Inhibition Of Viability Research Articles

Photoactive metabolite mediated photodynamic therapy of Rhabdomyosarcoma cell lines using medicinal plants and Doxorubicin co-treatments

BackgroundMedicinal plant-mediated combinational therapies have gained importance globally due to minimal side effects and enhanced treatment outcomes compared to single-drug modalities. We aimed to analyze the cytotoxic potential of each conventional treatment i.e., photodynamic therapy (PDT), chemotherapy (doxorubicin hydrochloride; Dox-HCl) with or without various concentrations of medicinal plant extracts (PE) on soft tissue cancer Rhabdomyosarcoma (RD) cell line.MethodsThe Rhabdomyosarcoma (RD) cell line was cultured and treated with Photosensitizer (Photosense (AlPc4)), Chemo (Dox-HCl), and their combinations with different concentrations of each plant extract i.e., Thuja occidentalis, Moringa oleifera, Solanum surattense. For the source of illumination, a Diode laser (λ = 630 nm ± 1 nm, Pmax = 1.5 mW) was used. Photosensitizer uptake time (∼ 45 min) was optimized through spectrophotometric measurements (absorption spectroscopy). Drug response of each treatment arm was assessed post 24 h of administration using 3-(4, 5-dimethyl-2-thiazolyl)-2, 5- 5-diphenyl-2 H- tetrazolium bromide (MTT) assay.ResultsPE-mediated Chemo-Photodynamic therapy (PDT) exhibited synergistic effects (CI < 1). Moreover, Rhabdomyosarcoma culture pretreated with various plant extracts for 24 h exhibited significant inhibition of cell viability however most effective outcomes were shown by low and high doses of Moringa oleifera compared to other plant extracts. Post low doses treated culture with all plant extracts followed by PDT came up with more effectiveness when compared to all di-therapy treatments.ConclusionThe general outcome of this work shows that the ethanolic plant extracts (higher doses) promote the death of cancerous cells in a dose-dependent way and combining Dox-HCl and photo-mediated photodynamic therapy can yield better therapeutic outcomes.

Copper-induced renal toxicity controlled by period1 through modulation of Atox1 in mice.

Copper (Cu) is known to induce oxidative stress and apoptosis in the liver, kidney, and brain. We previously demonstrated the molecular mechanism underlying the Cu-induced hepatic diurnal variation. However, the cellular molecule(s) involved in Cu-induced renal chronotoxicity remain unknown. In this study, we aimed to elucidate the molecular mechanisms underlying Cu-induced diurnal toxicity in the kidneys. We evaluated cell viability and clock gene expression levels in mouse renal cortex tubular cells (MuRTE61 cells) after Cu treatment. We also examined the Cu homeostasis- and apoptosis-related gene levels after period 1 (Per1) overexpression in MuRTE61 cells. Cu treatment decreased MuRTE61 cell viability in a dose-dependent manner. It increased the Per1 expression levels after 24 h. Notably, Per1 overexpression alleviated the Cu-induced inhibition of MuRTE61 cell viability. Moreover, Per1 overexpression downregulated the cleaved caspase-3 and reduced Cu levels by upregulating the antioxidant 1 copper chaperone (Atox1) levels. These results suggest that Cu-induced renal toxicity is associated with Per1 expression via the regulation of the copper chaperone, Atox1.

Synergistic anticancer effects of omega-3 fatty acids (EPA/DHA) and anticancer drug Doxorubicin against human lung adenocarcinoma

Single agent therapy in the treatment of cancer did not provide satisfactory clinical outcome in a long term due to drug resistance, cumulative toxicity and suboptimal efficacy. Therefore, combination therapies are apt to improve the quality of life in patients. In the present study, combinatorial effect of EPA and/or DHA with doxorubicin (Dox) was evaluated for their potential synergistic effect on human lung adenocarcinoma (A549) cells. The respective IC50 values obtained for EPA, DHA and Dox were 170, 140 and 2.5 μmol/L and thus EPA and DHA were separately combined with Dox at constant ratio of 68:1 and 56:1 respectively, depending on the ratio of their IC50 values. Different combinations of drugs revealed dose-dependent inhibition of cell viability and IC50 values were reduced to nearly 3-fold (58.75 μmol/L) and 4.5-fold (31.5 μmol/L) in EPA-Dox and DHA-Dox combination respectively. Combination index (CI) revealed strong synergism (CI < 0.5) in DHA-Dox (0.49) and moderate synergism (CI > 0.5) in EPA-Dox (0.79) at 0.5 Fa (Fraction affected), which is further confirmed by dose reduction index and isobologram. The mechanisms underlying synergistic interactions involve intracellular ROS generation and MMP depolarisation which causes changes in the cell morphology viz. Nuclear fragmentation/condensation, membrane blebbing and early/late apoptotic cells. Moreover, cell cycle arrest was observed at the S and G2/M phase, with more prominent effect in G2/M phase. No toxicity was imposed on non-cancerous HEK-293 T cells. These findings suggest that EPA-Dox and DHA-Dox could be explored further for animal studies and clinical trials for lung adenocarcinoma treatment.

Evaluation of Boric Acid Treatment on microRNA-127-5p and Metastasis Genes Orchestration of Breast Cancer Stem Cells.

Coregulation of microRNAs (miRNAs) and cancer stem cells (CSCs) is very important in carcinogenesis. miR-127-5p is known to be downregulated in breast cancer. In this study, we aimed to investigate how boric acid (BA), known for its previously unstudied anti-cancer properties, would affect the expression of miR127-5p and genes responsible for breast cancer stem cells (BC-SCs) metastasis. BC-SCs were isolated from human breast cancer cells (MCF-7) by immunomagnetic cell separation and characterized with flow cytometry and sphere formation. The viability of BC-SCs and the determination of its IC50 value in response to boric acid (BA) were assessed via the MTT assay. Boric acid exhibited dose- and time-dependent inhibition of cell viability in cells. The IC50 doses of boric acid in MCF-7 cells and BC-SCs were 45.69mM and 41.27mM, respectively. The impact of BA on the expression of metastatic genes and miR127-5p was elucidated through RT-qPCR analysis. While the expression of the COL1A1 (p < 0.05) and VIM (p < 0.01) was downregulated, the expression of the miR-127-5p, ZEB1 (p < 0.01), CDH1 (p < 0.05), ITGB1 (p < 0.05), ITGA5 (p < 0.05), LAMA5 (p < 0.01), and SNAIL (p < 0.05), was up-regulated in dose-treated BC-SCs (p < 0.001) to the RT-qPCR results. Our findings suggest that boric acid could induce miR-127-5p expression. However, it cannot be said that it improves the metastasis properties of breast cancer stem cells.

The Megacomplex protects ER-alpha from degradation by Fulvestrant in epithelial ovarian cancer

Ovarian cancer, a significant contributor to cancer-related mortality, exhibits limited responsiveness to hormonal therapies targeting the estrogen receptor (ERα). This study aimed to elucidate the mechanisms behind ERα resistance to the therapeutic drug Fulvestrant (ICI182780 or ICI). Notably, compared to the cytoplasmic version, nuclear ERα was minimally degraded by ICI, suggesting a mechanism for drug resistance via the protective confines of the nuclear substructures. Of these substructures, we identified a 1.3MDa Megacomplex comprising transcription factors ERα, FOXA1, and PITX1 using size exclusion chromatography (SEC) in the ovarian cancer cell line, PEO4. ChIP-seq revealed these factors colocalized at 6,775 genomic positions representing sites of Megacomplex formation. Megacomplex ERα exhibited increased resistance to degradation by ICI compared to cytoplasmic and nuclear ERα. A small molecule inhibitor of active chromatin and super-enhancers, JQ1, in combination with ICI significantly enhanced ERα degradation from Megacomplex as revealed by SEC and ChIP-seq. Interestingly, this combination degraded both the cytoplasmic as well as nuclear ERa. Pathway enrichment analysis showed parallel results for RNA-seq gene sets following Estradiol, ICI, or ICI plus JQ1 treatments as those defined by Megacomplex binding identified through ChIP-seq. Furthermore, similar pathway enrichments were confirmed in mass-spec analysis of the Megacomplex macromolecule fractions after modulation by Estradiol or ICI. These findings implicate Megacomplex in ERα-driven ovarian cancer chromatin regulation. This combined treatment strategy exhibited superior inhibition of cell proliferation and viability. Therefore, by uncovering ERα’s resistance within the Megacomplex, the combined ICI plus JQ1 treatment elucidates a novel drug treatment vulnerability.

Quercetin attenuated ropivacaine-induced neurotoxicity in SH-SY5Y cells through upregulating Pim1 and enhancing autophagy

BackgroundRopivacaine (Rop) is a local anesthetic that is widely used but is also potentially harmful. Quercetin (Quer) is a flavonoid component found in many plants and traditional Chinese medicines. It possesses anti-oxidant, anti-inflammatory, antitumor, and neuroprotective properties as a pharmaceutical. In Rop-induced neurotoxicity, the functions and molecular basis of Quer remain unclear. MethodsCell viability and proliferation were assessed using CCK-8 and 5-ethynyl-2′-deoxyuridine (EdU) incorporation assays, respectively. Apoptosis and autophagy were defined by both morphological criteria and markers such as caspase3 cleavage and monodansylcadaverine (MDC) staining. Western blot and immunofluorescence staining were evaluated. The target proteins were then predicted using molecular docking and validated at the cellular and protein levels. ResultsQuer was shown to significantly reduce Rop-induced viability inhibition and apoptosis in SH-SY5Y cells in a dose-dependent manner. Moreover, Quer reduced Rop-induced cytotoxicity by stimulating autophagy in SH-SY5Y cells by targeting the Pim1 protein, which was accomplished via the AMPK/mTOR pathway. ConclusionQuer relieved Rop-induced neurotoxicity in SH-SY5Y cells through upregulating Pim1 and enhancing autophagy, indicating that Quer may have potential therapeutic applications in the treatment of Rop-induced neurotoxicities.

CHRDL2 activates the PI3K/AKT pathway to ameliorate glucocorticoid-induced damages to bone microvascular endothelial cells (BMECs)

Steroid-induced avascular necrosis of the femoral head (ANFH) is characterized by the death of bone tissues, leading to the impairment of normal reparative processes within micro-fractures in the femoral head. Glucocorticoid (GCs)-induced bone microvascular endothelial cell (BMEC) damage has been reported to contribute to ANFH development. In this study, differentially expressed genes (DEGs) between necrosis of the femoral head (NFH) and normal samples were analyzed based on two sets of online expression profiles, GSE74089 and GSE26316. Chordin-like 2 (CHRDL2) was found to be dramatically downregulated in NFH samples. In GCs-stimulated BMECs, cellular damages were observed alongside CHRDL2 down-regulation. GCs-caused cell viability suppression, cell apoptosis promotion, tubule formation suppression, and cell migration suppression were partially abolished by CHRDL2 overexpression but amplified by CHRDL2 knockdown; consistent trends were observed in GCs-caused alterations in the protein levels of VEGFA, VEGFR2, and BMP-9 levels, and the ratios of Bax/Bcl-2 and cleaved-caspase3/Caspase3. GC stimulation significantly inhibited PI3K and Akt phosphorylation in BMECs, whereas the inhibitor effects of GCs on PI3K and Akt phosphorylation were partially attenuated by CHRDL2 overexpression but further amplified by CHRDL2 knockdown. Moreover, CHRDL2 overexpression caused improvement in GCs-induced damages to BMECs that were partially eliminated by PI3K inhibitor LY294002. In conclusion, CHRDL2 is down-regulated in NFH samples and GCs-stimulated BMECs. CHRDL2 overexpression could improve GCs-caused BMEC apoptosis and dysfunctions, possibly via the PI3K/Akt pathway.

Integrated Metabolomics and Transcriptomics Analysis of Anacardic Acid Inhibition of Breast Cancer Cell Viability.

Anacardic acid (AnAc) inhibits the growth of estrogen receptor α (ERα)-positive MCF-7 breast cancer (BC) cells and MDA-MB-231 triple-negative BC (TNBC) cells, without affecting primary breast epithelial cells. RNA sequencing (seq) and network analysis of AnAc-treated MCF-7 and MDA-MB-231 cells suggested that AnAc inhibited lipid biosynthesis and increased endoplasmic reticulum stress. To investigate the impact of AnAc on cellular metabolism, a comprehensive untargeted metabolomics analysis was performed in five independent replicates of control versus AnAc-treated MCF-7 and MDA-MB-231 cells and additional TNBC cell lines: MDA-MB-468, BT-20, and HCC1806. An analysis of the global metabolome identified key metabolic differences between control and AnAc-treated within each BC cell line and between MCF-7 and the TNBC cell lines as well as metabolic diversity among the four TNBC cell lines, reflecting TNBC heterogeneity. AnAc-regulated metabolites were involved in alanine, aspartate, glutamate, and glutathione metabolism; the pentose phosphate pathway; and the citric acid cycle. Integration of the transcriptome and metabolome data for MCF-7 and MDA-MB-231 identified Signal transduction: mTORC1 downstream signaling in both cell lines and additional cell-specific pathways. Together, these data suggest that AnAc treatment differentially alters multiple pools of cellular building blocks, nutrients, and transcripts resulting in reduced BC cell viability.

Comparison of Anticancer Activity Between Thymoquinone and Tamoxifen , Thymoquinone + Tamoxifen 0n Mcf -7 Cell Line of Human Breast Cancer –An Invitro Study.

This study aims at evaluating the anticancer effect on the MCF-7 (Michigan Cancer Foundation-7) cell line of human breast cancer using Thymoquinone and Tamoxifen alone as well as in combination therapy by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) Test. NCCS in Pune provided the MCF-7 cell line. The cells were kept at 37°C in a humidified medium of 50µg/ml CO2 in Minimal Essential Medium added with 10percent FBS (Foetal Bovine Serum), streptomycin (100µg/ml), as well as penicillin (100U/ml). MTT-(3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl tetrazolium bromide) test was conducted on MCF-7 cell line for Thymoquinone and Tamoxifen as sole and combination therapy. Measurements were performed using UV (Ultra-Violet)-spectrophotometer at 570-nanometre absorbance and the content needed for a 50 percent inhibitory concentration (IC50) was calculated and evaluated graphically. IC 50 of Thymoquinone on MCF 7 was found to be at 31.2 µg/ml and Tamoxifen was at 62.5 µg/ml were as in combination therapy the IC 50 was found to be at 7.8 µg/ml. There is a remarkable reduction in concentration to achieve IC 50 percentage in combination therapy with a comparison with individual therapy. Therefore, the combination therapy of Thymoquinone and Tamoxifen on the MCF-7 cell line is more efficacious when compared to individual treatment on cell viability inhibition.

Novel ferrocene cyclopalladated compounds: Synthesis, and in‐vitro antitumor activity study

AbstractMetal complexes have a significant impact on the treatment of human cancer. However, the scarcity of these compounds, resulting from their limited synthesis, hinders the comprehensive investigation of their anticancer mechanisms. Organic palladium compounds, known for their distinctive stability and properties, are thus an essential area of research in the development of anti‐tumor therapy. In our study, we synthesized two novel ferrocene cyclopalladated compounds (C2 and C4). Its configuration was thoroughly characterized by employing 1H, 13C NMR, ESI‐MS, and elemental analysis techniques. The molecular structures were determined by X‐ray single‐crystal diffraction. In an in vitro anticancer study, it was observed that both C2 and C4 exhibited excellent suppression of viability in various tumor cell lines. These compounds showed better potency than cisplatin and demonstrated lower toxicity in normal cells. Particularly, C4 displayed approximately 22 times greater potency than cisplatin in suppressing melanoma cells (B16F10). Our study suggests that ferrocene cyclopalladated compounds have the potential to be promising candidates for the development of innovative anticancer drugs.

Evaluation of the synergistic effects of curcumin-resveratrol co-loaded biogenic silica on colorectal cancer cells.

Colorectal cancer (CRC) remains a significant global health concern, being the third most diagnosed cancer in men and the second most diagnosed cancer in women, with alarming mortality rates. Natural phytochemicals have gained prominence among various therapeutic avenues explored due to their diverse biological properties. Curcumin, extracted from turmeric, and resveratrol, a polyphenol found in several plants, have exhibited remarkable anticancer activities. However, their limited solubility and bioavailability hinder their therapeutic efficacy. To enhance the bioavailability of these compounds, nanomaterials work as effective carriers with biogenic silica (BS) attracting major attention owing to their exceptional biocompatibility and high specific surface area. In this study, we developed Curcumin-resveratrol-loaded BS (Cur-Res-BS) and investigated their effects on colorectal cancer cell lines (HCT-116 and Caco-2). Our results demonstrated significant concentration-dependent inhibition of cell viability in HCT-116 cells and revealed a complex interplay of crucial proto-onco or tumor suppressor genes, such as TP53, Bax, Wnt-1, and CTNNB1, which are commonly dysregulated in colorectal cancer. Notably, Cur-Res-BS exhibited a synergistic impact on key signaling pathways related to colorectal carcinogenesis. While these findings are promising, further investigations are essential to comprehensively understand the mechanisms and optimize the therapeutic strategy. Moreover, rigorous safety assessments and in vitro studies mimicking the in vivo environment are imperative before advancing to in vivo experiments, ensuring the potential of Cur-Res-BS as an efficient treatment for CRC.

LINC00894 inhibited neuron cellular apoptosis and regulated activating transcription factor 3 expression

LINC00894 may be associated with synaptic function, but its biology function in neural cells is still unknown. In this study, LINC00894 knockdown decreased the EdU incorporated into newly synthesized DNA and cell viability in MTT or CCK-8 assay in HEK-293T and BE(2)-M17 (M17) neuroblastoma cells. And LINC00894 knockdown increased cellular apoptosis in Annexin V-FITC staining, the expression of activated Caspase3 and the level of reactive oxygen species (ROS) both in HEK-293T and M17 cells. Moreover, LINC00894 also protected cells from hydrogen peroxide induced apoptosis in in vitro models. Utilizing RNA sequencing (RNA-seq) integrated with quantitative reverse transcription polymerase chain reaction (RT-qPCR) and immunoblot, we identified that LINC00894 affected activating transcription factor 3 (ATF3) expression in HEK-293T, M17, and SH-SY5Y neuroblastoma cells. Finally, we found that ectopic expression of ATF3 restored cell proliferation and inhibited cell apoptosis in LINC00894 downregulated M17 cells. While knockdown of ATF3 also significantly increased the cell viability inhibition and apoptosis promotion induced by LINC00894 knockdown in M17 cells. Our results from in vitro models revealed that LINC00894 could promote neuronal cell proliferation and inhibit cellular apoptosis by affecting ATF3 expression.

Chlormequat Chloride Inhibits TM3 Leydig Cell Growth via Ferroptosis-Initiated Inflammation.

Ferroptosis hallmarked by lipid peroxidation and iron homeostasis imbalance is involved in the occurrence and development of various diseases. The plant growth regulator chlormequat chloride (CCC) can contribute to the causality and exacerbation of reproductive disorders. However, the mechanism by which CCC may cause Leydig cell attenuation remains poorly understood. In this study, TM3 Leydig cells were used to investigate the inhibitory effect of CCC on cell growth and its possible mechanism. The results showed that CCC caused apoptosis, pyroptosis, ferroptosis and necroinflammation in TM3 cells. By comparing the effects of ferroptosis inhibitor Ferrostatin-1 (Fer-1) and pan-Caspase inhibitor Z-VAD-FMK (ZVF) on lipid peroxidation and Caspase-mediated regulated cell death (RCD), we found that Fer-1 was better at rescuing the growth of TM3 cells than ZVF. Although ZVF reduced mitochondrial ROS level and inhibited the activation of Caspase3 and Caspase1, it could not significantly ameliorate lipid peroxidation and the levels of IL-1β and HMGB1 like Fer-1. Therefore, ferroptosis might be a key non apoptotic RCD mode responsible for CCC-driven inflammation, leading to weakened viability and proliferation of TM3 cells. In addition, overexpression of ferritin light chain (FTL) promoted the resistance of TM3 cells to CCC-induced ferroptosis-mediated inflammation and to some extent improved the inhibition of viability and proliferation. Altogether, ferroptosis-initiated inflammation might play a key role in CCC-impaired TM3 cell growth.

Oleanolic acid conjugated chitosan nanocomplex exerts anti-tumor effects by inhibiting autophagy in lung cancer cells through the signal transducers and activators of transcription 3/B cell lymphoma-2 signaling pathway.

The current study reveals the anticancer potential of oleanolic acid conjugated chitosan nanocomplex (OAC) in lung cancer (LC). Cell counting kit-8 (CCK-8) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) assay were used to detect cell viability, 5-ethynyl-2'-deoxyuridine (EdU) assay to detect cell proliferation, flow cytometry and TUNEL assay to detect cell apoptosis in A549 (ATCC®CCL-185™) and NCIH460 cells. Transwell evaluated cell migration and invasion ability, transmission electron microscopy and immunofluorescence observed autophagy, and Western blotting detected apoptosis- and autophagy-associated proteins. OAC inhibited LC cell viability, migration, and invasion, and induced apoptosis and autophagy depending on the concentration. The phosphorylation of signal transducers and activators of transcription 3 (STAT3) in cells was weakened after OAC treatment. STAT3 activation restored the inhibition of cell viability and induction of apoptosis by OAC. We conclude that OAC induces apoptosis and inhibits cell viability, which may be related to the STAT inactivation. Therefore, OAC is a promising compound for LC therapy.

Comparing invitro cytotoxic drug sensitivity in colon and pancreatic cancer using 2D and 3D cell models: Contrasting viability and growth inhibition in clinically relevant dose and repeated drug cycles.

In vitro drug screening that is more translatable to the invivo tumor environment can reduce both time and cost of cancer drug development. Here we address some of the shortcomings in screening and show how treatment with 5-fluorouracil (5-FU) in 2D and 3D culture models of colorectal cancer (CRC) and pancreatic ductal adenocarcinomas (PDAC) give different responses regarding growth inhibition. The sensitivity of the cell lines at clinically relevant 5-FU concentrations was monitored over 4 days of treatment in both 2D and 3D cultures for CRC (SW948 and HCT116) and PDAC (Panc-1 and MIA-Pa-Ca-2) cell lines. The 3D cultures were maintained beyond this point to enable a second treatment cycle at Day 14, following the timeline of a standard clinical 5-FU regimen. Evaluation after one cycle did not reveal significant growth inhibition in any of the CRC or PDAC 2D models. By the end of the second cycle of treatment the CRC spheroids reached 50% inhibition at clinically achievable concentrations in the 3D model, but not in the 2D model. The PDAC models were not sensitive to clinical doses even after two cycles. High content viability metrics point to even lower response in the resistant PDAC models. This study reveals the limitations of testing drugs in 2D cancer models and short exposure in 3D models, and the importance of using appropriate growth inhibition analysis. We found that screening with longer exposure and several cycles of treatment in 3D models suggests a more reliable way to assess drug sensitivity.

Synthesis and biological properties of axially bis − (3,4,5-trimethoxybenzyloxy) phthalocyaninato silicon (IV)

In this study, bis − (3,4,5-trimethoxybenzyloxy) phthalocyaninato silicon (IV) was obtained from the reaction of 3,4,5-trimethoxybenzyl alcohol with SiPcCl2. This phthalocyanine was characterized using 1H NMR, FTIR, UV–vis and mass spectra. 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, antidiabetic, deoxyribonucleic acid (DNA) cutting, biofilm inhibition, anti-microbial and antimicrobial photodynamic therapy (aPDT) activities of newly synthesized bis − (3,4,5-trimethoxybenzyloxy) phthalocyaninato silicon (IV) molecule were studied. The best activities were 41.58 % at 100 mg/L for antioxidant and 41.66 % for antidiabetic at 400 mg/L concentration. The molecule degraded the biofilm matrix formed by Pseudomonas aeruginosa and Staphylococcus aureus as 78.61 % and 89.26 %, at 50 mg/L concentration, respectively. It was observed that E. coli, which was used as a model microorganism, was inhibited at a level close to 100 % even at the lowest concentration of 50 mg/L. While double strand break was observed at 50 mg/L DNA cutting activity, it was determined that DNA was reduced to nucleotides at 100 and 200 mg/L. The Pc also displayed effective antimicrobial and aPDT abilities against pathogens. With the application of aPDT, the effectiveness of antimicrobial activity increased 2 to 4 times. These increase rates are very important. The main conclusion of the study was that the newly synthesized compound exhibited various effective biological activities such as effective antioxidant, antidiabetic, DNA cleavage, antimicrobial, aPDT, biofilm inhibition and microbial cell viability inhibition.

Exploring the antifungal, antibiofilm and antienzymatic potential of Rottlerin in an in vitro and in vivo approach

Candida species have been responsible for a high number of invasive infections worldwide. In this sense, Rottlerin has demonstrated a wide range of pharmacological activities. Therefore, this study aimed to evaluate the antifungal, antibiofilm and antivirulence activity of Rottlerin in vitro against Candida spp. and its toxicity and antifungal activity in vivo. Rottlerin showed antifungal activity against all yeasts evaluated, presenting Minimum Inhibitory and Fungicidal Concentration (MIC and MFC) values of 7.81 to > 1000 µg/mL. Futhermore, it was able to significantly inhibit biofilm production, presenting Biofilm Inhibitory Concentration (MICB50) values that ranged from 15.62 to 250 µg/mL and inhibition of the cell viability of the biofilm by 50% (IC50) from 2.24 to 12.76 µg/mL. There was a considerable reduction in all hydrolytic enzymes evaluated, with emphasis on hemolysin where Rottlerin showed a reduction of up to 20%. In the scanning electron microscopy (SEM) analysis, Rottlerin was able to completely inhibit filamentation by C. albicans. Regarding in vivo tests, Rottlerin did not demonstrate toxicity at the therapeutic concentrations demonstrated here and was able to increase the survival of C. elegans larvae infected. The results herein presented are innovative and pioneering in terms of Rottlerin’s multipotentiality against these fungal infections.

Competitive Inhibition and Synergistic Effects of Nutraceutical and Metabolite Molecules on Anti-Acetylcholinesterase Activity

The rapidly increasing prevalence of Alzheimer's disease (AD) poses a significant global public health threat. While medications such as Donepezil, Galantamine, and Rivastigmine are used, their serious side effects and limited healing fail to provide a definite cure. Consequently, combination therapies are being explored to enhance the efficacy of existing drugs. This study aims to evaluate the anti-acetylcholinesterase activities of previously identified nutraceutical and metabolite compounds, namely Queuine, Etoperidone, and Thiamine. Combined use of Queuine with Donepezil, Etoperidone, and Thiamine on acetylcholinesterase enzyme inhibition is also evaluated. The effects of the drug combinations on cell viability and acetylcholinesterase inhibition were investigated by using safe doses determined for each drug. The cytotoxic effect of drug combinations was investigated on the SH-SY5Y cell line using the RTCA method. All the individual or drug combinations were non-toxic to neuronal cells. Anti-acetylcholinesterase activities were estimated by Ellman’s method yielding the inhibition percentages as 70%, 61%, 45%, and 51% for Donepezil, Etoperidone, Queuine, and Thiamine, respectively. When drug combinations were analyzed, competitive inhibition resulted for Queuine+Donepezil and Queuine+Thiamine, the enzyme inhibition percentages being diminished to 47% and 21%, respectively. A significant synergistic effect was observed for Queuine+Etoperidone with the highest inhibition of 74%. This study provides the first evidence of the nutraceutical molecule Queuine's impact on acetylcholinesterase inhibition and the synergistic effect of Queuine and Etoperidone as a potent drug combination surpassing the effectiveness of Donepezil. Queuine and Etoperidone synergism may serve as a potential AD treatment by further in vivo validations.

Efficacy and Safety of Asparagusic Acid against Echinococcus multilocularis In Vitro and in a Murine Infection Model.

Alveolar echinococcosis (AE) stands as a perilous zoonotic affliction caused by the larvae of Echinococcus multilocularis. There is an imperative need to explore novel therapeutic agents or lead compounds for the treatment of AE. Asparagusic acid, characterized by its low toxicity and possessing antimicrobial, antioxidant, and anti-parasitic attributes, emerges as a promising candidate. The aim of this study was to investigate the in vivo and in vitro efficacy of asparagusic acid against E. multilocularis. Morphological observations, scanning electron microscopy, ROS assays, mitochondrial membrane potential assays, and Western blot were used to evaluate the in vitro effects of asparagusic acid on protoscoleces. The effects of asparagusic acid on vesicles were assessed via PGI release, γ-GGT release, and transmission electron microscopy observations. CellTiter-Glo assays, Caspase3 activity assays, flow cytometry, and Western blot were used for an evaluation of the effect of asparaginic acid on the proliferation and apoptosis of germinal cells. The in vivo efficacy of asparagusic acid was evaluated in a murine AE model. Asparagusic acid exhibited a pronounced killing effect on the protoscoleces post-treatment. Following an intervention with asparagusic acid, there was an increase in ROS levels and a decline in mitochondrial membrane potential in the protoscolex. Moreover, asparagusic acid treatment resulted in the upregulation of PGI and γ-GGT release in metacestode vesicles, concomitant with the inhibition of germinal cell viability. Furthermore, asparagusic acid led to an enhanced relative expression of Caspase3 in the culture supernatant of both the protoscoleces and germinal cells, accompanied by an increase in the proportion of apoptotic germinal cells. Notably, asparagusic acid induced an augmentation in Bax and Caspase3 protein expression while reducing Bcl2 protein expression in both the protoscoleces and germinal cells. In vitro cytotoxicity assessments demonstrated the low toxicity of asparagusic acid towards normal human hepatocytes and HFF cells. Additionally, in vivo experiments revealed that asparagusic acid administration at doses of 10 mg/kg and 40 mg/kg significantly reduced metacestode wet weight. A histopathological analysis displayed the disruption of the germinal layer structure within lesions post-asparagusic acid treatment, alongside the preservation of laminated layer structures. Transmission electron microscopy further revealed mitochondrial swelling and heightened cell necrosis subsequent to the asparagusic acid treatment. Furthermore, asparagusic acid promoted Caspase3 and Bax protein expression while decreasing Bcl2 protein expression in perilesional tissues. Subsequently, it inhibited the expression of Ki67, MMP2, and MMP9 proteins in the perilesional tissues and curbed the activation of the PI3K/Akt signaling pathway within the lesion-host microenvironmental tissues. Asparagusic acid demonstrated a pronounced killing effect on E. multilocularis, suggesting its potential as a promising therapeutic agent for the management of AE.

Polyphyllin I Mitigated IL-1β-Induced Chondrocytes Damage through Downregulating TWIST1 Expression.

Osteoarthritis (OA) is a chronic joint disease characterized by the degradation of articular cartilage. Polyphyllin I (PPI) has anti-inflammatory effects in many diseases. However, the mechanism of PPI in OA remains unclear. <P> </P> Methods: HC-a cells treated with IL-1β were identified by immunofluorescence staining and microscopic observation. The expression of collagen II and DAPI in HC-a cells was detected by immunofluorescence. The effects of gradient concentration of PPI on IL-1β-induced cell viability, apoptosis, senescence, and inflammatory factor release were detected by MTT, flow cytometry, SA-β-Gal assay and ELISA, respectively. Expressions of apoptosis-related genes, extracellular matrix (ECM)- related genes, and TWIST1 were determined by qRT-PCR and western blot as needed. The above-mentioned experiments were conducted again after TWIST1 overexpression in IL-1β-induced chondrocytes. <P> </P> Results: IL-1β reduced the number of chondrocytes and the density of collagen II. PPI (0.25, 0.5, 1 µmol/L) had no effect on cell viability, but it dose-dependently elevated the inhibition of cell viability regulated by IL-1β. The elevation of cell apoptosis, senescence and expression of IL-6 and TNF-α were suppressed by PPI in a dosedependent manner. Additionally, PPI reduced the expression of cleaved caspase-3, bax, MMP-3, and MMP-13 and promoted the expression of collagen II. TWIST1 expression was diminished by PPI. TWIST1 overexpression reversed the abovementioned effects of PPI on chondrocytes. <P> </P> Conclusion: PPI suppressed apoptosis, senescence, inflammation, and ECM degradation of OA chondrocytes by downregulating the expression of TWIST1.