Drug efflux transporters, in particular P-glycoprotein (Pgp), limit the success of chemotherapy. We previously found that synthetic doxorubicin conjugated with nitric oxide (NO)-releasing group ...overcomes resistance by inducing a NO-mediated inhibition of Pgp. Here we produced the first liposomal formulations of this nitrooxy-doxorubicin decorated with folic acid (FA), termed LNDF, in order to improve their active targeting against Pgp-expressing tumors. Folate was inserted onto liposomes surface using two different methods and the formulations were compared with respect to their technological features and in vitro behavior. By analyzing human and murine breast cancer cells with different expression of FA receptor (FAR) and Pgp, we demonstrated that LNDF are internalized in a FAR-dependent manner and achieve maximal anti-tumor efficacy against FAR-positive/Pgp-positive cells. Upon uptake of LNDF, nitrooxy-doxorubicin was delivered within nucleus, where it induced cell cycle arrest and DNA damages, and mitochondria, where it impaired the mitochondrial energy metabolism and triggered mitochondria-dependent apoptosis. LNDF reduced the growth of FAR-positive/Pgp-positive tumors and prevented tumor formation in mice, whereas doxorubicin and Caelyx® failed. LNDF cardiotoxicity was comparable to Caelyx®. The sensitivity to LNDF was maintained in tumors exposed to repeated cycles of the drug and in cells derived from the exposed tumors, excluding the onset of secondary resistance.
By combining an innovative multitarget cargo drug, conceived to achieve high efficacy against Pgp-expressing cells, and appropriate strategies of liposome formulation and decoration, we produced a therapeutic tool that may represent a significant advancement in the treatment of FAR-positive/Pgp-positive tumors.
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Doxorubicin is one of the most effective drugs for the first-line treatment of high-grade osteosarcoma. Several studies have demonstrated that the major cause for doxorubicin resistance in ...osteosarcoma is the increased expression of the drug efflux transporter ABCB1/P-glycoprotein (Pgp). We recently identified a library of H
2
S-releasing doxorubicins (Sdox) that were more effective than doxorubicin against resistant osteosarcoma cells. Here we investigated the molecular mechanisms of the higher efficacy of Sdox in human osteosarcoma cells with increasing resistance to doxorubicin. Differently from doxorubicin, Sdox preferentially accumulated within the endoplasmic reticulum (ER), and its accumulation was only modestly reduced in Pgp-expressing osteosarcoma cells. The increase in doxorubicin resistance was paralleled by the progressive down-regulation of genes of ER-associated protein degradation/ER-quality control (ERAD/ERQC), two processes that remove misfolded proteins and protect cell from ER stress-triggered apoptosis. Sdox, that sulfhydrated ER-associated proteins and promoted their subsequent ubiquitination, up-regulated ERAD/ERQC genes. This up-regulation, however, was insufficient to protect cells, since Sdox activated ER stress-dependent apoptotic pathways, e.g., the C/EBP-β LIP/CHOP/PUMA/caspases 12-7-3 axis. Sdox also promoted the sulfhydration of Pgp that was subsequently ubiquitinated: this process further enhanced Sdox retention and toxicity in resistant cells. Our work suggests that Sdox overcomes doxorubicin resistance in osteosarcoma cells by at least two mechanisms: it induces the degradation of Pgp following its sulfhydration and produces a huge misfolding of ER-associated proteins, triggering ER-dependent apoptosis. Sdox may represent the prototype of innovative anthracyclines, effective against doxorubicin-resistant/Pgp-expressing osteosarcoma cells by perturbing the ER functions.
Two novel NO photodonors (NOPDs) based on BODIPY and Rhodamine antennae activatable with the highly biocompatible green light are reported. Both NOPDs exhibit considerable fluorescence emission and ...release NO with remarkable quantum efficiencies. The combination of the photoreleasing and emissive performance for both compounds is superior to those exhibited by other NOPDs based on similar light‐harvesting centres, making them very intriguing for image‐guided phototherapeutic applications. Preliminary biological data prove their easy visualization in cell environment due to the intense green and orange‐red fluorescence and their photodynamic action on cancer cells due to the NO photo‐liberated.
BODIPY and Rhodamine‐based NO photodonors activatable by the highly biocompatible green light exhibit superior combination of fluorescence emission and NO release performances making them very intriguing for image‐guided phototherapeutic applications.
Human hemoglobin (Hb) is the preferred iron source of Staphylococcus aureus. This pathogenic bacterium exploits a sophisticated protein machinery called Iron-regulated surface determinant (Isd) ...system to bind Hb, extract and internalize heme, and finally degrade it to complete iron acquisition. IsdB, the surface exposed Hb receptor, is a proven virulence factor of S. aureus and the inhibition of its interaction with Hb can be pursued as a strategy to develop new classes of antimicrobials. To identify small molecules able to disrupt IsdB:Hb protein-protein interactions (PPIs), we carried out a structure-based virtual screening campaign and developed an ad hoc immunoassay to screen the retrieved set of commercially available compounds. Saturation-transfer difference (STD) NMR was applied to verify specific interactions of a sub-set of molecules, chosen based on their efficacy in reducing the amount of Hb bound to IsdB. Among molecules for which direct binding was verified, the best hit was submitted to ITC analysis to measure the binding affinity to Hb, which was found to be in the low micromolar range. The results demonstrate the viability of the proposed in silico/in vitro experimental pipeline to discover and test IsdB:Hb PPI inhibitors. The identified lead compound will be the starting point for future SAR and molecule optimization campaigns.
Multidrug resistance (MDR) is a critical hindrance to the success of cancer chemotherapy. The main thing responsible for MDR phenotypes are plasma-membranes associated with adenosine triphosphate ...(ATP) Binding Cassette (ABC) drug efflux transporters, such as the P-glycoprotein (Pgp) transporter that has the broadest spectrum of substrates. Curcumin (CURC) is a Pgp inhibitor, but it is poorly soluble and bioavailable. To overcome these limitations, we validated the efficacy and safety of CURC, loaded in biocompatible solid lipid nanoparticles (SLNs), with or without chitosan coating, with the goal of increasing the stability, homogeneous water dispersibility, and cellular uptake. Both CURC-loaded SLNs were 5-10-fold more effective than free CURC in increasing the intracellular retention and toxicity of doxorubicin in Pgp-expressing triple negative breast cancer (TNBC). The effect was due to the decrease of intracellular reactive oxygen species, consequent inhibition of the Akt/IKKα-β/NF-kB axis, and reduced transcriptional activation of the Pgp promoter by p65/p50 NF-kB. CURC-loaded SLNs also effectively rescued the sensitivity to doxorubicin against drug-resistant TNBC tumors, without signs of systemic toxicity. These results suggest that the combination therapy, based on CURC-loaded SLNs and doxorubicin, is an effective and safe approach to overcome the Pgp-mediated chemoresistance in TNBC.
Physiologically, smooth muscle cells (SMC) and nitric oxide (NO) produced by endothelial cells strictly cooperate to maintain vasal homeostasis. In atherosclerosis, where this equilibrium is altered, ...molecules providing exogenous NO and able to inhibit SMC proliferation may represent valuable antiatherosclerotic agents. Searching for dual antiproliferative and NO-donor molecules, we found that furoxans significantly decreased SMC proliferation in vitro, albeit with different potencies. We therefore assessed whether this property is dependent on their thiol-induced ring opening. Indeed, while furazans (analogues unable to release NO) are not effective, furoxans' inhibitory potency parallels with the electron-attractor capacity of the group in 3 of the ring, making this effect tunable. To demonstrate whether their specific block on G1-S phase could be NO-dependent, we supplemented SMCs with furoxans and inhibitors of GMP- and/or of the polyamine pathway, which regulate NO-induced SMC proliferation, but they failed in preventing the antiproliferative effect. To find the real mechanism of this property, our proteomics studies revealed that eleven cellular proteins (with SUMO1 being central) and networks involved in cell homeostasis/proliferation are modulated by furoxans, probably by interaction with adducts generated after degradation. Altogether, thanks to their dual effect and pharmacological flexibility, furoxans may be evaluated in the future as antiatherosclerotic molecules.
Cannabinoid type 1 (hCB1) and type 2 (hCB2) receptors are pleiotropic and crucial targets whose signaling contributes to physiological homeostasis and its restoration after injury. Being ...predominantly expressed in peripheral tissues, hCB2R represents a safer therapeutic target than hCB1R, which is highly expressed in the brain, where it regulates processes related to cognition, memory, and motor control. The development of hCB2R ligands represents a therapeutic opportunity for treating diseases such as pain, inflammation and cancer. Identifying new selective scaffolds for cannabinoids and determining the structural determinants responsible for agonism and antagonism are priorities in drug design. In this work, a series of
-1,3-dialkyl(aryl)-2-oxoimidazolidin-4-ylidene-aryl(alkyl)sulfonamides is designed and synthesized and their affinity for human hCB1R and hCB2R is determined. Starting with a scaffold selected from the NIH Psychoactive Drug Screening Program Repository, through a combination of molecular modeling and structure-activity relationship studies, we were able to identify the chemical features leading to finely tuned hCB2R selectivity. In addition, an in silico model capable of predicting the functional activity of hCB2R ligands was proposed and validated. The proposed receptor activation/deactivation model enabled the identification of four pure hCB2R-selective agonists that can be used as a starting point for the development of more potent ligands.
Multidrug resistant cancer cells are hard to eradicate for the inefficacy of conventional anticancer drugs. Besides escaping the cytotoxic effects of chemotherapy, they also bypass the ...pro-immunogenic effects induced by anticancer drugs: indeed they are not well recognized by host dendritic cells and do not elicit a durable anti-tumor immunity. It has not yet been investigated whether multidrug resistant cells have a different ability to induce immunosuppression than chemosensitive ones. We addressed this issue in human and murine chemosensitive and multidrug resistant cancer cells.
We found that the activity and expression of indoleamine 2,3-dioxygenase 1 (IDO1), which catalyzes the conversion of tryptophan into the immunosuppressive metabolite kynurenine, was higher in all the multidrug resistant cells analyzed and that IDO1 inhibition reduced the growth of drug-resistant tumors in immunocompetent animals. In chemoresistant cells the basal activity of JAK1/STAT1 and JAK1/STAT3 signaling was higher, the STAT3 inhibitor PIAS3 was down-regulated, and the autocrine production of STAT3-target and IDO1-inducers cytokines IL-6, IL-4, IL-1β, IL-13, TNF-α and CD40L, was increased. The disruption of the JAK/STAT signaling lowered the IDO1 activity and reversed the kynurenine-induced pro-immunosuppressive effects, as revealed by the restored proliferation of T-lymphocytes in STAT-silenced chemoresistant cells.
Our work shows that multidrug resistant cells have a stronger immunosuppressive attitude than chemosensitive cells, due to the constitutive activation of the JAK/STAT/IDO1 axis, thus resulting chemo- and immune-evasive. Disrupting this axis may significantly improve the efficacy of chemo-immunotherapy protocols against resistant tumors.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Diabetes and cancer are common, chronic, and potentially fatal diseases that frequently co-exist. Observational studies clearly indicate that the risk of several types of cancer is increased in ...diabetic patients and a number of cancer types have shown a higher mortality rate in patients with hyperglycemic associated pathologies. This scenario could be due, at least in part, to a lower efficacy of the cancer treatments which needs to be better investigated. Here, we evaluated the effects of a prolonged exposure to high glucose (HG) to the response to chemotherapy on human colon adenocarcinoma HT29 and LOVO cell lines. We observed that hyperglycemia protected against the decreased cell viability and cytotoxicity and preserved from the mitochondrial DNA lesions induced by doxorubicin (DOX) and 5-fluorouracil (5-FU) treatments by lowering ROS production. In HT29 cells the amount of intracellular DOX and its nuclear localization were not modified by HG incubation in terms of Pgp, BCRP, MRP1, 5 and 8 activity and gene expression. On the contrary, in LOVO cells, the amount of intracellular DOX was significantly decreased after a bolus of DOX in HG condition and the expression and activity of MPR1 was increased, suggesting that HG promotes drug chemoresistance in both HT29 and LOVO cells, but in a different way. In both cell types, HG condition prevented the susceptibility to apoptosis by decreasing the ratio Bax/Bcl-2 and Bax/Bcl-XL and diminished the level of cytosolic cytochrome c and the cleavage of full length of PARP induced by DOX and 5-FU. Finally, hyperglycemia reduced cell death by decreasing the cell percentage in sub-G1 peak induced by DOX (via a cell cycle arrest in the G2/M phase) and 5-FU (via a cell cycle arrest in the S phase) in HT29 and LOVO cells. Taken together, our data showed that a prolonged exposure to HG protects human colon adenocarcinoma cells from the cytotoxic effects of two widely used chemotherapeutic drugs, impairing the effectiveness of the chemotherapy itself.
To improve the biological and toxicological properties of Mefenamic acid (MA), the galactosylated prodrug of MA named MefeGAL was included in polymeric solid dispersions (PSs) composed of ...poly(glycerol adipate) (PGA) and Pluronic® F68 (MefeGAL-PS). MefeGAL-PS was compared with polymeric solid formulations of MA (MA-PS) or a mixture of equal ratio of MefeGAL/MA (Mix-PS).
The in vitro and in vivo pharmacological and toxicological profiles of PSs have been investigated. In detail, we evaluated the anti-inflammatory (carrageenan-induced paw edema test), analgesic (acetic acid-induced writhing test) and ulcerogenic activity in mice after oral treatment. Additionally, the antiproliferative activity of PSs was assessed on in vitro models of colorectal and non-small cell lung cancer.
When the PSs were resuspended in water, MefeGAL’s, MA’s and their mixture’s apparent solubilities improved due to the interaction with the polymeric formulation. By comparing the in-vivo biological performance of MefeGAL-PS with that of MA, MefeGAL and MA-PS, it was seen that MefeGAL-PS exhibited the same sustained and delayed analgesic and anti-inflammatory profile as MefeGAL but did not cause gastrointestinal irritation. The pharmacological effect of Mix-PS was present from the first hours after administration, lasting about 44 hours with only slight gastric mucosa irritation. In-vitro evaluation indicated that Mix-PS had statistically significant higher cytotoxicity than MA-PS and MefeGAL-PS.
These preliminary data are promising evidence that the galactosylated prodrug approach in tandem with a polymer-drug solid dispersion formulation strategy could represent a new drug delivery route to improve the solubility and biological activity of NSAIDs.
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•Galactosylated prodrug and polymeric solid dispersion strategies were applied.•Combined drug delivery strategies reduced the ulcerogenicity of mefenamic acid.•By applying two approaches, the biological activity of mefenamic acid was extended.•The concomitant use of two strategies boosted the cytotoxicity of mefenamic acid.
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