of reduced glutathione levels. Analysis of the cellular metabolic viability revealed that each strain was affected differently by the critical metals, which could be related to the distinct metal uptakes. Strain A3242 accumulated more Ga and In in comparison to strain B2A1Ga1, and showed lower metabolic activity. Understanding the biological response of the two metal resistant strains of S. fonticola to stress induced by Ga and In will tackle the current gap of information related with bacteria-critical metals interactions.

Abstract 5276: Enhancement of cisplatin activity against triple negative breast cancer cells by atorvastatin

Abstract Breast cancer (BC) is the second most common kind of cancer in the world and the most common among women. Its frequency rate is becoming alarming, being this way a major challenge to global health. It is worth mentioning that the lack of effective therapeutic options, in particular for certain subtypes, such as triple-negative tumor, still present a challenge for clinicians who often have to resort to highly unspecific cytotoxic therapies. Unfortunately, most patients relapse after a period of remission and eventually tumors becomes refractory to frontline therapy. This makes it desirable to identify drugs which synergize with chemotherapeutics and potentially reduce the dose of them that is necessary to treat patients. In the current financial and economical context, drug repositioning is gaining increasing interest as an alternative strategy for drug development. Cancer cells frequently exhibit specific alterations in their metabolic activity. Thus, targeting lipid metabolism in cancer cells could have therapeutic benefits and it does appear as promising auxiliary strategies in the fight against cancer. The present work aimed to investigate the effect of atorvastatin (ATV) on paclitaxel (PTX), cisplatin (CDDP) and olaparib (OLAP) cytotoxic on human BC cell lines MDA-MB-231 and MCF-7, and the mechanisms by which this interaction occurs. ATV decreased cellular metabolic viability (CMV; MTT assay) in a dose-time-dependent manner, especially in MDA-MB-231 being the maximum effect observed in with ATV 100µM (- 90%; p<0.001). In addition, ATV 5µM potentiates PTX and CDDP activities in MDA-MB-231, but not in MCF-7. Moreover, ATV had synergistic inhibitory effects with OLAP decreasing cellular metabolic viability in MDA-MB-231 (- 90%; p<0.001). . Cell cycle distribution analyzed by flow cytometry indicated that coadministration of ATV and CDDP in MDAM-MB-231 result in cell cycle arrest at G0/G1 phase as compared with that of the control and individual drug-treated cells. Altogether, our data suggest that association of ATV with conventional antineoplastic drugs seems to be beneficial, especially when associated with CISP for the MDA-MB-231 lineage. In any event, our study opens a new avenue in the fight against BC while possibly introducing a low cost substance in the portfolio of anticancer drugs and offering a promising strategy to increase the efficacy of them in breast cancer. Note: This abstract was not presented at the meeting. Citation Format: Diandra Zipinotti dos Santos, Isabella dos Santos Guimarães, Nayara Gusmão Tessarollo, Taciane Barbosa Henriques Barbosa Henriques, Paulo Cilas Lyra Junior, Marcele L L. de Souza, Maria C. Gomes, Ian Victor Silva, Leticia B. A. Rangel. Enhancement of cisplatin activity against triple negative breast cancer cells by atorvastatin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5276.

Abstract 2626: Novel naphtoquinone PIC21 targets PI3K in triple negative breast cancer cell line

Abstract Background: Triple negative breast cancer (TNBC) is a heterogeneous subgroup (ER-, PR-, and HER2-) of aggressive breast cancer, which poor prognosis is partially due to chemoresistance to available drugs. We have synthesized and screened 43 novel naphtoquinone-derived drugs (patent-protected), rationally designed to act through multiple pathways to avoid tumor chemoresistance, in MDA-MB231 by cellular metabolic viability and IC50 calculation. Of these, the most promising drugs PIC20 and PIC21 showed significantly higher AE than cisplatin, doxorubicin and paclitaxel in the lineage. Methods: Based on the crystalline structure of protein deposited on Protein Data Bank ( 1E7U, PI3K), and PIC20 and PIC21 tridimensional structures, computational molecular dock studies were conducted to investigate the molecules PIC20 e PIC21 tridimensional conformation and bounding energy to PI3K (Autodock Vina software). In vitro PI3K inhibition by PIC21 in MDA-MB231 was assessed using the Flowcellect PI3K/Mapk Dual Pathway Activation and Cancer Marker Detection. For PI3K/Akt and MAPK signaling pathways, cells were pre treated with PIC21 (0,1mM, 2h) or Wortmannin (Wort) (0,1μM, 30 min). Cells were then treated with insulin (0,6μM, 5 min) for stimulation of PI3K/Akt and MAPK pathways and incubated with Anti-phospho-Akt1/PKBα Alexa Fluor® 488 Conjugated Antibody and Anti-phospho-Erk1/2- R- Phycoerythrin conjugated antibody. A FACSCanto II cytometer was used for the flow cytometric analysis. FSC and SSC were used to establish size gates and exclude cellular debris from the analysis. In each measurement, a minimum of 30000 events were analyzed. Data were acquired and analyzed using BD FACSDiva and DeNovo FCS software. Findings: PIC21 targeting of PI3K was confirmed by docking studies, which data demonstrated that the interaction energy (E) for PI3K and PIC21, and PI3K inhibitors, LY294002 and Wort, was: PIC20 (E=-8.9), PIC21(E=-8.2), LY294002 (E=-9.5), Wort (E=-8.8). Cell treatment with PIC21 and Wort decreased the pAkt+ cell population (control=23.02%, PIC21 treated=6.63%, Wort treated=4.20%), whereas the pERK+ cell population increased (control=0,58%, PIC21 treated=6,41%, Wort treated=10,14%. This is due to the pathways crosstalk, by which pAkt inhibits the MAPK/ERK pathway. Conclusions: Our data strongly points to PIC21 as a novel PI3K inhibitor. A high prevalence of PI3K mutation, leading to its constitutive activation, has been described in TNBC, favoring tumor cells growth, proliferation, metabolism, and survival. Therefore, PIC21 opens a new avenue to overcome TNBC dramatic epidemiological scenario, bringing hope to improve patients overall outcome and quality of life. Citation Format: Renata D. Daltoe, Klesia P. Madeira, Murilo F. Cerri, João F. Allochio Filho, Maicon Delarmelina, Marcella L. Porto, Silvana S. Meirelles, Silvana S. Meirelles, Ian V. Silva, Sandro J. Greco, Leticia B A Rangel. Novel naphtoquinone PIC21 targets PI3K in triple negative breast cancer cell line. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2626. doi:10.1158/1538-7445.AM2014-2626

Pretreatment with mechano-growth factor E peptide protects bone marrow mesenchymal cells against damage by fluid shear stress

Improper fluid shear stress (FSS) can cause serious damages to bone marrow mesenchymal stem cells (MSCs). Mechano-growth factor (MGF) E peptide pretreatment was proposed to protect MSCs against FSS damage in this study. MSCs were exposed to FSS for 30min after they were pretreated with MGF E peptide for 24h. Then, the effects of MGF E peptide on the viability, proliferation and cell apoptosis of MSCs were investigated. MGF E peptide pretreatment could recover the cellular metabolic activity of MSCs reduced by 72dynecm(-2) FSS and had a synergistic effect with FSS on the cellular metabolic viability of MSCs under 24 and 72dynecm(-2) FSS. These results suggested that MGF E peptide pretreatment could be an effective method for the protection of FSS damage in bone tissue engineering.

Abstract P6-11-13: In vitro antineoplastic evaluation of rationally designed naphtoquinone-derived drugs in triple-negative breast cancer cell line

Abstract Background: Breast cancer (BC) comprises multiple diseases harboring different genetic alterations, which subtypes respond differently to treatment, and are associated to distinct clinical outcomes. Likewise, triple negative breast cancer (TNBCs) are a heterogeneous subgroup of BC, immunophenotypically negative for estrogen and progesterone receptor, and HER2, that account for 10–15% of all invasive BC, affecting mostly African-American and Hispanic pre-menopausal women. Considering that TNBC have poor prognosis, and cannot be effectively treated with current targeted therapies, the discovery of new treatment options is imperative. Methods: Novel naphtoquinone-derived drugs were rationally designed to act through multiple pathways aiming the avoidance of drug-resistant phenotype acquisition by tumor cells, and were synthesized following high efficiency and low cost method. Drugs antineoplastic efficacy (AE) was accessed in claudin-low TNBC cell line, MDA-MB-231, through the evaluation of cellular metabolic viability (CMV) (MTT method). Drugs structures are protected by patent. Cells were cultured in RPMI media supplemented with 10% (v/v) FBS and antibiotics until subconfluence; then, 1.5×105 cells/well were subcultured for 72h prior to treatment with drugs (10−4, 10−5, 10−6, 10−7, and 10−8 M). After 24h, CMV was assessed. Experiments in which the lineage was treated with cisplatin, doxorubicin or paclitaxel were run in parallel. The mean and standard-deviation of the absorbancies were used to calculate CMV and drugs IC50 (PrismaGraphPad version 5.1). Findings: We screened the AE of 43 novel naftoquinones-derived drugs in MDA-MB-231 lineage; seven have decreased its CMV by, at least, 50%, as: PIC1 (IC50 1.15×10−4M; CMV decrease of 50%); PIC6 (IC50 4.24×10−5M; CMV decrease of 70%); PIC10 (IC50 5.07×10−5M; CMV decrease of 70%); PIC 20 (IC50 1.38×10−5M; CMV decrease of 90%); PIC21 (IC50 5.00×10−5M; CMV decrease of 70%); S5 (IC50 7.26×10−5M; CMV decrease of 60%); M20 (IC50 7.94×10−5M; CMV decrease of 60%). Their AE was significantly higher than that of cisplatin (IC50 1.56×10−4M; CMV decrease < 10%), doxorubicin (IC50 1.76×10−4M; CMV decrease of 38%), and paclitaxel (IC50 5.05×10−7M; CMV decrease of 20%). Interpretation: Altogether, our results present potential novel antineoplastic drugs to treat TNBC from a panel of in house rationally designed naftoquinones-derived compounds, developing an innovative and economically viable project. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-11-13.

Abstract 161: The role of mTOR in the cisplatin resistant phenotype in ovarian cancer lineage

Abstract Background Ovarian cancer (OVCA) is the second cause of gynecological-cancer deaths in Brazil, being frequently associated to chemotherapy resistance, a characteristic usually associated to PI3K/Akt/mTOR. We herein present data concerning the role of mTORC2 in cisplatin-resistant phenotype acquisition by OVCA cells. Methods Antineoplastic efficacy of cisplatin, doxorubicin, paclitaxel, and rapamycin was accessed, in different combinations, in the OVCA lineage OVCAR3 (cisplatin-resistant, advanced serous OVCA), through cellular metabolic viability (CMV) (MTT method). Cells were cultured in RPMI media supplemented with 10% (v/v) FBS and antibiotics until subconfluence. To characterize the role of mTORC1 or mTORC2 in OVCAR3 MCV, 1.5x105 cells/well were treated according to: EA1) Inhibition of mTORC1: Drugs at IC50 for 24h; EA2) Inhibition of mTORC1 and mTORC2: Rapamycin 100nM for 72h, followed by drugs at IC50 for 24h. Modulation of MTOR expression by PKA, PKC, and PI3K was investigated by real time RT-PCR. Statistical analysis was performed using PrismaGraphPad version 5.1. Findings OVCAR3 CMV was not significantly changed by cisplatin (IC50 0,08mM) nor paclitaxel (IC50 0,06nM) or rapamycin (IC50 0,02nM) (CMV 95%, 100%, 100%, respectively), but by doxorubicin (IC50 0,02nM) (CMV 60%), especially in combination with cisplatin (CMV 31%), following EA1. Considering the irreversible doxorubicin-induced cardiotoxicity, we pursued with EA2. Intriguingly, OVCAR3 cisplatin sensitivity was, at least partially, recovered (CMV 65%). No addictive effect was observed with cisplatin/doxorubicin following EA2 (CMV 60%), suggesting that OVCA-resistant patient might benefit from less toxic rapamycin-based chemotherapy, leaving doxorubicin for palliative final-staged disease management. Finally, neither PKA nor PI3K modulate MTOR expression; however, PKC suppresses its transcription by 4.42-fold. Interpretation Altogether, our data suggest that mTORC2, but not mTORC1, might influence cisplatin sensitivity in OVCA. Moreover, PKC inhibits the expression of MTORC in cisplatin-resistant OVCA. We propose that the inhibition of mTORC2 followed by cisplatin treatment might lead to a conjunction of autophagic cell death and apoptosis in OVCA. Of remarkable clinical interest, we point to the urge of better design clinical trials, in which not only novel drugs are tested, but also the chronological order of drugs administration to the patient is seen as crucial for treatment efficacy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 161. doi:1538-7445.AM2012-161

Abstract 924: In vitro study of the potential antineoplastic effect of synthetic naphthoquinones in cisplatin-resistant ovarian cancer lineage

Abstract Background: Cancer development has been associated with alterations in polyamine biosynthesis and metabolism, which induce cell proliferation, angiogenesis, expression of genes related to tumor invasion and metastasis; whereas inhibit apoptosis. Based on the strong rational to develop novel polyamine depleting molecules, and adding the strategy to have substances that can control cancer through different cellular pathways aiming to bypass the acquisition of drug resistant phenotype by cancer cells; this work aimed to screen, in an ovarian cancer (OVCA) line, 40 novel rationally developed potential anti-cancer compounds, following rapid, high efficient, and low cost synthetic methodologies, then confirmed by spectroscopic techniques. OVCA is the most lethal gynecological malignancy, with high rates of chemoresistance and disease relapse; therefore, supporting the urge to generate novel anti-OVCA agents. Methods: Novel naphthoquinone-derived compounds were rationally designed to act through multiple cellular pathways aiming the avoidance of drug resistant phenotype acquisition by cancer cells, and were synthesized by rapid, efficient and low cost synthetic method. Drugs antineoplastic efficacy (AE) was accessed in OVCAR3, through the evaluation of cellular metabolic viability (CMV) (MTT method). Drugs structures are protected by patent. Cells were cultured in RPMI media supplemented with 10% (v/v) FBS, antibiotics and antifungics, in 5% CO2, until subconfluence; then, 1.5x105 cells/well were subcultured for 72h prior to treatment with drugs in different concentrations (10−4, 10−5, 10−6, 10−7, and 10−8 M). After 24h, CMV was assessed. Experiments in which the lineage was treated with cisplatin, doxorubicin or paclitaxel were run in parallel. The mean and standard-deviation of the absorbancies were used to calculate CMV and drugs IC50 (PrismaGraphPad version 5.1). Findings: We have screened the AE of 40 novel naphtoquinone-derived drugs in OVCAR3; five have decreased its CMV by, at least, 70%, namely: M8 (IC50 1.64x10−5M; CMV decrease of 90%); M10 (IC50 1.37x10−5M; CMV decrease of 96%); M14 (IC50 1.45x10−5M; CMV decrease of 85%); PIC10 (IC50 9.13x10−6M; CMV decrease of 95%); PIC20 (IC50 5.31x10−5M; CMV decrease of 70%). The IC50 for cisplatin, the gold therapy against OVCA was 3.87x10−5M and CMV decrease was 85%. Interpretation: We herein present novel drugs to treat OVCA; whereas PIC 10 is more potent that cisplatin, M8, M10, M14, PIC10 and PIC20 seem to have similar or higher antineoplastic efficacy in treating cisplatin-resistant OVCA. We strongly believe that the present pre-clinical research project is innovative, as it introduces novel anti-OVCA drugs, economically viable and socially important, as it might bring hope to put OVCA treatment in a perspective in which the disease control is a real possibility. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 924. doi:1538-7445.AM2012-924