Photosensitization of pancreatic cancer cells by cationic alkyl-porphyrins in free form or engrafted into POPC liposomes: The relationship between delivery mode and mechanism of cell death

Cationic porphyrins bearing an alkyl side chain of 14 (2b) or 18 (2d) carbons dramatically inhibit proliferation of pancreatic cancer cells following treatment with light. We have compared two different ways of delivering porphyrin 2d: either in free form or engrafted into palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine liposomes (L-2d). Cell cytometry shows that while free 2d is taken up by pancreatic cancer cells by active (endocytosis) and passive (membrane fusion) transports, L-2d is internalized solely by endocytosis. Confocal microscopy showed that free 2d co-localizes with the cell membrane and lysosomes, whereas L-2d partly co-localizes with lysosomes and ER. It is found that free 2d inhibits the KRAS-Nrf2-GPX4 axis and strongly triggers lipid peroxidation, resulting in cell death by ferroptosis. By contrast, L-2d does not affect the KRAS-Nrf2-GPX4 axis and activates cell death mainly through apoptosis. Overall, our study demonstrates for the first time that cationic alkyl porphyrins, which have a IC50 ~ 23 nM, activate a dual mechanism of cell death, ferroptosis and apoptosis, where the predominant form depends on the delivery mode

Potent Apoptotic Response Induced by Chloroacetamidine Anthrathiophenediones in Bladder Cancer Cells

We previously found that two neighboring G-quadruplexes behave as a molecular switch controlling the expression of <i>HRAS</i> (Cogoi, S.; Schekotikhin, A. E.; Xodo, L. E. Nucl. Acids Res. 2014, DOI: 10.1093/nar/gku574). In this study we have designed anthrathiophenediones with two chloroacetamidine-containing side chains (CATDs) as G-quadruplex binders and have examined their anticancer activity in T24 bladder cancer cells bearing mutant <i>HRAS</i> and in T24 xenografts. The designed CATDs (<b>3a</b>–<b>e</b>), bearing alkyl side chains of different length, penetrate T24 cancer cells more than their analogues with guanidine-containing side chains. The lead compounds <b>3a</b> and <b>3c</b> inhibit <i>HRAS</i> expression, metabolic activity, and colony formation in T24 cancer cells. They also activate a strong apoptotic response, as indicated by PARP-1, caspases 3/7, and annexin V/propidium iodide assays. Apoptosis occurs under conditions where cyclin D1 is down-regulated and the cell cycle arrested in G2 phase. Finally, compound <b>3a</b> inhibits the growth of T24 xenografts and increases the median survival time of nude mice

RNA G-Quadruplexes in Kirsten Ras (KRAS) Oncogene as Targets for Small Molecules Inhibiting Translation

The human KRAS transcript contains a G-rich 5\ue2\u80\ub2-UTR sequence (77% GC) harboring several G4 motifs capable to form stable RNA G-quadruplex (RG4) structures that can serve as targets for small molecules. A biotin-streptavidin pull-down assay showed that 4,11-bis(2-aminoethylamino)anthra[2,3-b]furan-5,10-dione (2a) binds to RG4s in the KRAS transcript under low-abundance cellular conditions. Dual-luciferase assays demonstrated that 2a and its analogue 4,11-bis(2-aminoethylamino)anthra[2,3-b]thiophene-5,10-dione (2b) repress translation in a dose-dependent manner. The effect of the G4-ligands on Panc-1 cancer cells has also been examined. Both 2a and 2b efficiently penetrate the cells, suppressing protein p21KRAS to <10% of the control. The KRAS down-regulation induces apoptosis together with a dramatic reduction of cell growth and colony formation. In summary, we report a strategy to suppress the KRAS oncogene in pancreatic cancer cells by means of small molecules binding to RG4s in the 5\ue2\u80\ub2-UTR of mRNA

Evaluation of Toxic Properties of New Glycopeptide Flavancin on Rats

Glycopeptide antibiotics have side effects that limit their clinical use. In view of this, the development of glycopeptides with improved chemotherapeutic properties remains the main direction in the search for new antibacterial drugs. The objective of this study was to evaluate the toxicological characteristics of new semi-synthetic glycopeptide flavancin. Acute and chronic toxicity of antibiotic was evaluated in Wistar rats. The medium lethal dose (LD50) and the maximum tolerated doses (MTD) were calculated by the method of Litchfield and Wilcoxon. In the chronic toxicity study, the treatment regimen consisted of 15 daily intraperitoneal injections using two dosage levels: 6 and 10 mg/kg/day. Total doses were equivalent to MTD or LD50 of flavancin, respectively. The study included assessment of the body weight, hematological parameters, blood biochemical parameters, urinalysis, and pathomorphological evaluation of the internal organs. The results of the study demonstrated that no clinical-laboratory signs of toxicity were found after 15 daily injections of flavancin at a total dose close to the MTD or LD50. The pathomorphological study did not reveal any lesions on the organ structure of animals after low-dose administration of flavancin. Thus, flavancin favorably differs in terms of toxicological properties from the glycopeptides currently used in the clinic

Essential Oil from Melaleuca leucadendra: Antimicrobial, Antikinetoplastid, Antiproliferative and Cytotoxic Assessment

Essential oils (EOs) are known for their use in cosmetics, food industries, and traditional medicine. This study presents the chemical composition and therapeutic properties against kinetoplastid and eukaryotic cells of the EO from Melaleucaleucadendra (L.) L. (Myrtaceae). Forty-five compounds were identified in the oil by GC-MS, containing a major component the 1,8-cineole (61%). The EO inhibits the growth of Leishmania amazonensis and Trypanosoma brucei at IC50 values &lt;10 &mu;g/mL. However, 1,8 cineole was not the main compound responsible for the activity. Against malignant (22Rv1, MCF-7, EFO-21, including resistant sublines MCF-7/Rap and MCF-7/4OHTAMO) and non-malignant (MCF-10A, J774A.1 and peritoneal macrophage) cells, IC50 values from 55 to 98 &mu;g/mL and from 94 to 144 &mu;g/mL were obtained, respectively. However, no activity was observed on Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Aspergillus niger, Candida parapsilosis, Microsporum canis, or Trypanosoma cruzi. The EO was able to control the lesion size and parasite burden in the model of cutaneous leishmaniasis in BALB/c mice caused by L. amazonensis compared to untreated animals (p &lt; 0.05) and similar with those treated with Glucantime&reg; (p &gt; 0.05). This work constitutes the first evidence of antiproliferative potentialities of EO from M. leucadendra growing in Cuba and could promote further preclinical investigations to confirm the medical value of this plant, in particular for leishmaniasis treatment

Photodynamic Therapy for ras-Driven Cancers: Targeting G-Quadruplex RNA Structures with Bifunctional Alkyl-Modified Porphyrins

Designing small molecules able to break down G4 structures in mRNA (RG4s) offers an interesting approach to cancer therapy. Here, we have studied cationic porphyrins (CPs) bearing an alkyl chain up to 12 carbons, as they bind to RG4s while generating reactive oxygen species upon photoirradiation. Fluorescence-activated cell sorting (FACS) and confocal microscopy showed that the designed alkyl CPs strongly penetrate cell membranes, binding to KRAS and NRAS mRNAs under low-abundance cell conditions. In Panc-1 cells, alkyl CPs at nanomolar concentrations promote a dramatic downregulation of KRAS and NRAS expression, but only if photoactivated. Alkyl CPs also reduce the metabolic activity of pancreatic cancer cells and the growth of a Panc-1 xenograft in SCID mice. Propidium iodide/annexin assays and caspase 3, caspase 7, and PARP-1 analyses show that these compounds activate apoptosis. All these data demonstrate that the designed alkyl CPs are efficient photosensitizers for the photodynamic therapy of ras-driven cancers

Engagement with tNOX (ENOX2) to Inhibit SIRT1 and Activate p53-Dependent and -Independent Apoptotic Pathways by Novel 4,11-Diaminoanthra[2,3-b]furan-5,10-diones in Hepatocellular Carcinoma Cells

Hepatocellular carcinoma (HCC) is the most frequent primary malignancy of the liver and is among the top three causes of cancer-associated death worldwide. However, the clinical use of chemotherapy for HCC has been limited by various challenges, emphasizing the urgent need for novel agents with improved anticancer properties. We recently synthesized and characterized a series of 4,11-diaminoanthra[2,3-b]furan-5,10-dione derivatives that exhibit potent apoptotic activity against an array of cancer cell lines, including variants with multidrug resistance. Their effect on liver cancer cells, however, was unknown. Here, we investigated three selected 4,11-diaminoanthra[2,3-b]furan-5,10-dione derivatives (compounds 1⁻3) for their cytotoxicity and the underlying molecular mechanisms in wild-type or p53-deficient HCC cells. Cytotoxicity was determined by WST-1 assays and cell impedance measurements and apoptosis was analyzed by flow cytometry. The interaction between compounds and tumor-associated NADH oxidase (tNOX, ENOX2) was studied by cellular thermal shift assay (CETSA). We found that compound 1 and 2 induced significant cytotoxicity in both HepG2 and Hep3B lines. CETSA revealed that compounds 1 and 2 directly engaged with tNOX, leading to a decrease in the cellular NAD⁺/NADH ratio. This decreased the NAD⁺-dependent activity of Sirtuin 1 (SIRT1) deacetylase. In p53-wild-type HepG2 cells, p53 acetylation/activation was enhanced, possibly due to the reduction in SIRT1 activity, and apoptosis was observed. In p53-deficient Hep3B cells, the reduction in SIRT1 activity increased the acetylation of c-Myc, thereby reactivating the TRAIL pathway and, ultimately leading to apoptosis. These compounds thus trigger apoptosis in both cell types, but via different pathways. Taken together, our data show that derivatives 1 and 2 of 4,11-diaminoanthra[2,3-b]furan-5,10-diones engage with tNOX and inhibit its oxidase activity. This results in cytotoxicity via apoptosis through tNOX-SIRT1 axis to enhance the acetylation of p53 or c-Myc in HCC cells, depending on their p53 status

Engagement with tNOX (ENOX2) to Inhibit SIRT1 and Activate p53-Dependent and -Independent Apoptotic Pathways by Novel 4,11-Diaminoanthra[2,3-<i>b</i>]furan-5,10-diones in Hepatocellular Carcinoma Cells

Hepatocellular carcinoma (HCC) is the most frequent primary malignancy of the liver and is among the top three causes of cancer-associated death worldwide. However, the clinical use of chemotherapy for HCC has been limited by various challenges, emphasizing the urgent need for novel agents with improved anticancer properties. We recently synthesized and characterized a series of 4,11-diaminoanthra[2,3-b]furan-5,10-dione derivatives that exhibit potent apoptotic activity against an array of cancer cell lines, including variants with multidrug resistance. Their effect on liver cancer cells, however, was unknown. Here, we investigated three selected 4,11-diaminoanthra[2,3-b]furan-5,10-dione derivatives (compounds 1&#8211;3) for their cytotoxicity and the underlying molecular mechanisms in wild-type or p53-deficient HCC cells. Cytotoxicity was determined by WST-1 assays and cell impedance measurements and apoptosis was analyzed by flow cytometry. The interaction between compounds and tumor-associated NADH oxidase (tNOX, ENOX2) was studied by cellular thermal shift assay (CETSA). We found that compound 1 and 2 induced significant cytotoxicity in both HepG2 and Hep3B lines. CETSA revealed that compounds 1 and 2 directly engaged with tNOX, leading to a decrease in the cellular NAD+/NADH ratio. This decreased the NAD+-dependent activity of Sirtuin 1 (SIRT1) deacetylase. In p53-wild-type HepG2 cells, p53 acetylation/activation was enhanced, possibly due to the reduction in SIRT1 activity, and apoptosis was observed. In p53-deficient Hep3B cells, the reduction in SIRT1 activity increased the acetylation of c-Myc, thereby reactivating the TRAIL pathway and, ultimately leading to apoptosis. These compounds thus trigger apoptosis in both cell types, but via different pathways. Taken together, our data show that derivatives 1 and 2 of 4,11-diaminoanthra[2,3-b]furan-5,10-diones engage with tNOX and inhibit its oxidase activity. This results in cytotoxicity via apoptosis through tNOX-SIRT1 axis to enhance the acetylation of p53 or c-Myc in HCC cells, depending on their p53 status