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Breast and lung cancers are the leading causes of cancer-related deaths in the world. Although considerable progress has been made in the field of cancer therapy, quest to discover potent, safe and cost-effective alternatives especially from natural sources is being pursued. Snake venom, which is a treasure trove of various peptides and proteins including natural toxins that specifically target tissues and receptors in the envenomated victims. Many such proteins are being explored for their therapeutic potential against various diseases including cancers. Here, we report the mechanism of cytotoxic activity of crude venom and a purified protein, Cytotoxin from the monocled cobra (Naja kaouthia), an elapid snake with neurotoxic venom prominently found in the North-East India. The crude venom showed significant cytotoxicity against breast (MCF-7and MDA-MB-231) and lung (A549, NCI-H522) cancer cell lines. Bioassay-guided fractionation using RP-HPLC showed highest cytotoxic activity in peak P9. Liquid chromatography-tandem mass spectrometry (ESI-LC-MS/MS) analysis was employed and the fraction is identified as Cytotoxin 10 which showed comparable cytotoxicity against the experimental cell lines. Cytotoxin 10 also exhibited apoptosis in MCF-7 and A549 cell lines using AO/EtBr and flow cytometry analysis. Expressions of apoptosis related proteins e.g. Bax, Bcl-2, Caspase-7 and PARP were also studied following Cytotoxin 10 treatment in both cell lines. Molecular docking experiments performed to investigate the interactions between Cytotoxin 10 and the apoptotic proteins revealed favourable binding scores compared to their corresponding inhibitors. Interestingly, Cytotoxin 10 inhibited migration and adhesion in a time and dose-dependent manner in both MCF-7 and A549 cells. This is the first report elucidating the mechanism of cytotoxic activity of Cytotoxin 10 purified from Naja kaouthia venom of North-East India origin and could pave the way for development of potential therapeutic strategies against breast and lung cancer., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Robin Doley reports financial support was provided by Tezpur University. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Oleanolic acid, a naturally occurring triterpenoid compound, has garnered significant attention in the scientific community due to its diverse pharmacological properties. Continuing our previous work on the synthesis of oleanolic acid dimers (OADs), a simple, economical, and safe acetylation reaction was performed. The newly obtained derivatives (AcOADs, 3a - 3n ) were purified using two methods. The structures of all acetylated dimers ( 3a - 3n ) were determined based on spectral methods (IR, NMR). For all AcOADs ( 3a - 3n ), the relationship between the structure and the expected directions of pharmacological activity was determined using a computational method (QSAR computational analysis). All dimers were also tested for their cytotoxic activity on the SKBR-3, SKOV-3, PC-3, and U-87 cancer cell lines. HDF cell line was applied to evaluate the Selectivity Index of the tested compounds. All cytotoxic tests were performed with the application of the MTT assay. Finally, all dimers of oleanolic acid were subjected to DPPH and CUPRAC tests to evaluate their antioxidant activity. The obtained results indicate a very high level of cytotoxic activity (IC 50 for most AcOADs below 5.00 µM) and a fairly high level of antioxidant activity (Trolox equivalent in some cases above 0.04 mg/mL)., Competing Interests: The authors declare no conflicts of interest.

In this paper, we provide an overview of mitochondrial bioenergetics and specific conditions that lead to the formation of non-bilayer structures in mitochondria. Secondly, we provide a brief overview on the structure/function of cytotoxins and how snake venom cytotoxins have contributed to increasing our understanding of ATP synthesis via oxidative phosphorylation in mitochondria, to reconcile some controversial aspects of the chemiosmotic theory. Specifically, we provide an emphasis on the biochemical contribution of delocalized and localized proton movement, involving direct transport of protons though the F o unit of ATP synthase or via the hydrophobic environment at the center of the inner mitochondrial membrane (proton circuit) on oxidative phosphorylation, and how this influences the rate of ATP synthesis. Importantly, we provide new insights on the molecular mechanisms through which cobra venom cytotoxins affect mitochondrial ATP synthesis, mitochondrial structure, and dynamics. Finally, we provide a perspective for the use of cytotoxins as novel pharmacological tools to study membrane bioenergetics and mitochondrial biology, how they can be used in translational research, and their potential therapeutic applications.

Background: Current immunotherapies with unexpected severe side effects and treatment resistance have not resulted in the desired outcomes for patients with melanoma, and there is a need to discover more effective medications. Cytotoxin (CTX) from Cobra Venom has been established to have favorable cytolytic activity and antitumor efficacy and is regarded as a promising novel anticancer agent. However, amphiphilic CTX with excellent anionic phosphatidylserine lipid-binding ability may also damage normal cells., Methods: We developed pH-responsive liposomes with a high CTX load (CTX@PSL) for targeted acidic-stimuli release of drugs in the tumor microenvironment. The morphology, size, zeta potential, drug-release kinetics, and preservation stability were characterized. Cell uptake, apoptosis-promoting effects, and cytotoxicity were assessed using MTT assay and flow cytometry. Finally, the tissue distribution and antitumor effects of CTX@PSL were systematically assessed using an in vivo imaging system., Results: CTX@PSL exhibited high drug entrapment efficiency, drug loading, stability, and a rapid release profile under acidic conditions. These nanoparticles, irregularly spherical in shape and small in size, can effectively accumulate at tumor sites (six times higher than free CTX) and are rapidly internalized into cancer cells (2.5-fold higher cell uptake efficiency). CTX@PSL displayed significantly stronger cytotoxicity (IC 50 0.25 μg/mL) and increased apoptosis in than the other formulations (apoptosis rate 71.78±1.70%). CTX@PSL showed considerably better tumor inhibition efficacy than free CTX or conventional liposomes (tumor inhibition rate 79.78±5.93%)., Conclusion: Our results suggest that CTX@PSL improves tumor-site accumulation and intracellular uptake for sustained and targeted CTX release. By combining the advantages of CTX and stimuli-responsive nanotechnology, the novel CTX@PSL nanoformulation is a promising therapeutic candidate for cancer treatment., Competing Interests: The authors declare no conflicts of interest., (© 2024 Lin et al.)

Purpose: The study aimed to address the non-specific toxicity of cytotoxins (CTX) in liver cancer treatment and explore their combined application with the photosensitizer Ce6, co-loaded into carbonized Zn/Co bimetallic organic frameworks. The goal was to achieve controlled CTX release and synergistic photodynamic therapy, with a focus on evaluating anti-tumor activity against human liver cancer cell lines (Hep G2)., Methods: Purified cobra cytotoxin (CTX) and photosensitizer Ce6 were co-loaded into carbonized Zn/Co bimetallic organic frameworks, resulting in RGD-PDA@C-ZIF@(CTX+Ce6). The formulation was designed with surface-functionalization using polydopamine and tumor-penetrating peptide RGD. This approach aimed to facilitate controlled CTX release and enhance the synergistic effect of photodynamic therapy. The accumulation of RGD-PDA@C-ZIF@(CTX+Ce6) at tumor sites was achieved through RGD's active targeting and the enhanced permeability and retention (EPR) effect. In the acidic tumor microenvironment, the porous structure of the metal-organic framework disintegrated, releasing CTX and Ce6 into tumor cells., Results: Experiments demonstrated that RGD-PDA@C-ZIF@(CTX+Ce6) nanoparticles, combined with near-infrared laser irradiation, exhibited optimal anti-tumor effects against human liver cancer cells. The formulation showcased heightened anti-tumor activity without discernible systemic toxicity., Conclusion: The study underscores the potential of utilizing metal-organic frameworks as an efficient nanoplatform for co-loading cytotoxins and photodynamic therapy in liver cancer treatment. The developed formulation, RGD-PDA@C-ZIF@(CTX+Ce6), offers a promising avenue for advancing the clinical application of cytotoxins in oncology, providing a solid theoretical foundation for future research and development., Competing Interests: The authors declare no competing interests in this work., (© 2024 Huang et al.)

This study focuses on understanding the structural and molecular changes in lipid membranes under the influence of six halogenated flavonoid derivatives differing in the number and position of substitution of chlorine and bromine atoms (D1-D6). Utilizing various analytical techniques, including fluorometric methods, dynamic light scattering (DLS), attenuated Fourier transform infrared spectroscopy (ATR- FTIR), and FT-Raman spectroscopy, the research aims to elucidate the mechanisms underlying the interaction of flavonoids with cell membranes. Additionally, the study includes in silico analyses to explore the physicochemical properties of these compounds and their potential pharmaceutical applications, along with toxicity studies to assess their effects on cancer, normal, and red blood cells. Our study showed the ability of halogenated derivatives to interact mostly with the outer part of the membrane, especially in the lipid heads region however, some of them were able to penetrate deeper into the membrane and affect the fluidity of hydrocarbon chains. The potential to reduce cancer cell viability, the lack of toxicity towards erythrocytes, and the favourable physicochemical and pharmacokinetic properties suggest these halogenated flavonoids potential candidates for exploring their potential for medical use., (© 2024. The Author(s).)

Snake venoms are a potential source of bioactive peptides, which have multiple therapeutic properties in treating diseases such as diabetes, cancer, and neurological disorders. Among bioactive peptides, cytotoxins (CTXs) and neurotoxins are low molecular weight proteins belonging to the three-finger-fold toxins (3FTxs) family composed of two β sheets that are stabilized by four to five conserved disulfide bonds containing 58-72 amino acid residues. These are highly abundant in snake venom and are predicted to have insulinotropic activities. In this study, the CTXs were purified from Indian cobra snake venom using preparative HPLC and characterized using high-resolution mass spectrometry (HRMS) TOF-MS/MS. Further SDS-PAGE analysis confirmed the presence of low molecular weight cytotoxic proteins. The CTXs in fractions A and B exhibited dose-dependent insulinotropic activity from 0.001 to 10 µM using rat pancreatic beta-cell lines (RIN-5F) in the ELISA. Nateglinide and repaglinide are synthetic small-molecule drugs that control sugar levels in the blood in type 2 diabetes, which were used as a positive control in ELISA. Concluded that purified CTXs have insulinotropic activity, and there is a scope to use these proteins as small molecules to stimulate insulinotropic activities. At this stage, the focus is on the efficiency of the cytotoxins to induce insulin. Additional work is ongoing on animal models to see the extent of the beneficial effects and efficiency to cure diabetes using streptozotocin-induced models., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The pathophysiology of lung cancer is dependent on the dysregulation in the apoptotic and autophagic pathways. The intricate link between apoptosis and autophagy through shared signaling pathways complicates our understanding of how lung cancer pathophysiology is regulated. As drug resistance is the primary reason behind treatment failure, it is crucial to understand how cancer cells may respond to different therapies and integrate crosstalk between apoptosis and autophagy in response to them, leading to cell death or survival. Thus, in this study, we have tried to evaluate the crosstalk between autophagy and apoptosis in A549 lung cancer cell line that could be modulated by employing a combination therapy of metformin (6 mM), an anti-diabetic drug, with gedunin (12 µM), an Hsp90 inhibitor, to provide insights into the development of new cancer therapeutics. Our results demonstrated that metformin and gedunin were cytotoxic to A549 lung cancer cells. Combination of metformin and gedunin generated ROS and promoted MMP loss and DNA damage. The combination further increased the expression of AMPKα1 and promoted the nuclear localization of AMPKα1/α2. The expression of Hsp90 was downregulated, further decreasing the expression of its clients, EGFR, PIK3CA, AKT1, and AKT3. Inhibition of the EGFR/PI3K/AKT pathway upregulated TP53 and inhibited autophagy. The combination was promoting nuclear localization of p53; however, some cytoplasmic signals were also detected. Further increase in the expression of caspase 9 and caspase 3 was observed. Thus, we concluded that the combination of metformin and gedunin upregulates apoptosis by inhibiting the EGFR/PI3K/AKT pathway and autophagy in A549 lung cancer cells., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Several studies have indicated the potential therapeutic outcomes of combining selective COX-2 inhibitors with tubulin-targeting anticancer agents. In the current study, a novel series of thiazolidin-4-one-based derivatives (7a-q) was designed by merging the pharmacophoric features of some COXs inhibitors and tubulin polymerization inhibitors. Compounds 7a-q were synthesized and evaluated for their cytotoxic activity against MCF7, HT29, and A2780 cancer cell lines (IC 50  = 0.02-17.02 μM). The cytotoxicity of 7a-q was also assessed against normal MRC5 cells (IC 50  = 0.47-13.46 μM). Compounds 7c, 7i, and 7j, the most active in the MTT assay, significantly reduced the number of HT29 colonies compared to the control. Compounds 7c, 7i, and 7j also induced significant decreases in the tumor volumes and masses in Ehrlich solid carcinoma-bearing mice compared to the control. The three compounds also exhibited significant anti-HT29 migration activity in the wound-healing assay. They have also induced cell cycle arrest in HT29 cells at the S and G 2 /M phases. In addition, they induced significant increases in both early and late apoptotic events in HT29 cells compared to the control, where 7j showed the highest effect. On the other hand, compound 7j (1 μM) displayed weak inhibitory activity against tubulin polymerization compared to colchicine (3 μM). On the other hand, compounds 7a-q inhibited the activity of COX-2 (IC 50  = 0.42-29.11 μM) compared to celecoxib (IC 50  = 0.86 μM). In addition, 7c, 7i, and 7j showed moderate inhibition of inflammation in rats compared to indomethacin, with better GIT safety profiles. Molecular docking analysis revealed that 7c, 7i, and 7j have higher binding free energies towards COX-2 than COX-1. These above results suggested that 7j could serve as a potential anticancer drug candidate., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Plasma membrane-induced protein folding and conformational transitions play a central role in cellular homeostasis. Several transmembrane proteins are folded in the complex lipid milieu to acquire a specific structure and function. Bacterial pore forming toxins (PFTs) are proteins expressed by a large class of pathogenic bacteria that exploit the plasma membrane environment to efficiently undergo secondary structure changes, oligomerize, and form transmembrane pores. Unregulated pore formation causes ion imbalance, leading to cell death and infection. Determining the free energy landscape of these membrane-driven-driven transitions remains a challenging problem. Although cholesterol recognition is required for lytic activity of several proteins in the PFT family of toxins, the regulatory role of cholesterol for the α-PFT, cytolysin A expressed by Escherichia coli remains unexplained. In a recent free energy computation, we showed that the β tongue, a critical membrane-inserted motif of the ClyA toxin, has an on-pathway partially unfolded intermediate that refolds into the helix-turn-helix motif of the pore state. To understand the molecular role played by cholesterol, we carry out string-method-based computations in membranes devoid of cholesterol, which reveals an increase of ∼30 times in the free energy barrier for the loss of β sheet secondary structure when compared with membranes containing cholesterol. Specifically, the tyrosine-cholesterol interaction was found to be critical to creating the unfolded intermediate. Cholesterol also increases the packing and hydrophobicity of the bilayer, resulting in enhanced interactions of the bound protein before complete membrane insertion. Our study illustrates that cholesterol is critical to catalyzing and stabilizing the membrane-inserted unfolded state of the β tongue motif of ClyA, opening up fresh insights into cholesterol-assisted unfolding of membrane proteins., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The semisynthesis of renieramycin-type derivatives was achieved under mild and facile conditions by attaching a 1,3-dioxole-bridged phenolic moiety onto ring A of the renieramycin structure and adding a 4'-pyridinecarbonyl ester substituent at its C-5 or C-22 position. These were accomplished through a light-induced intramolecular photoredox reaction using blue light (4 W) and Steglich esterification, respectively. Renieramycin M ( 4 ), a bis-tetrahydroisoquinolinequinone compound isolated from the Thai blue sponge ( Xestospongia sp.), served as the starting material. The cytotoxicity of the 10 natural and semisynthesized renieramycins against non-small-cell lung cancer (NSCLC) cell lines was evaluated. The 5- O -(4'-pyridinecarbonyl) renieramycin T ( 11 ) compound exhibited high cytotoxicity with half-maximal inhibitory concentration (IC 50 ) values of 35.27 ± 1.09 and 34.77 ± 2.19 nM against H290 and H460 cells, respectively. Notably, the potency of compound 11 was 2-fold more than that of renieramycin T ( 7 ) and equal to those of 4 and doxorubicin. Interestingly, the renieramycin-type derivatives with a hydroxyl group at C-5 and C-22 exhibited weak cytotoxicity. In silico molecular docking and dynamics studies confirmed that the mitogen-activated proteins, kinase 1 and 3 (MAPK1 and MAPK3), are suitable targets for 11 . Thus, the structure-cytotoxicity study of renieramycins was extended to facilitate the development of potential anticancer agents for NSCLC cells.

Levofloxacin, the optical S-(-) isomer of ofloxacin, is a broad-spectrum antibacterial agent widely used to control various infections caused by Gram-positive and Gram-negative bacteria. While the COOH group is necessary for antibacterial activity, its modification can offer anticancer activity to the fluoroquinolone framework. Therefore, several levofloxacin carboxamides 11a-j and 12 containing 5-substituted-1,3,4-thiadiazole residue were synthesized and screened in vitro for their anticancer activity. The in vitro MTT viability assay revealed that the most compounds had significant activity against cancer cells MCF-7, A549, and SKOV3. In particular, the 3-chloro- and 4-fluoro- benzyl derivatives (11b and 11h), with IC 50 values of 1.69-4.76 μM were as potent as or better than doxorubicin. It should be noted that the mother quinolone levofloxacin showed no activity on the tested cancer cell lines. The SAR analysis demonstrated that the 3-chloro or 4-fluoro substituent on the S-benzyl moiety had positive effect on the activity. Further in vitro evaluations of the most promising compounds 11b and 11h by flow cytometric analysis and comet test revealed the ability of compounds in the induction of apoptosis and blockage of the cell proliferation at the G1-phase by nuclear fragmentation and DNA degradation in cancer cells. The obtained results demonstrated that the alteration of 6-COOH functional group in the levofloxacin structure and conjugation with a proper heterocyclic pharmacophore is a good strategy to obtain new anticancer agents., (© 2023 Wiley Periodicals LLC.)

Metformin can enhance cancer cell chemosensitivity to anticancer drugs. IGF-1R is involved in cancer chemoresistance. The current study aimed to elucidate the role of metformin in osteosarcoma (OS) cell chemosensitivity modulation and identify its underlying mechanism in IGF-1R/miR-610/FEN1 signalling. IGF-1R, miR-610, and FEN1 were aberrantly expressed in OS and participated in apoptosis modulation; this effect was abated by metformin treatment. Luciferase reporter assays confirmed that FEN1 is a direct target of miR-610. Moreover, metformin treatment decreased IGF-1R and FEN1 but elevated miR-610 expression. Metformin sensitised OS cells to cytotoxic agents, while FEN1 overexpression partly compromised metformin's sensitising effects. Furthermore, metformin was observed to enhance adriamycin's effects in a murine xenograft model. Metformin enhanced OS cell sensitivity to cytotoxic agents via the IGF-1R/miR-610/FEN1 signalling axis, highlighting its potential as an adjuvant during chemotherapy.

Microglia are the resident immune cells of the brain which regulate both the innate and adaptive neuroimmune responses in health and disease. In response to specific endogenous and exogenous stimuli, microglia transition to one of their reactive states characterized by altered morphology and function, including their secretory profile. A component of the microglial secretome is cytotoxic molecules capable of causing damage and death to nearby host cells, thus contributing to the pathogenesis of neurodegenerative disorders. Indirect evidence from secretome studies and measurements of mRNA expression using diverse microglial cell types suggest different stimuli may induce microglia to secrete distinct subsets of cytotoxins. We demonstrate the accuracy of this hypothesis directly by challenging murine BV-2 microglia-like cells with eight different immune stimuli and assessing secretion of four potentially cytotoxic molecules, including nitric oxide (NO), tumor necrosis factor α (TNF), C-X-C motif chemokine ligand 10 (CXCL10), and glutamate. Lipopolysaccharide (LPS) and a combination of interferon (IFN)-γ plus LPS induced secretion of all toxins studied. IFN-β, IFN-γ, polyinosinic:polycytidylic acid (poly I:C), and zymosan A upregulated secretion of subsets of these four cytotoxins. LPS and IFN-γ, alone or in combination, as well as IFN-β induced toxicity of BV-2 cells towards murine NSC-34 neuronal cells, while ATP, N-formylmethionine-leucyl-phenylalanine (fMLP), and phorbol 12-myristate 13-acetate (PMA) did not affect any parameters studied. Our observations contribute to a growing body of knowledge on the regulation of the microglial secretome, which may inform future development of novel therapeutics for neurodegenerative diseases, where dysregulated microglia are key contributors to pathogenesis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Oncological diseases are difficult to treat even with strong drugs due to development the multidrug resistance (MDR) of cancer cells. A strategy is proposed to increase the efficiency and selectivity of cytotoxic agents against cancer cells to engage the differences in the morphology and microenvironment of tumor and healthy cells, including the pH, membrane permeability, and ion channels. Using this approach, we managed to develop enhanced formulations of cytotoxic agents with adjuvants (which are known as efflux inhibitors and as ion channel inhibitors in tumors)-with increased permeability in A549 and a protective effect on healthy HEK293T cells. The composition of the formulation is as follows: cytotoxic agents (doxorubicin (Dox), paclitaxel (Pac), cisplatin) + adjuvants (allylbenzenes and terpenoids) in the form of inclusion complexes with β-cyclodextrin. Modified cyclodextrins make it possible to obtain soluble forms of pure substances of the allylbenzene and terpenoid series and increase the solubility of cytotoxic agents. A comprehensive approach based on three methods for studying the interaction of drugs with cells is proposed: MTT test-quantitative identification of surviving cells; FTIR spectroscopy-providing information on the molecular mechanisms inaccessible to study by any other methods (including binding to DNA, surface proteins, or lipid membrane); confocal microscopy for the visualization of observed effects of Dox accumulation in cancer or healthy cells depending on the drug formulation as a direct control of the correctness of interpretation of the results obtained by the two other methods. We found that eugenol (EG) and apiol increase the intracellular concentration of cytostatic in A549 cells by 2-4 times and maintain it for a long time. However, an important aspect is the selectivity of the enhancing effect of adjuvants on tumor cells in relation to healthy ones. Therefore, the authors focused on adjuvant's effect on the control healthy cells (HEK293T): EG and apiol demonstrate "protective" properties from cytostatic penetration by reducing intracellular concentrations by about 2-3 times. Thus, a combined formulation of cytostatic drugs has been found, showing promise in the aspects of improving the efficiency and selectivity of antitumor drugs; thereby, one of the perspective directions for overcoming MDR is suggested., Competing Interests: The authors declare no conflict of interest.

This study describes, to some extent, the VCC contribution as an early stimulation of the macrophage lineage. Regarding the onset of the innate immune response caused by infection, the β form of IL-1 is the most important interleukin involved in the onset of the inflammatory innate response. Activated macrophages treated in vitro with VCC induced the activation of the MAPK signaling pathway in a one-hour period, with the activation of transcriptional regulators for a surviving and pro-inflammatory response, suggesting an explanation inspired and supported by the inflammasome physiology. The mechanism of IL-1β production induced by VCC has been gracefully outlined in murine models, using bacterial knockdown mutants and purified molecules; nevertheless, the knowledge of this mechanism in the human immune system is still under study. This work shows the soluble form of 65 kDa of the Vibrio cholerae cytotoxin (also known as hemolysin), as it is secreted by the bacteria, inducing the production of IL-1β in the human macrophage cell line THP-1. The mechanism involves triggering the early activation of the signaling pathway MAPKs pERK and p38, with the subsequent activation of (p50) NF-κB and AP-1 (cJun and cFos), determined by real-time quantitation. The evidence shown here supports that the monomeric soluble form of the VCC in the macrophage acts as a modulator of the innate immune response, which is consistent with the assembly of the NLRP3 inflammasome actively releasing IL-1β.

Highly efficient methodology was developed for the construction of functionalized Kojic acid involving Click linker via 1,3-dipolar cycloaddition and their cytotoxicity against MCF-7, MIAPaCa-2 and DU145 mammalian cell lines were evaluated. Preliminary studies on structure-activity-relationship (SAR) revealed that substitution at C-2 of kojic acid as well as C-5 of 1,2,3-triazole motif played a major role in the activity profile. Kojic acid 1,2,3-triazole analogue 3 b containing an alkyl chain ( n  = 6) exhibited two fold potent activity than the parent compound, kojic acid against MCF-7 and MIA PaCa-2 cell lines. It induced apoptosis in these cell lines via ID1/PARP1 mediated pathway. The structures of the new analogues of kojic acid 1,2,3-triazole were confirmed by the detailed spectroscopic data analysis.

A new series of 2,3-diaryl-1,3thiazolidin-4-one derivatives was designed, synthesized, and evaluated for their cytotoxicity and COXs inhibitory activities. Among these derivatives, compounds 4 k and 4j exhibited the highest inhibitory activities against COX-2 at IC 50 values of 0.05 and 0.06 μM, respectively. Compounds 4a, 4b, 4e, 4 g, 4j, 4 k, 5b, and 6b, which exhibited the highest inhibition% against COX-2, were evaluated for their anti-inflammatory activity in rats. Results showed 41.08-82.00 % inhibition of paw edema thickness by the test compounds compared to celecoxib (inhibition% = 89.51 %). In addition, compounds 4b, 4j, 4 k, and 6b exhibited better GIT safety profiles compared to celecoxib and indomethacin. The four compounds were also evaluated for their antioxidant activity. The results revealed the highest antioxidant activity for 4j (IC 50  = 45.27 μM) comparable to torolox (IC 50  = 62.03 μM). The antiproliferative activity of the new compounds was evaluated against HePG-2, HCT-116, MCF-7, and PC-3 cancer cell lines. The results showed the highest cytotoxicity for compounds 4b, 4j, 4 k, and 6b (IC 50  = 2.31-27.19 μM), with 4j being the most potent. Mechanistic studies revealed the ability of 4j and 4 k by inducing marked apoptosis and cell cycle arrest at the G1 phase in HePG-2 cancer cells. These biological results may also suggest a role for COX-2 inhibition in the antiproliferative activity of these compounds. The results of the molecular docking study for 4 k and 4j into the active site of COX-2 revealed good fitting and correlation with the results of the in vitro COX‑2 inhibition assay., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Cholesterol-dependent cytolysins (CDCs) are proteinaceous toxins widely distributed in gram-positive pathogenic bacteria. CDCs can be classified into three groups (I-III) based on the mode of receptor recognition. Group I CDCs recognize cholesterol as their receptor. Group II CDC specifically recognizes human CD59 as the primary receptor on the cell membrane. Only intermedilysin from Streptococcus intermedius has been reported as a group II CDC. Group III CDCs recognize both human CD59 and cholesterol as receptors. CD59 contains five disulfide bridges in its tertiary structure. Therefore, we treated human erythrocytes with dithiothreitol (DTT) to inactivate CD59 on membranes. Our data showed that DTT treatment caused a complete loss of recognition of intermedilysin and an anti-human CD59 monoclonal antibody. In contrast, this treatment did not affect the recognition of group I CDCs, judging from the fact that DTT-treated erythrocytes were lysed with the same efficiency as mock-treated human erythrocytes. The recognition of group III CDCs toward DTT-treated erythrocytes was partially reduced, and these results are likely due to the loss of human CD59 recognition. Therefore, the degree of human CD59 and cholesterol requirements of uncharacterized group III CDCs frequently found in Mitis group streptococci can be easily estimated by comparing the amounts of hemolysis between DTT-treated and mock-treated erythrocytes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Steroidal heterocyclic compounds constitute interesting and promising scaffolds for drug discovery as they have displayed diverse chemical reactivity and several types of biological activities. This study is a concise report on the most recent advancements in the chemistry of the steroid skeleton, including reactions at the A, B, and D ring systems. The modern synthetic methods for the steroidal nitrogen-containing six-membered heterocyclic derivatives from 3-keto-, 6-keto-, 17-keto-, and 20-keto-steroids, as well as 2-Aldo-, 4-Aldo-, 6-Aldo-, and 16-Aldo-steroids, are discussed. However, some other methods for the synthesis of steroidal N-containing 6-membered heterocyclic derivatives are also included. These compounds have shown therapeutic potential as cytotoxic agents against various cell lines and have also shown antiproliferative, anti-inflammatory, and antioxidant activities. Therefore, they could be used as prospective candidates for the development of various medications. This paper not only describes synthetic details involved in creating N-containing 6-membered heterocyclic steroid derivatives, but also provides a brief overview of the medicinal applications of these compounds. This information will be highly useful for the medicinal chemists conducting research in this field., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The plasma membrane (PM) protects cells from extracellular threats and supports cellular homeostasis. Some pathogens produce pore-forming toxins (PFTs) that disrupt PM integrity by forming transmembrane pores. High PFT concentrations cause massive damage leading to cell death and facilitating infection. Sub-lytic PFT doses activate repair mechanisms to restore PM integrity, support cell survival and limit disease. Shedding of extracellular vesicles (EVs) has been proposed as a key mechanism to eliminate PFT pores and restore PM integrity. We show here that cholesterol-dependent cytolysins (CDCs), a specific family of PFTs, are at least partially eliminated through EVs release, and we hypothesize that proteins important for PM repair might be included in EVs shed by cells during repair. To identify new PM repair proteins, we collected EVs released by cells challenged with sub-lytic doses of two different bacterial CDCs, listeriolysin O and pneumolysin, and determined the EV proteomic repertoire by LC-MS/MS. Intoxicated cells release similar EVs irrespectively of the CDC used. Also, they release more and larger EVs than non-intoxicated cells. A cluster of 70 proteins including calcium-binding proteins, molecular chaperones, cytoskeletal, scaffold and membrane trafficking proteins, was detected enriched in EVs collected from intoxicated cells. While some of these proteins have well-characterized roles in repair, the involvement of others requires further study. As proof of concept, we show here that Copine-1 and Copine-3, proteins abundantly detected in EVs released by intoxicated cells, are required for efficient repair of CDC-induced PM damage. Additionally, we reveal here new proteins potentially involved in PM repair and give new insights into common mechanisms and machinery engaged by cells in response to PM damage., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Pore-forming toxins (PFTs) are important virulence factors produced by many pathogenic bacteria. Here, we show that the Vibrio cholerae toxin MakA is a novel cholesterol-binding PFT that induces non-canonical autophagy in a pH-dependent manner. MakA specifically binds to cholesterol on the membrane at pH < 7. Cholesterol-binding leads to oligomerization of MakA on the membrane and pore formation at pH 5.5. Unlike other cholesterol-dependent cytolysins (CDCs) which bind cholesterol through a conserved cholesterol-binding motif (Thr-Leu pair), MakA contains an Ile-Ile pair that is essential for MakA-cholesterol interaction. Following internalization, endosomal acidification triggers MakA pore-assembly followed by ESCRT-mediated membrane repair and V-ATPase-dependent unconventional LC3 lipidation on the damaged endolysosomal membranes. These findings characterize a new cholesterol-binding toxin that forms pores in a pH-dependent manner and reveals the molecular mechanism of host autophagy manipulation., (© 2022 Jia et al.)

Novel halogenated phenoxychalcones 2a-f and their corresponding N -acetylpyrazolines 3a-f were synthesised and evaluated for their anticancer activities against breast cancer cell line (MCF-7) and normal breast cell line (MCF-10a), compared with staurosporine. All compounds showed moderate to good cytotoxic activity when compared to control. Compound 2c was the most active, with IC 50 = 1.52 µM and selectivity index = 15.24. Also, chalcone 2f showed significant cytotoxic activity with IC 50 = 1.87 µM and selectivity index = 11.03. Compound 2c decreased both total mitogen activated protein kinase (p38α MAPK) and phosphorylated enzyme in MCF-7 cells, suggesting its ability to decrease cell proliferation and survival. It also showed the ability to induce ROS in MCF-7 treated cells. Compound 2c exhibited apoptotic behaviour in MCF-7 cells due to cell accumulation in G2/M phase and elevation in late apoptosis 57.78-fold more than control. Docking studies showed that compounds 2c and 2f interact with p38alpha MAPK active sites.

Multidrug resistance (MDR) is a leading cause for treatment failure in cancer patients. One of the reasons of MDR is drug efflux by ATP-binding cassette (ABC) transporters in eukaryotic cells especially ABCB1 (P-glycoprotein). In this study, certain novel 1,2,5-trisubstituted benzimidazole derivatives were designed utilising ligand based pharmacophore approach. The designed benzimidazoles were synthesised and evaluated for their cytotoxic activity towards doxorubicin-sensitive cell lines (CCRF/CEM and MCF7), as well as against doxorubicin-resistant cancer cells (CEM/ADR 5000 and Caco-2). In particular, compound VIII showed a substantial cytotoxic effect in all previously mentioned cell lines especially in doxorubicin-resistant CEM/ADR5000 cells (IC 50 = 8.13 µM). Furthermore, the most promising derivatives VII , VIII and XI were tested for their ABCB1 inhibitory action in the doxorubicin-resistant CEM/ADR 5000 subline which is known for overexpression of ABCB1 transporters. The results showed that compound VII exhibited the best ABCB1 inhibitory activity at three tested concentrations (22.02 µM (IC 50 ), 50 µM and 100 µM) in comparison to verapamil as a reference ABCB1 inhibitor. Such inhibition resulted in a synergistic effect and a massive decrease in the IC 50 of doxorubicin (34.5 µM) when compound VII was used in a non-toxic dose in combination with doxorubicin in doxorubicin-resistant cells CEM/ADR 5000 (IC 50(Dox+VII) = 3.81 µM). Molecular modelling studies were also carried out to explain the key interactions of the target benzimidazoles at the ABCB1 binding site. Overall the obtained results from this study suggest that 1,2,5-trisubstituted benzimidazoles possibly are promising candidates for further optimisation and development of potential anticancer agents with ABCB1 inhibitory activity and therefore overcome MDR in cancer cells.

This study outlines a number of studies of dichloroacetic acid (DCA) and some of its derivatives. Although DCA has low cytotoxic potencies, various structural modifications are described which result in potent cytotoxins. In particular, hybrid molecules created from DCA and other bioactive molecules whose modes of action differ from DCA are particularly promising as candidate anticancer agents. Considerable emphasis in this review is placed on various series of compounds that incorporate both platinum and DCA into their structures. In addition, the importance of the formulation of some of the bioactive compounds described herein is revealed., (© 2022 The Authors. Archiv der Pharmazie published by Wiley-VCH GmbH on behalf of Deutsche Pharmazeutische Gesellschaft.)

Seaweeds are a great source of compounds with cytotoxic properties with the potential to be used as anticancer agents. This study evaluated the cytotoxic and proteasome inhibitory activities of 12 R -hydroxy-bromosphaerol, 12 S -hydroxy-bromosphaerol, and bromosphaerol isolated from Sphaerococcus coronopifolius . The cytotoxicity was evaluated on malignant cell lines (A549, CACO-2, HCT-15, MCF-7, NCI-H226, PC-3, SH-SY5Y, and SK-MEL-28) using the MTT and LDH assays. The ability of compounds to stimulate the production of hydrogen peroxide (H 2 O 2 ) and to induce mitochondrial dysfunction, the externalization of phosphatidylserine, Caspase-9 activity, and changes in nuclear morphology was also studied on MCF-7 cells. The ability to induce DNA damage was also studied on L929 fibroblasts. The proteasome inhibitory activity was estimated through molecular docking studies. The compounds exhibited IC 50 values between 15.35 and 53.34 µM. 12 R -hydroxy-bromosphaerol and 12 S -hydroxy-bromosphaerol increased the H 2 O 2 levels on MCF-7 cells, and bromosphaerol induced DNA damage on fibroblasts. All compounds promoted a depolarization of mitochondrial membrane potential, Caspase-9 activity, and nuclear condensation and fragmentation. The compounds have been shown to interact with the chymotrypsin-like catalytic site through molecular docking studies; however, only 12 S -hydroxy-bromosphaerol evidenced interaction with ALA20 and SER169, key residues of the proteasome catalytic mechanism. Further studies should be outlined to deeply characterize and understand the potential of those bromoditerpenes for anticancer therapeutics.

In this study, a series of coumarin derivatives, either alone or as hybrids with cinnamic acid, were synthesized and evaluated for their cytotoxicity against a panel of cancer cells using the MTT assay. Then, the most active compounds were inspected for their mechanism of cytotoxicity by cell-cycle analysis, RT-PCR, DNA fragmentation, and Western blotting techniques. Cytotoxic results showed that compound ( 4 ) had a significant cytotoxic effect against HL60 cells (IC 50 = 8.09 µM), while compound ( 8b ) had a noticeable activity against HepG2 cells (IC 50 = 13.14 µM). Compounds ( 4 ) and ( 8b ) mediated their cytotoxicity via PI3K/AKT pathway inhibition. These results were assured by molecular docking studies. These results support further exploratory research focusing on the therapeutic activity of coumarin derivatives as cytotoxic agents.

Host-associated bacteria are increasingly being recognized as underexplored sources of bioactive natural products with unprecedented chemical scaffolds. A recently identified example is the plant-root-associated marine bacterium Gynuella sunshinyii of the chemically underexplored order Oceanospirillales. Its genome contains at least 22 biosynthetic gene clusters, suggesting a rich and mostly uncharacterized specialized metabolism. Here, in silico chemical prediction of a non-canonical polyketide synthase cluster has led to the discovery of janustatins, structurally unprecedented polyketide alkaloids with potent cytotoxicity that are produced in minute quantities. A combination of MS and two-dimensional NMR experiments, density functional theory calculations of 13 C chemical shifts and semiquantitative interpretation of transverse rotating-frame Overhauser effect spectroscopy data were conducted to determine the relative configuration, which enabled the total synthesis of both enantiomers and assignment of the absolute configuration. Janustatins feature a previously unknown pyridodihydropyranone heterocycle and an unusual biological activity consisting of delayed, synchronized cell death at subnanomolar concentrations., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: The lack of drug targets is an obstacle to the treatment of patients with triple-negative breast cancer (TNBC). At present, non-specific cytotoxic drugs are first-line agents, but the development of resistance is a major problem with these agents. The epidermal growth factor receptor (EGFR) is a potential target in some TNBCs, because its tyrosine kinase activity drives tumorigenesis. Thus, small molecule inhibitors of the EGFR in combination with cytotoxic agents could be important for the treatment of TNBCs., Methods: The present study evaluated the efficacies of clinically approved EGFR inhibitors in combination with the cytotoxic agent ixabepilone in parental and docetaxel-resistant MDA-MB-231 cells (231C and TXT cells, respectively). Cell viability was assessed using MTT reduction assays, cell death pathways were evaluated using annexin V/7-aminoactinomycin D staining and flow cytometry and Western immunoblotting was used to assess the expression of pro- and anti-apoptotic proteins in cells., Results: Ixabepilone and the EGFR inhibitors gefitinib and vandetanib inhibited 231C and TXT cell proliferation, but the alternate EGFR inhibitors erlotinib and lapatinib were poorly active. Using combination analysis, ixabepilone/vandetanib was synergistic in both cell types, whereas the ixabepilone/gefitinib combination exhibited antagonism. By flow cytometry, ixabepilone/vandetanib enhanced 231C and TXT cell death over that produced by the single agents and also enhanced caspase-3 cleavage and the pro/anti-apoptotic Bcl-2 protein ratios over ixabepilone alone., Conclusions: These findings suggest that the ixabepilone/vandetanib combination may have promise for the treatment of patients with drug-resistant TNBC., (© 2022. The Author(s).)

Pancreatic cancer is one of the leading causes of cancer-related deaths worldwide. Unfortunately, therapeutic gains in the treatment of other cancers have not successfully translated to pancreatic cancer treatments. Management of pancreatic cancer is difficult due to the lack of effective therapies and the rapid development of drug resistance. The cytotoxic agent gemcitabine has historically been the first-line treatment, but combinations of other immunomodulating and stroma-depleting drugs are currently undergoing clinical testing. Moreover, the treatment of pancreatic cancer is complicated by its heterogeneity: analysis of genomic alterations and expression patterns has led to the definition of multiple subtypes, but their usefulness in the clinical setting is limited by inter-tumoral and inter-personal variability. In addition, various cell types in the tumor microenvironment exert immunosuppressive effects that worsen prognosis. In this review, we discuss current perceptions of molecular features and the tumor microenvironment in pancreatic cancer, and we summarize emerging drug options that can complement traditional chemotherapies.

NCI-60 growth inhibition and gene expression profiles were analyzed using Pearson correlation and functional enrichment computational tools to demonstrate critical mechanistic differences between a nucleolus-targeting platinum-acridine anticancer agent (PA) and other DNA-directed chemotherapies. The results support prior experimental data and are consistent with DNA being a major target of the hybrid agent based on the negative correlations observed between its potency and expression levels of genes implicated in DNA double-strand break (DSB) repair. Gene ontology terms related to RNA processing, including ribosome biogenesis, are also negatively enriched, suggesting a mechanism by which these processes render cancer cells more resistant to the highly cytotoxic agent. The opposite trend is observed for oxaliplatin and other DNA-targeted drugs. Significant functional interactions exist between genes/gene products involved in ribosome biogenesis and DSB repair, including the ribosomal protein (RPL5)-MDM2-p53 surveillance pathway, as a response to the nucleolar stress produced by PAs., (© 2022 Wiley-VCH GmbH.)

Microtubule targeting agents (MTAs) have attracted extensive attention for cancer treatment. However, their clinical efficacies are limited by intolerable toxicities, inadequate efficacy and acquired multidrug resistance. The combination of MTAs with other antineoplastics has become an efficient strategy to lower the toxicities, overcome resistance and improve the efficacies for cancer treatment. In this article, we review the combinations of MTAs with some other anticancer drugs, such as cytotoxic agents, kinases inhibitors, histone deacetylase inhibitors, immune checkpoints inhibitors, to overcome these obstacles. We strongly believe that this review will provide helpful information for combination therapy based on MTAs., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

αKlotho is a transmembrane protein acting as a co-receptor for FGF23, a bone hormone regulating renal phosphate and vitamin D metabolism. αKlotho expression is controlled by PPARγ. Soluble αklotho (sKL) regulates cellular signaling impacting stress resistance and death. αKlotho deficiency causes early onset of aging-associated diseases while its overexpression markedly increases lifespan. Cellular stress due to cytotoxic therapeutics or apoptosis induction through caspase activation or serum deficiency may result in cell death. Owing to αklotho's role in cellular stress and aging, this study explored the effect of cytotoxic agents or apoptosis stimulants on cellular αklotho expression. Experiments were performed in renal MDCK, NRK-52E and HK-2 cells. Gene expression was determined by qRT-PCR, sKL by ELISA, apoptosis and necrosis by annexin V binding and a fluorescent DNA dye, and cell viability by MTT assay. Cytostatic drugs cisplatin, paclitaxel, and doxorubicin as well as apoptosis induction with caspase 3 activator PAC-1 and serum deprivation induced αklotho and PPARG gene expression while decreasing viability and proliferation and inducing apoptosis of MDCK and NRK-52E cells to a variable extent. PPARγ antagonism attenuated up-regulation of αklotho in MDCK cells. In HK-2 cells, αklotho gene expression and sKL protein were down-regulated by chemotherapeutics. SKL serum levels in patients following chemotherapy were not significantly changed. In summary, potentially fatal stress results in up-regulation of αKlotho gene expression in MDCK and NRK-52E cells and down-regulation in HK-2 cells. These results indicate that different renal cell lines may exhibit completely different regulation of αklotho.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal types of cancer, mainly due to its delayed diagnosis and lack of effective therapeutic options. Therefore, it is imperative to find novel treatment options for PDAC. Here, we tested a series of conventional chemotherapeutics together with anthracycline compounds as single agents or in combination, determining their effectivity against established commercial and patient-derived, low-passage PDAC cell lines. Proliferation and colony formation assays were performed to determine the anticancer activity of anthracyclines; aclarubicin and doxorubicin, on commercial and patient-derived, low-passage PDAC cell lines. In addition, the effect of standard-of-care drugs gemcitabine and individual components of FOLFIRINOX were also investigated. To evaluate which mechanisms of cell death were involved in drug response, cleavage of poly(ADP-ribose)polymerase was evaluated by western blot. Aclarubicin showed superior antitumor activity compared to other anthracyclines and standard of care drugs (gemcitabine and individual components of FOLFIRINOX) in a patient-derived, low-passage PDAC cell line and in commercial cell lines. Importantly, the combination of gemcitabine and aclarubicin showed a synergistic effect at a dose range where the single agents by themselves were ineffective. In parallel, evaluation of the antitumor activity of aclarubicin demonstrated an apoptotic effect in all PDAC cell lines. Aclarubicin is cytotoxic for commercial and patient-derived low-passage PDAC cell lines, at doses lower than peak serum concentrations for patient treatment. Our findings support a (re)consideration of aclarubicin as a backbone of new combination regimens for pancreatic cancer patients., (Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc.)

Pore-forming toxins (PFTs) produced by pathogenic bacteria serve as prominent virulence factors with potent cell-killing activity. Most of the β-barrel PFTs form transmembrane oligomeric pores in the membrane lipid bilayer in the presence of cholesterol. The pore-formation mechanisms of the PFTs highlight well-orchestrated regulated events in the membrane environment, which involve dramatic changes in the protein structure and organization. Also, concerted crosstalk between protein and membrane lipid components appears to play crucial roles in the process. Membrane-damaging lesions formed by the pore assembly of the PFTs would also be expected to impose drastic alterations in the membrane organization, details of which remain obscure in most of the cases. Prior reports have established that aqueous interfaces of liquid crystals (LCs) offer promise as responsive interfaces for biomolecular events (at physiologically relevant concentrations), which can be visualized as optical signals. Inspired by this, herein, we sought to understand the lipid membrane interactions of a β-barrel PFT i.e. , Vibrio cholerae cytolysin (VCC), using LC-aqueous interfaces. Our results show the formation of dendritic patterns upon the addition of VCC to the lipid embedded with cholesterol over the LC film. In contrast, we did not observe any LC reorientation upon the addition of VCC to the lipid-laden LC-aqueous interface in the absence of cholesterol. An array of techniques such as polarizing optical microscopy (POM), atomic force microscopy (AFM), and fluorescence measurements were utilized to decipher the LC response to the lipid interactions of VCC occurring at these interfaces. Altogether, the results obtained from our study provide a novel platform to explore the mechanistic aspects of the protein-membrane interactions, in the process of membrane pore-formation by the membrane-damaging PFTs.

Addressing the increasing incidences of cancer worldwide along with the multifaceted problem of drug resistance via development of new anticancer agents has become an essential goal. Due to the known cytotoxic effects and reported Akt inhibitory potential of azaindoles we designed a new framework incorporating the structural features of rosuvastatin and 5- or 7-azaindole. The framework was used to construct a library of small molecules for further pharmacological evaluation. The design was supported by the docking studies of two representative molecules in silico. A one-pot sonochemical approach was established for the synthesis of these rosuvastatin based azaindoles that involved the coupling-cyclization of a rosuvastatin derived terminal alkyne with appropriate 3-iodopyridine derivatives under Pd/Cu-catalysis. When tested using an MTT assay, some of the synthesized compounds showed desirable cytotoxic effects against three cancer cell lines e.g. HCT 116, Hep G2 and PA-1 but no significant effects against the non-cancerous HEK cell line. According to the SAR the 5-azaindole ring appeared to be marginally better than the 7-azaindole whereas the activity was varied with the variation of sulfonamide moiety attached to the N-1 atom of the azaindole ring. Among all the groups present in the sulfonamide moiety the p-MeC 6 H 4 group appeared to be most effective in terms of activity. While 3b and 5b were identified as initial hit molecules the compound 5b (in addition to 3b) also showed significant inhibition of Akt1 in vitro that was reflected by its strong interaction with Akt1 in silico (with the docking score -11.7 kcal/mol) involving two H-bonding interactions with Ser7 and Asp439 residues. Further, a reasonable ADME was predicted for 5bin silico. Being a potent inhibitor (MIA Paca-2 IC 50  = 18.79 ± 0.17 nM) and with NOAEL (No Observed Adverse Effect Level) > 100 µM in Zebrafish, 5b emerged as a promising compound., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The detailed mycochemical analysis of the n -hexane extract of Pholiota populnea led to the isolation of four new lanostane diesters, named pholiols A-D ( 1 - 4 ), together with an acyclic triterpene, (3 S ,6 E ,10 E ,14 E ,18 E ,22 S )-2,3,22,23-tetrahydroxy-2,6,10,15,19,23-hexamethyl-6,10,14,18-tetracosatetraene ( 5 ), ergosterol ( 6 ), and 3β-hydroxyergosta-7,22-diene ( 7 ). The isolation was carried out by multistep flash chromatography, and the structures were elucidated using extensive spectroscopic analyses, including 1D and 2D NMR and MS measurements. The isolated metabolites ( 1 - 6 ) were investigated for cytotoxic activity against Colo205 and Colo320 colon adenocarcinoma and nontumoral MRC-5 cell lines. Among the tested compounds, ergosterol ( 6 ) showed substantial cytotoxic activity against all cell lines with IC 50 values of 4.9 μM (Colo 205), 6.5 μM (Colo 320), and 0.50 μM (MRC) with no tumor cell selectivity. A P-glycoprotein efflux pump modulatory test on resistant Colo320 cells revealed that pholiols A ( 1 ) and B ( 2 ) and linear triterpene polyol 5 have the capacity to inhibit the efflux-pump overexpressed in the cells. Moreover, the drug interactions of triterpenes with doxorubicin were studied by the checkerboard method on Colo 320 cells. Pholiols B ( 2 ) and D ( 4 ) interacted in synergistic and acyclic triterpene 5 in a very strong synergistic manner; the combination index (CI) values at 50% of the growth inhibition dose (ED 50 ) were found to be 0.348, 0.660, and 0.082, respectively. Our results indicate that P. populnea is a promising source for finding new triterpenes with significant chemosensitizing activity on cancer cells.

Due to their manifold biological activities, natural products such as triterpenoids have advanced to represent excellent leading structures for the development of new drugs. For this reason, we focused on the syntheses and cytotoxic evaluation of derivatives obtained from gypsogenin, hederagenin, and madecassic acid, cytotoxicity increased-by and large-from the parent compounds to their acetates. Another increase in cytotoxicity was observed for the acetylated amides (phenyl, benzyl, piperazinyl, and homopiperazinyl), but a superior cytotoxicity was observed for the corresponding rhodamine B conjugates derived from the (homo)-piperazinyl amides. In particular, a madecassic acid homopiperazinyl rhodamine B conjugate 24 held excellent cytotoxicity and selectivity for several human tumor cell lines. Thus, this compound was more than 10,000 times more cytotoxic than parent madecassic acid for A2780 ovarian cancer cells. We assume that the presence of an additional hydroxyl group at position C-6 in derivatives of madecassic, as well as the (2α, 3β) configuration of the acetates in ring A, had a beneficial effect onto the cytotoxicity of the conjugates, as well as onto tumor/non-tumor cell selectivity.

Squamous cell carcinoma is the major form of malignancy that arises in head and neck cancer. The modest improvement in the 5‑year survival rate underpins its complex etiology and provides the impetus for the discovery of new therapeutics. The present study describes the discovery of an indole‑based small molecule (24a) that was a potent cytotoxic agent with antiproliferative and pro‑apoptotic properties against a pharyngeal carcinoma cell line, Detroit 562, effectively killing the cells at a half‑maximal inhibitory concentration of 0.03 µM, as demonstrated using cell proliferation studies. The antiproliferative property of 24a was demonstrated by its ability to promote G 2 /M blockade, as assessed by cell cycle analysis using flow cytometry and the monitoring of real‑time cell cycle progression by the fluorescence ubiquitination‑based cell cycle indicator. This pro‑apoptotic property is supported by the promotion of TUNEL‑staining and increase in the activities of caspases‑3/7 and ‑6, in addition to the expression of death receptors and the cleavage of poly (ADP‑ribose) polymerase 1 protein as demonstrated by western blotting. Given that Detroit 562 lacks functional p53, it is suggested that 24a acts independently of the tumor suppressor.

A series of novel oleanolic acid (OA)-linked disulfide, thioether, or selenium ether derivatives was synthesized, and their antiproliferative activity was evaluated against human liver cancer (BEL-7402 and HepG-2), colon cancer (HCT116), and normal liver (L02) cell lines using methyl thiazolyl tetrazolium assay (MTT). Preliminary bioassay results revealed that OA derivatives modified at the C3-OH position, i. e., compound a4 containing sulfide ether, exhibited the best antiproliferative activity against BEL-7402 cells, with an IC 50 value of 5.70±0.82 μM. Further flow cytometry assays revealed that compound a4 exerted its antiproliferative effects by inducing cell cycle arrest in the G2/M phase leading to apoptosis. Moreover, compared with the lead compound OA and the positive control drug 5-fluorouracil (5-FU), the OA derivatives demonstrated potent antiproliferative activities against the cancer cell lines., (© 2022 Wiley-VHCA AG, Zurich, Switzerland.)

Background: Previous studies had reported on the cytotoxic activities of generic antibiotics such as doxycycline (DOXY) and azithromycin (AZI) in multiple types of human cancers. Given that resistance to standard anti-glioblastoma multiforme (GBM) drug [temozolomide (TMZ)] is common and inevitable, alternative candidates are greatly needed., Purpose and Method: The present study was undertaken to explore the cytotoxicity and anticancer effects of DOXY and AZI on human GBM U87 cells via 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), Hoechst, Annexin V-FITC/PI, and clonogenic assays. CompuSyn software was used to determine the combination index (CI) for DOXY+AZI., Result: Individual treatment with DOXY and AZI decreased U87 cell viability in dose- and time-dependent, and quantitatively comparable to TMZ. Nevertheless, combinations of both antibiotics evidenced antagonistic behaviour in U87 cells. Increased apoptotic event was also observed with the individual treatment of DOXY and AZI. Furthermore, the proliferative and clonogenic capability of 21-day survived U87 cells was completely terminated by DOXY and AZI, but not TMZ., Conclusion: The antiproliferative and apoptosis-inducing activity exhibited by both antibiotics against U87 cells demonstrates their potential as a likely alternative to combat GBM. It would be interesting to find out more about their molecular players and cytotoxic effects in different types of GBM cells, including glioma stem cells (GSCs).

Chromatographic investigation of the aerial parts of the Rhazya stricta (Apocynaceae) resulted in the isolation of two new monoterpene indole alkaloids, 6- nor -antirhine- N 1 -methyl ( 1 ) and razyamide ( 2 ), along with six known compounds, eburenine ( 3 ), epi -rhazyaminine ( 4 ), rhazizine ( 5 ), 20- epi -sitsirikine ( 6 ), antirhine ( 7 ), and 16- epi -stemmadenine- N -oxide ( 8 ). The chemical structures were established by various spectroscopic experiments. Compounds 1 - 8 exhibited cytotoxic effects against three cancer cells with IC 50 values ranging between 5.1 ± 0.10 and 93.2 ± 9.73 µM against MCF-7; 5.1 ± 0.28 and 290.2 ± 7.50 µM against HepG2, and 3.1 ± 0.17 and 55.7 ± 4.29 µM against HeLa cells. Compound 2 showed the most potent cytotoxic effect against all cancer cell lines (MCF-7, HepG2 and HeLa with IC 50 values = 5.1 ± 0.10, 5.1 ± 0.28, and 3.1 ± 0.17 µM, respectively). Furthermore, compound 2 revealed a significant increase in the apoptotic cell population of MCF-7, HepG2, and HeLa cells, with 31.4 ± 0.2%, 29.2 ± 0.5%, and 34.9 ± 0.6%, respectively. Compound 2 decreased the percentage of the phagocytic pathway on HepG2 cells by 15.0 ± 0.1%. These findings can explain the antiproliferative effect of compound 2 .

Mangrove endophytic fungi are tolerant to numerous stresses and are inevitably capable of exhibiting excellent biological activity by producing impressive numbers of metabolites with special biological functions, based on previous work on the biological potential of mangrove-derived endophytic fungi. To obtain marked antimicrobial and cytotoxic fermentation products of culturable endophytic fungi from mangrove forests, our research evaluated the antimicrobial and cytotoxic activities of crude extracts of endophytic fungi from Rhizophora stylosa and Rhizophora mucronata. Forty-six fungal isolates were cultured on four different media, namely, dextrose agar (PDA), Czapek's agar (CZA), rice medium (RM) and grain medium (GM) and harvested by ethyl acetate solvent at 40 days. The extracts were tested for antimicrobial activity by the microdilution method against the gram-negative bacteria Pseudomonas adaceae (PA), gram-positive bacteria Enterococcus faecalis (EF), methicillin-resistant Staphylococcus aureus (MRSA) and pathogenic fungus Monilia albicans (MA). The cytotoxic activity of the extracts was evaluated by MTT assay using A549 human lung cancer cells, HeLa human cervical carcinoma cells, and HepG2 human hepatocellular cells. The results showed that rice medium could promote the secretion of antimicrobial and antitumour secondary metabolites of endophytic fungi in comparison with other cultivation media. Seventeen strains (68%) from R. stylosa exhibited inhibitory effects on indicators, especially N. protearum HHL46, which could inhibit the growth of four microbes with MIC values reaching 0.0625 mg/mL. Fifteen strains (71.4%) from R. mucronata displayed activities against human pathogenic microbes; in particular, Pestalotiopsis sp. HQD6 and N. protearum HQD5 could resist the growth of four microbes with MIC values ranging from 0.015 to 1 mg/mL. In the cytotoxicity assay, the extracts of 10 strains (40%), 9 strains (40%) and 13 strains (52%) of R. stylosa and 13 strains (61.9%), 10 strains (47.6%) and 10 strains (47.6%) of R. mucronata displayed cytotoxicity against A549, HeLa and HepG2 cancer cells with cell viability values ≤ 50%. Neopestalotiopsis protearum HHL46, Phomopsis longicolla HHL50, Botryosphaeria fusispora HQD83, Fusarium verticillioides HQD48 and Pestalotiopsis sp. HQD6 displayed significant antitumour activity with IC 50 values below 20 μg/mL. These results highlighted the antimicrobial and antitumour potential of endophytic fungi from R. stylosa and R. mucronata and the possibility of exploiting their antimicrobial and cytotoxic agents., (© 2022. The Author(s).)

Three new polyhydroxylated oleanane triterpenoids, cissatriterpenoid A-C ( 1 - 3 ), along with one known analogue ( 4 ), were isolated from the whole plant of Cissampelos pareira var. hirsuta . Their chemical structures were elucidated by extensive spectroscopic data (IR, HR-ESI-MS, 1 H-NMR, 13 C-NMR, DEPT, 1 H- 1 H COSY, HSQC, HMBC, NOESY) and the microhydrolysis method. The isolation of compounds 1 - 4 represents the first report of polyhydroxylated oleanane triterpenoids from the family Menispermaceae. All isolated compounds were evaluated for their cytotoxicity against five human cancer cell lines, and the inhibitory activity against NO release in LPS-induced RAW 264.7 cells. Compound 3 showed the most potent cytotoxic activities against the A549, SMMC-7721, MCF-7, and SW480 cell lines, with IC 50 values of 17.55, 34.74, 19.77, and 30.39 μM, respectively, whereas three remaining ones were found to be inactive. The preliminary structure-activity relationship analysis indicated that the γ-lactone ring at C-22 and C-29, and the olefinic bond at C-12 and C-13 were structurally required for the cytotoxicity of polyhydroxylated oleanane triterpenoids against these four cell lines. Based on lipid-water partition coefficients, compound 3 is less lipophilic than 1 and 4 , which agrees with their cytotoxic activities. This confirms the potential of C. pareira var. hirsuta in the tumor treatment.

We report a screening study aimed to assess for the first time the air- and water-stability and the biological potential of simple metal-carbamates. These molecular metallic species are based on elements belonging to the groups 4-5, 7-9 and 11, and tin, and are easily available from inexpensive reagents. Complexes [Ag(O 2 CNEt 2 )] (13-Ag) and [Au(O 2 CNMe 2 )(PPh 3 )] (14-Au) resulted substantially stable in aqueous media and exhibited a potent in vitro cytotoxicity. Especially 13-Ag revealed a significant selectivity against the A549 lung adenocarcinoma and the A2780 ovarian cancer cell lines with respect to the noncancerous HEK293 cell line. Generation of ROS (reactive oxygen species) and mitochondrial membrane depolarization were recognized for 13-Ag and 14-Au; notwithstanding, the cell death mechanism is different in the two cases: apoptosis and cell cycle arrest in G0/G1 phase for 13-Ag; necroptosis and cell cycle arrest in S phase for 14-Au. Both 13-Ag and 14-Au are endowed with antibacterial activity, which is relatively stronger for 13-Ag towards Gram negative and for 14-Au towards Gram positive strains, respectively., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The CDK4/6 inhibitor palbociclib, combined with endocrine therapy, has been shown to be effective in postmenopausal women with estrogen receptor-positive, HER2-negative advanced or metastatic breast cancer. However, palbociclib is not as effective in the highly aggressive, triple-negative breast cancer that lacks sensitivity to chemotherapy or endocrine therapy. We hypothesized that conjugation of the near-infrared dye MHI-148 with palbociclib can produce a potential theranostic in triple-negative, as well as estrogen receptor-positive, breast cancer cells. In our study, the conjugate was found to have enhanced activity in all mammalian cell lines tested in vitro. However, the conjugate was cytotoxic and did not induce G1 cell cycle arrest in breast cancer cells, suggesting its mechanism of action differs from the parent compound palbociclib. The study highlights the importance of investigating the mechanism of conjugates of near-infrared dyes to therapeutic compounds, as conjugation can potentially result in a change of mechanism or target, with an enhanced cytotoxic effect in this case.

A series of quaternary diammonium salts derivatives of 1,4:3,6-dianhydro-l-iditol were synthesized, using isommanide (1,4:3,6-dianhydro-d-mannitol) as a starting material. Both aromatic (pyridine, 4-( N , N -dimethylamino)pyridine (DMAP), (3-carboxamide)pyridine; N -methylimidazole) and aliphatic (trimethylamine, N , N -dimethylhexylamine, N , N -dimethyloctylamine, N , N -dimethyldecylamine) amines were used, giving eight gemini quaternary ammonium salts (QAS). All salts were tested for their antimicrobial activity against yeasts, Candida albicans and Candida glabrata , as well as bacterial Staphylococcus aureus and Escherichia coli reference strains. Moreover, antibacterial activity against 20 isolates of S. aureus collected from patients with skin and soft tissue infections ( n = 8) and strains derived from subclinical bovine mastitis milk samples ( n = 12) were evaluated. Two QAS with octyl and decyl residues exhibited antimicrobial activity, whereas those with two decyl residues proved to be the most active against the tested pathogens, with MIC of 16-32, 32, and 8 µg/mL for yeast, E. coli, and S. aureus reference and clinical strains, respectively. Only QAS with decyl residues proved to be cytotoxic in MTT assay against human keratinocytes (HaCaT), IC 50 12.8 ± 1.2 μg/mL. Ames test was used to assess the mutagenic potential of QAS, and none of them showed mutagenic activity in the concentration range 4-2000 µg/plate.

Liquid chromatography electrospray ionization tandem mass spectrometric (LC-ESI-MS/MS) qualitative and quantitative analysis of different extracts from the aerial parts and roots of Alchemilla acutiloba led to the identification of phenolic acids and flavonoids. To the best of our knowledge, isorhamnetin 3-glucoside, kaempferol 3-rutinoside, narcissoside, naringenin 7-glucoside, 3- O -methylquercetin, naringenin, eriodictyol, rhamnetin, and isorhamnetin were described for the first time in Alchemilla genus. In addition, the antioxidant, anti-inflammatory and cytotoxic activity of all extracts were evaluated. The results clearly showed that among analyzed extracts, the butanol extract of the aerial parts exhibited the highest biological activity comparable with the positive controls used.

A ferrofluid with 1,2-Benzenediol-coated iron oxide nanoparticles was synthesized and physicochemically analyzed. This colloidal system was prepared following the typical co-precipitation method, and superparamagnetic nanoparticles of 13.5 nm average diameter, 34 emu/g of magnetic saturation, and 285 K of blocking temperature were obtained. Additionally, the zeta potential showed a suitable colloidal stability for cancer therapy assays and the magneto-calorimetric trails determined a high power absorption density. In addition, the oxidative capability of the ferrofluid was corroborated by performing the Fenton reaction with methylene blue (MB) dissolved in water, where the ferrofluid was suitable for producing reactive oxygen species (ROS), and surprisingly a strong degradation of MB was also observed when it was combined with H 2 O 2 . The intracellular ROS production was qualitatively corroborated using the HT-29 human cell line, by detecting the fluorescent rise induced in 2,7-dichlorofluorescein diacetate. In other experiments, cell metabolic activity was measured, and no toxicity was observed, even with concentrations of up to 4 mg/mL of magnetic nanoparticles (MNPs). When the cells were treated with magnetic hyperthermia, 80% of cells were dead at 43 °C using 3 mg/mL of MNPs and applying a magnetic field of 530 kHz with 20 kA/m amplitude.

Linear cationic venom peptides are antimicrobial peptides (AMPs) that exert their effects by damaging cell membranes. These peptides can be highly specific, and for some, a significant therapeutic value was proposed, in particular for treatment of bacterial infections. A prolific source of novel AMPs are arthropod venoms, especially those of hitherto neglected groups such as pseudoscorpions. In this study, we describe for the first time pharmacological effects of AMPs discovered in pseudoscorpion venom. We examined the antimicrobial, cytotoxic, and insecticidal activity of full-length Checacin1, a major component of the Chelifer cancroides venom, and three truncated forms of this peptide. The antimicrobial tests revealed a potent inhibitory activity of Checacin1 against several bacteria and fungi, including methicillin resistant Staphylococcus aureus (MRSA) and even Gram-negative pathogens. All peptides reduced survival rates of aphids, with Checacin1 and the C-terminally truncated Checacin1 1-21 exhibiting effects comparable to Spinosad, a commercially used pesticide. Cytotoxic effects on mammalian cells were observed mainly for the full-length Checacin1. All tested peptides might be potential candidates for developing lead structures for aphid pest treatment. However, as these peptides were not yet tested on other insects, aphid specificity has not been proven. The N- and C-terminal fragments of Checacin1 are less potent against aphids but exhibit no cytotoxicity on mammalian cells at the tested concentration of 100 µM.