Time-dependent Cytotoxicity Research Articles

Mesoporous silica nano-adjuvant triggers pro-inflammatory responses in Caco-2/peripheral blood mononuclear cell (PBMC) co-cultures.

The aim of this study was to evaluate the cytotoxicity and immune-stimulatory effect of Mesoporous silica nanoparticle (MSN) Nano-adjuvant on pro-inflammatory cytokines and pattern recognition receptors (PRR) genes expression in Caco-2/PBMC co-culture model. MSNs were synthesized and characterized by scanning electron microscope (SEM), Brunauer Emmett Teller (BET) and Barrett Joyner Halenda (BJH) techniques. The BET specific surface area of MSNs was around 947 m2/g and the total pore volume and average pore diameter were 1.5 cm3/g and 8.01 nm, respectively. At the concentration of 10 µg/mL, MSN showed a low and time-dependent cytotoxicity on Caco-2 cells, while no cytotoxic effect was observed for 0.1 and 1 µg/mL concentrations after 24, 48 and 72 h. The expression of pro-inflammatory cytokines genes (IL-1, IL-8 and TNF-α) in co-cultures treated with different concentrations of MSN showed a dose-dependent significant increase up to 17.44, 2.722 and 4.34 folds, respectively, while the expression augmentation of IL-1 gene was significantly higher than the others. This indicates slight stimulation of intestinal inflammation. Different concentrations of MSN significantly increased TLR4 and NOD2 expression to 4.14 and 2.14 folds, respectively. NOD1 was not affected significantly. It can be concluded that MSN might increase protective immune responses against antigens as a vaccine adjuvant candidate. It seems that stimulation of TNF-α, IL-1, and IL-8 expression in enterocytes probably transpires through the agonistic activity of MSN for TLRs including TLR4, while NOD2-associated signaling pathways are also involved. This study provides an overall picture of MSN as a novel and potent oral adjuvant for mucosal immunity.

The Antiseptic and Antineoplastic Agent Taurolidine Modulates Key Leukocyte Functions

Although taurolidine is known to exert a wide spectrum of biological actions, its effects on immune cells have not been characterized in detail. In this study, we investigated the ex vivo effects of taurolidine on relevant innate and adaptive immune cell functions. Leukocyte functions in whole blood were assessed following treatment with various taurolidine concentrations. Viability of peripheral blood mononuclear cells (PBMCs) and granulocytes was measured using the WST-1 assay. PBMC function was assessed by measuring TNFα and IFNγ production after stimulation with lipopolysaccharide (LPS) or Candida, respectively. Reactive oxygen species (ROS) production by granulocytes was measured in whole blood using luminol-enhanced chemiluminescence. Granulocyte degranulation and activation were evaluated by membrane expression of degranulation (CD63, CD66B) and adhesion markers (CD62L, CD11b) using immunofluorescent staining followed by flow-cytometric analysis. Taurolidine decreased viability of PBMCs and granulocytes: after 2 h, IC50 concentrations were 500 and 520 μg/ml, respectively. Following prolonged exposure (≥24 h) of PBMCs, the IC50 concentrations declined to 40 μg/ml. PBMC cytokine production significantly decreased at taurolidine concentrations below the cytotoxic threshold, whereas no changes in ROS production were observed. The expression of all granulocyte adhesion and degranulation markers increased at concentrations higher than 500 μg/ml (the cytotoxic level of taurolidine). Taurolidine exhibits a dose- and time-dependent cytotoxicity toward PBMCs and granulocytes. The effects on PBMCs, as exemplified by a decrease in cytokine production, occurred below the toxic threshold, whereas granulocyte function (ROS production) remained unchanged at these taurolidine concentrations. Granulocyte activation and degranulation markers only increased at cytotoxic taurolidine concentrations.

Extracts of Tagetes erecta exhibit potential cytotoxic and antitumor activity that could be employed as a promising therapeutic agent against cancer: A study involving in vitro and in vivo approach

Background and purposeGlobally, the burden of cancer is increasing consistently. Modern cancer therapies include lots of toxicity in the non-targeted organs reducing the life expectancy of the patients. Therefore, the development of safer alternative medicines with less toxicity and high efficacy is of immense importance. The present study was designed to evaluate the anticancer activity of a medicinal plant, “Tagetes erecta L.” (TE), in established cancer cell lines in vitro and in animal models in vivo. MethodsGC–MS analysis was performed to reveal the bioactive compounds present in the leaves of TE. To evaluate the antitumor potential of aqueous extract of TE (AETE), EAC (Ehrlich ascites carcinoma) induced solid tumor model was prepared in Swiss albino mice. The effect of AETE on body weight (BW), tumor weight (TW), and tumor volume (TV) was assessed in control and tumor-bearing animals. Further, the survival of the experimental animals was also monitored to assess the effect of AETE on life span. To determine the cytotoxicity induced by AETE, trypan blue dye exclusion assay, MTT assay, and LDH release assay was performed in EAC and HEp-2 cell lines by in vitro methods. Further, cell cycle assay and apoptosis assay was employed to ascertain the AETE-induced cytotoxicity is due to apoptosis or not. In addition, we have done a mitochondrial membrane potential assay using a flow cytometer to understand the mechanism of apoptosis. ResultsGC–MS analysis revealed the presence of hexadecanoic acid, Linolenic acid, Quinic acid, 2,3- dihydro benzofuran (Coumaran), and β-stigmasterol as major bioactive compounds in TE leaves. Results in animal studies showed that AETE treatment potentially reduced the TW and TV and increased the life span in EAC-induced tumor-bearing animals. Side effect analysis was also done in normal Swiss albino mice that confirmed the lack of toxicity of AETE. Studies in cancer cell lines indicated dose and time-dependent cytotoxicity in HEp-2 and EAC cells. Flow cytometric analysis established significant induction of apoptosis in EAC cells without arresting the cell cycle. In addition, AETE treatment led to a significant increase in cells with depolarised mitochondrial membrane potential. ConclusionThe present study indicated that AETE potentially inhibits tumor progression without disturbing normal body physiology. Thus, we conclude that AETE can be used as a potential therapeutic agent against cancer.

In vitro effects of cannabidiol and its main metabolites in mouse and human Sertoli cells

Cannabidiol (CBD) is a major cannabinoid present in extracts of the plant Cannabis sativa (marijuana). While the therapeutic effects of CBD on epilepsy have been demonstrated, less is understood regarding its potential adverse effects. Recent studies revealed that CBD induced toxicity in the male reproductive system of animal models. In this study, we used TM4, an immortalized mouse Sertoli cell line, and primary human Sertoli cells to evaluate the toxicities of CBD and its main metabolites, 7-carboxy-CBD and 7-hydroxy-CBD. CBD induced concentration- and time-dependent cytotoxicity in mouse and human Sertoli cells, which mainly resulted from the inhibition of the G1/S-phase cell cycle transition. CBD also inhibited DNA synthesis and downregulated key cell cycle proteins. Moreover, CBD reduced the mRNA and protein levels of a functional marker, Wilms’ tumor 1. Similar to CBD, 7-carboxy-CBD and 7-hydroxy-CBD inhibited cellular proliferation and decreased DNA synthesis. 7-Carboxy-CBD was less cytotoxic than CBD, while 7-hydroxy-CBD showed comparable cytotoxicity to CBD in both mouse and human Sertoli cells. Compared to mouse Sertoli cells, CBD, 7-hydroxy-CBD, and 7-carboxy-CBD were more cytotoxic in human Sertoli cells. Our results indicate that CBD and its main metabolites can inhibit cell proliferation in mouse and human Sertoli cells.

Chimaphila umbellata extract exerts anti-proliferative effect on human breast cancer cells via RIP1K/RIP3K-mediated necroptosis

BackgroundChimaphila umbellata (CU) is a small perennial plant whose roots and leaves are used in the preparation of homeopathic medicines. In this study, the anti-proliferative and apoptotic effects of CU extract, obtained from its homeopathic mother tincture, were evaluated in human breast cancer cell lines. MethodsMTT assay was used to evaluate the dose- and time-dependent cytotoxicity of the extract in MCF-7 and HEK293 cells. The anti-proliferative effect of the extract was evaluated using the clonogenic and wound healing assays. The anti-angiogenic activity of the drug was evaluated using the chick chorioallantoic membrane assay. The mode of cell death was analyzed using Annexin V and PI staining assays through flow cytometry. In addition, the expression patterns of associated genes were evaluated using immunoblot analysis. ResultsCU extracts exerted anti-proliferative effect in breast cancer cell line, i.e., MCF-7 cells, by inhibiting their growth and migration. The extract also demonstrated significant anti-angiogenic ability, limiting the de-novo blood vessel growth and development in chick embryos. The extract was found to increase caspase-independent necroptosis by involving RIP1/RIP3 kinases and MLKL proteins. This finding was further confirmed by using NEC-1 (an inhibitor of necroptosis), which significantly abolished the CU extract-induced necroptotic effect. ConclusionsThe CU extract exhibits great potential in preventing breast cancer and thus warrants further investigations in vivo.

Zinc Chloride: Time-Dependent Cytotoxicity, Proliferation and Promotion of Glycoprotein Synthesis and Antioxidant Gene Expression in Human Keratinocytes.

Simple SummaryZinc ions are involved in the biology of cell growth, proliferation, differentiation or apoptosis by regulating many biological molecules, such as transcription factors, enzymes and growth factors. In this study, the time-dependent cytotoxicity, cell proliferation and gene expression in human keratinocytes HaCaT cells were evaluated when exposed to ZnCl2. The results of this study showed non-cytotoxic effects up to 10 µg/mL after 24 h, no significant effect on cell proliferation when exposed to 5 or 1 µg/mL ZnCl2 at 72 h and upregulation of eight genes, with great potential in the biomedical field, particularly for regenerative-medicine applications and wound healing.The use of ionic metals such as zinc (Zn2+) is providing promising results in regenerative medicine. In this study, human keratinocytes (HaCaT cells) were treated with different concentrations of zinc chloride (ZnCl2), ranging from 1 to 800 µg/mL, for 3, 12 and 24 h. The results showed a time–concentration dependence with three non-cytotoxic concentrations (10, 5 and 1 µg/mL) and a median effective concentration value of 13.5 µg/mL at a cell exposure to ZnCl2 of 24 h. However, the zinc treatment with 5 or 1 µg/mL had no effect on cell proliferation in HaCaT cells in relation to the control sample at 72 h. The effects of the Zn2+ treatment on the expression of several genes related to glycoprotein synthesis, oxidative stress, proliferation and differentiation were assessed at the two lowest non-cytotoxic concentrations after 24 h of treatment. Out of 13 analyzed genes (superoxide dismutase 1 (SOD1), catalase (CAT), matrix metallopeptidase 1 (MMP1), transforming growth factor beta 1 (TGFB1), glutathione peroxidase 1 (GPX1), fibronectin 1 (FN1), hyaluronan synthase 2 (HAS2), laminin subunit beta 1 (LAMB1), lumican (LUM), cadherin 1 (CDH1), collagen type IV alpha (COL4A1), fibrillin (FBN) and versican (VCAN)), Zn2+ was able to upregulate SOD1, CAT, TGFB1, GPX1, LUM, CDH1, FBN and VCAN, with relative expression levels of at least 1.9-fold with respect to controls. We found that ZnCl2 promoted glycoprotein synthesis and antioxidant gene expression, thus confirming its great potential in biomedicine.

Body fluids from the rat exposed to chlorpyrifos induce cytotoxicity against the corresponding tissue\u2212derived cells in vitro

BackgroundThis study aims to establish an in vitro monitoring approach to evaluate the pesticide exposures. We studied the in vitro cytotoxicity of three different body fluids of rats to the respective corresponding tissue-derived cells.MethodsWistar rats were orally administrated daily with three different doses of chlorpyrifos (1.30, 3.26, and 8.15 mg/kg body weight/day, which is equal to the doses of 1/125, 1/50, and 1/20 LD50, respectively) for consecutive 90 days. Blood samples as well as 24-hour urine and fecal samples were collected and processed. Then, urine, serum, and feces samples were used to treat the correspondent cell lines, i.e., T24 bladder cancer cells, Jurkat lymphocytes, and HT-29 colon cancer cells respectively, which derived from the correspondent tissues that could interact with the respective corresponding body fluids in organism. Cell viability was determined by using MTT or trypan blue staining.ResultsThe results showed that urine, serum, and feces extract of the rats exposed to chlorpyrifos displayed concentration- and time-dependent cytotoxicity to the cell lines. Furthermore, we found that the cytotoxicity of body fluids from the exposed animals was mainly due to the presence of 3, 4, 5-trichloropyrindinol, the major toxic metabolite of chlorpyrifos.ConclusionsThese findings indicated that urine, serum, and feces extraction, especially urine, combining with the corresponding tissue-derived cell lines as the in vitro cell models could be used to evaluate the animal exposure to pesticides even at the low dose with no apparent toxicological signs in the animals. Thus, this in vitro approach could be served as complementary methodology to the existing toolbox of biological monitoring of long-term and low-dose exposure to environmental pesticide residues in practice.

Mechanisms of Apoptotic Cell Death by Stainless Steel Nanoparticle Through Reactive Oxygen Species and Caspase-3 Activities on Human Liver Cells.

Background: Nanoparticles are widely used in pharmaceutical, agriculture, and food processing industries and in many other fields. However, the effect of stainless steel nanoparticles (SSNPs) remains unclear. So in this study, we evaluate the effect of SSNPs’ toxicity on human liver (CHANG and HuH-7) cell lines over 24 and 48 h. Methods: We have analyzed the quality, shape, and size of SSNPs using x-ray diffraction (XRD), energy dispersive x-ray (EDX) scanning electron microscope (SEM), and transmission electron microscope (TEM). The cytotoxicity and cell growth were determined by using the MTT and wound healing tests. The oxidative stress parameters were determined by measuring ROS generation and antioxidant enzymes, such as glutathione (GSH) and superoxide dismutase (SOD), due to SSNP exposure on human liver cell lines over 24 and 48 h. The confirmation of the apoptotic effect of SSNPs on livers cells was determined by the Western blot analysis for the expression of apoptotic proteins, such as Bax, bcl2, and p53, and real-time PCR for the expression of apoptotic genes, such as Bax, bcl2, caspase-3, and p53. Results: We have observed the dose- and time-dependent cytotoxicity and apoptosis of SSNPs on both cells. The results showed that SSNPs induced cell toxicity, inhibited cell growth, GSH, and increased generation of intracellular ROS and SOD levels at higher concentrations of exposure in both cells. SSNPs showed an apoptotic activity with upregulation of Bax, caspase-3, and p53 and downregulation of the bcl2 gene expression in CHANG and HuH-7 cell lines. Moreover, the immunoblotting assay confirmed the apoptotic activity of SSNPs in cells. Conclusion: In conclusion, these findings demonstrated that SSNPs showed toxic effects on human liver cells via activating the caspase-3 activity and they induced more toxicity in HuH-7 cells than in CHANG cells.

Hybrid derivative of cathelicidin and human beta defensin-2 against Gram-positive bacteria: A novel approach for the treatment of bacterial keratitis

Bacterial keratitis (BK) is a major cause of corneal blindness globally. This study aimed to develop a novel class of antimicrobial therapy, based on human-derived hybrid host defense peptides (HyHDPs), for treating BK. HyHDPs were rationally designed through combination of functional amino acids in parent HDPs, including LL-37 and human beta-defensin (HBD)-1 to -3. Minimal inhibitory concentrations (MICs) and time-kill kinetics assay were performed to determine the concentration- and time-dependent antimicrobial activity and cytotoxicity was evaluated against human corneal epithelial cells and erythrocytes. In vivo safety and efficacy of the most promising peptide was examined in the corneal wound healing and Staphylococcus aureus (ATCC SA29213) keratitis murine models, respectively. A second-generation HyHDP (CaD23), based on rational hybridization of the middle residues of LL-37 and C-terminal of HBD-2, was developed and was shown to demonstrate good efficacy against methicillin-sensitive and methicillin-resistant S. aureus [MIC = 12.5–25.0 μg/ml (5.2–10.4 μM)] and S. epidermidis [MIC = 12.5 μg/ml (5.2 μM)], and moderate efficacy against P. aeruginosa [MIC = 25-50 μg/ml (10.4–20.8 μM)]. CaD23 (at 25 μg/ml or 2× MIC) killed all the bacteria within 30 min, which was 8 times faster than amikacin (25 μg/ml or 20× MIC). After 10 consecutive passages, S. aureus (ATCC SA29213) did not develop any antimicrobial resistance (AMR) against CaD23 whereas it developed significant AMR (i.e. a 32-fold increase in MIC) against amikacin, a commonly used treatment for BK. Pre-clinical murine studies showed that CaD23 (0.5 mg/ml) achieved a median reduction of S. aureus bioburden by 94% (or 1.2 log10 CFU/ml) while not impeding corneal epithelial wound healing. In conclusion, rational hybridization of human-derived HDPs has led to generation of a potentially efficacious and safe topical antimicrobial agent for treating Gram-positive BK, with no/minimal risk of developing AMR.

Targeting Autophagy Triggers Apoptosis and Complements the Action of Venetoclax in Chronic Lymphocytic Leukemia Cells.

Simple SummaryVenetoclax is an antagonist of the antiapoptotic protein Bcl-2, and is currently approved for treatment of chronic lymphocytic leukemia (CLL). Recently, clinical use has shown that patients develop resistance to venetoclax. Therefore, the demand for novel targets for treatment of CLL remains high. One such target is autophagy, an evolutionarily old system for degradation of long-lived proteins and organelles that recovers the energy for normal cellular functions. Here, the antileukemic potential of different autophagy inhibitors was evaluated in patient-derived CLL cells. Among these, inhibitors of the AMPK/ULK1 pathway and late-stage autophagy were the most potent, with selective cytotoxic activities seen. They also show activity against CLL cells with unfavorable genetic characteristics. These inhibitors complement the cytotoxic action of venetoclax. In conclusion, targeting autophagy shows potential as a novel approach for treatment of patients with CLL.Continuous treatment of patients with chronic lymphocytic leukemia (CLL) with venetoclax, an antagonist of the anti-apoptotic protein Bcl-2, can result in resistance, which highlights the need for novel targets to trigger cell death in CLL. Venetoclax also induces autophagy by perturbing the Bcl-2/Beclin-1 complex, so autophagy might represent a target in CLL. Diverse autophagy inhibitors were assessed for cytotoxic activities against patient-derived CLL cells. The AMPK inhibitor dorsomorphin, the ULK1/2 inhibitor MRT68921, and the autophagosome–lysosome fusion inhibitor chloroquine demonstrated concentration-dependent and time-dependent cytotoxicity against CLL cells, even in those from hard-to-treat patients who carried del(11q) and del(17p). Dorsomorphin and MRT68921 but not chloroquine triggered caspase-dependent cell death. According to the metabolic activities of CLL cells and PBMCs following treatments with 10 µM dorsomorphin (13% vs. 84%), 10 µM MRT68921 (7% vs. 78%), and 25 µM chloroquine (41% vs. 107%), these autophagy inhibitors are selective toward CLL cells. In these CLL cells, venetoclax induced autophagy, and addition of dorsomorphin, MRT68921, or chloroquine showed potent synergistic cytotoxicities. Additionally, MRT68921 alone induced G2 arrest, but when combined with venetoclax, it triggered caspase-dependent cytotoxicity. These data provide the rationale to target autophagy and for autophagy inhibitors as potential treatments for patients with CLL.

Carbon Nanofibers versus Silver Nanoparticles: Time-Dependent Cytotoxicity, Proliferation, and Gene Expression.

Carbon nanofibers (CNFs) are one-dimensional nanomaterials with excellent physical and broad-spectrum antimicrobial properties characterized by a low risk of antimicrobial resistance. Silver nanoparticles (AgNPs) are antimicrobial metallic nanomaterials already used in a broad range of industrial applications. In the present study these two nanomaterials were characterized by Raman spectroscopy, transmission electron microscopy, zeta potential, and dynamic light scattering, and their biological properties were compared in terms of cytotoxicity, proliferation, and gene expression in human keratinocyte HaCaT cells. The results showed that both AgNPs and CNFs present similar time-dependent cytotoxicity (EC50 of 608.1 µg/mL for CNFs and 581.9 µg/mL for AgNPs at 24 h) and similar proliferative HaCaT cell activity. However, both nanomaterials showed very different results in the expression of thirteen genes (superoxide dismutase 1 (SOD1), catalase (CAT), matrix metallopeptidase 1 (MMP1), transforming growth factor beta 1 (TGFB1), glutathione peroxidase 1 (GPX1), fibronectin 1 (FN1), hyaluronan synthase 2 (HAS2), laminin subunit beta 1 (LAMB1), lumican (LUM), cadherin 1 CDH1, collagen type IV alpha (COL4A1), fibrillin (FBN), and versican (VCAN)) treated with the lowest non-cytotoxic concentrations in the HaCaT cells after 24 h. The AgNPs were capable of up-regulating only two genes (SOD1 and MMP1) while the CNFs were very effective in up-regulating eight genes (FN1, MMP1, CAT, CDH1, COL4A1, FBN, GPX1, and TGFB1) involved in the defense mechanisms against oxidative stress and maintaining and repairing tissues by regulating cell adhesion, migration, proliferation, differentiation, growth, morphogenesis, and tissue development. These results demonstrate CNF nanomaterials’ unique great potential in biomedical applications such as tissue engineering and wound healing.

3,3′-Diselenodipropionic acid (DSePA) induces reductive stress in A549 cells triggering p53-independent apoptosis: A novel mechanism for diselenides

The aim of present study was to investigate the anticancer mechanisms of 3,3′-diselenodipropionic acid (DSePA), a redox-active organodiselenide in human lung cancer cells. DSePA elicited a significant concentration and time-dependent cytotoxicity in human lung cancer cell line A549 than in normal WI38 cells. The cytotoxic effect of DSePA was preceded by an acute decrease in the level of basal reactive oxygen species (ROS) and a concurrent increase in levels of reducing equivalents (like GSH/GSSG and NADH/NAD) within cells. Further, a series of experiments were performed to measure the markers of intrinsic (Bax, cytochrome c and caspase-9), extrinsic (TNFR, FADR and caspase-8) and endoplasmic reticulum (ER) stress (protein ubiquitylation, calcium flux, Bip, CHOP and caspase-12) pathways in DSePA treated cells. DSePA treatment significantly increased the levels of all the above markers. Moreover, DSePA did not alter the expression and phosphorylation (Ser15) of p53 but caused a significant damage to mitochondria. Pharmacological modulation of GSH level by BSO and NAC in DSePA treated cells led to partial abrogation and augmentation of cell kill respectively. This established the role of reductive stress as a trigger for the apoptosis induced by DSePA treatment. Finally, in vitro anticancer activity of DSePA was also corroborated by its in vivo efficacy of suppressing the growth of A549 derived xenograft tumor in SCID mice. In conclusion, above results suggest that DSePA induces apoptosis in a p53 independent manner by involving extrinsic and intrinsic pathways together with ER stress which can an interesting strategy for lung cancer therapy.

Nanoparticles of cisplatin augment drug accumulations and inhibit multidrug resistance transporters in human glioblastoma cells

BackgroundCisplatin (CSP) is a potent anticancer drug widely used in treating glioblastoma multiforme (GBM). However, CSP's clinical efficacy in GBM contrasted with low therapeutic ratio, toxicity, and multidrug resistance (MDR). Therefore, we have developed a system for the active targeting of cisplatin in GBM via cisplatin loaded polymeric nanoplatforms (CSP-NPs). MethodsCSP-NPs were prepared by modified double emulsion and nanoprecipitation techniques. The physiochemical characterizations of CSP-NPs were performed using zeta sizer, scanning electron microscopy (SEM), drug release kinetics, and drug content analysis. Cytotoxicity, induction of apoptosis, and cell cycle-specific activity of CSP-NPs in human GBM cell lines were evaluated by MTT assay, fluorescent microscopy, and flow cytometry. Intracellular drug uptake was gauged by fluorescent imaging and flow cytometry. The potential of CSP-NPs to inhibit MDR transporters were assessed by flow cytometry-based drug efflux assays. ResultsCSP-NPs have smooth surface properties with discrete particle size with required zeta potential, polydispersity index, drug entrapment efficiency, and drug content. CSP-NPs has demonstrated an ‘initial burst effect’ followed by sustained drug release properties. CSP-NPs imparted dose and time-dependent cytotoxicity and triggered apoptosis in human GBM cells. Interestingly, CSP-NPs significantly increased uptake, internalization, and accumulations of anticancer drugs. Moreover, CSP-NPs significantly reversed the MDR transporters (ABCB1 and ABCG2) in human GBM cells. ConclusionThe nanoparticulate system of cisplatin seems to has a promising potential for active targeting of cisplatin as an effective and specific therapeutic for human GBM, thus eliminating current chemotherapy's limitations.

Antioxidative, cytotoxic, and anti-metastatic potentials of Laurencia obtusa and Ulva lactuca seaweeds

Objective: To investigate the antioxidant and cytotoxic properties of the most active extracts from Ulva lactuca and Laurencia obtusa against colon and cervical cancer cell lines. Methods: The antioxidant radical scavenging activity of the algal extracts was estimated using the 2,2-diphenyl-1-picrylhydrazyl colorimetric assay. Moreover, the cytotoxic potential of these bioactive extracts was studied against HCT-116 and HeLa cells by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide assay. Wound healing analysis was adopted to evaluate the anti-metastatic effects of protein extracts from both algae. Cell cycle analysis was performed by flow cytometry, and apoptotic bodies were detected using 4’,6-diamidino-2-phenylindole fluorescent staining. Amino acids of hydrolyzed protein extract were separated and identified chromatographically by high performance liquid chromatography. Results: The green algae Ulva lactuca had higher lipid content than Laurencia obtusa, whereas the latter had higher protein content with high antioxidant capacity. Protein extracts had significant dose- and time-dependent cytotoxicity and anti-metastatic activity against HCT-116 cells. Protein b extracts of both algae obtained from the chloroform:hexane solvent lipid-free residue caused morphological changes and induced apoptosis of HCT-116 cells. Further analysis revealed that apoptosis induced upon Laurencia obtusa protein b treatment was triggered via the ROS pathway, causing a significant increase in the sub-G1 population. Glycine and arginine (5.94 and 5.47 μM, respectively) were the most common amino acids in Laurencia obtusa protein b extract. Conclusions: Proteins of Laurencia obtusa and Ulva lactuca could be considered as an adjuvant nutraceutical source of pro-apoptotic and anti-metastatic biomolecules against colon cancer.

Silencing Oncogene Translation Using Pateamine a Analogues As a Novel Therapeutic Strategy for c-Myc Driven Lymphoma

IntroductionThe oncogene c-Myc is a master transcription factor that is associated with pathogenesis and chemo-resistance in a vast array of human cancers including aggressive lymphoma. Although the therapeutic benefit of targeting c-Myc has been demonstrated in many preclinical studies, direct inhibitors of c-Myc have not been successfully developed for the clinic. On the other hand, translation of c-Myc is highly dependent on the eukaryotic translation initiation factor 4F (eIF4F), a complex comprised of the eIF4A, eIF4G, and eIF4E subunits. These results suggest that silencing the translation of c-Myc via inhibiting eIF4F or its subunits may be a valid alternative strategy for indirectly targeting the “undruggable” c-Myc oncogene. In the current project, we have investigated the pharmacological activity and mechanism of Pateamine A (PatA) analogues in lymphoma cells in vitro . PatA was originally purified as a marine natural product and demonstrated anti-proliferation activity. One of the analogues, Biotin-DMDA-PatA conjugate (B-DMDA-PatA) pulled down eIF4A, specifically inhibits de novo protein synthesis with much less effect on de novo RNA synthesis, and specifically inhibits cap dependent translation. DMDA-PatA acts by decreasing the interaction between eIF4A and eIF4G. Our hypothesis is the following: If PatA analogues preferentially inhibit the translation of oncogenes such as c-Myc, then PatA analogues will be highly effective for c-Myc driven cancers and potentially safe for animals and humans.MethodsMZ-735 is a recently developed and optimized PatA analogue, based on insights from DMDA-PatA. Both compounds were synthesized at the CPRIT Synthesis and Drug-Lead Discovery Laboratory at Baylor University. Cytotoxicity was evaluated in lymphoma cell lines using CellTiter-Glo (Promega®). The cells were exposed to the PatA analogues or the vehicle control on either continuous exposure or on a treatment (progressively decreasing exposure)-washout schedule. To evaluate the toxicity of PatA analogues to normal cells, cytotoxicity in peripheral blood mononuclear cells (PBMC) of healthy donors were tested. Immunoblot was used to study the molecular effects of MZ-735 on translation of oncogenes, particularly that of c-Myc, in lymphoma cell lines representing DLBCL, DHL, mantle cell lymphoma (MCL), cutaneous T cell lymphoma (CTCL), anaplastic large cell lymphoma (ALCL), and adult T cell lymphoma/leukemia (ATL). To further investigate the broad translational downregulation of poorly understood network of oncogenes by MZ-735, we are conducting in parallel proteomic and RNAseq studies in 3 cell lines representing DLBCL (no MYC translocation), DHL (with MYC and BCL2 translocation), and MCL (with CCND1 translocation). Finally, the efficacy and safety of MZ-735 will be investigated in xenograft mouse models of DHL.ResultsDMDA-PatA and MZ-735 were studied in 13 lymphoma cell lines. DMDA-PatA and MZ-735 exhibited concentration- and time-dependent cytotoxicity in lymphoma cell lines at low nanomolar concentrations (Figure 1A). The IC20 and IC50 values of each lymphoma cell lines are shown in Table 1. MZ-735 was more effective in B cell lymphoma than in T cell lymphoma cells. MZ-735 was significantly more potent than DMDA-PatA in all tested cell lines. Remarkably, a brief treatment by MZ-735 for as short as 3 hours followed by washout of the compound produced comparable cytotoxicity to that of continuous exposure of the compound without washout. Importantly, DMDA-PatA and MZ-735 did not show significant cytotoxic effect in PBMC from healthy donors (Figure 1B). MZ-735 markedly repressed the protein level of c-Myc in all tested lymphoma cell lines, and effectively suppressed the protein level of cyclin D1 in MCL cells (Figure 2). The expression of the MDM2 oncogene was suppressed in a way similar to C-MYC .ConclusionThese results suggest that the PatA analogue MZ-735 has potent pharmacological activity in lymphoma cell lines, and acts by suppressing the production of critical oncogenes, including C-MYC, CCND1, and MDM2 , but is not toxic to normal PBMC. MZ-735 may cause rapid and irreversible modification of the translation apparatus, which is not affected by drug washout, leading to rapid commitment to growth arrest and death of cancer cells. MZ-735 is a promising and novel treatment for C-MYC- and CCND1 -driven aggressive lymphoma. [Display omitted] DisclosuresO'Connor:Trillium Therapeutics: Research Funding; Celgene: Honoraria, Research Funding.

Biocompatibility and Antibiofilm Properties of Calcium Silicate-Based Cements: An In Vitro Evaluation and Report of Two Clinical Cases.

Simple SummaryCalcium silicate-based cements are successfully applied in the different fields of endodontics and vital pulp therapy. To better assess the properties of these bioactive materials, the present in vitro and in vivo study aimed to compare the biocompatibility and antibiofilm properties of ProRoot MTA and Biodentine. Human osteogenic sarcoma (Saos-2) cells were cultured in the presence of both materials and evaluated. Moreover, the bioactive cements were in vivo applied to perform vital pulp therapy on immature permanent teeth affected by reversible pulpitis. Saos-2 cells’ viability was slightly greater in the presence of ProRootMTA than Biodentine and cells would grow in a better way on ProRootMTA disks than on Biodentine ones. Moreover, ProRootMTA showed a powerful antibiofilm effect towards Streptococcus mutans. The in vitro results were clinically supported by a 100% success rate after 2 years of follow-up.Calcium silicate-based cements have reached excellent levels of performance in endodontics, providing predictable and successful results. To better assess the properties of these bioactive materials, the present study aimed to compare the biocompatibility and antibiofilm properties of ProRoot MTA and Biodentine. Human osteogenic sarcoma (Saos-2) cells were cultured on ProRoot MTA and Biodentine samples or in the presence of both cement extracts. Cell viability assay, measurement of reactive oxygen species (ROS), immunofluorescence analysis, as well as morphological evaluations were conducted. Moreover, Streptococcus mutans was used to assess the biofilm forming ability on ProRoot MTA and Biodentine disks. Finally, both cements were applied in vivo to treat immature permanent teeth affected by reversible pulpitis. Results: Cell viability assay demonstrated that Saos-2 cells had a dose- and time-dependent cytotoxicity to both analyzed cements, although cells exposed to ProRoot MTA showed a better cell vitality than those exposed to Biodentine (p < 0.001). Both cements demonstrated ROS production while this was greater in the case of Biodentine than ProRoot MTA (p < 0.001). Immunofluorescence images of the cytoskeleton and focal adhesions showed no differences in Saos-2 cells grown in the presence of ProRoot MTA eluate; whereas in the Biodentine groups, cells showed a morphology and focal adhesions more similar to that of the control sample, as the eluate concentration decreased. Morphological analysis revealed that Saos-2 cells were more flattened and exhibited better spreading when attached to ProRoot MTA disks than to Biodentine ones. The antibiofilm properties showed a time-dependent powerful inhibition of S. mutans superficial colonization and an antibiofilm effect of both cements. Clinically, complete root formation of the treated elements was achieved using the two studied cements, showing stable results over time. ProRoot MTA and Biodentine was demonstrated to be biocompatible and to possess antibiofilm properties. Their clinical application in vital pulp therapy provided successful outcomes after 2 years of follow-up.

In vitro and in silico anticancer potential analysis of Streptomyces sp. extract against human lung cancer cell line, A549.

During our previous investigation, bioactive compounds present in the extract of Streptomyces sp. strain 196 were characterized using LC-MS/MS and 1H NMR studies. These compounds were K-252-C aglycone indolocarbazole alkaloid, decoyinine, and cycloheximide; the study of these natural drugs against lung carcinoma is still limited. Focus of the current investigation was to study the anticancer effect of strain 196 extract on lung cancer cells (A549). During in vitro studies, anti-proliferative effect of extract was studied using MTT assay in A549 cells. Effect of extract on cell survival was further evaluated using colony assay. Cell death was qualitatively assessed using apoptosis assay. The aftereffect of extract treatment on metastatic potential of cancerous cells was studied using wound closure assay. Effect of extract on the morphology and cytoskeletal arrangement of A549 cells was studied using phalloidin staining. The extract demonstrated concentration and time-dependent cytotoxicity with IC50 value at 0.5 mg/ml (6 h) and 0.15 mg/ml (24 h). The proliferation and metastatic potential of cells, as characterized by MTT and migration assay, decreased over time in a concentration-dependent manner. Discrete changes in cellular morphology were noted as a result of the induced cytotoxicity. Apoptosis assay demonstrated 98.7% cell death at highest concentration of extract (1 mg/ml). During in silico studies, molecular docking revealed that strain 196 compounds are efficiently binding to mutant EGFR form (T790M/L858R) with release of binding energy (∆G) between - 5 and - 6.9 kcal/Mol. In conclusion, strain 196 extract could be a source of therapeutic drugs to treat lung carcinoma.

Cannabinoids pharmacological effects are beyond the palliative effects: CB2 cannabinoid receptor agonist induced cytotoxicity and apoptosis in human colorectal cancer cells (HT-29)

Colorectal cancer (CRC) is between the top three occurring cancers worldwide. The anticancer effects of Cannabinoid receptor 2 (CB2) agonist (GW833972A) in the presence and absence of its inverse agonist (SR144528) on Human colorectal adenocarcinoma cells (HT-29) was investigated. Following cell viability assays on HT-29 and HFF cells, the molecular mechanism(s) of cytotoxicity and apoptotic pathways of cell death were analyzed. The anticancer effects of CB2 agonist were measured with tumor cell migration and colony-forming assays. Real-time PCR and Western blotting techniques were used to examine any alterations in the expression of apoptotic genes. A concentration and time-dependent cytotoxicity of CB2 agonist with IC50 value of 24.92 ± 6.99μM was obtained. The rate of lipid peroxidation was elevated, while the TNF-α concentration was declined, significantly (p < 0.05). CB2 agonist (50μM) reduced the colony-forming capability by 83% and tumor cell migration by 50%. Apoptotic effects of CB2 agonist were revealed with the increase of apoptotic cells in Acridine orange/Ethidium bromide staining, clear DNA fragmentation, pro-apoptotic genes and proteins upregulation (Caspase-3 and p53), and significant downregulation of anti-apoptotic Bcl-2. All assessments demonstrated that CB2 agonist-induced effects were reversed by CB2 inverse agonist. These data suggest that CB2 agonists at micro-molar concentrations might be considered in the CRC treatment, and their effectiveness attributes to the apoptosis induction via upregulation of caspase-3 and p53 and downregulation of Bcl-2.

Cytotoxic Effect of Silver Nanoparticles on Colorectal Adenocarcinoma Cell Line

Silver nanoparticles (Ag-NPs) with antimicrobial activity are by far the most commercialized nano-compound. However, there is limited information about antitumor potential. Therefore, the present study was aimed to observe the effect of small-dimensional Ag-NPs (20Nm) on human epithelial colorectal adeno-carcinoma (Caco-2) cells. Caco-2 cells were exposed to Ag-NPs with different concentrations (1–15μg/mL) for 24 and 48 hours and cytotoxicity was analyzed by MTT assay and IC50 was calculated. In addition, flow cytometry was used to determine the effect of Ag-NPs on the cell cycle progression and on apoptosis. An MTT assay revealed that following exposure to Ag-NPs the viability of Caco-2 cells decreased, and the viability decreased further with increasing exposure time. The IC50 was 7.85 µg/ml and 4.92 µg/ml at 24 and 48 h respectively after exposure. The flow cytometry technique revealed that Ag-NPs induced a significant increase in the percentage of cells in G1 phase of the cell cycle and of apoptotic cells. In conclusion, Ag-NPs exhibited dose- and time-dependent cytotoxicity, and these results have signposted that Ag-NPs were promising nanoparticles which could be a potential candidate in treating colorectal cancer.

Multi-Layer Graphene Oxide in Human Keratinocytes: Time-Dependent Cytotoxicity, Proliferation, and Gene Expression

Few-layer graphene oxide (GO) has shown no or very weak cytotoxicity and anti-proliferative effects in a wide range of cell lines, such as glioma cells and human skin HaCaT cells at concentrations up to 100 µg/mL. However, as multi-layer GO has hardly been explored in the biomedical field, in this study, this other type of GO was examined in human keratinocyte HaCaT cells treated with different concentrations, ranging from 0.01 to 150 µg/mL, for different periods of time (3, 12, and 24 h). The results revealed a time–concentration dependence with two non-cytotoxic concentrations (0.01 and 0.05 µg/mL) and a median effective concentration value of 4.087 µg/mL at 24 h GO exposure. Contrary to what has previously been reported for few-layer GO, cell proliferation of the HaCaT cells in contact with the multi-layer GO at 0.01 μg/mL showed identical proliferative activity to an epidermal growth factor (1.6-fold greater than the control group) after 96 h. The effects of the multi-layer GO on the expression of 13 genes (SOD1, CAT, MMP1, TGFB1, GPX1, FN1, HAS2, LAMB1, LUM, CDH1, COL4A1, FBN, and VCAN) at non-cytotoxic concentrations of GO in the HaCaT cells were analyzed after 24 h. The lowest non-cytotoxic GO concentration was able to upregulate the CAT, TGFB1, FN1, and CDH1 genes, which confirms multi-layer GO’s great potential in the biomedical field.