HeLa Research Articles

Cordycepin ameliorates autoimmunity by promoting STING degradation via autophagy pathway.

Stimulator of interferon response cGAMP interactor 1 (STING), a central hub protein of cyclic GMP-AMP synthase (cGAS)-STING signalling pathway, has a crucial role in regulating type I interferons (IFNs) production and response. Recent studies indicate that excessive activation of STING is strongly associated with autoimmune diseases, including systemic lupus erythematosus (SLE). Searching immunomodulators that negatively regulate STING might greatly contribute to the suppression of autoimmunity. The peripheral blood mononuclear cells (PBMCs) of SLE patients, Hela cells, L929 cells and bone marrow-derived macrophages (BMDMs) from mice were used as in vitro models. While, Trex1 KO mouse autoimmune disease model was used as in vivo model. After treatment with cordycepin, a nucleoside from Cordyceps mushrooms, type I IFNs production and response were determined by western blotting, real-time polymerase chain reaction (PCR), dual-luciferase assay, enzyme-linked immunosorbent assay (ELISA), haematoxylin-eosin staining and RNA-seq. Cordycepin inhibited type I IFNs production and response in human and murine systems following cGAS-STING signalling activation. Importantly, cordycepin markedly attenuates the autoinflammatory and autoimmune responses in Trex1 KO BMDMs and Trex1 KO mice. Furthermore, cordycepin effectively suppressed the production of type I IFNs and interferon-stimulated genes (ISGs) in the PBMCs of SLE patients. Mechanistically, cordycepin promoted STING degradation via autophagy pathway upon DNA stimulation. This study shows that cordycepin promotes STING autophagic degradation to alleviate autoimmunity upon DNA stimulation. Cordycepin might be a potential therapeutic candidate for alleviating aberrant type I IFNs in autoimmune and autoinflammatory diseases.

Endogenous interactomes of MFN1 and MFN2 provide novel insights into interorganelle communication and autophagy

ABSTRACT MFN1 (mitofusin 1) and MFN2 are key players in mitochondrial fusion, endoplasmic reticulum (ER)-mitochondria juxtaposition, and macroautophagy/autophagy. However, the mechanisms by which these proteins participate in these processes are poorly understood. Here, we studied the interactomes of these two proteins by using CRISPR-Cas9 technology to insert an HA-tag at the C terminus of MFN1 and MFN2, and thus generating HeLa cell lines that endogenously expressed MFN1-HA or MFN2-HA. HA-affinity isolation followed by mass spectrometry identified potential interactors of MFN1 and MFN2. A substantial proportion of interactors were common for MFN1 and MFN2 and were regulated by nutrient deprivation. We validated novel ER and endosomal partners of MFN1 and/or MFN2 with a potential role in interorganelle communication. We characterized RAB5C (RAB5C, member RAS oncogene family) as an endosomal modulator of mitochondrial homeostasis, and SLC27A2 (solute carrier family 27 (fatty acid transporter), member 2) as a novel partner of MFN2 relevant in autophagy. We conclude that MFN proteins participate in nutrient-modulated pathways involved in organelle communication and autophagy.

Discovery of a Novel Co-crystal of Chrysin and Oroxylin a with Anticancer Properties from Leaves of Oroxylum indicum.

As the number of new cancer cases increases every year, there is a necessity to develop new drugs for the treatment of different types of cancers. Plants' resources are considered to be huge reservoirs for therapeutic agents in nature. Among all the medicinal plants, Oroxylum indicum is one of the most widely used medicinal plants in India, China, and Southeast Asian countries. Combinatorial drug treatment, on the other hand, is favored over single drug treatment in order to target multiple biomolecular moieties that help in the growth and development of cancer. Therefore, combinatorial drug treatment using a co-crystal of multiple drugs gives researchers an idea of the development of a new type of drug for targeting multiple targets. In this study, a new co-crystal of chrysin and oroxylin A was isolated from the leaves of O. indicum, and its anticancer properties were studied in cervical cancer cells HeLa. This study was conducted with the aim of identifying new anticancer compounds from the leaves of Oroxylum indicum and studying the anticancer properties of the isolated compound. In this study, we elucidated the structure of a new co-crystal compound, which was isolated from the leaf extract of Oroxylum indicum. The apoptosis induction mechanism of the newly discovered co-crystal in HeLa cells was also studied. A crystal compound from the chloroform extract of leaves of Oroxylum Indicum was isolated by solvent fractionation and chromatographic methods involving HPLC. The molecular structure of the isolated crystal was elucidated by Single Crystal-XRD, FT-IR analysis, and further determined by LC-MS. The antiproliferative activity was carried out using an MTT assay and fluorescence microscopy, and the mechanism of apoptosis was determined using Western blotting techniques. The novel co-crystal consists of two active pharmaceutical ingredients (APIs) in a 1:1 ratio, i.e., oroxylin A and chrysin. The isolated new co-crystal induced death in HeLa cells with a very low IC50 value of 8.49μM. It induced caspase-dependent apoptosis in HeLa cells by activation of Caspase-3 through inhibition of ERKs and activation of p38 of MAPK cell signalling pathway. This study presents the first report on the discovery of a naturally occurring co-crystal of chrysin and oroxylin A and the involvement of ERKs and p38 of MAPK pathways in the induction of apoptosis in HeLa cells by the co-crystal. Our study sheds light on the development of a co-crystal of chrysin and oroxylin A in a specific ratio of 1:1 for combination therapy of the two APIs. The purified co-crystal was found to be more efficient compared to the compounds present individually. Further analysis of the physiochemical properties and molecular mechanisms of the isolated co-crystal in different cancer cells is warranted for its application in therapeutics.

Antiproliferative Activity and Molecular Docking Analyses of Sesquiterpene Lactones Obtained from Activity-Directed Isolation of Centaurea saligna (K.Koch) Wagenitzin Neoplastic Cells.

Secondary metabolites obtained from plants are among the most commonly encountered chemotherapeutics used in cancer treatment. Plants contain thousands of metabolites; therefore, it is important to reach the compound primarily responsible for activity by fractionating plant extracts through activity-guided isolation. The cytotoxic activities of C. saligna fractions, sub-fractions, and all pure compounds obtained from the plant were investigated in vitro using MCF-7 (human breast cancer), HeLa (human cervical cancer), and PC-3 (prostate cancer) cell lines. Eighteen compounds were isolated from C. saligna, comprising eight sesquiterpene lactones, three flavonoids, five lignans, and two phenolic compounds, with their structures elucidated through 1H-NMR, 13C-NMR, and HMBC spectroscopic techniques. The molecular docking scores of the pure compounds obtained from these sub-fractions were determined using both AutoDock and AutoDock Vina programs. It has been proven that the affinities of linichlorin B and aguerin B for Bcl-2 are higher than those of other compounds, considering the calculated Ki values and placement scores. Notable activities of linichlorin B, cynaropicrin, and aguerin B (with IC50 values of 13.67μg/ml, 6.79μg/ml, and 3.46μg/ml, respectively) were detected in the PC-3 cell line; aguerin B demonstrated activity most comparable to the standard anticancer agent doxorubicin. Likewise, linichlorin B, aguerin B, and cynaropicrin demonstrated notable efficacy in the HeLa and MCF-7 cell lines, as reported by the American National Cancer Institute. Aguerin B, linichlorin B, and cynaropicrin are projected to serve as promising novel chemotherapeutic agents for cancer therapy.

Bioinformatic Analysis of Actin-Binding Proteins in the Nucleolus During Heat Shock

Background/Objectives: Actin plays a crucial role not only in the cytoplasm, but also in the nucleus, influencing various cellular behaviors, including cell migration and gene expression. Recent studies reveal that nuclear actin dynamics is altered by cellular stresses, such as DNA damage; however, the effect of heat shock on nuclear actin dynamics, particularly in the nucleolus, remains unclear. This study aims to elucidate the contribution of nucleolar actin to cellular responses under heat shock conditions. Methods: Nuclear actin dynamics in response to heat shock were investigated using nAC-GFP, a GFP-tagged actin chromobody, to visualize nuclear actin in HeLa cells. Bioinformatic analyses were also performed. Results: Heat shock induced the reversible assembly of nAC-GFP in the nucleolus, with disassembly occurring upon recovery in a heat shock protein (Hsp) 70-dependent manner. Because the nucleolus, formed via liquid–liquid phase separation (LLPS), sequesters misfolded proteins under heat shock to prevent irreversible aggregation, we hypothesized that nucleolar actin-binding proteins might also be sequestered in a similar manner. Using several databases, we identified 47 actin-binding proteins localized in the nucleolus and determined the proportion of intrinsically disordered regions (IDRs) known to promote LLPS. Our analysis revealed that many of these 47 proteins exhibited high levels of IDRs. Conclusions: The findings from our bioinformatics analysis and further cellular studies may help elucidate new roles for actin in the heat shock response.

New thiazolidin-4-ones as anti-cervical cancer agents targeting EGFR: design, synthesis, and computational studies.

A new series of 3,4-dihydronaphthalen-1(2 h)-ylidene)hydrazineylidene)-5-substituted thiazolidin-4-one derivatives were designed and synthesized. The new compounds were screened for in vitro antitumor activity against Hela cancer cell line. The compounds 7b, 7 h, and 7i produced more potent cytotoxicity than doxorubicin with IC50 values of 1.83 ± 0.1, 2.54 ± 0.14, 2.75 ± 0.15, and 3.63 ± 0.2 μM, respectively. They also showed a promising safety profile against WI-38 normal cells. In addition, compound 7b produced a promising multi-kinase inhibition against EGFR (WT) while being very selective toward the mutant forms (L858R and T790M) with IC50 values of 0.099 ± 0.006, 0.064 ± 0.006, and 0.026 ± 0.007 μM, respectively, in comparison to gefitinib and osimertinib. A study of the cell cycle in Hela cells showed that 7b arrests cell cycle in the pre-G1 phase and causes early and late apoptosis. Eventually, the molecular docking results showed that 7b had good-binding interactions with EGFRWT, EGFRL858R, and EGFRT790M. Compound 7b was predicted to have promising oral absorption, good drug-likeness, and low toxicity risks in humans. Moreover, MD simulations confirmed the stable complexes of 7b with EGFRWT, EGFRL858R, and EGFRT790M (with RMSD 0.12-0.35 nm, RMSF 0.2-0.55 nm, SASA 140-150, and Rg 1.80-2.00 nm).

Microgels of N-Isopropylacrylamide Copolymerized with an Amphiphilic Acid for the Delivery of Doxorubicin

This study aims to design microgels that are thermo- and pH-sensitive for controlled doxorubicin (Dox) release in response to tumor microenvironment changes. N-isopropylacrylamide (NIPAAm) is widely used for thermoresponsive tumor-targeted drug delivery systems for the release of therapeutic payloads in response to temperature changes. Herein, a NIPAAm microgel (MP) that is responsive to temperature and pH was designed for the smart delivery of Dox. MP was made from NIPAAm, and polyethylene glycol methyl ether methacrylate (PEGMA) was copolymerized with 5%, 10%, or 15% mol of methacryloylamido hexanoic acid, (CAM5) an amphiphilic acid. We characterized the microgels using FTIR-ATR, DLS, and FESEM. The MP 10% CAM5 exhibited a particle size of 268 nm, with a transition temperature of 44 °C. MP had a drug loading capacity of 13% and entrapment efficiency of 87%. Nearly 100% of the Dox was released at pH 5 and 42 °C, compared to 30% at pH 7.4 and 37 °C. MP 10% CAM5 showed cytocompatibility in HeLa cells using the MTT assay. However, the cell viability assay showed that dox-MP was twice as effective as free Dox. Specifically, 3 μg/mL of free Dox resulted in 74% cell viability, while the same doses of Dox in NP reduced it to 35%. These results are promising for the future tumor-targeted delivery of antineoplastic-drugs, as they may reduce the side effects of Dox.

Transcriptional regulation in the absence of inositol trisphosphate receptor calcium signaling

The activation of IP3 receptor (IP3R) Ca2+ channels generates agonist-mediated Ca2+ signals that are critical for the regulation of a wide range of biological processes. It is therefore surprising that CRISPR induced loss of all three IP3R isoforms (TKO) in HEK293 and HeLa cell lines yields cells that can survive, grow and divide, albeit more slowly than wild-type cells. In an effort to understand the adaptive mechanisms involved, we have examined the activity of key Ca2+ dependent transcription factors (NFAT, CREB and AP-1) and signaling pathways using luciferase-reporter assays, phosphoprotein immunoblots and whole genome transcriptomic studies. In addition, the diacylglycerol arm of the signaling pathway was investigated with protein kinase C (PKC) inhibitors and siRNA knockdown. The data showed that agonist-mediated NFAT activation was lost but CREB activation was maintained in IP3R TKO cells. Under base-line conditions transcriptome analysis indicated the differential expression of 828 and 311 genes in IP3R TKO HEK293 or HeLa cells, respectively, with only 18 genes being in common. Three main adaptations in TKO cells were identified in this study: 1) increased basal activity of NFAT, CREB and AP-1; 2) an increased reliance on Ca2+- insensitive PKC isoforms; and 3) increased production of reactive oxygen species and upregulation of antioxidant defense enzymes. We suggest that whereas wild-type cells rely on a Ca2+ and DAG signal to respond to stimuli, the TKO cells utilize the adaptations to allow key signaling pathways (e.g., PKC, Ras/MAPK, CREB) to transition to the activated state using a DAG signal alone.

DL-CSPF: deep-learning-based cell segmentation with a physical framework for digital holographic microscopy

Digital holographic microscopy (DHM) offers label-free, full-field imaging of live-cell samples by capturing optical path differences to produce quantitative phase images. Accurate cell segmentation from phase images is crucial for long-term quantitative analysis. However, complicated cellular states (e.g., cell adhesion, proliferation, and apoptosis) and imaging conditions (e.g., noise and magnification) pose significant challenge to the accuracy of cell segmentation. Here, we introduce DL-CSPF, a deep-learning-based cell segmentation method with a physical framework designed for high-precision live-cell analysis. DL-CSPF utilizes two neural networks for foreground-background segmentation and cell detection, generating foreground edges and “seed points.” These features serve as input for a marker-controlled watershed algorithm to segment cells. By focusing on foreground edges and “seed points”, which have lower information entropy than complete cell contours, DL-CSPF achieves accurate segmentation with a reduced dataset and without manual parameter tuning. We validated the feasibility and generalization of DL-CSPF using various open-source and DHM-collected datasets, including HeLa, pollen, and COS-7 cells. Long-term live-cell imaging results further demonstrate that DL-CSPF reliably characterized and quantitatively analyzed the morphological metrics across the cellular lifecycle, rendering it a promising tool for biomedical research.

Amino acids modulate liquid–liquid phase separation in vitro and in vivo by regulating protein–protein interactions

Liquid-liquid phase separation (LLPS) is an intracellular process widely used by cells for many key biological functions. It occurs in complex and crowded environments, where amino acids (AAs) are vital components. We have found that AAs render the net interaction between proteins more repulsive. Here, we find that some AAs efficiently suppress LLPS in test tubes (in vitro). We then screen all the proteinogenic AAs and find that three specific AAs, including proline, glutamine, and glycine, significantly suppressed the formation of stress granules (SGs) in U2OS and HeLa cell lines (in vivo) irrespective of stress types. We also observe the effect in primary fibroblast cells, a viable cell model for neurodegenerative disorders. Kinetic studies by live-cell microscopy show that the presence of AAs not only slows down the formation but also decreases the saturating number and prevents the coalescence of SGs. We finally use sedimentation-diffusion equilibrium analytical ultracentrifuge (SE-AUC) to demonstrate that the suppression effects of AAs on LLPS may be due to their modulation in protein-protein and RNA-RNA interactions. Overall, this study reveals an underappreciated role of cellular AAs, which may find biomedical applications, especially in treating SG-associated diseases.

Differentiation-inducing factor-1 inhibits EMT by proteasomal degradation of TAZ and YAP in cervical and colon cancer cell lines.

We previously reported differentiation-inducing factor-1 (DIF-1) activated glycogen synthase kinase-3 (GSK-3) in various mammalian cells. GSK-3 has been proposed to regulate a number of signaling pathway including TAZ/YAP signaling pathway. To clarify the effect of DIF-1 on TAZ/YAP signaling pathway, we examined whether DIF-1 affect the expression levels of TAZ and YAP. We found that DIF-1-induced proteasomal- and GSK-3-dependent degradation of both TAZ and YAP in human cervical cancer cell line HeLa in a time- and dose-dependent manner. As TAZ/YAP signaling pathway is well known to accelerate the epithelial-mesenchymal transition (EMT) of the cancer cell, we examined the effect of TAZ/YAP signaling pathway on EMT-related proteins. Knockdown of TAZ and YAP proteins by siRNA significantly reduced the expression of fibronectin, vimentin, and Snail. We also found that DIF-1 suppressed the expression levels of TAZ/YAP target gene products and EMT-related protein. Further, overexpression of TAZ and YAP attenuated the inhibitory effects of DIF-1 on these protein expressions. Migration and trans-well invasion assays revealed that DIF-1 significantly inhibited HeLa cell migration and invasion. DIF-1-induced proteasomal- and GSK-3-dependent degradation of TAZ and YAP proteins and inhibition of cell migration and invasion were also observed in human colon cancer cell line HCT-116. These results suggest that DIF-1 inhibits the TAZ/YAP signaling pathway via GSK-3 activation. Further, it has been suggested that the inhibition of EMT induced by DIF-1 is involved with the suppression of TAZ/YAP signaling pathway.

Single‐Cell Secretome Profiling Enabled by Selective Enrichment and NanoLC‐MS/MS Analysis

AbstractRecent advances in single‐cell proteomics enable the direct profiling of thousands of proteins from a single mammalian cell. However, due to the bottlenecks in detecting low‐abundance secreted proteins and extracellular vesicle (EV) proteins (collectively referred to as the secretome) against a background of high‐abundance proteins in serum‐containing culture medium, the comprehensive investigation of the secretome at the single‐cell level using nanoLC‐MS/MS still remains challenging. Herein, we report a novel single‐cell secretome profiling (SCSP) method by integrating the metabolic labeling of newly synthesized proteins, click chemistry‐based enrichment, and in situ digestion of the labeled secretome in an alkyne‐functionalized capillary micro‐reactor, followed by nanoLC‐MS/MS analysis. By this method, an average of 389 protein groups were quantified from the secretome of single HeLa cells (n=17), with a total of 752 protein groups confidently identified in the single‐cell secretome, which is a significant increase compared to the previously reported targeted analysis limited to dozens of secreted proteins by antibody recognition. These results indicated that our developed SCSP method would provide a powerful tool to gain insights into secretion heterogeneity and intercellular communication at the single‐cell level.

Combination of Vitex pseudo-negundo methanolic-extract with cisplatin can induce antioxidant activity and apoptosis in HeLa and Caski cells

BackgroundCisplatin-based chemotherapy as a common therapeutic regimen for cervical cancer patients, is becoming more and more ineffective due to high resistance. This urges the need for introducing novel metabolics such as botanical drugs with the capacity to increase the cisplatin effectiveness. In that regard, here we investigated the anticancer effects of the Cisplatin-Vitex pseudo-Negundo combination in cervical cancer cell lines.Method and MaterialV. pseudo-Negundo fruits were dried and extracted methanolic fraction. The MTT assay was performed to evaluate cytotoxicity of both drugs in CaSki and HeLa cells. Then, apoptosis, ROS production, and cell cycling were assessed by flow cytometry assay in cells treated with V. pseudo-Negundo and Cisplatin and their combination. Also, the rate of cell migration and colony formation were measured, using wound healing and colony formation assay, respectively. Also, the expression level of related genes (CD133, BAX, BCL2, Casp-3/8/9, MMP-3) was evaluated using the RT-PCR method.ResultsThe obtained results established that the V. pseudo-Negundo plant has medicinal properties to induce apoptotic and antioxidant signals. The combination treatment of methanol extraction and Cisplatin had a cytotoxic effect on cervical cancer cell lines (HeLa and CaSki) compared to monotherapy. Also, combination therapy resulted in an increased apoptosis rate and diminished ROS production in both CaSki and HeLa cell lines. Furthermore, V. pseudo-Negundo and Cisplatin combination therapy leads to cell cycle arrest in the G2-M and G0-G1 phase in HeLa and CaSki cell lines, respectively. Moreover, combination therapy decreased the colony formation and cell motility in both cell lines and upregulated caspases gene expression.ConclusionThe combination of V. pseudo-Negundo with Cisplatin therapy results in a significant anti-cancer and antioxidant effect compared to cisplatin, representing a promising candidate for future clinical investigations.

Ellagic Acid Potentiates the Inhibitory Effects of Fluconazole Against Candida albicans

Background/Objectives: Antifungal resistance to azoles, coupled with the increasing prevalence of Candida albicans infections, represents a significant public health challenge and has driven the search for new natural compounds that can act as alternatives or adjuvants to the current antifungals. Ellagic acid (EA) has demonstrated antifungal activity; however, its effects are not fully understood. In this study, we investigated the in vitro anti-Candida activity of EA and its ability to potentiate the effects of fluconazole (FLZ) on C. albicans. Methods: The Minimum Inhibitory Concentration (MIC) of EA was determined by broth microdilution and its interaction with FLZ was assessed using a checkerboard assay. Additionally, we examined the effects of EA on yeast-to-hypha transition, inhibition of biofilm formation, time–kill kinetics, hemolytic activity, and cytotoxicity in HeLa ATCC® CCL-2™ cells. Results: EA exhibited MIC values ranging from 250 to 2000 µg/mL and showed synergistic and additive interactions with FLZ, resulting in a marked reduction in the MIC values of FLZ (up to 32-fold) and EA (up to 16-fold). In the time–kill assay, the most effective combinations were 4× EA MIC, 2× EA MIC, and FIC EA + FLZ, which showed fungicidal activity. Furthermore, EA did not show hemolytic activity and demonstrated low and dose-dependent cytotoxicity in HeLa cells, with no cytotoxic effects observed in combination with FLZ. EA and the synergistic combination of EA and FLZ interfered with both the yeast-to-hypha transition process in C. albicans cells and biofilm formation. In addition to its antifungal efficacy, EA demonstrated a favorable safety profile at the concentrations used. Conclusions: This study presents promising results regarding the potential use of EA in combination with FLZ for the treatment of C. albicans infections.

Effect of Cherenkov light on cell survival in x-ray irradiation of LINAC based on Monte Carlo simulation and cell survival measurements

Cherenkov radiation is emitted during x-ray irradiation in a linear accelerator (LINAC). Cherenkov light contains many short wavelength components, including ultraviolet (UV) light, which is well-known for its bactericidal effects. A similar phenomenon is probable for human cancer cells. To assess the effect of Cherenkov light on cell death in x-ray irradiation, we employed simulations and UV cell survival data. We measured the survival rates of HeLa cells exposed to 254 nm (UVC) and 310 nm (UVB) light to determine the 50% lethal dose (LD50) required to kill 50% of the cells. For other wavelengths, we utilized literature values to establish the relationship between wavelength and LD50. Due to the broad range of the Cherenkov light spectrum, we need LD50 as a function of wavelength to estimate cell survival solely from Cherenkov light. A Monte Carlo simulation was used to calculate the fluence distribution of Cherenkov light in a 300 mm3phantom comprised of soft tissue, both with and without absorption of visible light. The latter scenario is considered to be most influenced by Cherenkov light. By combining the fluence distribution and the wavelength-LD50 relationship, we determined the distribution of the survival rate. Our findings indicate that, in the absence of absorption, a radiation dose of approximately 90 Gy or greater is necessary for Cherenkov light to have any effect. As a result, the impact of Cherenkov light on cell survival can be considered negligible for typical dose of 2 Gy.

Epsilon Toxin from Clostridium perfringens Induces the Generation of Extracellular Vesicles in HeLa Cells Overexpressing Myelin and Lymphocyte Protein

Epsilon toxin (ETX) from Clostridium perfringens is a pore-forming toxin (PFT) that crosses the blood–brain barrier and binds to myelin structures. In in vitro assays, ETX causes oligodendrocyte impairment, subsequently leading to demyelination. In fact, ETX has been associated with triggering multiple sclerosis. Myelin and lymphocyte protein (MAL) is widely considered to be the receptor for ETX as its presence is crucial for the effects of ETX on the plasma membrane of host cells that involve pore formation, resulting in cell death. To overcome the pores formed by PFTs, some host cells produce extracellular vesicles (EVs) to reduce the amount of pores inserted into the plasma membrane. The formation of EVs has not been studied for ETX in host cells. Here, we generated a highly sensitive clone from HeLa cells overexpressing the MAL-GFP protein in the plasma membrane. We observed that ETX induces the formation of EVs. Moreover, the MAL protein and ETX oligomers are found in these EVs, which are a very useful tool to decipher and study the mode of action of ETX and characterize the mechanisms involved in the binding of ETX to its receptor.

Nimotuzumab and irinotecan synergistically induce ROS-mediated apoptosis by endoplasmic reticulum stress and mitochondrial-mediated pathway in cervical cancer.

Irinotecan (CPT-11), a chemotherapeutic agent used to treat several types of cancer, induces cytotoxic effects on healthy cells. The epidermal growth factor receptor (EGFR) plays a crucial role in various forms of cancer. Nimotuzumab (NmAb), a monoclonal antibody that targets the EGFR, is utilized in some countries to treat malignancies that have an overexpression of EGFR. Yet, there is a lack of literature on the potential anticancer properties of the CPT-11 and NmAb combination on in vitro human cervical cancer cells. This study investigates the apoptosis mode of the CPT-11 and NmAb combination on cervical HeLa cancer cells. The Annexin V/propidium iodide staining examination demonstrated that the combination of CPT-11 and NmAb resulted in a decrease in the number of viable cells and more potent induction of cell apoptosis than the effects of CPT-11 or NmAb alone in HeLa cells. Furthermore, the combined treatment resulted in elevated levels of reactive oxygen species (ROS) and Ca2+ compared to the treatment with CPT-11 or NmAb alone. Cells that were pretreated with N-acetyl-l-cysteine, a substance that scavenges ROS, and then treated with CPT-11, NmAb, or a combination of CPT-11 and NmAb exhibited higher numbers of viable cells compared to those treated with CPT-11 or NmAb alone. The combination of CPT-11 and NmAb resulted in significantly higher caspase-3, -8, and -9 activity levels than CPT-11 or NmAb alone, as measured by flow cytometer assay. The combination of CPT-11 and NmAb in HeLa cells resulted in elevated endoplasmic reticulum stress-, mitochondria-, and caspase-mediated proteins compared to treatment with CPT-11 or NmAb alone. According to these observations, NmAb enhances the effectiveness of CPT-11 in fighting cancer by stimulating cell death in the HeLa cells. Therefore, NmAb has the potential to improve the efficacy of CPT-11 as a future cervical cancer treatment in humans.

Human immunodeficiency virus-1 induces host genomic R-loops and preferentially integrates its genome near the R-loop regions

Although HIV-1 integration sites favor active transcription units in the human genome, high-resolution analysis of individual HIV-1 integration sites has shown that the virus can integrate into a variety of host genomic locations, including non-genic regions. The invisible infection by HIV-1 integrating into non-genic regions, challenging the traditional understanding of HIV-1 integration site selection, is more problematic because they are selected for preservation in the host genome during prolonged antiretroviral therapies. Here, we showed that HIV-1 integrates its viral genome into the vicinity of R-loops, a genomic structure composed of DNA-RNA hybrids. VSV-G-pseudotyped HIV-1 infection initiates the formation of R-loops in both genic and non-genic regions of the host genome and preferentially integrates into R-loop-rich regions. Using a HeLa cell model that can independently control transcriptional activity and R-loop formation, we demonstrated that the exogenous formation of R-loops directs HIV-1 integration-targeting sites. We also found that HIV-1 integrase proteins physically bind to the host genomic R-loops. These findings provide novel insights into the mechanisms underlying retroviral integration and the new strategies for antiretroviral therapy against HIV-1 latent infection.

Phytochemical Analysis, Isolation, and Characterization of Gentiopicroside from Gentiana kurroo and Cytotoxicity of Biosynthesized Silver Nanoparticles Against HeLa Cells.

Gentiana kurroo Royle, a critically endangered Himalayan herb, is valued in treating leucoderma, syphilis, bronchial asthma, hepatitis, etc. The current investigation performed quantitative and qualitative phytochemical analysis of G. kurroo root extracts prepared in chloroform, methanol, and ethyl acetate. The phenolic and flavonoid contents were the highest in methanol and chloroform extract, respectively. Several pharmacologically important compounds were identified through gas chromatography-mass spectrometry. Antioxidant analysis revealed methanolic extract to be the most efficient scavenger of 2,2-diphenyl-1-picrylhydrazyl (IC50 = 114µgmL-1), hydrogen peroxide (IC50 = 109.9µgmL-1), and superoxide (IC50 = 74.63µgmL-1) radicals. Gentiopicroside was isolated from the methanolic root extract through silica-gel column-chromatography, and the characterization of concentrated fractions was achieved employing various analytical techniques. Pertaining to silver nanoparticle (GkAgNPs) synthesis, different physicochemical parameters were optimized and it was observed that root extract treated with silver-nitrate (0.5mM) at 60°C and incubated in dark for at least 120min after initial color change, yielded GkAgNPs optimally. GkAgNPs were anisotropic and polydisperse and exhibited characteristic surface plasmon resonance (424nm), crystalline face-centered cubic geometry, size (50-300nm), and zeta-potential (- 16.3mV). FT-IR spectra indicated the involvement of phenols and flavonoids in AgNPs synthesis. GkAgNPs were evidenced as strongly cytotoxic (IC50 = 1.964µgmL-1) against HeLa cells and also showed deformed cellular morphology, a significant reduction in viable cell counts and colony-forming efficiency (4.08%). The findings suggest potential applications in drug development for treating serious human diseases. To the best of our knowledge, this study represents the first report on the isolation of gentiopicroside, the bio-fabrication of GkAgNPs using G.kurroo root extract, and their strong bioefficacy against HeLa cells.

Fluorescent superparamagnetic magnetite-silica nanocomposites as carriers of a platinum diimine complex for photodynamic therapy.

Novel fluorescent superparamagnetic nanocomposites have been fabricated by introduction of the coumarin group on the surface of amine-functionalized magnetite-silica nanocomposites, and characterized by X-ray diffraction, Fourier transform infrared spectroscopy, transmission electron microscopy, fluorescence spectra, dynamic light scattering and vibrating sample magnetometer techniques. The nanocomposites were employed as delivery vehicles of a photoactive platinum diimine complex. The cellular uptake and photocytotoxicity of the photosensitizer-loaded nanocomposites in HeLa cells (human cervical cancer line) or HL-7702 cells (human liver cell line) have been studied by fluorescence spectra and cell viability assay, respectively. The results suggest that the nanocomposites can be used to monitor the cellular uptake of the photosensitizer, and can significantly enhance the photocytotoxicity of the photosensitizer towards cancer cells when employed as carriers of the photosensitizer. Also, the photosensitizer-loaded nanocomposites are almost nontoxic to human normal cells either in the dark or after irradiation.