betadex and Melanoma

Injectable stimuli-responsive hydrogels could offer an opportunity for local administration at the tumor site and a sustained drug release. In this paper, a copolymer of azobenzene derivative and N-isopropyl acrylamide (NIPAM) was synthesized, which are performed as light- and thermo-sensitive parts, respectively. The DAS@SCD/NIPAZO hydrogel was prepared upon the establishment of host-guest interactions between the hydrophobic core of CD and azobenzene moiety. The LCST of the synthesized copolymer was modified from 31.3 °C to 36.5 °C by the incorporation of the hydrophilic host moieties of the modified starch into the NIPAM copolymer structure. The LCST-based property of the hydrogel made it syringable in low temperatures and switch to a gel state after local injection. The drug release profile of the hydrogel was explored in four different conditions involving two distinct temperatures combined with two different light wavelengths to examine the light- and thermo-sensitivity of the hydrogel. Moreover, a Paclitaxel-loaded hydrogel was prepared to study the in vitro efficiency of the sample and was investigated by MTT assay against the cancerous fibroblastic cells (A-431), which revealed a sharp decline in cell viability under 365 nm light irradiation; furthermore, to evaluate the in vivo effects of the PTX-loaded hydrogel, histological studies based on staining techniques were carried out.

Topics: beta-Cyclodextrins; Humans; Hydrogels; Melanoma; Paclitaxel; Polymers; Temperature

Combining photodynamic therapy (PDT) with natural killer (NK) cell-based immunotherapy has shown great potential against cancers, but the shedding of NK group 2, member D ligands (NKG2DLs) on tumor cells inhibited NK cell activation in the tumor microenvironment. Herein, we assembled microenvironment-/light-responsive bio-nanosystems (MLRNs) consisting of SB-3CT-containing β-cyclodextrins (β-CDs) and photosensitizer-loaded liposomes, in which SB-3CT was considered to remodel the tumor microenvironment. β-CDs and liposomes were linked by metalloproteinase 2 (MMP-2) responsive peptides, enabling sequential release of SB-3CT and chlorin e6 triggered by the MMP-2-abundant tumor microenvironment and 660 nm laser irradiation, respectively. Released SB-3CT blocked tumor immune escape by antagonizing MMP-2 and promoting the NKG2D/NKG2DL pathway, while liposomes were taken up by tumor cells for PDT. MLRN-mediated photo-immunotherapy significantly induced melanoma cell cytotoxicity (83.31%), inhibited tumor growth (relative tumor proliferation rate: 1.13% of that of normal saline) in the xenografted tumor model, and enhanced tumor-infiltrating NK cell (148 times) and NKG2DL expression (9.55 and 16.52 times for MICA and ULBP-1, respectively), achieving a synergistic effect. This study not only provided a simple insight into the development of new nanomedicine for programed release of antitumor drugs and better integration of PDT and immunotherapy but also a novel modality for clinical NK cell-mediated immunotherapy against melanoma.

Topics: Antineoplastic Agents; beta-Cyclodextrins; Cell Line, Tumor; Enzyme Inhibitors; Heterocyclic Compounds, 1-Ring; Humans; Immunotherapy; Liposomes; Matrix Metalloproteinase 2; Melanoma; NK Cell Lectin-Like Receptor Subfamily K; Photosensitizing Agents; Saline Solution; Sulfones; Triazenes; Tumor Microenvironment

Topics: Antineoplastic Agents; beta-Cyclodextrins; Biological Availability; Cations; Cell Line, Tumor; Curcumin; Humans; Melanoma; Starch

Harman, a natural β-carboline alkaloid, has recently gained considerable interest due to its anticancer properties. However, its physicochemical characteristics and poor oral bioavailability have been limiting factors for its pharmaceutical development. In this paper, we described the complexation of harman (HAR) with β-cyclodextrin (βCD) as a promising alternative to improve its solubility and consequently its cytotoxic effect in chemoresistant melanoma cells (A2058 cell line). Inclusion complexes (βCD-HAR) were prepared using a simple method and then characterized by FTIR, NMR and SEM techniques. Through in silico studies, the mechanism of complexation of HAR with βCD was elucidated in detail. Both HAR and βCD-HAR promoted cytotoxicity, apoptosis, cell cycle arrest and inhibition of cell migration in melanoma cells. Interestingly, complexation of HAR with βCD enhanced its pro-apoptotic effect by increasing of caspase-3 activity (p < 0.05), probably due to an improvement in HAR solubility. In addition, HAR and βCD-HAR sensitized A2058 cells to vemurafenib, dacarbazine and 5FU treatments, potentializing their cytotoxic activity. These findings suggest that complexation of HAR with natural polymers such as βCD can be useful to improve its bioavailability and antimelanoma activity.

Topics: Antineoplastic Agents; Apoptosis; beta-Cyclodextrins; Cell Line, Tumor; Cell Movement; Cell Survival; Drug Resistance, Neoplasm; Harmine; Humans; Melanoma; Molecular Dynamics Simulation; Mutation; Proto-Oncogene Proteins B-raf; Skin Neoplasms

The constitutive expression of Major Histocompatibility Complex (MHC) class II molecules is restricted to professional Antigen-Presenting Cells (APCs), nevertheless almost 50% of melanomas express constitutively the MHC class II molecules. Therefore, in two MHC class II constitutive expressing melanoma cell lines we studied the signalling mediated by the HLA-DR molecules in the aim to understand the consequence of class II mediated signalling on metastatic dissemination of melanoma. In particular, we reported that the HLA-DR mediated signalling play a new role in melanoma progression, increasing the migration and invasion of melanoma cells. Furthermore, we showed that the HLA-DR mediated signalling increases the expression and the lipid raft localisation of class II molecules, PD-L1 receptor, Integrin and CAM adhesion receptors, FAK, AKT and STAT3 signalling proteins. We also showed that the HLA-DR mediated signalling increases the activation of FAK, AKT, ERK, PKC and STAT3 signalling proteins and the expression of ILK, PAX, BRAF, ERK and PKC. Indeed, the results showed suggest that the HLA-DR mediated signalling provides a platform useful to frustrate an effective anti-tumour response and to increase melanoma migration and metastatic dissemination of this cancer.

Topics: B7-H1 Antigen; beta-Cyclodextrins; Cell Line, Tumor; Cell Movement; HLA-DR Antigens; Humans; Integrins; Kinetics; Melanoma; Membrane Microdomains; Neoplasm Invasiveness; Platelet Glycoprotein GPIb-IX Complex; Signal Transduction

Application of electric pulses (electroporation/electropermeabilization) is an effective method for gene transfer (i.e. gene electrotransfer (GET)) in vitro and in vivo. Currently, the mechanisms by which the DNA enters the cell are not yet fully understood. Experimental evidence is building up that endocytosis is the main mechanism by which the DNA, which is later expressed, enters the cell. Therefore the aim of our study was to elucidate whether inhibitors of endocytosis, methyl-β-cyclodextrin (MβCD), Concanavalin A (ConA) and Dynasore, can impair the transfection efficacy of GET in vitro in B16F1 murine melanoma and in vivo in m. tibialis cranialis in mice. We show that MβCD--general inhibitor of endocytosis--can almost prevent GET of EGFP-N1 plasmid in vitro, that ConA--inhibitor of clathrin mediated endocytosis--also abrogates GET but to a lesser extent, and when using Dynasore--reversible inhibitor of dynamin--there is no effect on GET efficacy, if endocytosis is blocked for only 5 min after GET. Moreover, MβCD also reduced GET efficacy in vivo in m. tibialis cranialis and this effect was long lasting. The results of this study show that endocytosis is probably the main mechanism of entrance of DNA after GET in vitro and also in vivo.

Topics: Animals; beta-Cyclodextrins; Concanavalin A; DNA; Electroporation; Endocytosis; Female; Gene Transfer Techniques; Hydrazones; Melanoma; Mice, Inbred C57BL; Muscles; Plasmids; Transfection; Tumor Cells, Cultured

Melanoma is a life-threatening disorder and its incidence is increasing gradually. Despite the numerous treatment approaches, conventional systemic chemotherapy has not reduced the mortality rate among melanoma patients, probably due to the induction of toxicity to normal tissues. Recently, we have developed folate-conjugated methyl-β-cyclodextrin (FA-M-β-CyD) and clarified its potential as a new antitumor agent involved in autophagic cell death. However, it remains uncertain whether FA-M-β-CyD exerts anticancer effects against melanomas. Therefore, in this study, we investigated the effects of FA-M-β-CyD on the folate receptor-α (FR-α)-expressing melanoma cell-selective cytotoxic effect. FA-M-β-CyD showed cytotoxic effects in Ihara cells, a human melanoma cell line expressing FR-α. In sharp contrast to methyl-β-cyclodextrin, FA-M-β-CyD entered Ihara cells [FR-α(+)] through FR-α-mediated endocytosis. Additionally, FA-M-β-CyD elicited the formation of autophagosomes in Ihara cells. Notably, FA-M-β-CyD suppressed melanoma growth in BALB/c nude recombinase-activating gene-2 (Rag-2)/Janus kinase 3 (Jak3) double deficient mice bearing Ihara cells. Therefore, these results suggest that FA-M-β-CyD could be utilized as a potent anticancer agent for melanoma chemotherapy by regulating autophagy.

Topics: Animals; Antineoplastic Agents; Autophagy; beta-Cyclodextrins; Cyclodextrins; Drug Combinations; Endocytosis; Folic Acid; Folic Acid Transporters; Humans; Melanoma; Mice, Inbred BALB C; Mice, Nude; Phagosomes

Despite modern advances in treatment, skin cancer is still one of the most common causes of death in the western countries. Chemotherapy plays an important role in melanoma management. Tamoxifen has been used either alone or in- combination with other chemotherapeutic agents to treat melanoma. However, response rate of tamoxifen as a single agent has been comparatively low. In the present study, we investigated whether treatment with methyl-β-cyclodextrin (MCD), a cholesterol depleting agent, increases the efficacy of tamoxifen in melanoma cells.. This was a two-part study that incorporated in vitro effects of tamoxifen and MCD combination by analyzing cell survival, apoptosis and cell cycle analysis and in vivo antitumor efficacy on tumor isografts in C57BL/6J mice.. MCD potentiated tamoxifen induced anticancer effects by causing cell cycle arrest and induction of apoptosis. Sensitization to tamoxifen was associated with down regulation of antiapoptotic protein Bcl-2, up-regulation of proapoptotic protein Bax, reduced caveolin-1 (Cav-1) and decreased pAkt/pERK levels. Co-administration of tamoxifen and MCD caused significant reduction in tumor volume and tumor weight in mice due to enhancement of drug uptake in the tumor. Supplementation with cholesterol abrogated combined effect of tamoxifen and MCD.. Our results emphasize a potential synergistic effect of tamoxifen with MCD, and therefore, may provide a unique therapeutic window for improvement in melanoma treatment.

Topics: Animals; Antineoplastic Agents, Hormonal; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; beta-Cyclodextrins; Cell Line, Tumor; Cell Survival; Cholesterol; Drug Synergism; Gene Expression Regulation, Neoplastic; Humans; Melanoma; Mice; Mice, Inbred C57BL; Neoplasm Transplantation; Tamoxifen; Xenograft Model Antitumor Assays

Although cyclin dependent kinase (CDK)-2 is known to be dispensable for the growth of most tumors, it is thought to be important for the proliferation of melanoma cells, where its expression is controlled by the melanocyte-lineage specific transcription factor MITF. Treatment of a panel of melanoma cells with the CDK inhibitor dinaciclib led to a concentration-dependent inhibition of growth under both 2D adherent and 3D organotypic cell culture conditions. Dinaciclib targeted melanoma cell lines regardless of cdk2 or MITF levels. Inhibition of growth was associated with a rapid induction of G2/M cell arrest and apoptosis. Treatment of human melanoma mouse xenografts with dinaciclib led to tumor regression associated with reduced retinoblastoma protein phosphorylation and Bcl-2 expression. Further mechanistic studies revealed that dinaciclib induces p53 expression whilst simultaneously downregulating the expression of the anti-apoptotic factors Mcl-1 and XIAP. To clarify the role of p53 activation in the dinaciclib-induced cell death, we generated melanoma cell lines in which p53 expression was knocked down using a shRNA lentiviral vector. Knockdown of p53 completely abolished the induction of apoptosis seen following dinaciclib treatment as shown by a lack of annexin-V staining and caspase-3 cleavage. Altogether, these data show that dinaciclib induces apoptosis in a large panel of melanoma cell lines through a mechanism requiring p53 expression.

Topics: 2-Hydroxypropyl-beta-cyclodextrin; Animals; Apoptosis; beta-Cyclodextrins; Blotting, Western; Bridged Bicyclo Compounds, Heterocyclic; Cell Culture Techniques; Cell Line, Tumor; Cyclic N-Oxides; Cyclin-Dependent Kinase 2; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Indolizines; Melanoma; Mice; Mice, SCID; Pyridinium Compounds; Signal Transduction; Spheroids, Cellular; Tumor Suppressor Protein p53

4-Hydroxynonenal (HNE) is the most studied end product of the lipoperoxidation process, by virtue of its relevant biological activity. The antiproliferative and proapoptotic effects of HNE have been widely demonstrated in a great variety of tumor cell types in vitro. Thus, it might represent a promising new molecule in anticancer therapy strategies. However, the extreme reactivity of this aldehyde, as well as its insolubility in water, a limiting factor for drug bioavailability, and its rapid degradation by specific enzymes represent major obstacles to its possible in vivo application. Various strategies can used to overcome these problems. One of the most attractive strategies is the use of nanovehicles, because loading drugs into nanosized structures enhances their stability and solubility, thus improving their bioavailability and their antitumoral effectiveness. Several natural or synthetic polymers have been used to synthesize nanosized structures and, among them, β-cyclodextrin (βCD) polymers are playing a very important role in drug formulation by virtue of the ability of βCD to form inclusion compounds with a wide range of solid and liquid molecules by molecular complexation. Moreover, several βCD derivatives have been designed to improve their physicochemical properties and inclusion capacities. Here we report that the inclusion complex of HNE with a derivative of βCD, the βCD-poly(4-acryloylmorpholine) conjugate (PACM-βCD), enhances the aldehyde stability. Moreover, the inclusion of HNE in PACM-βCD potentiates its antitumor effects in several tumor cell lines and in a more complex system, such as a human reconstructed skin carrying melanoma tumor cells.

Topics: Aldehydes; Antineoplastic Agents; beta-Cyclodextrins; Cell Culture Techniques; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cell Survival; Drug Carriers; Drug Screening Assays, Antitumor; Drug Stability; Humans; Inhibitory Concentration 50; Melanoma

Lipid rafts are related to cell surface receptor function. Integrin is a major surface receptor protein in cell adhesion and migration on the extracellular matrix (ECM). Here, we showed that lipid rafts played a critical role in human melanoma A375 cell spreading and migration on fibronectin; an important component of the ECM that interacts with β1 integrin. We found that the disruption of lipid rafts did not markedly inhibit the expression and activation of β1 integrin. By coimmunoprecipitation and mass spectrometry, we investigated the influence of lipid rafts on the β1 integrin complex and identified nucleolin as a potential lipid-raft-dependent β1-integrin-interacting protein. Upon confirmation of the interaction between β1 integrin and nucleolin, further studies revealed that nucleolin colocalized with β1 integrin in lipid rafts and raft disruption interrupted their association. In addition, knockdown of nucleolin markedly attenuated A375 cell spreading and migration on fibronectin. Taken together, we demonstrated that nucleolin is a critical lipid-raft-dependent β1-integrin-interacting protein in A375 cell spreading and migration on fibronectin.

Topics: Amino Acid Sequence; beta-Cyclodextrins; Binding Sites; Cell Line, Tumor; Cell Movement; Cell Proliferation; Fibronectins; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Integrin beta1; Mass Spectrometry; Melanoma; Membrane Microdomains; Molecular Sequence Data; Nucleolin; Phosphoproteins; RNA-Binding Proteins

HLA-G is a non-classical HLA class I molecule with tolerogenic properties and restricted tissue distribution. The expression of HLA-G can be induced by tumors thus providing an efficient way to escape the anti-tumoral immune response. Although lipid rafts regulate diverse immunological mechanisms their relationship with HLA-G remains controversial. Our results show that HLA-G-mediated inhibition of both the interaction between NK and tumor cells, and of intracellular calcium flux in NK cells conjugated to their target cells were independent of lipid raft integrity. In addition, cytotoxicity assays indicated that HLA-G continued to efficiently inhibit NK-cell cytolytic function in several different tumor cells independently of lipid raft integrity. Confocal microscopy with 3D reconstruction combined with biochemical analysis showed that HLA-G was mainly localized outside the lipid rafts of tumor cells after cross-linking with specific antibody and remained excluded from lipid rafts during interaction with the ILT2 inhibitory receptor of NK cells. This study indicates that the inhibitory function of HLA-G is uncoupled from lipid raft organization, further distinguishing HLA-G from classical HLA molecules and providing novel information in the understanding of tumor immune escape mechanism mediated through HLA-G.

Topics: Antigens, CD; beta-Cyclodextrins; Cell Line, Tumor; Cytotoxicity, Immunologic; Flow Cytometry; HLA-G Antigens; Humans; Killer Cells, Natural; Leukocyte Immunoglobulin-like Receptor B1; Melanoma; Membrane Microdomains; Microscopy, Confocal; Receptors, Immunologic; Tumor Escape

Various analogs of curcumin show high in vitro cytotoxic activity and are potential candidates for treating a deadly skin disease, melanoma. Due to the low solubility of the drugs, a new delivery agent, namely a cationic gemini surfactant-conjugated β-cyclodextrin, was designed to incorporate novel drug candidates of the 1,5-diaryl-3-oxo-1,4-pentadienyl family. Based on physicochemical parameters, such as particle size and zeta potential, a schematic model for the potential interaction of the drug with the delivery agent was developed. The drug formulations were highly efficient in inhibiting the growth of melanoma cells, with IC(50) values significantly lower than melphalan, the drug currently used for the treatment of in-transit melanoma. CDgemini formulations showed excellent cellular selectivity, triggering apoptosis in the A375 cell line while showing no cytotoxicity to healthy human epidermal keratinocytes. The goal is to develop this novel nanoparticle approach into a non-invasive therapy for in-transit melanoma metastasis that lacks adequate treatment to date.

Topics: Antineoplastic Agents; Apoptosis; beta-Cyclodextrins; Cell Line, Tumor; Curcumin; Drug Carriers; Drug Delivery Systems; Drug Design; Humans; Inhibitory Concentration 50; Keratinocytes; Melanoma; Nanoparticles; Particle Size; Quaternary Ammonium Compounds; Solubility

Aging and pathophysiological conditions are linked to membrane changes which modulate membrane-controlled molecular switches, causing dysregulated heat shock protein (HSP) expression. HSP co-inducer hydroxylamines such as BGP-15 provide advanced therapeutic candidates for many diseases since they preferentially affect stressed cells and are unlikely have major side effects. In the present study in vitro molecular dynamic simulation, experiments with lipid monolayers and in vivo ultrasensitive fluorescence microscopy showed that BGP-15 alters the organization of cholesterol-rich membrane domains. Imaging of nanoscopic long-lived platforms using the raft marker glycosylphosphatidylinositol-anchored monomeric green fluorescent protein diffusing in the live Chinese hamster ovary (CHO) cell plasma membrane demonstrated that BGP-15 prevents the transient structural disintegration of rafts induced by fever-type heat stress. Moreover, BGP-15 was able to remodel cholesterol-enriched lipid platforms reminiscent of those observed earlier following non-lethal heat priming or membrane stress, and were shown to be obligate for the generation and transmission of stress signals. BGP-15 activation of HSP expression in B16-F10 mouse melanoma cells involves the Rac1 signaling cascade in accordance with the previous observation that cholesterol affects the targeting of Rac1 to membranes. Finally, in a human embryonic kidney cell line we demonstrate that BGP-15 is able to inhibit the rapid heat shock factor 1 (HSF1) acetylation monitored during the early phase of heat stress, thereby promoting a prolonged duration of HSF1 binding to heat shock elements. Taken together, our results indicate that BGP-15 has the potential to become a new class of pharmaceuticals for use in 'membrane-lipid therapy' to combat many various protein-misfolding diseases associated with aging.

Topics: Acetylation; Animals; beta-Cyclodextrins; CHO Cells; Cholesterol; Cricetinae; Cricetulus; Gene Expression Regulation; Green Fluorescent Proteins; Heat-Shock Proteins; Heat-Shock Response; HEK293 Cells; Humans; Melanoma; Membrane Lipids; Membrane Microdomains; Mice; Molecular Dynamics Simulation; Nanostructures; Oximes; Piperidines; rac1 GTP-Binding Protein; Signal Transduction; Stress, Physiological; Temperature

The basis of photodynamic therapy (PDT) is the phototoxicity resulting from co-action of light, sensitizer and oxygen. In this study we demonstrate in vitro phototoxicity measurement on G361 cell lines using ZnTPPS(4) sensitizer bound to cyclodextrin hpbetaCD. We have proved its photodamage effect on cancer cell lines in the visible region of spectrum. We used the halogen lamp (24V/250W) as a source of radiation. After 24h incubation of cell cultures with 10 microM ZnTPPS(4) and 1mM cyclodextrine hpbetaCD, the cells were irradiated for 7.5 min at the total irradiation dose of 12.5 Jcm(-2). Analysis of DNA damage in the cell line after PDT was proved by comet assay and using inversion fluorescent microscope with image analysis. This treatment method gave rise to DNA damage. The used radiation dose of visible light in the absence of sensitizers does not induce DNA breaks in tumour cells. In conclusion, binding of ZnTPPS(4) sensitizer to cyclodextrin hpbetaCD may improve the efficacy of PDT for the treatment of malign melanoma.

Topics: beta-Cyclodextrins; Cell Line, Tumor; Comet Assay; DNA Damage; DNA, Single-Stranded; Humans; Light; Melanoma; Metalloporphyrins; Photochemotherapy; Photosensitizing Agents; Time Factors

Recent data suggest that membrane microdomains or rafts that are rich in sphingolipids and cholesterol are important in signal transduction and membrane trafficking. Two models of raft structure have been proposed. One proposes a unique role for glycosphingolipids (GSL), suggesting that GSL-head-group interactions are essential in raft formation. The other model suggests that close packing of the long saturated acyl chains found on both GSL and sphingomyelin plays a key role and helps these lipids form liquid-ordered phase domains in the presence of cholesterol. To distinguish between these models, we compared rafts in the MEB-4 melanoma cell line and its GSL-deficient derivative, GM-95. Rafts were isolated from cell lysates as detergent-resistant membranes (DRMs). The two cell lines had very similar DRM protein profiles. The yield of DRM protein was 2-fold higher in the parental than the mutant line, possibly reflecting cytoskeletal differences. The same amount of DRM lipid was isolated from both lines, and the lipid composition was similar except for up-regulation of sphingomyelin in the mutant that compensated for the lack of GSL. DRMs from the two lines had similar fluidity as measured by fluorescence polarization of diphenylhexatriene. Methyl-beta-cyclodextrin removed cholesterol from both cell lines with the same kinetics and to the same extent, and both a raft-associated glycosyl phosphatidylinositol-anchored protein and residual cholesterol showed the same distribution between DRMs and the detergent-soluble fraction after cholesterol removal in both cell lines. Finally, a glycosyl phosphatidylinositol-anchored protein was delivered to the cell surface at similar rates in the two lines, even after cholesterol depletion with methyl-beta-cyclodextrin. We conclude that GSL are not essential for the formation of rafts and do not play a major role in determining their properties.

Topics: Alkaline Phosphatase; Animals; beta-Cyclodextrins; Biological Transport; Cell Membrane; Cholesterol; Cyclodextrins; Detergents; Female; Glycosphingolipids; Glycosylphosphatidylinositols; Humans; Isoenzymes; Kinetics; Melanoma; Membrane Fluidity; Membrane Lipids; Membrane Proteins; Protein Structure, Tertiary; Tumor Cells, Cultured

Topics: Animals; Benzaldehydes; beta-Cyclodextrins; Lasers; Lasers, Solid-State; Melanoma; Mice; Neodymium; Neoplasms; Neoplasms, Experimental; Radiation Effects; Research

Topics: Animals; Benzaldehydes; beta-Cyclodextrins; Lasers; Lasers, Solid-State; Melanoma; Mice; Neoplasms; Neoplasms, Experimental; Photomicrography; Research