Natural Inhibitors of P-glycoprotein in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) remains an insidious neoplasm due to the percentage of patients who develop resistance to both classic chemotherapy and emerging drugs. Multidrug resistance (MDR) is a complex process determined by multiple mechanisms, and it is often caused by the overexpression of efflux pumps, the most important of which is P-glycoprotein (P-gp). This mini-review aims to examine the advantages of using natural substances as P-gp inhibitors, focusing on four molecules: phytol, curcumin, lupeol, and heptacosane, and their mechanism of action in AML.

Exploring the cellular uptake of hectorite clay mineral and its drug carrier capabilities.

In the last years, the use of clay minerals for pharmaceutical purposes has increased due to their interesting properties. Hectorite (Ht) is a clay belonging to the smectite group which has attracted attention for applications in biology, tissue engineering and as drug carrier and delivery system. However, the mechanisms involved in Ht cellular uptake and transport into cells, are still unclear. Herein, we used a labeled Ht (Ht/1Cl) to study both the cellular uptake, by confocal laser scanning microscopy, and internalization pathways involved in the cellular uptake, by various endocytosis-inhibiting studies and fluorescence microscopy. These studies highlighted that Ht can penetrate the cellular membrane, localizing mainly in the cytoplasm. The main intracellular transport mechanisms are the ATP-dependent ones and those where filaments and microtubules are involved. Finally, as proof of concept for the potential of Ht as carrier system, we envisaged the covalent grafting of the anticancer molecule methotrexate (MTX), chosen as model, to obtain the Ht-MTX nanomaterial. The covalent linkage was confirmed by several techniques and the morphology of the obtained nanomaterial was imaged by SEM and TEM investigations. The kinetic release of the drug from the Ht-MTX nanomaterial in physiological conditions was studied as well. Furthermore, cytotoxic studies on different cell lines, namely, HL-60, HL-60R, MCF-7, 5637, UMUC3 and RT112 showed that Ht could be a promising material for anticancer therapy.

A Curcumin-BODIPY Dyad and Its Silica Hybrid as NIR Bioimaging Probes.

In this paper we describe the synthesis of a novel bichromophoric system in which an efficient photoinduced intercomponent energy transfer process is active. The dyad consists of one subunit of curcumin and one of BODIPY and is able to emit in the far-red region, offering a large Stokes shift, capable of limiting light scattering processes for applications in microscopy. The system has been encapsulated in MCM-41 nanoparticles with dimensions between 50 and 80 nm. Both the molecular dyad and individual subunits were tested with different cell lines to study their effective applicability in bioimaging. MCM-41 nanoparticles showed no reduction in cell viability, indicating their biocompatibility and bio-inertness and making them capable of delivering organic molecules even in aqueous-based formulations, avoiding the toxicity of organic solvents. Encapsulation in the porous silica structure directed the location of the bichromophoric system within cytoplasm, while the dyad alone stains the nucleus of the hFOB cell line.

Antitumor Effect of Glandora rosmarinifolia (Boraginaceae) Essential Oil through Inhibition of the Activity of the Topo II Enzyme in Acute Myeloid Leukemia.

It was previously shown that the antitumor and cytotoxic activity of the essential oil (EO) extracted from the aerial parts of Glandora rosmarinifolia appears to involve a pro-oxidant mechanism in hepatocellular carcinoma (HCC) and in triple-negative breast cancer (TNBC) cell lines. Its most abundant compound is a hydroxy-methyl-naphthoquinone isomer. Important pharmacological activities, such as antitumor, antibacterial, antifungal, antiviral and antiparasitic activities, are attributed to naphthoquinones, probably due to their pro-oxidant or electrophilic potential; for some naphthoquinones, a mechanism of action of topoisomerase inhibition has been reported, in which they appear to act both as catalytic inhibitors and as topoisomerase II poisons. Our aim was to evaluate the cytotoxic activity of the essential oil on an acute myeloid leukemia cell line HL-60 and on its multidrug-resistant (MDR) variant HL-60R and verify its ability to interfere with topoisomerase II activity. MTS assay showed that G. rosmarinifolia EO induced a decrease in tumor cell viability equivalent in the two cell lines; this antitumor effect could depend on the pro-oxidant activity of EO in both cell lines. Furthermore, G. rosmarinifolia EO reduced the activity of Topo II in the nuclear extracts of HL-60 and HL-60R cells, as inferred from the inability to convert the kinetoplast DNA into the decatenated form and then not inducing linear kDNA. Confirming this result, flow cytometric analysis proved that EO induced a G0-G1 phase arrest, with cell reduction in the S-phase. In addition, the combination of EO with etoposide showed a good potentiation effect in terms of cytotoxicity in both cell lines. Our results highlight the antitumor activity of EO in the HL-60 cell line and its MDR variant with a peculiar mechanism as a Topo II modulator. Unlike etoposide, EO does not cause stabilization of a covalent Topo II-DNA intermediate but acts as a catalytic inhibitor. These data make G. rosmarinifolia EO a potential anticancer drug candidate due to its cytotoxic action, which is not affected by multidrug resistance.

Novel insights on [1,2]oxazolo[5,4-e]isoindoles on multidrug resistant acute myeloid leukemia cell line.

A series of [1,2]oxazolo[5,4-e]isoindole derivatives was evaluated against HL-60 cell line and its multidrug resistance (MDR) variant, HL-60R, resistant to doxorubicin and to other P-gp substrates by overexpressing the efflux pump. They displayed antiproliferative activities, with IC50 values ranging from 0.02 to 5.5 µM. In particular, the newly synthesized compound 4k produced synergistic effects in terms of cell growth inhibition and cell death induction either in combination with a Vinca alkaloid, Vinblastine, and a Taxane, Paclitaxel in HL-60R cells. The study of the mechanism of action indicated that all compounds showed antimitotic activity through inhibition of tubulin polymerization. Thus, [1,2]oxazoles could represent a valuable tool to overcome MDR mechanism, confirming the potential use of this class of compounds.

Synthesis, In Vitro and In Silico Analysis of New Oleanolic Acid and Lupeol Derivatives against Leukemia Cell Lines: Involvement of the NF-κB Pathway.

Oleanolic acid (OA) and Lupeol (LU) belong to the class of natural triterpenes and are endowed with a wide range of biological activities, including cytotoxicity toward several cancer cell lines. In this context, we investigated a set of compounds obtained from the two natural precursors for the cytotoxicity against leukemia HL60 cells and the multidrug-resistant (MDR) variant HL60R. Six new semi-synthetic triterpenes have been synthetized, fully characterized, and were investigated together with other triterpenes compounds for their pharmacological mechanism of action. The interaction of the more cytotoxic compounds with the nuclear factor kappa B (NF-κB) pathway has been also evaluated with the aid of docking. The lupane-like compounds were more active than the precursor, while the oleane-like compounds showed more complex behavior. Both OA and LU derivatives possess a similar interaction pattern with the p65 subunit of NF-κB, justifying the similar trend in their ability to inhibit the binding of p65 to DNA. Further, some of the derivatives tested were able to increase IκB-α levels preventing the translocation of NF-κB to the nucleus. In conclusion, this study offers a deeper insight on the pharmacological action of triterpenes toward leukemia cells, and it improves the background useful for the development of new anti-cancer drugs.

Phytol and Heptacosane Are Possible Tools to Overcome Multidrug Resistance in an In Vitro Model of Acute Myeloid Leukemia.

Drug resistance is the ability of cancer cells to gain resistance to both conventional and novel chemotherapy agents, and remains a major problem in cancer therapy. Resistance mechanisms are multifactorial and involve more strictly pharmacological factors, such as P-glycoprotein (P-gp) and biological factors such as inhibitor of apoptosis proteins (IAPs) and the nuclear factor-kappa B (NF-κB) pathway. Possible therapeutic strategies for the treatment of acute myeloid leukemia (AML) have increased in recent years; however, drug resistance remains a problem for most pa-tients. Phytol and heptacosane are the major compounds of Euphorbia intisy essential oil (EO) which were demonstrated to inhibit P-gp in a multidrug resistant in vitro model of AML. This study investigated the mechanism by which phytol and heptacosane improve P-gp-mediated drug transport. Phytol suppresses the P-gp expression via NF-κB inhibition and does not seem to act on the efflux system. Heptacosane acts as a substrate and potent P-gp inhibitor, demonstrating the ability to retain the substrate doxorubicin inside the cell and enhancing its cytotoxic effects. Our results suggest that these compounds act as non-toxic modulators of P-gp through different mechanisms and are able to revert P-gp-mediated drug resistance in tumor cells.

Prodrug based on halloysite delivery systems to improve the antitumor ability of methotrexate in leukemia cell lines.

The prodrug approach, as well as the development of specific systems able to deliver a chemotherapeutic agent in the target site, decreasing the side effects often associated with its administration, are still a challenging. In this context, both methotrexate drug molecules (MTX) and biotin ligand moieties, whose receptors are overexpressed on the surface of several cancer cells, were loaded on halloysite nanotubes (HNTs) to develop nanomaterial based on multifunctional and "smart" delivery systems. To highlight the crucial role played by biotin, carrier systems based on HNTs and MTX were also synthetized. In detail, several approaches were envisaged: i) a supramolecular interaction between the clay and the drug; ii) a covalent grafting of the drug onto the HNTs external surface and, iii) a combination of both approaches. The nanomaterials obtained were characterized by thermogravimetric analysis, FT-IR, and UV-vis spectroscopies, DLS and ζ-potential measurements and the morphologies were imaged by HAADF/STEM investigations. Kinetic release experiments at different pH conditions were also performed. Finally, as a proof-of-concept application of our pro-drug delivery systems based on HNTs in cancer therapy, the cytotoxic effects were evaluated on acute myeloid leukemia cell lines, HL60 and its multidrug resistance variant, HL60R. The obtained results showed that both the MTX prodrug system and the biotinylated ones played a crucial role in the biological activity and, they are promising agents for the cancer treatments.

Patterns of Innate or Acquired Resistance to Anticancer Drugs: Our Experience to Overcome It.

Drug resistance, which is often of a multiple type, can be defined as the ability of cancer cells to obtain resistance to both conventional and novel chemotherapy agents. It remains a major problem to solve in cancer therapy. The mechanisms of resistance are multifactorial, and in our cellular models of acute myeloid leukemia, hepatocellular carcinoma, and triple-negative breast cancer, it involves the NF-κB pathway. In our opinion, multitarget molecules can be considered as privileged compounds capable of attacking and reversing the resistant phenotype. In the phenomena of both innate and acquired drug resistance that we have been studying since 1998 to today and up to 2016 under the guidance of Professor Natale D'Alessandro, more strictly pharmacological factors are certainly involved. These factors include P-glycoprotein and biological factors such as inhibitory proteins; apoptosis; the Raf-1 kinase inhibitor protein, an important tumor suppressor and metastasis inhibitor, which enhances drug-induced apoptosis of cancer cells; and Yin Yang, a transcription factor involved in drug resistance.

In Vitro Modulation of P-Glycoprotein Activity by Euphorbia intisy Essential Oil on Acute Myeloid Leukemia Cell Line HL-60R.

Euphorbia species have a large spectrum of traditional medicinal uses. We tested the biological activities of the essential oil (EO) of Euphorbia intisy Drake in an acquired multidrug resistance leukemia model to assess whether the EO obtained by hydrodistillation of stems was able to reverse the resistant phenotype. HL-60R cell lines are characterized by the overexpression of P-glycoprotein (P-gp), inhibitors of apoptosis proteins (IAPs) and constitutive expression of NF-κB. EO chemical composition was determined by GC/MS analysis; cytotoxic activity of EO by MTS assay alone or in combination with doxorubicin; pro-apoptotic effect and doxorubicin accumulation were analyzed by flow cytometry; P-gp ATPase activity was measured by P-gp-Glo™ assay systems kit. The ability to inhibit NF-κB and its target genes was also assessed. E. intisy EO exhibited a comparable cytotoxic effect and ability to block P-gp in both the HL-60 and its MDR variant HL-60R. In addition, EO suppressed P-gp protein expression and significantly downregulated MDR1 mRNA level, as well as some IAPs proteins, probably through the inhibition of NF-κB. Our results suggest that E. intisy EO could reverse P-gp-mediated drug resistance in tumor cells acting as a chemosensitizing agent.

NF-κB and Disease.

The role of NF-κB in all diseases characterized by an inflammatory process, from cancer to autoimmune diseases, is known, but-precisely because it is involved in many diseases-this transcriptional factor continues to attract scientific research and the new knowledge that emerges is fundamental in highlighting the therapeutic potential that this factor can have in the various diseases in which it is involved [...].

Can NF-κB Be Considered a Valid Drug Target in Neoplastic Diseases? Our Point of View.

Multidrug resistance (MDR), of the innate and acquired types, is one of major problems in treating tumor diseases with a good chance of success. In this review, we examine the key role of nuclear factor-kappa B (NF-κB) to induce MDR in three tumor models characterized precisely by innate or acquired MDR, in particular triple negative breast cancer (TNBC), hepatocellular carcinoma (HCC), and acute myeloid leukemia (AML). We also present different pharmacological approaches that our group have employed to reduce the expression/activation of this transcriptional factor and thus to restore chemo-sensitivity. Finally, we examine the latest scientific evidence found by other groups, the most significant clinical trials regarding NF-κB, and new perspectives on the possibility to consider this transcriptional factor a valid drug target in neoplastic diseases.

Synthesis of Curcumin Derivatives and Analysis of Their Antitumor Effects in Triple Negative Breast Cancer (TNBC) Cell Lines.

We analyzed antitumor effects of a series of curcumin analogues. Some of them were obtained by reaction of substitution involving the two phenolic OH groups of curcumin while the analogues with a substituent at C-4 was prepared following an original procedure that regards the condensation of benzenesulfenic acid onto the nucleophilic central carbon of the curcumin skeleton. We analyzed cytotoxic effects of such derivatives on two TNBC (triple negative breast cancer) cell lines, SUM 149 and MDA-MB-231, but only three of them showed an IC50 in a lower micromolar range with respect to curcumin. We also focused on these three derivatives that in both cell lines exhibited a higher or at least equivalent pro-apoptotic effect than curcumin. The analysis of molecular mechanisms of action of the curcumin derivatives under study has highlighted that they decreased NF-κB transcriptional factor activity, and consequently the expression of some NF-κB targets. Our data confirmed once again that curcumin may represent a very good lead compound to design analogues with higher antitumor capacities and able to overcome drug resistance with respect to conventional ones, even in tumors difficult to treat as TNBC.

Antitumor Mechanism of the Essential Oils from Two Succulent Plants in Multidrug Resistance Leukemia Cell.

Drug resistance remains a major challenge in the treatment of cancer. The multiplicity of the drug resistance determinants raises the question about the optimal strategies to deal with them. Essential oils showed to inhibit the growth of different tumor cell types. Essential oils contain several chemical classes of compounds whose heterogeneity of active moieties can help prevent the development of drug resistance. In the present paper, we analyzed, by gas chromatography-mass spectrometry the chemical composition of the essential oil of the leaves of Kalanchoe beharensis obtained by hydrodistillation and compared the chemical composition of its essential oil with that of Cyphostemma juttae. Our results demonstrated the anticancer and proapoptotic activities of both species against acute myeloid leukemia on an in vitro model and its multidrug resistant variant involving NF-κB pathway. The essential oils of both species produced a significant decrease in many targets of NF-κB both at mRNA and protein levels. The results corroborate the idea that essential oils may be a good alternative to traditional drugs in the treatment of cancer, especially in drug resistant cancer.

Essential Oil Composition of Alluaudia procera and in Vitro Biological Activity on Two Drug-Resistant Models.

Drug resistance is a major obstacle in antibiotic and antitumor chemotherapy. In response to the necessity to find new therapeutic strategies, plant secondary metabolites including essential oils (EOs) may represent one of the best sources. EOs in plants act as constitutive defenses against biotic and abiotic stress, and they play an important role in the pharmacology for their low toxicity, good pharmacokinetic and multitarget activity. In this context, natural products such as EOs are one of the most important sources of drugs used in pharmaceutical therapeutics. The aim of this paper was to identify the chemical composition of the essential oil of Alluaudia procera leaves, obtained by hydrodistillation and analysed by gas chromatography-mass spectrometry, and to verify its biological activities on acute myeloid leukemia cancer cell HL60 and its multidrugresistant variant HL60R and the Gram-positive Staphylococcus aureus exhibiting multi-antibiotic resistance. We speculate that cytotoxic and antibiotic effects observed in the tested resistant models may be due to the coordinate activities of forty compounds detected or to the C16 macrocyclic lactones which are the major ones (30%). Our data confirm the possibility of using EOs as therapeutic strategies in resistant models is due to the heterogeneous composition of the oils themselves.

Targeting multiple myeloma with natural polyphenols.

Multiple myeloma (MM) is still an incurable hematologic malignancy. Although new therapeutic strategies have been developed to target different pathways in malignant cells, such as proliferation, differentiation, and apoptosis, better survival rates have also been achieved by the introduction of autologous stem cell transplantation (ASCT). Hematopoietic stem cell transplantation and novel targeted agents, such as proteasome inhibitors, monoclonal antibodies, immunomodulatory drugs, check-point inhibitors and epigenetic modulators, have significantly achieved long remission time and increased survival rates. However, most patients relapse, develop resistance, and eventually die because of refractory disease. All these issues highlight the need to investigate newer therapeutic targets to improve patient outcomes. Natural products play an important role in anti-tumor drug discovery, for this reason, in the investigation of novel natural anti-MM agents, we focused on natural polyphenols. Moreover, plant extracts show no or low toxicity towards normal cells and some of them have also a favorable pharmacokinetic profile. The biological activities of plant extracts are mainly due to their content in polyphenols, flavonoids, and terpenoids. Numerous studies showed that polyphenols, generally recognized as antioxidants, possess anticancer and pro-apoptosis properties. Other studies reported the potential clinical applications of flavonoids for their well-known protective and therapeutic effects against cancer, cardiovascular, and neurodegenerative diseases. The combination of plant extracts with anti-cancer drugs may offer a relevant advantage for therapeutic efficacy by sensitizing malignant cells to drugs and overcoming drug-induced resistance in cancer. For all these reasons, a significant number of polyphenolic compounds isolated from plants are still used nowadays in cancer clinical practice in combination with other drugs, also against hematologic malignancies.

Cytotoxicity of oleanolic and ursolic acid derivatives toward hepatocellular carcinoma and evaluation of NF-κB involvement.

Oleanolic and ursolic acids are two ubiquitous isomeric triterpene phytochemicals known for their anticancer activity. A set of derivatives of the two compounds with a modified oxidation state and lipophylicity at C-3 and C-28 positions, were prepared and tested as anticancer agents versus the lines HepG2, Hep3B and HA22T/VGH of hepatocarcinoma, a strongly aggressive tumor that is not responsive toward the standard therapies. New derivatives containing a three carbons side chain on the C-3 position were synthetized in both stereoisomeric forms by the Barbier-Grignard procedure and three of them were found to be active toward all of the three targets. The implication of the transcriptional nuclear factor NF-κB in the mechanism of action was assessed for the more active compounds in the set, as hepatocellular carcinoma (HCC) cyto-types are known to overexpress NF-κB.

Essential oil of Cyphostemma juttae (Vitaceae): Chemical composition and antitumor mechanism in triple negative breast cancer cells.

The genus Cyphostemma (Planch.) Alston (Vitaceae) includes about 150 species distributed in eastern and southern Africa and Madagascar. Some species are used in traditional medicine and their biological activities, including antiproliferative effects against cancer cell lines, have been demonstrated. To date no investigations on Cyphostemma essential oils have been carried out. Essential oils, which play important roles in plant defenses have been demonstrated to be active in the treatment of several human diseases and to enhance bioavability of other drugs. The aim of this paper was to identify the chemical composition of the essential oil of the leaves of Cyphostemma juttae (Dinter & Gilg) Desc. and to verify some biological activities on two triple negative breast cancer cell lines (MDA-MB-231, SUM 149), characterized by the over-expression of the transcription factor NF-κB. In the essential oil, obtained by hydrodistillation and analysed by gas chromatography-mass spectrometry, 39 compounds were detected and with phytol (30%) dominating the chemical composition. C. juttae essential oil reduced cell growth and showed a pro-oxidant activity in both cell lines. Moreover, C. juttae essential oil caused a substantial decrease of NF-κB activation and consequently a significant reduction of some NF-κB target genes. The present study shows for the first time the cytotoxic properties of C. juttae essential oil and highlight its availability to interfere with NF-κB pathway, suggesting a potential therapeutic use in triple negative breast cancers (TNBCs) of this essential oil.

Chemical composition, in vitro antitumor and pro-oxidant activities of Glandora rosmarinifolia (Boraginaceae) essential oil.

The biological properties of essential oils have been demonstrated in the treatment of several diseases and to enhance the bioavailability of other drugs. In natural habitats the essential oils compounds may play important roles in the protection of the plants as antibacterials, antivirals, antifungals, insecticides and also against herbivores by reducing their appetite for such plants or by repelling undesirable others. We analyzed by gas-chromatography mass spectrometry the chemical composition of the essential oil of aerial parts of Glandora rosmarinifolia (Ten.) D.C. Thomas obtained by hydrodistillation and verified some biological activities on a panel of hepatocellular carcinoma cell lines (HA22T/VGH, HepG2, Hep3B) and triple negative breast cancer cell lines (SUM 149, MDA-MB-231). In the essential oil we detected 35 compounds. The results of the biological assays indicate that essential oil of G. rosmarinifolia induces cell growth inhibition at concentration-dependent way in all cell line models. This oil does not seem to possess antioxidant activity, while the cytotoxicity of G. rosmarinifolia essential oil appeared to involve, at least in part, a pro-oxidant mechanism. Our results show for the first time the antitumoral and pro-oxidant activities of G. rosmarinifolia essential oil and suggest that it may represent a resource of pharmacologically active compounds.

Potential Therapeutic Applications of MDA-9/Syntenin-NF-κB-RKIP Loop in Human Liver Carcinoma.

BACKGROUND: Overexpression of MDA-9/Syntenin occurs in multiple human cancer cell lines and is associated with higher grade of tumor classification, invasiveness and metastasis. In some cases, its role in cancer biology depends on relationships between MDA-9/Syntenin and NF-κB. OBJECTIVE: This study aims to analyze the presence of a regulation loop like that between MDA-9/Syntenin - NF-κB - RKIP in human liver carcinoma. METHODS: Transient transfection was performed with siRNA anti-MDA-9/Syntenin. Expression of different factors was evaluated by Real time-PCR and Western blotting, while NF-κB activation by TransAM assay. Invasion capacity was analyzed by Matrigel Invasion Assay and the effects of agents on cell viability were examined by MTS assay. RESULTS: We have examined basal expression of MDA-9/Syntenin in three cell lines of human liver carcinoma (HA22T/VGH, Hep3B and HepG2). In all cell lines there was an inverse relationship between MDA-9/Syntenin and RKIP expression levels, and a positive correlation between MDA-9/Syntenin expression and NF-κB activation levels. By silencing with a siRNA anti-MDA-9/Syntenin we observed in all cell lines a very strong increase of RKIP at mRNA level. Interestingly, in all cell lines, inhibition of MDA- 9/Syntenin expression induced NF-κB downregulation and contemporary a reduction in invasion ability MMP-2 dependent. Finally, we showed a good additive effect of MDA- 9/Syntenin siRNA when associated with Curcumin or Doxorubicin on cell growth inhibition. CONCLUSION: Our data confirm the key role of MDA-9/Syntenin in HCC biology. The presence of a regulation loop among MDA-9/Syntenin, NF-κB and RKIP provide new pharmacological approaches.