2-(Methyl(phenyl)amino)-N-(phenyloxyphenyl)acetamide structural motif representing a framework for selective SIRT2 inhibition.

The mammalian cytoplasmic protein SIRT2, a class III histone deacetylase family member, possesses NAD+-dependent lysine deacetylase/deacylase activity. Dysregulation of SIRT2 has been implicated in the pathogenesis of several diseases, including neurological and metabolic disorders and cancer; thus, SIRT2 emerges as a potential therapeutic target. Herein, we identified a series of diaryl acetamides (ST61-ST90) by the structural optimization of our hit STH2, followed by enhanced SIRT2 inhibitory potency and selectivity. Among them, ST72, ST85, and ST88 selectively inhibited SIRT2 with IC50 values of 9.97, 5.74, and 8.92 µM, respectively. Finally, the entire study was accompanied by in silico prediction of binding modes of docked compounds and the stability of SIRT2-ligand complexes. We hope our findings will provide substantial information for designing selective inhibitors of SIRT2.

In silico approach reveals N-(5-phenoxythiophen-2-yl)-2-(arylthio)acetamides as promising selective SIRT2 inhibitors: the case of structural optimization of virtual screening-derived hits.

Epigenetic modifications play an essential role in tumor suppression and promotion. Among the diverse range of epigenetic regulators, SIRT2, a member of NAD+-dependent protein deacetylates, has emerged as a crucial regulator of cellular processes, including cell cycle progression, DNA repair, and metabolism, impacting tumor growth and survival. In the present work, a series of N-(5-phenoxythiophen-2-yl)-2-(arylthio)acetamide derivatives were identified following a structural optimization of previously reported virtual screening hits, accompanied by enhanced SIRT2 inhibitory potency. Among the compounds, ST44 and ST45 selectively inhibited SIRT2 with IC50 values of 6.50 and 7.24 µM, respectively. The predicted binding modes of the two compounds revealed the success of the optimization run. Moreover, ST44 displayed antiproliferative effects on the MCF-7 human breast cancer cell line. Further, the contribution of SIRT2 inhibition in this effect of ST44 was supported by western blotting, affording an increased α-tubulin acetylation. Furthermore, molecular dynamics (MD) simulations and binding free energy calculations using molecular mechanics/generalized born surface area (MM-GBSA) method evaluated the accuracy of predicted binding poses and ligand affinities. The results revealed that ST44 exhibited a remarkable level of stability, with minimal deviations from its initial docking conformation. These findings represented a significant improvement over the virtual screening hits and may contribute substantially to our knowledge for further selective SIRT2 drug discovery.Communicated by Ramaswamy H. Sarma.

Cuproptosis as the new kryptonite of cancer: a copper-dependent novel cell death mechanism with promising implications for the treatment of hepatocellular carcinoma.

Copper is an essential element for critical cellular functions such as mitochondrial respiration, cholesterol biosynthesis and immune response. Altered copper homeostasis has been associated with various disorders, including cancer. The copper overload is known to contribute to tumorigenesis, angiogenesis and metastasis, and recently it has been suggested that the elevated level of this element may also create vulnerability to a novel cell death mechanism, named cuproptosis. Excessive amount of copper in mitochondria binds to lipoylated enzymes of the TCA cycle and forms insoluble oligomers. The aggregation of these oligomers and subsequent iron-sulfur cluster protein loss results in proteotoxic stress and eventual cell death. Hepatocellular carcinoma is a common malignancy with a low survival rate, despite the available treatment options. The discovery of cuproptosis led many researchers to explore its potential use in hepatocellular cancer therapy due to the rich mitochondria content of hepatic cells. In this regard, a number of genomic studies were conducted to discover several cuproptosis-related genes and explored their association with prognosis, survival and immunotherapy response. This review brings together the available data on the relationship between cuproptosis and hepatocellular cancer for the first time, and highlights some of the potential biomarkers or target molecules that may be useful in the treatment.

Etoposide in combination with erastin synergistically altered iron homeostasis and induced ferroptotic cell death through regulating IREB2/FPN1 expression in estrogen receptor positive-breast cancer cells.

AIM: Ferroptosis is an iron-dependent cell death mechanism that substantially differs from apoptosis. Since its mechanism involves increased oxidative stress and rich iron content, cancer cells are particularly vulnerable to ferroptotic death compared to healthy tissues. In the present study, the effect of etoposide in combination with a ferroptotic agent, erastin, was investigated in breast cancer. MAIN METHODS: Cell viability was assessed by the MTT assay. Oxidative stress, lipid peroxidation and glutathione peroxidase activity were detected using the relevant kits. Intracellular iron levels were measured by HPLC. Ferroptosis markers were explored by western blotting. KEY FINDINGS: Results demonstrated that although etoposide didn't induce a significant cell death up to 50 µM in MCF-7 cells, with the addition of erastin, a significant synergistic activity was achieved at a dose as low as 1 µM (p < 0.05), contrary to normal breast epithelial cells. This cytotoxic effect was blocked by ferrostatin-1, which is a specific inhibitor of ferroptosis. The combined treatment of etoposide and erastin synergistically induced oxidative stress and lipid peroxidation, while suppressing glutathione peroxidase activity. More importantly, the combination treatment synergistically increased iron accumulation, which was associated with altered expression of IREB2/FPN1. Additionally, ferroptosis-regulating proteins ACSF2 and GPX4 were altered more potently by the combination treatment, compared to untreated cells and erastin treatment alone (p < 0.05). SIGNIFICANCE: In conclusion, this is the first study that reports enhanced cytotoxicity of etoposide, in combination with erastin, in ER-positive breast cancer cells via activation of ferroptotic pathways, and offers a new perspective for future regimens.

Investigation on formulation parameters of donepezil HCl loaded solid lipid nanoparticles

Abstract Donepezil-HCl is a member of the acetylcholinesterase inhibitors that is indicated for the symptomatic treatment of Alzheimer's disease (AD) and has many side effects. In this study, to reduce the side effects of Donepezil-HCl and increase the penetration of the drug through the blood-brain barrier, we aimed to design a solid lipid nanoparticle (SLN) formulation. The effects of the different formulation parameters, such as homogenization speed, sonication time, lipid and drug concentration, surfactant type and concentration, and volume of the aqueous phase, were assessed for optimization. The particle size and PDI increased with increasing lipid concentration but decreased with increasing amounts of surfactant (Tween 80) and co-surfactant (lecithin). When the homogenization rate and sonication time increased, the particle size decreased and the encapsulation efficiency increased. The optimized formulation exhibited particle size, PDI, encapsulation efficiency, and zeta potential of 87.2±0.11 nm; 0.22±0.02; 93.84±0.01 %; -17.0±0.12 mV respectively. The in vitro release investigation revealed that approximately 70% of Donepezil-HCl was cumulatively released after 24 hours. TEM analysis proved that spherical and smooth particles were obtained and formulations had no toxic effect on cells. The final optimized formulation could be a candidate for Donepezil-HCl application in Alzheimer's treatment with reduced side effects and doses for patients

Atorvastatin induces downregulation of matrix metalloproteinase-2/9 in MDA-MB-231 triple negative breast cancer cells.

Matrix metalloproteinases (MMPs) are a family of endopeptidases, mainly responsible of extracellular tissue remodeling. Abundant expression of MMPs leads to a number of tumorigenic processes including proliferation, angiogenesis, metastasis and invasion. Therefore, suppressing MMP expression is particularly important in cancer. Atorvastatin is a member of statin family, with cholesterol-lowering properties. Recently, it has emerged as a potential anticancer agent. Multiple researchers have reported promising results of atorvastatin use in cancer therapies. However, its effect on the expression of matrix metalloproteinases in breast cancer is unknown. In the present study, we have confirmed the apoptotic activity of atorvastatin on highly metastatic MDA-MB-231 triple negative breast cancer cells and investigated the gene expression of MMP-2/9. In this regard, MTT analysis was performed to evaluate cytotoxicity. Apoptotic activity was assessed by Annexin V binding and multicaspase assays. Western blot analysis was used to detect the apoptosis-related proteins. RT-PCR analysis was performed to evaluate the mRNA expression levels of MMP-2/9. Results indicated that atorvastatin reduces cell viability significantly at 5 µM after 48 h of treatment (p < 0.0001). It also induces caspase-dependent apoptosis, alters the expression of Bax and Bcl-2 in favour of apoptosis and stimulates cell cycle arrest at S phase (p < 0.05). Moreover, atorvastatin downregulates the mRNA expression of MMP-2 and MMP-9 significantly (p < 0.05). In conclusion, these results demonstrate for the first time that atorvastatin inhibits MMP-2 and MMP-9 gene expression in MDA-MB-231 cells, in addition to inducing caspase-dependent apoptosis.

Box-Behnken design optimization and in vitro cell based evaluation of piroxicam loaded core-shell type hybrid nanocarriers for prostate cancer.

In the present research, piroxicam entrapped core-shell lipid-polymer hybrid nanocarriers were developed and also evaluated in terms of nanoparticle features and cell-based in vitro efficacy on prostate cancer cells. Box-Behnken optimization approach was implemented to evaluate the impact of the input variables, namely phospholipid/PLGA ratio, total lipids/lecithin molar ratio, and piroxicam concentration, on two output variables: particle size and entrapment efficiency. Surface charge, size distribution, morphological structure of particles, drug release profiles, presence of outer lipid shell, thermal profile and possible interactions and storage stability of core-shell nanocarriers of piroxicam were studied as particle features. Cell viability, apoptosis and cell cycle arrest studies were utilized for in vitro cell-based evaluation of the core-shell nanosystems. The hybrid nanocarrier formulation with a particle size of 119.2 nm and an entrapment efficiency of 91.7% at the center point of the design was selected as the optimized formulation according to the desired function (d) method applied within the scope of the Box-Behnken design approach and RSM strategy. The cell viability and apoptosis experiments were performed on the optimized nanocarrier. In conclusion, this study demonstrates that the optimized core-shell nanoformulation of piroxicam is a more promising strategy in the treatment of prostate cancer compared to the pure molecule.

Hit evaluation results in 5-benzyl-1,3,4-thiadiazole-2-carboxamide based SIRT2-selective inhibitor with improved affinity and selectivity.

Sirtuin 2 (SIRT2), member of sirtuin family, belongs to class III histone deacetylases (HDACs) and is majorly cytosolic with occasional nuclear translocation. The enzymatic activity of SIRT2 is dependent on nicotinamide adenine dinucleotide (NAD+) and SIRT2 regulates post-translational modifications that are responsible for deacetylation of lysine residues in histone and non-histone substrates. SIRT2, thus affects most likely multiple cellular processes, such as signaling, gene expression, aging, autophagy, and has been identified as potential drug target in relation to inflammation, neurodegenerative diseases and cancer. Therefore, probing potential selective inhibitors is essential for the accurate understanding of enzyme functions. Here, we report a series of heteroaryl-2-carboxamide hybrids bearing substituted benzyl or substituted phenoxy group at the 5-position of the central heterocyclic ring. The synthesized compounds were screened against SIRT1-3 and MCF-7 human breast cancer cell line to evaluate their biological activity. The best SIRT2 inhibition profiles were displayed by ST29 (SIRT2 IC50 = 38.69 µM) and ST30 (SIRT2 IC50 = 43.29 µM) with excellent selectivity against SIRT2 over SIRT1 and SIRT3. Molecular docking study of the synthesized compounds into SIRT2 active site was performed to rationalize the remarkable SIRT2 inhibitory activity. Furthermore, we performed all-atom, explicit-solvent molecular dynamics (MD) simulations and end-point binding free energy calculations using molecular mechanics/generalized Born surface area (MM/GBSA) method to evaluate whether this design strategy was successfully deployed. The results implied that the binding poses and ligand affinities were predicted without significant loss of accuracy. Conclusively, the developed chemotypes were advocated as promising leads for SIRT2 inhibition and required further investigation for SIRT2-targeted drug discovery and development.

Ferroptosis: A Trusted Ally in Combating Drug Resistance in Cancer.

Ferroptosis, which is an iron-dependent, non-apoptotic cell death mechanism, has recently been proposed as a novel approach in cancer treatment. Bearing distinctive features and its exclusive mechanism have put forward the potential therapeutic benefit of triggering this newly discovered form of cell death. Numerous studies have indicated that apoptotic pathways are often deactivated in resistant cells, leading to a failure in therapy. Hence, alternative strategies to promote cell death are required. Mounting evidence suggests that drug-resistant cancer cells are particularly sensitive to ferroptosis. Given that cancer cells consume a higher amount of iron than healthy ones, ferroptosis not only stands as an excellent alternative to trigger cell death and reverse drug-resistance, but also provides selectivity in therapy. This review focuses specifically on overcoming drugresistance in cancer through activating ferroptotic pathways and brings together the relevant chemotherapeutics-based and nanotherapeutics-based studies to offer a perspective for researchers regarding the potential use of this mechanism in developing novel therapeutic strategies.

Potentiation of the Effect of Lonidamine by Quercetin in MCF-7 human breast cancer cells through downregulation of MMP-2/9 mRNA Expression.

Combination therapies are becoming increasingly important to develop an effective treatment in cancer. Lonidamine is frequently used in cancer treatment, but it's often preferred to be used in combination with other drugs because of its side effects. In the present study, the efficacy of the combination of lonidamine with quercetin, a flavonoid of natural origin, on human MCF-7 breast cancer cells was evaluated. The results showed that the combined use of the compounds significantly increased cytotoxicity compared to administration alone (p<0.0001). In addition, while lonidamine induced a cell cycle arrest in the G2/M phase, administration of quercetin and its combination with lonidamine arrested the cell division at S point, indicating the synergistic strength of quercetin on cytotoxicity. The combination of quercetin and lonidamine significantly induced apoptosis of MCF-7 cells (p<0.0001) and increased caspase levels (p<0.0001). In this study, the combination of quercetin and lonidamine has been evaluated for the first time and the combination treatment decreased MMP-2/-9 mRNA expression more potently than the effects of the compounds alone. The results showed that lonidamine was more effective when combined with quercetin, and their combination may be a candidate for a novel strategy of treatment for breast cancer.

Encapsulation of cucurbitacin B into lipid polymer hybrid nanocarriers induced apoptosis of MDAMB231 cells through PARP cleavage.

In the present study, we developed the lipid polymer hybrid nanoparticles of cucurbitacin B (CuB) and evaluated its effects on triple negative breast cancer cells. The 32 factorial design was utilized to understand the influence of input variables including PEG-conjugated phospholipid/biodegradable polymer ratio and the total lipids/lecithin molar percentage ratio. The hybrid formulation at the center point of design was specified as optimal hybrid nanocarrier due to its superior features. CuB loaded nanoparticles (CuB-NP) inhibited cell growth through a cell cycle arrest at G0/G1 phase. The studies investigating the efficacy of CuB-NP on apoptosis of cancer cells showed that the annexin v-bound cell population was 20.66 ± 1.99%, and the depolarized cell population was higher in CuB-NP treated cells. The pro-apoptotic bax, Iκb-α and cleaved PARP levels increased in CuB-NP treated cells, while anti-apoptotic Bcl-2 and NF-κB levels decreased. The caspase (+) cell population was higher in nanoparticle-treated group as 14.20 ± 0.56% and the findings obtained from the caspase assay were also compatible with western blot data. Overall, both CuB and CuB-NP demonstrated anticancer activity, while the lipid polymer hybrid nanoparticle formulation of CuB indicated that the nanoparticle formulation has more promising effect for the treatment of breast cancer.

The Trinity of Matrix Metalloproteinases, Inflammation, and Cancer: A Literature Review of Recent Updates.

The critical link between cancer and inflammation has been known for many years. This complex network was further complexed by revealing the association of the matrix metalloproteinase family members with inflammatory cytokines, which were previously known to be responsible for the development of metastasis. This article summarizes the current studies which evaluate the relationship between cancer and inflammatory microenvironment as well as the roles of MMPs on invasion and metastasis together.

The combined treatment of brassinin and imatinib synergistically downregulated the expression of MMP-9 in SW480 colon cancer cells.

In cancer treatment, which is a major cause of mortality today, combination studies with clinically used chemotherapeutics are becoming increasingly important as much as investigating the effects of novel natural compounds. In this context, phytoalexins constitute an important group due to their unique structure. Brassinin is an essential indole phytoalexin and is a biosynthetic precursor for other phytoalexins. The purpose of this study was to evaluate the anticancer effects of brassinin in combination with imatinib in SW480 cells. In the study, it was observed that brassinin-imatinib combination significantly increased cytotoxicity compared with the single treatment of both compounds and inhibited cell cycle at G0/G1 phase. Annexin V binding and fluorescence imaging assays showed that the combination of brasinin-imatinib induces apoptosis in a dose-dependent manner. Furthermore, the effect of brassinin on the activity of MMP-9 in SW480 cells was evaluated for the first time, and it was detected that MMP-9 activity was significantly reduced. The combination of brassinin-imatinib was found to inhibit MMP-9 activity as well as relative MMP-9 gene expression on a higher level compared with control and compounds alone. Our findings have revealed that the combination of brassinin-imatinib synergistically induces cytotoxicity and apoptosis in SW480 cells. The findings on MMP-9 downregulation have also revealed the anti-metastatic potential of treatment.