SPRTN is involved in hepatocellular carcinoma development through the ER stress response.

Endoplasmic reticulum (ER) stress, prompted by the accumulation of misfolded or unfolded proteins, triggers the activation of the unfolded protein response (UPR) pathway to restore ER homeostasis. This stress response is implicated in the development of hepatocellular carcinoma (HCC). A biallelic mutation in SPRTN is currently the only known single-gene mutation implicated in the early onset of HCC. However, the exact mechanism linking SPRTN mutations to HCC remains unclear. In our study, we analyzed SPRTN and UPR in 21 human HCC tissue samples using RT-qPCR, immunoblot, and immunohistochemistry. We found alterations in the expression levels of SPRTN and UPR-related genes and proteins in HCC samples. The impact of SPRTN on the ER stress response was assessed in SPRTN-depleted HepG2 cells through RNA sequencing, pull-down assay, comet assay, and mitotic index calculation. We demonstrated that SPRTN interacts with the UPR sensor GRP78. Furthermore, we observed a decrease in SPRTN levels during ER stress, and increased sensitivity to ER stress in SPRTN-depleted cells. These findings suggest an essential role for SPRTN in the ER stress response and provide new insights into HCC pathogenesis. This newly discovered function of SPRTN could significantly enhance our understanding and treatment of HCC.

Chemical Profiling and Bioactivity Assessment of Helichrysum italicum (Roth) G. Don. Essential Oil: Exploring Pure Compounds and Synergistic Combinations.

Helichrysum italicum (Roth) G. Don., immortelle, is a plant species used in ethnomedicine and the food industry as a spice added to food, beverages, and bakery products. It has been shown to possess various biological activities, such as antioxidant and antibacterial activity, making it useful as a natural preservative. We investigated the phytochemical profile and biological activity of H. italicum essential oils from wild-grown plant material collected from natural habitats in the Republic of Croatia and Bosnia and Herzegovina. Using high-resolution scanning electron microscopy (SEM), a visual investigation of plant organs (stem, leaf, and flower) was performed, confirming the presence of essential oil reservoirs on the surface of all examined plant organs. Essential oils were isolated by hydrodistillation in the Clevenger apparatus. The chemical composition of the essential oils was determined using the GC-MS analytical technique. Cytotoxic activity tests were performed in vitro on three cell lines: skin (fibroblast), lung, and breast cancer. Using statistical tools, the synergistic and selective effects of H. italicum essential oil on healthy and tumor cells were correlated to chemical composition and cytotoxic activity. The synergistic and antagonistic effects of H. italicum essential oil's individual components were simulated by testing pure compounds and their mixture of cytotoxic activity on fibroblasts and breast cancer cells. The results confirm that essential oil's biological activity is much greater than the sum of the effects of its components. The present data are novel contributions to the body of knowledge on the biological activity of this species used in the food industry.

Seasonality of Polyaromatic Hydrocarbons (PAHs) and Their Derivatives in PM2.5 from Ljubljana, Combustion Aerosol Source Apportionment, and Cytotoxicity of Selected Nitrated Polyaromatic Hydrocarbons (NPAHs).

Airborne particulate matter (PM) is a vector of many toxic pollutants, including polyaromatic hydrocarbons (PAHs) and their derivatives. Especially harmful is the fine fraction (PM2.5), which penetrates deep into the lungs during inhalation and causes various diseases. Amongst PM2.5 components with toxic potential are nitrated PAHs (NPAHs), knowledge of which is still rudimentary. Three of the measured NPAHs (1-nitropyrene (1-nP), 9-nitroanthracene (9-nA), and 6-nitrochrysene (6-nC)) were detected in ambient PM2.5 from Ljubljana, Slovenia, along with thirteen non-nitrated PAHs. The highest concentrations of pollutants, which are closely linked with incomplete combustion, were observed in the cold part of the year, whereas the concentrations of NPAHs were roughly an order of magnitude lower than those of PAHs throughout the year. Further on, we have evaluated the toxicity of four NPAHs, including 6-nitrobenzo[a]pyrene (6-nBaP), to the human kidney cell line, HEK293T. The most potent was 1-nP (IC50 = 28.7 µM), followed by the other three NPAHs, whose IC50 was above 400 or 800 µM. According to our cytotoxicity assessment, atmospheric 1-nP is the most harmful NPAH among the investigated ones. Despite low airborne concentrations of NPAHs in ambient air, they are generally considered harmful to human health. Therefore, systematic toxicological assessment of NPAHs at different trophic levels, starting with cytotoxicity testing, is necessary in order to accurately evaluate their threat and adopt appropriate abatement strategies.

Chemical Composition and Biological Activity of Salvia officinalis L. Essential Oil.

In our study, we investigated the chemical composition and cytotoxic activity of essential oils isolated from Dalmatian sage (Salvia officinalis L.) collected along the Adriatic coast of Croatia. Scanning electron microscopy (SEM) was used to examine the morphology of the stem and leaf surfaces. Essential oil excretory glands were detected on both the leaves and stem surfaces. The essential oils were isolated by hydrodistillation, and their chemical composition was determined by gas chromatography and mass spectrometry (GC-MS). Sage essential oils were mixtures of terpene compounds, among which the most common were: α- and ß-thujone, camphor, and 1,8-cineol. Cytotoxic activity was tested using MTS assay on multiple cell lines: normal and immortalized fibroblasts (HF77FA and HDF-Tert), immortalized lung line (BEAS-2B), and breast adenocarcinoma (MDA-MB-231). The growth of treated cells was determined relative to control conditions without treatment. The immortalized lung line was the least resistant to the activity of the essential oils, whereas immortalized fibroblasts were the most resistant. Statistical analysis has connected the cytotoxic effect and chemical composition of the studied essential oils. To the best of our knowledge, this work is the first testing of the cytotoxic activity of S. officinalis EO's on the BEAS-2B, HF77FA, and HDF-Tert cell lines. The presented data on essential oil chemical composition and cytotoxic effect on 4 types of human cells supports pharmacotherapeutic potential this plant is known to have.

Centaurea triumfetii essential oil chemical composition, comparative analysis, and antimicrobial activity of selected compounds.

The essential oils from the Centaurea genus are well known for their pharmacological properties. The most abundant and dominant chemical components in Centaurea essential oils are ß-caryophyllene, hexadecanoic acid, spathulenol, pentacosane, caryophyllene oxide, and phytol. However, whether these dominant components are the key drivers for observed antimicrobial activity remains unclear. Thus, the aim of this study was dual. Here we provide comprehensive, literature-based data to correlate the chemical compounds in Centaurea essential oils with the tested antimicrobial activity. Secondly, we characterized the essential oil of Centaurea triumfettii All. squarrose knapweed using coupled system gas chromatography-mass spectrometry and tested its phytochemicals for antimicrobial activity against E. coli and S. epidermis using disc diffusion assay and monitoring their growth in Muller Hinton broth. The most abundant compounds in C. triumfettii essential oil were hexadecanoic acid (11.1%), spathulenol (10.8%), longifolene (8.8%), germacrene D (8.4%), aromadendrene oxide (6.0%) and linoleic acid (5.3%). Based on our analysis of literature data from other Centaurea essential oils, they were positively correlated with antimicrobial activity. Using an agar disk diffusion method, tested chemical constituents did not show experimental evidence to support this positive correlation to antimicrobial activity when we tested them as pure components. The antibacterial effect of essential oil constituents may be related to a complex synergistic, rather than a single component as suggested by performed network pharmacology analysis, underlying the theoretical interactions between the essential oil phytochemicals listed as potentially responsible for antimicrobial activity and should be confirmed in further in-depth studies. This is the first report on the comparative analysis of Centaurea essential oils with good antimicrobial activity, as well as the first analysis of chemical components of the essential oil from C. triumfettii and the first report of antimicrobial activity of the representative, pure components: aromadendrene, germacrene D, spathulenol, longifolene, and the mixture of selected chemical compounds. This work contributes to the body of knowledge on the genus Centaurea and C. triumfettii species.

Phytochemical and Cytogenetic Study of Two Centaurea Species from Croatia: the Particular Case of Diploid and Tetraploid C. salonitana.

Natural wild populations of C. rupestris and C. salonitana were studied to determine possible relationships between the volatile oil (VO) composition and ploidy level. The chemical composition of the volatile oil was investigated using the GC/MS technique. The predominant components of the VO of diploid and tetraploid C. salonitana were hexadecanoic acid and α-linoleic acids, while in C. rupestris they were germacrene D and ß-caryophyllene in one population and heptacosane and germacrene D, in another. The nuclear DNA amounts (2 C DNA), determined by flow cytometry, were 3.54 pg for C. rupestris, 3.39 pg for the diploid and 6.79 pg for the tetraploid population of C. salonitana. Evidence that the degree of ploidy solely influences the chemical composition of the essential oil of C. salonitana was not found. The results presented are the first data to be reported on the DNA content of the studied Centaurea populations from Croatia, as well as on the chemical composition of C. salonitana volatile oil.

Mechanism of cis-Nerolidol-Induced Bladder Carcinoma Cell Death.

Nerolidol is a naturally occurring sesquiterpene alcohol with multiple properties, including antioxidant, antibacterial, and antiparasitic activities. A few studies investigating the antitumor properties of nerolidol have shown positive results in both cell culture and mouse models. In this study, we investigated the antitumor mechanism of cis-nerolidol in bladder carcinoma cell lines. The results of our experiments on two bladder carcinoma cell lines revealed that nerolidol inhibited cell proliferation and induced two distinct cell death pathways. We confirmed that cis-nerolidol induces DNA damage and ER stress. A mechanistic study identified a common cAMP, Ca2+, and MAPK axis involved in signal propagation and amplification, leading to ER stress. Inhibition of any part of this signaling cascade prevented both cell death pathways. The two cell death mechanisms can be distinguished by the involvement of caspases. The early occurring cell death pathway is characterized by membrane blebbing and cell swelling followed by membrane rupture, which can be prevented by the inhibition of caspase activation. In the late cell death pathway, which was found to be caspase-independent, cytoplasmic vacuolization and changes in cell shape were observed. cis-Nerolidol shows promising antitumor activity through an unorthodox mechanism of action that could help target resistant forms of malignancies, such as bladder cancer.

Direct and cost-effective method for histone isolation from cultured mammalian cells.

Histones are an essential part of nucleosomes that regulate chromatin structure and function. Histone exchanges and modifications represent a scaffold for DNA transcription, repair, and replication. Studying histones and histone code is an important and fast-developing branch of epigenetic science. Here we propose a fast, efficient, and versatile assay for nucleosomal histone isolation from mammalian cells, without the use of acids or high salt solutions which are common for other histone isolation techniques. All components used in the protocol are common and inexpensive laboratory chemicals. The protocol has been evaluated on six commonly used cell lines and two animal tissue samples. The mild extraction conditions preserve delicate histone epigenetic changes, allowing its downstream analyses. We have demonstrated the assays' successful application during changes in the transcriptional activity of histone genes, cell cycle transitions, and DNA-damaging conditions. Histone fractions, obtained by the protocol, can be used for further applications, such as electrophoresis, immunoblot, and mass spectrometry. Therefore, the new proposed nucleosomal histone isolation method is sensitive, specific, and suitable for downstream applications of various kinds.

SPRTN-dependent DPC degradation precedes repair of damaged DNA: a proof of concept revealed by the STAR assay.

DNA-protein crosslinks (DPCs), formed by the covalent conjugation of proteins to DNA, are toxic lesions that interfere with DNA metabolic processing and transcription. The development of an accurate biochemical assay for DPC isolation is a priority for the mechanistic understanding of their repair. Here, we propose the STAR assay for the direct quantification of DPCs, sensitive to physiologically relevant treatment conditions. Implementing the STAR assay revealed the formation of small cross-linked peptides on DNA, created by the proteolytic degradation of DPCs by SPRTN. The initial proteolytic degradation of DPCs is required for the downstream activation of DNA repair, which is mediated through the phosphorylation of H2Ax. This leads to the accumulation of DNA repair factors on chromatin and the subsequent complete removal of the cross-linked peptides. These results confirmed that the repair of DPCs is a two-step process, starting with proteolytic resection by SPRTN, followed by the repair of the underlying damage to the DNA.