Proteins In HeLa Cells Research Articles

Functionalized PDA/DEX-PEI@HA nanoparticles combined with sleeping-beauty transposons for multistage targeted delivery of CRISPR/Cas9 gene

CRISPR/Cas9 system has been used as the most powerful gene editing tool for precision medicine and advanced gene therapy. However, its wide applications are limited by the poor biosafety of lentivirus delivery vectors though with high-efficiency transduction. To construct a safer vector and promote genome integration, the CRISPR/Cas9 gene is cloned into a plasmid-based non-viral safe vector Sleeping-Beauty (SB) transposon in this study to obtain pT2SpCas9. Meanwhile, PDA/DEX-PEI@HA (PDPH) nanoparticles are constructed to facilitate the precise CRISPR/Cas9 targeting delivery, by using polydopamine (PDA) as the carrier, hyaluronic acid (HA) as the cell-targeting ligand and dexamethasone (DEX) as the nuclear localization signal (NLS). The results showed that PDPH could deliver pDNA efficiently into the cell and further into the nucleus. The transfection efficiency of PDPH is much higher than that of NPs without HA and DEX. Remarkably, the cytotoxicity of PDPH is negligible in comparison to PEI25k and PEI10k. Western blots showed that after the transfection of PDPH/pT2SpCas9-Nanog/SB11, Nanog protein in HeLa cells is knocked out, and the proliferation and migration abilities of tumor cells are significantly decreased. This study demonstrates that PDA/DEX-PEI25k@HA/pT2SpCas9 (PDPH25 K/pT2SpCas9) has the great potential as a non-viral gene vector for CRISPR/Cas9 delivery and clinical medication.

Characterizing microRNA-mediated modulation of gene expression noise and its effect on synthetic gene circuits.

MicroRNAs (miRNAs) have been shown to modulate gene expression noise, but less is known about how miRNAs with different properties may regulate noise differently. Here, we investigate the role of competing RNAs and the composition of miRNA response elements (MREs) in modulating noise. We find that weak competing RNAs could introduce lower noise than strong competing RNAs. In comparison with a single MRE, both repetitive and composite MREs can reduce the noise at low expression, but repetitive MREs can elevate the noise remarkably at high expression. We further observed the behavior of a synthetic cell-type classifier with miRNAs as inputs and find that miRNAs and MREs that could introduce higher noise tend to enhance cell state transition. These results provide a systematic and quantitative understanding of the function of miRNAs in controlling gene expression noise and the utilization of miRNAs to modulate the behavior of synthetic gene circuits.

Curcumin-Galactomannoside Complex inhibits the Proliferation of Human Cervical Cancer Cells: Possible Role in Cell Cycle Arrest and Apoptosis.

Background:Cervical cancer is the most common cancer and has the highest morbidity rate of gynaecological malignancies in women worldwide. So, the development of effective anti-cancer agents to treat this condition is vital. Considering the recent interest in free (unconjugated) curcuminoids delivery, the present study investigated the efficacy of a novel food-grade, free-curcuminoids (curcumin-galactomannoside complex; CGM) on cervical cancer cells (HeLa) of human origin. In this study, we examined the anticancer potential of CGM as well as its effects on the cell cycle and the apoptosis of HeLa cancer cell. Methods:Determination of anti-proliferative and apoptosis validation of CGM on HeLa cells was performed by 3-(4,5-Dimethylthiazol-2-yl)-2, 5,-diphenyltetrazolium bromide (MTT), acridine orange/propidium iodide and annexin-V-fluorescein isothiocyanate assays. Measurement of Reactive Oxygen Species (ROS) production, Caspase activities and protein expression experiments were performed to investigate the potential mechanisms of action in the apoptotic process. Results:The cytotoxic assays revealed that the CGM showed inhibition of cell survival and exhibited high cytotoxic activity against HeLa cells at 25 μg/mL. Further studies on morphological changes were done in CGM-treated cervical cancer cells contributing to apoptosis. Flow cytometry analysis with Annexin V-FITC and PI staining precisely indicated that CGM induced apoptosis in HeLa cell lines at 25 μg/mL. By the supplementation of CGM showed an increase in Bax and cleaved caspase-8 protein in HeLa cells after 48 h exposure. Conclusion:The evidence obtained from this study suggests that CGM is a potent and promising natural formulation against cervical cancer cells via induction of apoptosis through ROS mediated mitochondrial damage in HeLa cells. Hence, CGM could be further explored as a potential lead in treating cancer.

Glucocappasalin Induces G2/M-Phase Arrest, Apoptosis, and Autophagy Pathways by Targeting CDK1 and PLK1 in Cervical Carcinoma Cells.

Glucocappasalin (GCP), a natural product derived from the seeds of Descurainia sophia (L.) Webb. ex Prantl, exhibits potential antitumor activity in HeLa cervical carcinoma cells. In this study, we investigated the anti-cervical cancer property of GCP through the induction of cell cycle arrest, apoptosis, and autophagy in vitro and in vivo, and elucidated the underlying molecular mechanisms. We demonstrated that treatment with GCP inhibited the growth of HeLa, Siha, and Ca Ski cell lines in a dose-dependent manner, with HeLa cells displaying particular sensitivity to the GCP treatment. Subsequently, the expression of cyclin-dependent kinase 1 (CDK1) and polo like kinase 1 (PLK1) were evaluated in HeLa cells using the CDK1 kinase assay kit, the fluorescence polarization assay, real-time quantitative PCR, and western blotting. Our results demonstrate that GCP could be employed to attenuate the expression of CDK1 and PLK1 in a dose- and time-dependent manner. The complementary results obtained by flow cytometry and western blotting allowed us to postulate that GCP may exhibit its antitumor effects by inducing G2/M cell cycle arrest. Moreover, HeLa cells treated with GCP exhibited a loss in mitochondrial membrane potential, together with the activation of caspases 3 and 9, and poly ADP-ribose polymerase (PARP). Additionally, we found that GCP could increase the formation of acidic vesicular organelles (AVOs), as well as the levels of Beclin1, LC3-II, p62, and Atg5 proteins in HeLa cells. Further studies indicated that GCP triggered autophagy via the suppression of the PI3K/AKT/mTOR signaling pathways. The autophagy inhibitor 3-methyladenine (3-MA) was used to determine whether autophagy affects the apoptosis induced by GCP. Interestingly, the inhibition of autophagy attenuated apoptosis. In vivo anti-tumor experiments indicated that GCP (60 mg/kg, i.p.) markedly reduced the growth of HeLa xenografts in nude mice without apparent toxicity. Taken together, we demonstrate that GCP induces cell cycle G2/M-phase arrest, apoptosis, and autophagy by acting on the PI3K/AKT/mTOR signaling pathways in cervical carcinoma cells. Thus, GCP may represent a promising agent in the eradication of cervical cancer.

Alantolactone inhibits cervical cancer progression by downregulating BMI1

Cervical cancer is the second most common cancer in women. Despite advances in cervical cancer therapy, tumor recurrence and metastasis remain the leading causes of mortality. High expression of BMI1 is significantly associated with poor tumor differentiation, high clinical grade, and poor prognosis of cervical cancer, and is an independent prognostic factor in cervical carcinoma. Alantolactone (AL), a sesquiterpene lactone, exhibits potent anti-inflammatory and anticancer activities. In this paper, we investigated the mechanism of AL in reducing the proliferation, migration, and invasion of HeLa and SiHa cervical cancer cells as well as its promotion of mitochondrial damage and autophagy. BMI1 silencing decreased epithelial-mesenchymal transformation-associated proteins and increased autophagy-associated proteins in HeLa cells. These effects were reversed by overexpression of BMI1 in HeLa cells. Thus, BMI1 expression is positively correlated with invasion and negatively correlated with autophagy in HeLa cells. Importantly, AL decreased the weight, volume, and BMI1 expression in HeLa xenograft tumors. Furthermore, the structure of BMI1 and target interaction of AL were virtually screened using the molecular docking program Autodock Vina; AL decreased the expression of N-cadherin, vimentin, and P62 and increased the expression of LC3B and Beclin-1 in xenograft tumors. Finally, expression of BMI1 increased the phosphorylation of STAT3, which is important for cell proliferation, survival, migration, and invasion. Therefore, we suggest that AL plays a pivotal role in inhibiting BMI1 in the tumorigenesis of cervical cancer and is a potential therapeutic agent for cervical cancer.

Modeling the complete kinetics of coxsackievirus B3 reveals human determinants of host-cell feedback.

Complete kinetic models are pervasive in chemistry but lacking in biological systems. We encoded the complete kinetics of infection for coxsackievirus B3 (CVB3), a compact and fast-acting RNA virus. The model consists of separable, detailed modules describing viral binding-delivery, translation-replication, and encapsidation. Specific module activities are dampened by the type I interferon response to viral double-stranded RNAs (dsRNAs), which is itself disrupted by viral proteinases. The experimentally validated kinetics uncovered that cleavability of the dsRNA transducer mitochondrial antiviral signaling protein (MAVS) becomes a stronger determinant of viral outcomes when cells receive supplemental interferon after infection. Cleavability is naturally altered in humans by a common MAVS polymorphism, which removes a proteinase-targeted site but paradoxically elevates CVB3 infectivity. These observations are reconciled with a simple nonlinear model of MAVS regulation. Modeling complete kinetics is an attainable goal for small, rapidly infecting viruses and perhaps viral pathogens more broadly. A record of this paper's transparent peer review process is included in the Supplemental information.

NME4 Loss-of-Function Alters Mitochondria, Triggers Retrograde Signaling and Leads to Cellular Reprogramming

NME4 (or nucleoside diphosphate kinase D, NDPK-D, NM23-H4) is a mitochondrial protein with multiple key functions in bioenergetics and signaling. The hexameric NME4 is mainly localized in the intermembrane space, bound to the inner membrane. As we showed earlier, NME4 provides GTP to the mitochondrial GTPase OPA1, stimulates respiration, binds to cardiolipin and promotes its inter-membrane transfer to the mitochondrial surface. Here we deleted either its NDP kinase activity or its cardiolipin interaction and expressed these mutants or wild-type protein in HeLa cells that are almost devoid of endogenous NME4. Both mutations independently led to fragmentation of the mitochondrial network and loss of mitochondrial mass, consistent with disturbed GTP-fueling of the pro-fusion GTPase OPA1. NME4 loss-of-function mutants also showed increased ROS generation and a metabolic shift with reduced cellular respiration and increased aerobic glycolysis. The mitochondrial alterations triggered a specific change in cellular protein expression, with down-regulation of several mitochondrial proteins and altered cell signaling. At the cellular level, NME4 loss-of-function manifested in a morphotypic switch, characterized by inhibited intercellular adhesion, and increased migratory and invasive potential. These data suggest that NME4 loss of function leads to primary alterations in mitochondria that trigger retrograde signaling, finally leading to downstream events responsible for pro-invasive reprogramming of the cell.

Zinc oxide nanoparticles synthesized from Aspergillus terreus induces oxidative stress-mediated apoptosis through modulating apoptotic proteins in human cervical cancer HeLa cells.

This study was aimed to analyze the cytotoxicity of biogenic zinc oxide nanoparticles (ZnO NPs) in human cervical epithelial cancer HeLa. The ZnO NPs was synthesized from the culture filtrated of Aspergillus terreus, and examined by UV-spectroscopy, X-ray diffraction (XRD), transmission electron microscope (TEM), energy-dispersive X-ray (EDX) and Fourier transform infrared (FTIR) analysis. The cytotoxicity of synthesized ZnO NPs was analyzed by the MTT assay, and the expression of apoptotic proteins was examined by Western blot analyses. The ZnO NPs exhibited concentration-dependent cytotoxicity on HeLa cells and induced the apoptosis as evidenced by reduced superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) levels, and increased reactive oxygen species (ROS) and diminished mitochondrial membrane potential (MMP) was noticed in ZnO NPs treated HeLa cells. Western blot analyses explored that the Bcl-2 expression was significantly downregulated, whereas, the expression of p53, Bax, Caspase-3, Caspase-9 and Cytochrome-c were significantly upregulated in ZnO NPs treated cells. Consequently, the mycosynthesized ZnO NPs induces apoptosis in HeLa cells by persuading oxidative damage and modulating the apoptotic proteins. Therefore, A. terreus synthesized ZnO NPs could be used as an effective chemotherapeutic agent for cervical cancer treatment.

MiT/TFE family members suppress L-leucyl-L-leucine methyl ester-induced cell death.

Lysosomes are degradative organelles essential for cell homeostasis. However, various internal and external stimuli, including L-leucyl-L-leucine methyl ester (LLOMe), which is one of the common lysosomotropic agents, permeabilize the lysosomal membrane, leading to lysosome-dependent cell death because of leakage of lysosomal contents to the cytosol. The microphthalmia/transcription factor E (MiT/TFE) family members, which include transcription factor EB (TFEB), transcription factor E3 (TFE3), and microphthalmia-associated transcription factor (MITF), are master regulators of lysosomal biogenesis and are known to be involved in the lysosomal stress response. However, their protective effects against cell death associated with lysosomal-membrane damage are still poorly understood. In this study, we confirmed that LLOMe-induced lysosomal damage triggered nuclear translocation of TFEB/TFE3/MITF and increased the mRNA levels of their target genes encoding lysosomal hydrolases and lysosomal membrane proteins in HeLa cells. Furthermore, we revealed that TFEB/TFE3/MITF knockdown exacerbated LLOMe-induced cell death. However, TFEB overexpression only slightly attenuated LLOMe-induced cell death, despite enhanced LLOMe-induced increase in CTSD mRNA levels, implying that the endogenous levels of MiT/TFE family members might be sufficient to promote lysosomal biogenesis in response to lysosomal-membrane damage. Our results suggest that MiT/TFE family members suppress the cell death associated with lysosomal-membrane damage.

Generation of an Interactome for the Tetratricopeptide Repeat Domain of O-GlcNAc Transferase Indicates a Role for the Enzyme in Intellectual Disability.

The O-GlcNAc transferase (OGT) modifies nuclear and cytoplasmic proteins with β-N-acetyl-glucosamine (O-GlcNAc). With thousands of O-GlcNAc-modified proteins but only one OGT encoded in the mammalian genome, a prevailing question is how OGT selects its substrates. Prior work has indicated that the tetratricopeptide repeat (TPR) domain of OGT is involved in substrate selection. Furthermore, several variants of OGT causal for X-linked intellectual disability (XLID) occur in the TPR domain. Therefore, we adapted the BioID labeling method to identify interactors of a TPR-BirA* fusion protein in HeLa cells. We identified 115 interactors representing known and novel O-GlcNAc-modified proteins and OGT interactors (raw data deposited in MassIVE, Dataset ID MSV000085626). The interactors are enriched in known OGT processes (e.g., chromatin remodeling) as well as processes in which OGT has yet to be implicated (e.g., pre-mRNA processing). Importantly, the identified TPR interactors are linked to several disease states but most notably are enriched in pathologies featuring intellectual disability that may underlie the mechanism by which mutations in OGT lead to XLID. This interactome for the TPR domain of OGT serves as a jumping-off point for future research exploring the role of OGT, the TPR domain, and its protein interactors in multiple cellular processes and disease mechanisms, including intellectual disability.

Facile preparation of bifunctional adsorbents for efficiently enriching N-glycopeptides and phosphopeptides

In bottom-up strategy, specific enrichment of glycopeptides and phosphopeptides from complicated biological samples is a prerequisite for efficient identifying glycosylation and phosphorylation by mass spectrometry. Although there were a plethora of materials used as either hydrophilic interaction liquid chromatography (HILIC) or immobilized metal affinity chromatography (IMAC) adsorbents, even several bifunctional materials for simultaneous enrichment of glycopeptides and phosphopeptides, most of them are not easily commercialized as many other well-performing adsorbents due to the complicated preparation process. In our case, a one-step modification strategy was developed to prepare bifunctional adsorbents for HILIC and IMAC, employing O-phospho-l-serine as the modifier and poly(GMA-co-EDMA) microspheres, a kind of macroporous adsorption resin (MAR) with epoxy groups, as the matrix. The MARs were directly modified with O-phospho-l-serine under facile condition for HILIC strategy and further chelated with Ti4+ for IMAC strategy. A total of 522 unique N-glycopeptides and 442 unique N-glycosylation sites mapped to 275 N-glycoproteins was identified from HeLa cell proteins, showing excellent enrichment efficiency in HILIC. Additionally, 3141 unique phosphopeptides were unambiguously identified from 200 μg of digest of HeLa cell proteins, demonstrating great enrichment efficiency in IMAC. Moreover, these materials have been successfully applied in the analysis of multiple biological samples including human serum and milk, demonstrating their feasibility for real sample applications and potential business value.

In Situ Visualization of Proteins in Single Cells by Time-of-Flight-Secondary Ion Mass Spectrometry Coupled with Genetically Encoded Chemical Tags.

In situ visualization of proteins of interest in single cells is attractive in cell biology, molecular biology, and biomedicine fields. Time-of-flight-secondary ion mass spectrometry (ToF-SIMS) is a powerful tool for imaging small organic molecules in single cells, yet difficult to image biomacromolecules such as proteins and DNA. Herein, a universal strategy is reported to image specific proteins in single cells by ToF-SIMS following genetic incorporation of fluorine-containing unnatural amino acids as a chemical tag into the proteins via a genetic code expansion technique. The method was developed and validated by imaging a green fluorescence protein (GFP) in Escherichia coli (E. coli) and human HeLa cancer cells and then utilized to visualize the characteristic polar distribution of chemotaxis protein CheA in E. coli cells and the interaction between high-mobility group box 1 protein and cisplatin-damaged DNA in HeLa cells. The present work highlights the power of ToF-SIMS imaging combined with genetically encoded chemical tags for in situ visualization of specific proteins as well as the interactions between proteins and drugs or drug-damaged DNA in single cells.

Cytotoxicity Effect of Quercus infectoria Based Vaginal Cream on Hela Cells and its Preliminary in Vivo Toxicity Evaluation Towards Female Rats

Introduction: Cervical cancer is the third leading cause of cancer death among females in less developed countries. Drugs used in the treatment of cervical cancer were reported to exert a cytotoxic effect on the normal cells. This study aimed to determine the effectiveness of Quercus infectoria (QI) vaginal cream towards cervical cancer cell, HeLa and its toxicity effect on the female rat model. Methods: MTT assays were utilized to determine the median concentration (IC50) for cell cytotoxicity of QIA and QI vaginal cream against cervical cancer cells, HeLa. Expression of HPV E6 and E7 protein in HeLa cells treated with QI vaginal cream for 24 hours were conducted by Western blot analysis. In separate experiments, the toxicity of QI vaginal cream on a lower reproductive tract of the female rat model has been assessed by histopathological examination after application for three weeks. The antioxidant activity of QIA extract and QI vaginal cream were assessed by DPPH radical scavenging assay. Results: A moderate cytotoxicity activity exerted by QIA extract and QI vaginal cream against HeLa cell with IC50 values of 13.90 ± 2.27, and 20.80 ± 1.94 respectively. Furthermore, QI vaginal cream suppressed the expression of HPV E6 and E7. Daily application of QI vaginal did not exert any inflammation to the vaginal mucosa and cervix. QIA extract and QI vaginal cream demonstrated high DPPH radical scavenging activity. Conclusion: Formulated QI vaginal cream has cytotoxic effect on HeLa cells without causing an adverse effect on the lower reproductive tract in female rat model.

Proteomics Study of DNA-Protein Crosslinks in Methylmethanesulfonate and Fe2+-EDTA-Exposed Human Cells.

The formation of covalently bound DNA-protein crosslinks (DPCs) is linked to the pathophysiology of cancers and many other degenerative diseases. Knowledge of the proteins that were frequently involved in forming DPCs will improve our understanding of the etiological mechanism of diseases and facilitate the establishment of preventive measures and treatment methods. By using SDS-PAGE and nano-LC coupled Orbitrap LC-MS/MS analyses, we identified, for the first time, that the major DNA-cross-linked proteins in HeLa cells exposed to a methylating agent (methylmethanesulfonate) or hydroxyl free radicals are transcription-associated proteins. In particular, histone H2B3B and poly(rC) binding protein 2 were identified as the most frequent DPC-forming proteins.

Tyrosine-phosphorylation and activation of glucosylceramide synthase by v-Src: Its role in survival of HeLa cells against ceramide

Sphingolipids represent a family of cellular lipid-molecules that regulate physiological and pathophysiological processes. Glucosylceramide (GlcCer), the simplest glycosphingolipid (GSL), is synthesized from ceramide and UDP-glucose by GlcCer synthase (GCS). Both GlcCer (and resulting GSLs) and ceramide regulate various cellular functions including cell death and multiple drug resistance. Src family tyrosine kinases are up-regulated in various human cancer cells. We examined the effect of v-Src expression on GCS activity, the formation of 4-nitrobenzo-2-oxa-1,3-diazole (NBD)-labeled GlcCer from NBD-ceramide, and the effect of tyrosine132 mutation in GCS on ceramide-induced cytotoxicity in HeLa cells. Expression of v-Src increased the formation of NBD-GlcCer in both intact cells without marked changes in other sphingolipid metabolites and cell homogenates without changing affinities of NBD-ceramide and UDP-glucose. Expression of v-Src also increased tyrosine-phosphorylated levels in GCS proteins in HeLa and HEK293T cells. In HEK293T cells transiently expressing the GCS mutant, GCS-Y132F-HA, showing replacement of the tyrosine132 residue with phenylalanine, tyrosine-phosphorylated levels in GCS proteins were significantly lower than those in control cells expressing the GCS-wild-type-HA. The formation of NBD-GlcCer in HeLa cells stably expressing GCS-Y132F-HA was significantly lower than that in the control. Ceramide-induced cytotoxicity in HeLa-GCS-Y132F-HA cells was significantly greater than in the control. In this study, we showed for the first time that expression of v-Src up-regulated GCS activity via tyrosine phosphorylation of the enzyme in a post-translational manner. Mechanisms of Src-induced resistance to ceramide-induced cytotoxicity are discussed in relation to the Src-induced up-regulation of GCS activity.

Distinct Functions of Acyl/Alkyl Dihydroxyacetonephosphate Reductase in Peroxisomes and Endoplasmic Reticulum.

Plasmalogens are a subclass of ether glycerophospholipids characterized by a vinyl-ether bond at the sn-1 position of the glycerol backbone. Plasmalogen biosynthesis is initiated in peroxisomes. At the third step of plasmalogen synthesis, alkyl-dihydroxyacetonephosphate (DHAP) is enzymatically reduced to 1-alkyl-sn-glycero-3-phospate by acyl/alkyl DHAP reductase (ADHAPR), whose activity is found in both peroxisomes and microsomes. We herein show that knockdown of ADHAPR in HeLa cells reduced the synthesis of ethanolamine plasmalogen (PlsEtn), similar to the Chinese hamster ovary cell mutant FAA.K1B deficient in ADHAPR activity. Endogenous ADHAPR and ectopically expressed FLAG-tagged ADHAPR were localized to peroxisomes and endoplasmic reticulum (ER) as a type I integral membrane protein in HeLa cells. ADHAPR targets to peroxisomes via a Pex19p-dependent class I pathway. In addition, it is also inserted into the ER via the SRP-dependent mechanism. The ADHAPR mutant lacking the N-terminal domain preferentially targets to the ER, restoring the reduced level of PlsEtn synthesis in FAA.K1B cell. In contrast, the expression of full-length ADHAPR in the mutant cells elevates the synthesis of phosphatidylethanolamine, but not PlsEtn. Taken together, these results suggest that the third step of plasmalogen synthesis is mediated by ER-localized ADHAPR.

MicroRNA216b mediated downregulation of HSP27/STAT3/AKT signaling is critically involved in lambertianic acid induced apoptosis in human cervical cancers.

Since heat shock protein (HSP27) is a prognostic marker in cervical cancer, in the present study, the apoptotic mechanism of lambertianic acid (LA) was investigated in human cervical cancers in association with HSP27/STAT3/AKT signaling axis. LA exerted significant cytotoxicity, induced sub-G1 population, and increased the cleavage of Poly (ADP-ribose) polymerase (PARP) and cysteine aspartyl-specific protease 3 (caspase3) in HeLa and Caski cancer cells. Consistently, LA downregulated anti-apopotic genes such as B-cell lymphoma 2 (Bcl-2) and inhibitors of apoptosis proteins (c-IAP) in HeLa and Caski cells. Furthermore, LA-inhibited phosphorylation of HSP27, signal transducer, and activator of transcription 3 (STAT3) and Protein kinase B (AKT) through disturbing the binding of HSP27 with STAT3 or AKT in HeLa cells. Notably, LA upregulated the level of miR216b in HeLa and Caski cells. Consistently, miR216b mimic suppressed phosphorylation of HSP27 and reduced the expression of pro-PARP, while miR216b inhibitor reversed the ability of LA to attenuate phosphorylation of AKT, HSP27, and STAT3 and to reduce the expression of pro-PARP in HeLa cells. Overall, our findings suggest that miRNA216b mediated inhibition of HSP27/STAT3/ AKT signaling axis is critically involved in LA-induced apoptosis in cervical cancers.

In-cell destabilization of a homodimeric protein complex detected by DEER spectroscopy

The complexity of the cellular medium can affect proteins' properties, and, therefore, in-cell characterization of proteins is essential. We explored the stability and conformation of the first baculoviral IAP repeat (BIR) domain of X chromosome-linked inhibitor of apoptosis (XIAP), BIR1, as a model for a homodimer protein in human HeLa cells. We employed double electron-electron resonance (DEER) spectroscopy and labeling with redox stable and rigid Gd3+ spin labels at three representative protein residues, C12 (flexible region), E22C, and N28C (part of helical residues 26 to 31) in the N-terminal region. In contrast to predictions by excluded-volume crowding theory, the dimer-monomer dissociation constant KD was markedly higher in cells than in solution and dilute cell lysate. As expected, this increase was partially recapitulated under conditions of high salt concentrations, given that conserved salt bridges at the dimer interface are critically required for association. Unexpectedly, however, also the addition of the crowding agent Ficoll destabilized the dimer while the addition of bovine serum albumin (BSA) and lysozyme, often used to represent interaction with charged macromolecules, had no effect. Our results highlight the potential of DEER for in-cell study of proteins as well as the complexities of the effects of the cellular milieu on protein structures and stability.

Garcinone-E exhibits anticancer effects in HeLa human cervical carcinoma cells mediated via programmed cell death, cell cycle arrest and suppression of cell migration and invasion

Xanthones are an important class of natural compounds bearing huge bioactivity profiles. Garcinone-E is one among most active xanthones showing potential anticancer activity against various human cancer cell lines. Therefore, the current study was performed to explore the anticancer potency of naturally occurring garcinone-E xanthone against human HeLa cervical cancer cells. The underlying mechanism of action was also tried to be explored via testifying its induction of programmed cell death, arrest of cell cycle, suppression of cell migration, cell invasion and cell adhesion. MTT assay was implemented to estimate the viability of HeLa cells after garcinone-E exposure and clonogenic assay was used to analyze the effect on clonogenic potential. Acridine orange/ethidium bromine (AO/EB) staining assay was performed for monitoring of programmed cell death along with western blotting. Flow cytometric studies were carried out to analyze cell cycle check points. Transwell chambers assays were carried out for studying the impact of garcinone-E on migration and invasion potency of HeLa cells. Western blotting was used to study the expressions of apoptosis linked proteins in HeLa cells. Results indicated that garcinone-E remarkably decreased the viability to minimum in HeLa cells in both dose and time-reliant manner. The clonogenic capacity of HeLa cells was efficiently reduced by garcinone exposure. AO/EB staining showed that the anti-viability action of garcinone-E was apoptosis allied which was supported by western blotting as well. The cell cycle check points study indicated cell cycle arrest at G2/M-phase. HeLa cell migration and invasion were reduced efficiently after being subjected to garcinone-E treatment in a dose reliant fashion. In conclusion, garcinone-E has a remarkable potential to act as anti-cervical cancer chemopreventive provided further in vivo studies are required.

Importance of CNOT8 Deadenylase Subunit in DNA Damage Responses Following Ionizing Radiation (IR).

The Ccr4-Not protein complex (CNOT complex) is a key regulator of gene expression in eukaryotic cells. Ccr4-Not Complex is composed of at least nine conserved subunits in mammalian cells with two main enzymatic activities. CNOT8 is a subunit of the complex with deadenylase activity that interacts transiently with the CNOT6 or CNOT6L subunits. Here, we focused on the role of the human CNOT8 subunit in the DNA damage response (DDR). Cell viability was assessed to measure ATP level using a Cell Titer-Glo Luminescence reagent up to 4 days' post CNOT8 siRNA transfection. In addition, expression level of phosphorylated proteins in signalling pathways were detected by western blotting and immunofluorescence microscopy. CNOT8- depleted Hela cells post- 3 Gy ionizing radiation (IR) treatment were considered as a control. Our results from cell viability assays indicated a significant reduction at 72-hour post CNOT8 siRNA transfection (p= 0.04). Western blot analysis showed slightly alteration in the phosphorylation of DNA damage response (DDR) proteins in CNOT8-depleted HeLa cells following treatment with ionizing radiation (IR). Increased foci formation of γH2AX, RPA, 53BP1, and RAD51 foci was observed after IR in CNOT8-depleted cells compared to the control cells. We conclude that CNOT8 deadenylase subunit is involved in the cellular response to DNA damage.