Pretreatment Of HeLa Cells Research Articles

TUDCA inhibits EV71 replication by regulating ER stress signaling pathway and suppressing autophagy

Tauroursodeoxycholic acid (TUDCA) is a naturally occurring hydrophilic bile acid that alleviates endoplasmic reticulum (ER) stress and inhibits apoptosis, thereby protecting cells. Previous studies have shown that enterovirus 71 (EV71) infection modulates ER stress and induces autophagy to assist viral replication. This study observed the effects of TUDCA pretreatment on HeLa and Vero cells infected with EV71, finding that TUDCA inhibits EV71 replication in TUDCA pretreated HeLa and Vero cells in a dose-dependent manner. We found that TUDCA pretreatment inhibited EV71 replication by regulating three branches of UPR, that is up-regulated ATF6, down-regulated both PERK and IRE1. The results also indicated that autophagy which is downstream of UPR, was inhibited either. The results indicate that TUDCA inhibits EV71 replication by regulating UPR sensor proteins and autophagy following ER stress.

An in situ self‐assembled peptide derivative for inhibition of glutathione synthesis and selective enhancement of tumor radiotherapy

AbstractBackgroundInhibition of glutathione (GSH) synthesis in cancer cells considerably improves the efficacy of reactive oxygen species (ROS)‐related tumor therapy. Self‐assembled peptide derivatives can facilitate the efficient delivery and accumulation of small molecular drugs in cancer cells.MethodsSelf‐assembling modules were covalently linked to the GSH‐biosynthesis inhibitor l‐buthionine sulfoximine (BSO) by solid‐phase synthesis to form the self‐assembling peptide derivative Nap‐DFDFpY‐GG‐BSO (Nano‐BSO@ in situ). Subsequently, its enzyme‐instructed self‐assembly in vitro and on cell surfaces were confirmed, and its intracellular GSH depletion and radiotherapy‐sensitizing effects were determined.ResultsNano‐BSO@ in situ successfully self‐assembles into a hydrogel with a nanofibrous microstructure upon incubation with alkaline phosphatase (ALP) at a critical concentration of 9.84 μM. Furthermore, it selectively self‐assembles in situ on HeLa cells with high ALP expression. At a concentration of 50 μM, Nano‐BSO@ in situ decreases intracellular GSH levels by 80%, ∼2.3 times more than free BSO. Meanwhile, pretreatment of HeLa cells with 50 μM Nano‐BSO@ in situ for 24 h results in a radiotherapy sensitization enhancement ratio to γ‐rays of 2.09.ConclusionsA novel in situ self‐assembling peptide derivative for GSH depletion and selective enhancement of tumor radiotherapy was constructed. The excellent GSH‐depletion ability and remarkable radiotherapy‐enhancement performance indicate that Nano‐BSO@ in situ is a promising selective sensitizer for ROS‐related treatment of tumor cells with high ALP expression.

PINK1-Dependent Mitophagy Inhibits Elevated Ubiquitin Phosphorylation Caused by Mitochondrial Damage.

Ubiquitin phosphorylation by the mitochondrial protein kinase PTEN-induced kinase 1 (PINK1), upon mitochondrial depolarization, is an important intermediate step in the recycling of damaged mitochondria via mitophagy. As mutations in PINK1 can cause early-onset Parkinson's disease (PD), there has been a growing interest in small-molecule activators of PINK1-mediated mitophagy as potential PD treatments. Herein, we show that N6-substituted adenosines, such as N6-(2-furanylmethyl)adenosine (known as kinetin riboside) and N6-benzyladenosine, activate PINK1 in HeLa cells and induce PINK1-dependent mitophagy in primary mouse fibroblasts. Interestingly, pre-treatment of HeLa cells and astrocytes with these compounds inhibited elevated ubiquitin phosphorylation that is induced by established mitochondrial depolarizing agents, carbonyl cyanide m-chlorophenyl-hydrazine and niclosamide. Together, this highlights N6-substituted adenosines as progenitor PINK1 activators that could potentially be developed, in the future, as treatments for aged and sporadic PD patients who have elevated phosphorylated ubiquitin levels in the brain.

Apigenin targets protein and lipids of HeLa cells and exhibits protective efficacy against oxidative stress

Apigenin, non-toxic and non-mutagenic flavonoid, is an alternative to classical drugs that interact with membranes. Hence, the objects of the study were to examine its effects on liposomes made of egg yolk phosphatidylcholine (EYPC) with the use of 1H NMR, four-electrode BLM and EPR techniques, and determine its activity on lipids and proteins of human cervix carcinoma (HeLa) cells. In addition its protective efficacy against H2O2- induced oxidative shock was investigated. FTIR spectroscopy was applied to study molecular interactions with membrane lipids and proteins of HeLa cells. Microscopic techniques (SEM, light, and fluorescence), flow cytometer analysis, and NR assays were employed to reveal apigenin involvement in apoptosis in different cell conditions. Apigenin affected mainly the region of choline head groups and the hydrophobic core below this area. Simultaneously, the ordering effect was shown. The fingerprint region of lipids in the HeLa cells was found as a target for apigenin. Furthermore, in the amide I and amide II regions, a decrease in β-sheets and an increase in turns, loops, and unordered structures were noted. Apigenin reduced viability of cells and induced apoptosis. SEM observations revealed characteristic changes in morphology of the examined cells. Pretreatment of HeLa cells with apigenin protected them against H2O2-induced oxidative stress by the increase in glutathione content as well as superoxide dismutase and catalase levels. The consequences of molecular changes related to membranes and cancer cells make apigenin a unique and very interesting bio-compound with great significance for medical and biological applications.

Basic structure and cytocompatibility of giant membrane vesicles derived from paraformaldehyde-exposed human cells.

Exposure of cultured mammalian cells to paraformaldehyde (PFA) is an effective approach to induce membrane blebs, which is followed by their detachment from the cellular cortex to yield giant membrane vesicles in extracellular spaces. Although PFA-induced giant vesicles have attracted significant interest in the field of cell membrane dynamics, their biochemical components and cytocompatibility remain largely unknown. In this report, we exposed human cervical cancer HeLa cells to PFA under metal-free buffer conditions to produce giant vesicles. We analyzed the components and structure of the purified PFA-induced giant vesicles. Co-culturing PFA-induced giant vesicles with exponentially growing HeLa cells resulted in docking of a significant number of the giant vesicles to the cell surface with seemingly no cytotoxicity. Intriguingly, we found that pre-treatment of HeLa cells with peptide-N-glycosidase and neuraminidase was effective in facilitating cellular uptake of constituents residing inside the vesicles. The results revealed further details about the effect of PFA on cell membranes and provide insights for studying the interaction between PFA-induced giant vesicles and cultured cells.

Tumor microenvironment responsive nanogels as a smart triggered release platform for enhanced intracellular delivery of doxorubicin

The fabrication of novel and intelligent delivery systems that can effectively deliver therapeutics to the targeted site and release payload in enhanced/controlled manner is highly desired to overcome the multiple challenges in chemotherapy. The present article demonstrates the potential application of dual stimuli responsive nanogels as tumor microenvironment targeted drug delivery carrier. Disulfide cross-linked pH and redox responsive PEG-PDMAEMA nanogels were synthesized by atom transfer radical polymerization (ATRP). The nanogels were characterized by nuclear magnetic resonance (NMR) spectroscopy, Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD). The PEG-PDMAEMA nanogels exhibited dual stimuli-responsive release of the encapsulated model anticancer drug (doxorubicin, DOX) due to the acidic pH-response of dimethyl amine group in PDMAEMA and reductive cleavage of the disulfide linkages. A relatively higher release of DOX was observed from the nanogels at pH 5.0 than at pH 7.4. DOX release was further accelerated in tumor simulated environment of pH 5.0 and 10 mM glutathione (GSH). Confocal microscopy images revealed that DOX-loaded PEG-PDMAEMA nanogels can rapidly internalize and effectively deliver the drug into the cells. The nanogels exhibited higher cytotoxicity in GSH-OEt pretreated HeLa cells than untreated cells. The dual stimuli responsive nanogels synthesized in this study exhibited many favorable traits, such as pH and redox dependent controlled release of drug, biodegradability, biocompatibility, and enhanced cytotoxicity, which endow them as a promising candidate for anticancer drug delivery.

The Serratia grimesii outer membrane vesicles-associated grimelysin triggers bacterial invasion of eukaryotic cells.

Serratia grimesii are facultative pathogenic bacteria that can penetrate a wide range of host cells and cause infection, especially in immunocompromised patients. Previously, we have found that bacterial metalloprotease grimelysin is a potential virulence determinant of S. grimesii invasion (E. S. Bozhokina et al., (2011).Cell Biology International, 35(2), 111-118). Protease is characterized as an actin-hydrolyzing enzyme with a narrow specificity toward other cell proteins. It is not known, however, whether grimelysin is transported into eukaryotic cells. Here, we show, for the first time, that S. grimesii can generate outer membrane vesicles (OMVs) displayed specific proteolytic activity against actin, characteristic of grimelysin. The presence of grimelysin was also confirmed by the Western blot analysis of S. grimesii OMVs lysate. Furthermore, confocal microscopy analysis revealed that the S. grimesii grimelysin-containing OMVs attached to the host cell membrane. Finally, pretreatment of HeLa cells with S. grimesii OMVs before the cells were infected with bacteria increased the bacterial penetration several times. These data strongly suggest that protease grimelysin promotes S. grimesii internalization by modifying bacterial and/or host molecule(s) when it is delivered as a component of OMVs.

Curcumin ameliorates the targeted delivery of methotrexate intercalated montmorillonite clay to cancer cells

Montmorillonite Clay (MMT) is aimed to develop as an orally administrable drug delivery vehicle with enhanced efficacy. Aiming to enhance the therapeutic index of methotrexate, curcumin is concomitantly used with methotrexate in the present study. Being folate antagonist in nature, methotrexate is internalized into cells by folate receptor (FR); which is over-expressed in certain human cancer cells such as cervical carcinoma cells (HeLa). Firstly, montmorillonite Clay (MMT) is organically modified (OMMT) with cetyl trimethyl ammonium bromide (CTAB) and used to intercalate curcumin and methotrexate separately, designated as OMMT-Cur and OMMT-MTX, respectively. XRD pattern demonstrated successful intercalation of therapeutics and an increase in clay interlayer distance facilitated by CTAB. The dissolution kinetics of methotrexate follows Higuchi model for both Simulated Gastric Fluid (SGF) and Simulated Intestinal Fluid (SIF), while the release kinetics for curcumin fitted into Higuchi model for SGF and Hixson-Crowell model for SIF, respectively. OMMT-MTX are able to discriminate FR-positive HeLa cells from FR-negative breast cancer cells (MCF7); irrespective of alike cellular phenotypes. Further, the pre-treatment of HeLa cells with curcumin improves its sensitivity towards methotrexate causing a greater killing of the Hela cells. Together, the results propose the concomitant use of curcumin and methotrexate for successfully targeting highly invasive FR-positive carcinomas by means of folate receptor using MMTs.

Dietary oleic acid-induced CD36 promotes cervical cancer cell growth and metastasis via up-regulation Src/ERK pathway

Epidemiological and experimental studies have revealed strong associations between dietary lipids and cancer risk. However, the molecular mechanisms underlying the effects of dietary fatty acids on the genesis and progression of cancer have been poorly explored. In this study, we found that a high olive oil diet stimulated cervical cancer (CC) carcinogenesis, and oleic acid (OA), the main lipid in olive oil, was associated with increased malignancy in HeLa cells. OA up-regulated the expression of CD36, which is the best characterized fatty acid transporter. Inhibiting CD36 prevented the tumor-promoting effects of OA, while overexpressing CD36 mimicked the effects of OA. Clinically, CD36 expression was positively correlated with tumor progression and poor prognosis in patients with CC. Furthermore, OA induced Src kinase and downstream ERK1/2 pathway activation in a CD36-dependent manner. Pretreatment of HeLa cells with an Src kinase inhibitor largely blocked the tumor-promoting effect of OA. Our findings suggest that dietary OA exerts a stimulatory effect on CC growth and metastasis, and CD36 might be a promising therapeutic target that acts against CC through an Src/ERK-dependent signaling pathway.

Biological Activity of Flavonoids and Rare Sesquiterpene Lactones Isolated From Centaurea ragusina L.

The endemic Croatian species Centaurea ragusina L., like other species from the genus Centaurea, has been traditionally used in Croatia as an antibacterial agent and for the treatment of gastrointestinal and urogenital disorders. In several chromatographic steps, three flavonoids and three sesquiterpene lactones (STLs) were isolated and identified from the most active fractions of the ethanol extract. Two STLs, one for which we created the trivial name ragusinin, and hemistepsin A are here reported for the first time as constituents of the genus Centaurea. All six compounds were screened for their effect on several tumor and one normal cell lines. Among them, ragusinin showed the best bioactivity and high specificity to affect tumor murine SCCVII, human HeLa and Caco-2 cell lines, but not the viability of normal V79 fibroblasts. Due to these characteristics the action of ragusinin was investigated in more detail. Since DNA is the primary target for many drugs with antibacterial and anticancer activity, we studied its interaction with ragusinin. Rather moderate binding affinity to DNA excluded it as the primary target of ragusinin. Due to the possibility of STL interaction with glutathione (GSH), the ubiquitous peptide that traps reactive compounds and other xenobiotics to prevent damage to vital proteins and nucleic acids, its role in deactivation of ragusinin was evaluated. Addition of the GSH precursor N-acetyl-cysteine potentiated the viability of HeLa cells, while the addition of GSH inhibitor L-buthionine sulfoximine decreased it. Moreover, pre-treatment of HeLa cells with the inhibitor of glutathione-S-transferase decreased their viability indicating the detoxifying role of GSH in ragusinin treated cells. Cell death, derived by an accumulation of cells in a G2 phase of the cell cylce, was shown to be independent of poly (ADP-ribose) polymerase and caspase-3 cleavage pointing toward an alternative cell death pathway.

Augmented cytotoxic effects of paclitaxel by curcumin induced overexpression of folate receptor-α for enhanced targeted drug delivery in HeLa cells

BackgroundNew targeted therapies are intended to minimize the toxic effects and maximize destruction of tumor cells. Folate is a membrane-bound receptor that plays a vital role in the uptake of anti-folate molecules aimed for efficient drug delivery of anti-folate drugs. PurposeThe present study is aimed at the modulation of the expression of folate receptor by curcumin that enhances the intake, cytotoxicity and anticancer effects of paclitaxel in HeLa cells. Materials and methodsHeLa cells were pretreated with curcumin and treated with paclitaxel. We measured the cell viability, uptake of radiolabelled folic acid and paclitaxel, Folate receptor –alpha (FR-α) protein expression by immunocytochemistry and western blot and FR-α mRNA expression by qualitative and quantitative analysis. ResultsThis study shows that curcumin (10 - 50 µM) causes significantly increased cytotoxicity in a dose and time dependent manner. It also enhances the intake of radiolabeled folic acid and paclitaxel 3–4 folds in HeLa cells. The pretreatment of HeLa cells with curcumin shows statistically significant of cell death by paclitaxel. The quantitative RT-PCR demonstrates the expression of FR- α mRNA upon curcumin treatment. Furthermore, immunochemistry and western blotting analysis proved that curcumin enhances expression the FR- α in HeLa cells. ConclusionOur study proved that the molecular mechanism of curcumin enhances the upregulation of FR – α mRNA and protein expression in HeLa cells. Therefore, a combination of curcumin and paclitaxel at less concentration may be a targeting strategy for FR-targeted drug delivery providing a better therapeutic intervention of cancer.

A ruthenium polypyridyl intercalator stalls DNA replication forks, radiosensitizes human cancer cells and is enhanced by Chk1 inhibition.

Ruthenium(II) polypyridyl complexes can intercalate DNA with high affinity and prevent cell proliferation; however, the direct impact of ruthenium-based intercalation on cellular DNA replication remains unknown. Here we show the multi-intercalator [Ru(dppz)2(PIP)]2+ (dppz = dipyridophenazine, PIP = 2-(phenyl)imidazo[4,5-f][1,10]phenanthroline) immediately stalls replication fork progression in HeLa human cervical cancer cells. In response to this replication blockade, the DNA damage response (DDR) cell signalling network is activated, with checkpoint kinase 1 (Chk1) activation indicating prolonged replication-associated DNA damage, and cell proliferation is inhibited by G1-S cell-cycle arrest. Co-incubation with a Chk1 inhibitor achieves synergistic apoptosis in cancer cells, with a significant increase in phospho(Ser139) histone H2AX (γ-H2AX) levels and foci indicating increased conversion of stalled replication forks to double-strand breaks (DSBs). Normal human epithelial cells remain unaffected by this concurrent treatment. Furthermore, pre-treatment of HeLa cells with [Ru(dppz)2(PIP)]2+ before external beam ionising radiation results in a supra-additive decrease in cell survival accompanied by increased γ-H2AX expression, indicating the compound functions as a radiosensitizer. Together, these results indicate ruthenium-based intercalation can block replication fork progression and demonstrate how these DNA-binding agents may be combined with DDR inhibitors or ionising radiation to achieve more efficient cancer cell killing.

A dual-responsive nanocapsule via disulfide-induced self-assembly for therapeutic agent delivery.

One-step synthesis of fluorescent molecules (SNBDP) containing one disulfide bond and two o-nitrobenzyl groups was demonstrated via multi-component Passerini reaction. This hydrophobic SNBDP could self-assemble into nanocapsules (SNBDP NCs) in aqueous solution via disulfide-induced assembly. The obtained nanocapsules were stable in aqueous solution for several weeks and exhibited enhanced fluorescence when nanocapsules were destroyed due to disaggregation-induced emission. The nanocapsules not only were reduction-sensitive and light-responsive, but also could be endocytosed by HeLa cells for cellular imaging. The enhanced fluorescence in the glutathione (GSH) pretreated HeLa cells showed that the compound was reduction-sensitive in living cells. In vitro WST-8 assays showed the nanocapsules were biocompatible and could further be used as drug delivery carriers. Indocyanine green (ICG), a clinically approved NIR dye, was loaded into the nanocapsules (ICG@SNBDP NCs). ICG@SNBDP NCs showed enhanced photothermal efficacy compared with same concentration of free ICG under 808-nm laser irradiation. Consequently, ICG@SNBDP NCs upon NIR irradiation can effectively kill cancer cells through local hyperthermia. These results highlight the potential of disulfide-induced nanocapsules as smart nanoparticles for cellular imaging and therapeutic agent delivery.

Antiviral Phosphorodiamidate Morpholino Oligomers are Protective against Chikungunya Virus Infection on Cell-based and Murine Models.

Chikungunya virus (CHIKV) infection in human is associated with debilitating and persistent arthralgia and arthritis. Currently, there is no specific vaccine or effective antiviral available. Anti-CHIKV Phosphorodiamidate Morpholino Oligomer (CPMO) was evaluated for its antiviral efficacy and cytotoxcity in human cells and neonate murine model. Two CPMOs were designed to block translation initiation of a highly conserved sequence in CHIKV non-structural and structural polyprotein, respectively. Pre-treatment of HeLa cells with CPMO1 signficantly suppressed CHIKV titre, CHIKV E2 protein expression and prevented CHIKV-induced CPE. CPMO1 activity was also CHIKV-specific as shown by the lack of cross-reactivity against SINV or DENV replication. When administered prophylactically in neonate mice, 15 μg/g CPMO1v conferred 100% survival against CHIKV disease. In parallel, these mice demonstrated significant reduction in viremia and viral load in various tissues. Immunohistological examination of skeletal muscles and liver of CPMO1v-treated mice also showed healthy tissue morphology, in contrast to evident manifestation of CHIKV pathogenesis in PBS- or scrambled sCPMO1v-treated groups. Taken together, our findings highlight for the first time that CPMO1v has strong protective effect against CHIKV infection. This warrants future development of morpholino as an alternative antiviral agent to address CHIKV infection in clinical applications.

Dihydrolipoic but not alpha-lipoic acid affects susceptibility of eukaryotic cells to bacterial invasion

Sensitivity of eukaryotic cells to facultative pathogens can depend on physiological state of host cells. Previously we have shown that pretreatment of HeLa cells with N-acetylcysteine (NAC) makes the cells 2–3-fold more sensitive to invasion by the wild-type Serratia grimesii and recombinant Escherichia coli expressing gene of actin-specific metalloprotease grimelysin [1]. To evaluate the impact of chemically different antioxidants, in the present work we studied the effects of α-Lipoic acid (LA) and dihydrolipoic acid (DHLA) on efficiency of S. grimesii and recombinant E. coli expressing grimelysin gene to penetrate into HeLa and CaCo cells. Similarly to the effect of NAC, pretreatment of HeLa and CaCo cells with 0.6 or 1.25 mM DHLA increased the entry of grimelysin producing bacteria by a factor of 2.5 and 3 for the wild-type S. grimesii and recombinant E. coli, respectively. In contrast, pretreatment of the cells with 0.6 or 1.25 mM LA did not affect the bacteria uptake. The increased invasion of HeLa and CaCo cells correlated with the enhanced expression of E-cadherin and β-catenin genes, whereas expression of these genes in the LA-treated cells was not changed. Comparison of these results suggests that it is sulfhydryl group of DHLA that promotes efficient modification of cell properties assisting bacterial uptake. We assume that the NAC- and DHLA-induced stimulation of the E-cadherin-catenin pathway contributes to the increased internalization of the grimelysin producing bacteria within transformed cells.

Reduction-sensitive micelles with sheddable PEG shells self-assembled from a Y-shaped amphiphilic polymer for intracellular doxorubicine release

A new type of shell-sheddable micelles with disulfide linkages between the hydrophobic polyester core and hydrophilic poly(ethylene glycol) (PEG) shell was developed based on Y-shaped amphiphilic polymers mPEG-S-S-(PCL)2. The micelles were then used for the glutathione-mediated intracellular delivery of the anticancer drug doxorubicin (DOX) into tumor cells. The polymer could self-assemble into micelles with an average diameter of 135nm in aqueous solution and load DOX at a total content of 3.6%. The hydrophilic PEG shell of these micelles could be shed in the presence of reducing agent dithiothreitol (DTT), which resulted in size change of the micelles. In vitro release studies revealed that DOX-loaded mPEG-S-S-(PCL)2 micelles exhibited faster DOX release in the presence of DTT. MTT assay demonstrated that DOX-loaded mPEG-S-S-(PCL)2 micelles showed higher cytotoxicity against 10mM of glutathione monoester (GSH-OEt) pretreated HeLa cells than that of the non-pretreated ones. Confocal laser scanning microscopy and flow cytometry analyses indicated that DOX-loaded mPEG-S-S-(PCL)2 micelles were efficiently internalized into HeLa cells and exhibited faster DOX release in GSH-OEt-pretreated cells than in cells with no pretreatment. Endocytosis inhibition results proved that mPEG-S-S-(PCL)2 micelles entered the cells mainly through the clathrin-mediated endocytosis pathway, and caveolae-mediated endocytosis was involved to a small extent. These results indicate the great potential of the proposed Y-shaped reduction-sensitive polymer for application in effective intracellular anticancer drug delivery.

Redox-responsive micelles self-assembled from dynamic covalent block copolymers for intracellular drug delivery

Redox-responsive micelles self-assembled from dynamic covalent block copolymers with double disulfide linkage in the backbone have been developed successfully. The amphiphilic block copolymers PEG–PLA associated with complementary H-bonding sequences can self-assemble into spherical micelles in aqueous media with sizes from 34nm to 107nm with different molar mass of PEG and PLA. Moreover, in vitro drug release analyses indicate that reductive environment can result in triggered drug release profiles. The glutathione (GSH) mediated intracellular drug delivery was investigated against HeLa human cervical carcinoma cell line. Flow cytometry and fluorescence microscopy measurements demonstrated that the micelles exhibited faster drug release in glutathione monoester (GSH-OEt) pretreated HeLa cells than that in the nonpretreated cells. Cytotoxicity assay of DOX-loaded micelles indicated the higher cellular proliferation inhibition against 10mM of GSH-OEt pretreated HeLa cells than that of the nonpretreated ones. These reduction-responsive, biodegradable and biocompatibility micelles could provide a favorable platform to construct excellent drug delivery systems for cancer therapy.

Reduction-sensitive amphiphilic copolymers made via multi-component Passerini reaction for drug delivery

One-step synthesis of amphiphilic polymers containing disulfide bond within the hydrophobic backbone was demonstrated via multi-component Passerini reaction. The obtained polymer was self-assembled into micelles in aqueous solution. Curcumin (CUR), an effective and safe anticancer agent, which was limited by its water insolubility and poor bioavailability, was loaded into the micelles as a model drug. The nanoscale polymeric micelles were confirmed by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Faster intracellular CUR release was observed by confocal laser scanning microscopy (CLSM) in the HeLa cells pretreated with GSH than in the unpretreated ones. Micelles also loaded with NH2-BODIPY which was almost non-fluorescent and gave strong enhanced fluorescence under acid conditions. The phenomenon of the stronger enhanced fluorescence in the pretreated HeLa cells showed further that the obtained polymer was reduction-sensitive. In vitro MTT assays showed that the micelles were biocompatible and CUR-loaded micelles had higher cellular proliferation inhibition in contrast to free CUR toward HeLa cells. These results highlight the potential of using multi-component Passerini reaction to make functional copolymers as smart nanocarriers for drug delivery.

3-Acetyl-bis(2-chloro-4-nitrophenyl)triazene is a potent antitumor agent that induces oxidative stress and independently activates the stress-activated protein kinase/c-Jun NH2-terminal kinase pathway

Previously, we described the synthesis and biological activity of a new class of anticancer molecules that preferentially target malignant cells and may serve as potential antitumor agents. Among several synthesized agents, we selected 3-acetyl-1,3-bis(2-chloro-4-nitrophenyl)-1-triazene (8b) as a representative of the group of 4-nitro-substituted 1,3-diaryltriazenes. The aim of this study was to further investigate the mechanism of cell response to the 8b compound. The HeLa human cervical carcinoma cell line was used as an experimental model to further investigate the mechanism of cell response to the 8b compound. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay was used to address cell survival, and western blot (immunoblotting) was used for the expression of relevant proteins after 8b drug exposure. The pretreatment of HeLa cells with salubrinal, a specific inhibitor of endoplasmic reticulum (ER) stress, confirmed the importance of ER stress in apoptosis induced by 8b. We also demonstrate that 8b triggers the activation of stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) in a time-dependent and dose-dependent manner. Moreover, the inhibition of SAPK/JNK activity by JNK II before 8b treatment increased the survival rate of HeLa cells relative to survival in the presence of 8b alone, indicating the importance of this kinase in cell death. The simultaneous inhibition of ER stress induction and SAPK/JNK activation increased the survival of HeLa cells upon 8b treatment more than inhibition of both pathways independently, suggesting the separate triggering of both signaling pathways. Our data indicate that cytotoxic activity of the novel compound 8b is based on its ability to induce ER stress and SAPK/JNK signaling pathways independently, driving cells to cell death.

Novel hydroxyl-containing reduction-responsive pseudo-poly(aminoacid) via click polymerization as an efficient drug carrier

A novel biodegradable pseudo-poly(aminoacid) copolymer (mPEG–HRSCP–mPEG) was produced via a mechanism of step polymerization through Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) (click polymerization) using dialkyned-cystine and diazide monomers. The disulfide and hydroxyl groups were repeatedly arranged in the polymer backbone. The introduced hydroxyl groups could not only adjust the hydrophilicity of the amphiphilic polymer, but also improve the DOX loading efficiency and solubility. Compared with the unmodified one, the polymer containing hydroxyl groups has increased hydrophilicity with a larger critical aggregation concentration (CAC) value, lower water contact angle, higher drug loading content and drug loading efficiency. The pH and reduction sensitivities of this drug delivery system (DDS) were investigated using Dynamic Light Scattering (DLS) to monitor the changes of average diameters. DOX was loaded as a model drug and in vitro release experiments demonstrated that DOX release was accelerated by an addition of 10 mM glutathione (GSH) or under acidic conditions rather than under physiological conditions. Our study on in vitro anticancer efficiency showed that DOX-loaded nanoparticles had a higher cytotoxicity towards GSH pretreated HeLa cells. These newly designed copolymer nanoparticles can be used as novel and impactful pH and reduction dual-responsive nanocarriers for intelligent DOX delivery.