Raheleh Shakeri

The anticancer effects of six tert-butyl peresters derivatives were investigated against MDA‑MB‑231 (triple‑negative) and MCF‑7 (hormone receptor‑positive) breast cancer cells. Specifically, only the nitro‑substituted derivatives, including tert-butyl-4-nitroperbenzoate (1SS) and tert-butyl-3,5-dinitroperbenzoate (6SS), showed significant cytotoxicity against the cancer cells while displaying lower cytotoxicity against the non-cancerous L929 cell line. The triple‑negative cell line was consistently more sensitive. Compound 6SS was the more potent agent, with IC₅₀ values reaching 0.35 µg/mL in MDA‑MB‑231 monolayer cultures after 72 h (compared to 3.18 µg/mL for 1SS). Both compounds were cytotoxic in three‑dimensional (3D) spheroid models, although higher IC₅₀ values indicated greater resistance in the 3D environment. Caspase‑3/7 activity increased by 3.0‑fold (6SS) and 2.0‑fold (1SS) in MDA‑MB‑231 cells, and by 4.4‑fold (6SS) and 2.8‑fold (1SS) in MCF‑7 cells, suggesting that apoptosis is the primary cell death pathway. Neither compound inhibited the enzymatic activity of MMP‑9 or MMP‑2. Notably, while MMP‑9 appeared as a hub protein in the 1SS network, no direct enzymatic inhibition was detected, suggesting a non‑enzymatic or indirect regulatory mechanism. In silico predictions showed favorable drug‑likeness, high gastrointestinal absorption, absence of P‑glycoprotein substrate activity, and lower predicted acute toxicity (LD₅₀ of 914-1200 mg/kg) compared to doxorubicin (205 mg/kg), all of which await experimental confirmation. Network pharmacology predicted distinct target profiles: 1SS hubs include PTGS2, MMP9 and RELA (inflammation and tumor microenvironment), whereas 6SS targets EGFR, ERBB2, CASP3, PTGS2 and HSP90AA1 (proliferation, apoptosis and protein stability). Taken together, the dinitro derivative 6SS exhibits potent, multi‑targeted anti‑breast cancer activity through a proposed caspase‑dependent apoptotic mechanism, with a safety profile that supports further development. The online version contains supplementary material available at 10.1007/s13205-026-04893-x.

Investigation of toxicological profile and possible side effects of engineered nanomaterials (ENMs) is of high importance. Historically, two-dimensional (2D) cell culture was used to study the toxicity of the ENMs, but due to their inability to simulate in vivo cell behavior, three-dimensional (3D) cell culture systems have been developed. Nanotoxicity studies initiate with in vitro experiments and continue with in vivo studies, which are very challenging and sometimes accompanied by conflicting data due to the in vitro-in vivo gap. Thus, scientists are turning their attention to microfabrication techniques and engineered systems “called organ-on-a-chips”, which act as an intermediate between in vivo and in vitro systems. The present account tries to review the classical study models and suitably cover the emerging 3D culture models including scaffold-free and scaffold-based 3D cell cultures, 3D co-culture with direct contact and without cell-cell contact methods as well as microfluidic-based tissue chips and organoids. Overall, this review aims to give readers a better insight about the ENMs’ toxicology and fill the gaps between the knowledge and practical techniques. Hopefully, the presented information will resolve the issues of 2D in vitro cultures and display the clinically relevant responses to the concerns of therapeutic ENMs. Graphical Abstract

Breast cancer is one of the most prevalent cancers among women, characterized by the uncontrolled proliferation of breast epithelial cells. Chemotherapy, a common treatment for breast cancer, is associated with significant side effects, including damage to healthy proliferating cells and the development of drug resistance. These limitations highlight the need for novel therapeutic agents. Natural and plant‐based compounds are promising alternatives in this regard. Thymus daenensis Celak. ( T. daenensis ), a medicinal plant from the Lamiaceae family, is rich in bioactive compounds. In this study, the effects of various concentrations of T. daenensis essential oil on MDA‐MB‐231 and MCF‐7 human breast cancer cell lines were evaluated using the MTT assay in both monolayer (2D) and 3D cell cultures over 24 and 48 h. The finding revealed that T. daenensis essential oil caused cell death in a dose‐ and time‐exposure‐dependent manner. The IC ₅₀ values in 3D cultures were higher than those in monolayer cultures, indicating greater resistance of spheroid cells to cell death. The L929 cell line, used as a model for normal cells, showed higher resistance to the cytotoxic effects of the T. daenensis essential oil, suggesting its selective toxicity toward cancer cells. Furthermore, the effects of T. daenensis essential oil on the activity of extracellular matrix metalloproteinases (proMMP‐2 and −9), key players in cancer invasion and metastasis, were assessed via zymography. T. daenensis essential oil significantly reduced proMMP‐2 activity at concentrations of 0.2 and 0.5 mg/mL and proMMP‐9 activity at 0.5 mg/mL in the MDA‐MB‐231 cell line. gas chromatography‐mass spectrometry analysis identified thymol (32.79%) as the most abundant compound in T. daenensis essential oil, followed by carvacrol (8.307%), p‐cymene (7.024%), β‐caryophyllene (7.023%), and caryophyllene oxide (6.39%). These findings suggest that T. daenensis essential oil has potent inhibitory effects on breast cancer cell proliferation and MMP activity, making it a promising candidate for further research.

This study evaluated the anticancer potential of Satureja sahendica essential oil (SSEO) against MDA-MB-231 (breast cancer) and A549 (lung cancer) cell lines. The chemical composition of SSEO, determined by GC-MS, revealed thymol (57.11%) and γ-terpinene (25.27%) as the major constituents. The MTT assay demonstrated that SSEO induced concentration- and time-dependent cytotoxicity, with IC₅₀ values of 0.69 mg/mL (24 h) and 0.28 mg/mL (72 h) for MDA-MB-231 cells, and 0.59 mg/mL (24 h) and 0.37 mg/mL (72 h) for A549 cells. Apoptosis was identified as the primary mode of cell death, confirmed by acridine orange/ethidium bromide dual staining and Annexin V-FITC/propidium iodide flow cytometry. Flow cytometric analysis quantified early apoptosis, showing 41.2% of treated MDA-MB-231 cells and 35.5% of treated A549 cells in the Annexin V⁺/PI^- quadrant. To elucidate the molecular basis of these effects, a comprehensive in silico analysis, incorporating target prediction, protein-protein interaction network, and gene ontology enrichment, was performed. This approach identified key cancer-related proteins, including MAPK14, PIM1, CTSD, TOP2A, PDK1, KIFC1, and NF-κB, as potential targets of the major SSEO constituents. Additionally, the cytotoxic effect of SSEO was evaluated on the normal L929 cell line, where IC₅₀ values of 1.082 mg/mL (24 h) and 0.89 mg/mL (72 h) were observed, indicating notably lower toxicity toward normal cells compared to cancer cells. Collectively, these findings demonstrate that SSEO exerts cytotoxic effects primarily through the induction of apoptosis in both breast and lung cancer cells, highlighting its potential as a complementary anticancer agent.

هدف: هدف این مطالعه ارزیابی تاثیر ترکیبی دو داروی مسدودکننده­ی کانال­ کلسیمی شامل آملودیپین و دیلتیازم بر سمیت دوکسوروبیسین در رده­های سلولی سرطان پستان است. مواد و روش‫ها: سلول­های MDA-MB-231 و MCF-7 با غلظت­های مختلف آملودیپین، دیلتیازم به‫صورت تکی و در ترکیب با دوکسوروبیسین به‫مدت 48 و 72 ساعت تیمار شدند. بقای سلولی با تست MTT بررسی شد. مقادیر IC50 از منحنی دوز-پاسخ، محاسبه شد. اثر ترکیبی داروها با محاسبه شاخص ترکیبی (CI) مورد بررسی قرار گرفت. فعالیت کاسپاز-3/7 به‫عنوان نشانگر القای آپوپتوزیس با روش سنجش آنزیمی مبتنی بر سوبسترای رنگ­زا (Ac-DEVE-pNA) در لیز سلول­های تیمارشده اندازه­گیری شد. نتایج: نتایج نشان داد که آملودیپین و دیلتیازم با برابر با اثر مهاری بر تکثیر سلول­های سرطانی MDA-MB-231 و MCF-7 به‫صورت وابسته به غلظت و زمان دارند. ارزیابی فعالیت کاسپاز-3/7 در لیز سلول‌های تیمارشده با داروها نشان داد که آملودیپین باعث افزایش فعالیت کاسپاز-3/7 در هر رده­ی سلولی می‌شود که نشان‌دهنده القای آپوپتوزیس است. در سلول‌های تیمارشده با دیلتیازم، فعالیت کاسپاز-3/7 تنها در سلول‌های MCF-7 مشاهده شد، در حالی‫که فعالیت کاسپاز-3/7 در سلول‌های MDA-MB-231 کم‫تر از کنترل بود که نتیجه­گیری در این...

Caspase-9 is crucial for initiating apoptosis, and its activity is tightly regulated through various mechanisms, especially phosphorylation by kinases activated by extracellular growth factors, osmotic stress, or during mitosis. Mass spectrometric analyses have shown that residues S302 and S307 in human caspase-9 are prone to phosphorylation. To investigate the effects of phosphorylation at these sites, three phosphomimetic variants of recombinant caspase-9 were created: S302D, S307D, and the combined S302D/S307D variant. The QuickChange method was employed to generate these mutant constructs, which were expressed in Escherichia coli (E. coli) and purified using affinity chromatography. For enzymatic assays, the chromogenic substrate Ac-LEHD-pNA was utilized, and the temperature profiles of enzyme activity were assessed. Computational modeling was used to predict the structures of the mutants, allowing for comparison with the native enzyme. The results indicated that both the S302D and S302D/S307D variants exhibited complete loss of enzyme activity. In contrast, the S307D variant demonstrated a 10-fold increase in the Michaelis constant (K_m) for the substrate and a 4-fold increase in the maximum reaction rate (V_max) compared to the wild-type enzyme. Notably, the k_cat/K_m value for wild-type caspase-9 was three times greater than that of the S307D variant. The optimal temperature for wild-type activity was between 30 and 37 °C, while for the S307D variant, it ranged from 37 to 45 °C. Importantly, the S302 residue is essential for caspase-9 function; introducing a negative charge at this position leads to complete inactivation of the enzyme.

Background: Breast cancer is one of the most common cancers in women. Current treatments, such as chemotherapy and radiation therapy, often involve numerous side effects. Consequently, it is essential to discover new anticancer compounds that are safer and more effective. Objectives: The aim of this research is to investigate the anticancer effects of Hymenocrater longiflorus Benth. essential oil. Methods: The anticancer properties of H. longiflorus Benth. essential oil were investigated on MDA-MB-231 and MCF-7 breast cancer cells using MTT assay, double staining with acridine orange/ethidium bromide, and flow cytometry. The essential oil of H. longiflorus Benth. was analyzed using gas chromatography-mass spectrometry (GC-MS). Results: The essential oil significantly reduced the viability of breast cancer cells depending on the concentration (dose) and duration. Results from double fluorescent staining and flow cytometric analysis also indicated that apoptosis is the primary mechanism of cell death. Non-targeted GC-MS metabolomic data analysis of the essential oil revealed the presence of various anticancer compounds, including α-pinene, linalool, and caryophyllene, in the essential oil. Conclusions: In conclusion, the essential oil of H. longiflorus Benth. is a promising source of anticancer metabolites that can be further evaluated for drug therapy or as a co-treatment with conventional chemotherapeutic agents.

Background and Aim: Caspase-9 is a key enzyme in the intrinsic pathway of apoptosis that its activity is regulated by various mechanisms such as phosphorylation. It has been reported that phosphorylation of serine 310 in murine caspase-9 prevents enzyme processing. The role of this residue in human caspase-9 activity in not ...

Safranal, crocin, crocetin, and picrocrocin are major known compounds in the stigma extract of Crocus sativus with various medicinal properties. Crocus cancellatus is another Crocus species that grows extensively in Irans various regions, such as the Kurdistan province. The predominant metabolites and biological properties of C. cancellatus have not yet been investigated. The ingredients of the stigma ethanol extract of C. cancellatus were investigated using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography with tandem mass spectrometry (LC-MS). The ROIMCR approach was performed to analyze the LC-MS full scan data sets. This method searches the MS regions of interest (ROI) data in the m/z domain and analyses the results using the multivariate curve-resolution alternating least squares (MCR-ALS) chemometrics technique for simultaneous resolution of two extracts. Also, the antiproliferative properties of C. cancellatus against MDA-MB-231 and MCF-7 cancer cells were examined by MTT, dual acridine orange/ethidium bromide test, Annexin V-FITC/PI, and zymography. The GC-MS and LC-MS untargeted metabolomics data analysis of the extract indicated the presence of cytotoxic agents including safranal, crocin, picrocrocin, and crocetin in the stigma ethanol extract of C. cancellatus. Biological tests showed that the viability of MDA-MB-231 and MCF-7 cancer cells is decreased following C. cancellatus treatment in a time- and dose-dependent way in both monolayer and 3D cell cultures. The MCF-7 cell spheroids had greater resistance to the cytotoxic activity of the extract in 3D cell culture than the MDA-MB-231 cell spheroids. The morphological changes of the cells treated with C. cancellatus stigmas extract were indicative of apoptosis. Zymography analysis revealed a similar trend of matrix metallopeptidase-2 (MMP-2) and matrix metallopeptidase-9 (MMP-9) activity in the treated cells with C. cancellatus extract in comparison with doxorubicin treatment as a positive control. The findings of this research indicate that the ethanolic extract of C. cancellatus stigmas was a good source of bioactive metabolites with anticancer activity.