MANAGEMENT OF SKIN CANCER BY AGONISTS OF 5-HT1A ANDANTAGONISTS OF - - PowerPoint PPT Presentation

MANAGEMENT OF SKIN CANCER BY AGONISTS OF 5-HT1A ANDANTAGONISTS OF 5-HT2A RECEPTORS

ANA CATARINA MENEZES, SANDRA SIMÕES, HELENA OLIVEIRA, ANDREIA ASCENSO

SUPERVISORS: PROFESSORA DOUTORAANDREIAASCENSO & DOUTORA HELENA OLIVEIRA

PHOTOCARCINOGENESIS

Skin cancer is one of the most predominant form of human cancer and its incidence is rapidly increasing around the world in the past few decades Possible causes:

• Ionizing radiations
• Viruses
• Inflammation
• Genetic factors
• UV radiation
• Fig. 1. Exposure to UV radiation and development of melanoma and

nonmelanoma skin cancers (Menezes AC, et al. Molecular Neurobiology 2015.doi:10.1007/s12035-014-9068-z)

DNA DAMAGE OXIDATIVE STRESS IMMUNOSUPPRESSION

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PHOTOCARCINOGENESIS

• Fig. 2. Immunosuppression resulting from exposure to UVB

(Menezes AC, et al. Molecular Neurobiology 2015. doi:10.1007/s12035-014-9068-z)

The main target of carcinogenesis is the DNA

Cyclobutane-type pyrimidine dimers (CPD) 6-4 photoproducts

Immunosuppression

DNA damage Cis-urocanic acid (UCA) Free radicals Membrane lipid peroxidation

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MELANOMA

DNA photoproducts Gene mutations Immunosuppression Oxidative damage

• Fig. 3. Direct effects of UV exposure (adapted from

http://www.skincancer.org/)

• Several mechanisms are involved in chemoresistance of melanoma cells

Counteracting the harmful effects of DNA-damaging drugs Promoting antioxidant adaptive mechanisms

• Malignant melanoma is the deadliest form of skin cancer (80% of

deaths) and one of the most challenging malignancies to address therapeutically due to its metastatic potential

• Melanoma arises from epidermal melanocytes, which are the main

producers of serotonin in the skin and possess both 5-HT1/2A receptors

SEROTONIN:A KEY MEDIATOR BETWEENTHE SKIN ANDTHE NEUROENDOCRINE SYSTEM

• Serotonin (5-HT) is biosynthesized from L-tryptophan and is

widely distributed throughout the body, including the skin

• Serotonergic membrane-bound receptors are categorized into seven

general families from 5-HT1R to 5-HT7R, with at least 21 subtypes

T able 1. Serotonergic receptors or serotonin transporter (SERT) distribution in human skin of atopic dermatitis patients (Menezes AC, et al. Molecular Neurobiology 2015. doi:10.1007/s12035- 014-9068-z)

Modulation of immune cells function

5-HT1AR 5-HT2AR

Binding with cis-UCA

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THERAPEUTIC POTENTIAL OF SEROTONERGIC DRUGS IN THE PHOTOCARCINOGENESIS CONTEXT

• 5-HT1AR agonists and 5-HT2AR antagonists are capable of preventing UV- and cis-UCA-induced

immunosuppression and hence skin cancer induction and progression

• 5-HT2AR antagonists are also capable of inducing DNA repair by two different paths

Accelerating the repair of both CPD and 6-4 photoproducts Decreasing ROS-induced lesions

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• 1-(1-Naphthyl)piperazine (1-NPZ) is both an agonist of 5-HT1A and antagonist of 5-HT2A receptors with a

dual mechanism of action

AIMS

Assess the potential of a novel therapeutic strategy based on the topical delivery of 1-(1-Naphthyl)piperazine (1-NPZ), both an agonist and antagonist of serotonin receptors (i.e. 5-HT1/2A),towards the treatment of melanoma skin cancer

A) Cell Biology Studies B) Pharmaceutical Technology Studies

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EFFECT OF 1-(1-NAPHTHYL)PIPERAZINE ON HUMAN MNT

• 1 MELANOMA CELLS

ANA CATARINA MENEZES1, MANUELA CARVALHEIRO2, JOSÉ MIGUEL P . FERREIRA DE OLIVEIRA3, ANDREIA ASCENSO2, HELENAOLIVEIRA3

1Faculdade de Farmácia da Universidade de Lisboa – Lisbon,Portugal 2NanoBB Research Group of iMed.UL – Lisbon,Portugal 3Departamento de Biologia,CESAM,Universidade deAveiro –Aveiro,Portugal

Toxicology and Applied Pharmacology

METHODS

24-h treatment with 1-NPZ (0,IC20,IC50) Harvested and centrifuged Centrifugations Incubated for 20min with PI and RNase Flow cytometry 24-h treatment with 1-NPZ (0,IC20,IC50) Flow cytometry Incubated for 30min with DCFH-DA Harvested 24-h treatment with 1-NPZ (0,IC20,IC50) Harvested, centrifuged and resuspended in 1x binding buffer Flow cytometry Incubated for 15min with PI and AnnexinV-FITC +1x binding buffer 3/24-h treatment with 1-NPZ (0-300 µg/mL) Incubated for 4h with MTT solution Incubated for 2h with DMSO Absorbance at 570 nm MTT assay 24-h treatment with 1-NPZ (0,IC50) Washed and lysed in TRIzol reagent + Chloroform Centrifugations Nanodrop cDNA synthesis qR T

• PCR

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RESULTS– CELL VIABILITY ASSAY

• Fig. 4. Light microscopy images (100X) of MNT
• 1 cells exposed to 1-NPZ (0, 141.8 and

163.6 µM) for 24h 0 (Control) 141.8 µM (IC20) 163.6 µM (IC50)

20 40 80 100 120 50 100 200 250 300

Cell Viability(%)

150

[1-NPZ] µM 3 h 24 h

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• Fig. 5. Effects of 1-NPZ exposure on MNT
• 1 cell viability for 24h. Results are

represented as mean ± SD (n = 3). * p<0.05, **p<0.001

• Treatment of human MNT
• 1 melanoma cells with 1-NPZ caused distinctive morphological changes, such as roundness and flattening
• 1-NPZ exposure for either 3 or 24h significantly (p<0.001, p<0.05) inhibited the cell viability in a dose- and time-dependentmanner

* * ** ** ** ** **

CELL CYCLE ANALYSIS

• T

reatment

• f MNT
• 1

cells with 1-NPZ (IC50) induced a significant (p<0.05) S-phase delay of cell cycle progression

• The cellular response to DNA damage is a cell cycle arrest

via checkpoint mechanisms, which in turn depend on the type

• f damage and the phase of the cell cycle at which it occurs
• At IC50, the percentage of cells in sub-G1 phase significantly

(p<0.05) increased, which might be a sign ofapoptosis

10 30 60 70

Cell cycle distribution (%) Control IC20 IC50

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* Sub-G1 G0/G1 S G2/M

• Fig. 6. Effects of 1-NPZ exposure on MNT
• 1 cell cycle dynamics for 24h. Results are

represented as mean ± SD (n = 3). * p<0.05

ROS DETECTIONASSAY

15 10 5 25 30 35

DCF MFI Control IC20 IC50

• Fig. 7. DCF mean fluorescence intensity (MFI) after 24-h treatment.

Results are expressed as mean ± SD (n = 3). *p<0.05

*

• Fig. 8. DCF fluorescence intensity following treatment with 1-

NPZ on MNT-1 cells for 24h

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• Treatment of MNT
• 1 cells with 1-NPZ significantly (p<0.05) increased ROS levels in a dose-dependent manner
• These results supported the hypothesis that treatment with 1-NPZ would lead to an increase in ROS intracellular levels, which might play

an essential role in 1-NPZ-induced apoptosis in cells

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CELL APOPTOSIS ASSAY

• Treating cells with 1-NPZ caused a highly significant (p<0.001) reduction in viable and non-apoptotic cells as well as a highly significant

(p<0.001) increase in early apoptotic cells at both concentrations

Cells (%) Control IC20 IC50 * * Viable and non- Early apoptotic Late apoptotic Necrotic apoptotic * * ** **

100 90 80 70

**

60 50 40 30 20 10

• Fig. 9. Percentage of apoptotic cells after 24 h treatment. Results are expressed as mean ± SD (n = 3).

*p<0.05; **p<0.001

Control IC20 IC50

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• Fig. 10. Annexin V-FITC dot-plots gating (FL1 LOG vs FITC LOG) following treatment with 1-

NPZ on MNT-1 cells for 24h: Q1 – Necrotic cells (Annexin-FITC (-) and PI (+)); Q2 – Late apoptotic cells (Annexin-FITC (+) and PI (+)); Q3 – Early apoptotic cells (Annexin-FITC (+) and PI (-)); Q4 – Viable and non-apoptotic cells (Annexin-FITC (-) and PI(-))

GENE EXPRESSION ASSAY

a)

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b) c) **

• Fig. 11. COX-2 (a), IL12A (b) and PAK1 (c) expression after 24-h treatment with 1-NPZ on MNT-1 cells. Results are

represented as mean ± SD (n = 3). ** p<0.001

• mRNA expression of genes associated with UV-induced immunosuppression (COX-2 or PTGS2 and IL12A) increased following

exposure to 1-NPZ, being COX-2 significantly up-regulated(p<0.001)

• Exposure of MNT
• 1 cells to 1-NPZ induced a decrease in PAK1 expression, which in turn is related to chemoresistanceevents.
• The unexpected increase in COX-2 expression levels might be explained by a similar COX-2-dependent pathway underlying apoptosis.

CONCLUSIONS

This study showed that 1-NPZ was capable of inhibiting cellular growth by inducing S-phase cell cycle delay, ROS generation and apoptosis in human MNT

• 1 melanoma cells

1-NPZ treatment was capable of inducing changes on important signaling cascades attending to different expression levels of various genes in exposed cells

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DEVELOPMENT AND CHARACTERIZATION OF NOVEL 1-(1-NAPHTHYL)PIPERAZINE LOADED LIPID VESICLES FOR PREVENTION OF UV-INDUCED SKIN INFLAMMATION

ANA CATARINA MENEZES1, PATRÍCIA M. CAMPOS3, CARLA EULETÉRIO1,FABÍOLA SG PRAÇA3, MARIA VITÓRIA LB BENTLEY3, SANDRA SIMÕES,2 ANDREIA ASCENSO2

1Faculdade de Farmácia da Universidade de Lisboa – Lisbon,Portugal 2NanoBB Research Group of iMed.UL – Lisbon,Portugal 3School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil

European Journal of Pharmaceutics and Biopharmaceutics

INTRODUCTION

• For metastatic melanoma, nanomedicine offers a powerful

drug delivery system, allowing a site-specific drug delivery to cancer cells (↑ treatment efficiency)

*

Epidermis Dermis

Stratum

* * ** * * * * * * ** * * * *L * * * * * * * * **** * * * * * * * * * * * * * * * * * * * * * ** *

corneum

* * * * * * * * * * * * * *** * * * * * * ** * * * * * * * * * * **

*****

* * * * * * * * * I II III IV V VI * Drug L

Permeation enhancer

* * ** * * *

Drug-loaded vesicle

• Fig. 12. Skin drug delivery: I) drug delivery from a conventional delivery system; II) drug-

loaded vesicular carrier with non flexible membrane; III) drug delivery after carrier adsorption and/or fusion with the SC; IV) drug delivery from a formulation containing a permeation enhancer; V) drug delivery by means of deformable intact vesicle penetration into and through the intact skin; and VI) transappendageal penetration of drug-loaded deformable carriers. (Ascenso et al. Current drug delivery 2011, 8(6):640-60)

• Several drug nanodelivery systems have been developed over

the years for the treatment of skin cancer Liposomes Ultradeformable vesicles (UDV) ↑ Flexibility ↑ Skin penetration

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INTRODUCTION



Transethosomes (TE) emerged a few years ago and consist

• f

phospholipids, water, ethanol and an edge activator or a penetration enhancer (↑ skin permeation, ↑ superior characteristics)



Innovative vesicles containing DMSO (DM) were recently developed by

• ur research group. DMSO is a powerful aprotic solvent and one of the

most popular skin penetration enhancer



DMSO is generally used as a tissue/organ preservative, penetration enhancer and solubilizing agent. It has also biological activity, being a free radical scavenger and exhibiting anti-inflammatory analgesic effects

Edge activator (surfactant) Ethanol Water Lipid bilayer Citrate buffer Lipid bilayer DMSO

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METHODS

1-NPZ DMSO SPC Citrate buffer Stirring for 24 h Pressure filtration LIPID: SPC SURFACTANT: NaCo Dissolved in Ethanol Water bath 1-NPZ Water Stirring for 5 min Pressure filtration

Classic cold method

Vesicles size Zeta Deformability Rheology Phospholipid Drug pH Stress & PDI potential index content entrapment stability Skin Permeation yield

Physical characterization Chemical characterization

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Topical delivery In Vivo Study

MPO and Cytokines

RESULTS – VESICLES SIZE & ZETA POTENTIAL

• NPZ-TE showed a significant (p<0.05) smaller size compared to NPZ-DM (↓ SPC, ↑ ethanol)
• The negative charges were due to the presence of both ethanol and sodium cholate inTE and citrate ion in DM
• 1-NPZ loading decreased the absolute charge of both formulations due to the presence of a protonated amino group (pKa = 8.87)

in its structure

T able 2. Short composition and physical features of the prepared lipid vesicles. Values are expressed as mean ± SD (n = 3). Statistical analysis for both inter- and intra-groups: *p<0.05,**p<0.001

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** *

VESICLES DEFORMABILITY

• Fig. 13. Deformability index of NPZ-TE and NPZ-DM formulations

and respective controls (empty vesicles). TE diluted 1:10 and DM diluted 1:20.Values are expressed as mean ± SD (n = 3).**p<0.001

• There were no differences in flux and DI between the unloaded and 1-NPZ-loaded vesicles for both formulations,which means that

1-NPZ had no effect on these parameters

• Higher deformability ofTE could be associated with a lower percentage of SPC and with the synergistic presence of ethanol and sodium

cholate

T able 3. Flux under pressure, deformability index and viscosity of the lipid formulations. TE diluted 1:10 and DM diluted 1:20. Values are expressed as mean ± SD (n = 3). Statistical analysis for both inter- and intra-groups:**p<0.001

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PHOSPHOLIPID & DRUG ENTRAPMENTYIELDS

• Drug entrapment yield of both formulations was also very high (> 90%)
• In the case of NPZ-TE, drug loading might be increased due to its solubilization by the presence of both co-solvent (ethanol) and

surfactant

• Regarding NPZ-DM, the high value of this parameter is related with the extremely high solubility of 1-NPZ inDMSO

T able 4. Chemical characterization of the lipid formulations.Values are expressed as mean ± SD (n = 3).*p<0.05

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RHEOLOGY

• Both NPZ-TE and NPZ-DM showed a non-Newtonian time-independent pseudoplastic behaviour revealing a good spreadability, which

makes them quite suitable for topical application

• Fig. 14. Rheograms of NPZ-TE (a) and NPZ-DM (b) formulations

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TOPICAL DELIVERY

• NPZ-DM revealed a statistically significant increase in permeation through skin in comparison with NPZ-TE (p<0.05), thus improving the

permeation and skin delivery of 1-NPZ. => NPZ-DM was selected for In Vivo Study

• The best permeation profile was obtained for the solution of 1-NPZ dissolved in DMSO (p<0.001), showing the permeation

enhancement effect of this powerful solvent

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• Fig. 15. Skin permeation of 1-NPZ for both NPZ-TE and NPZ-DM formulations. Values are expressed as mean ± SD (n = 3). Statistical analysis: *p<0.05 vs NPZ-H2O and

NPZ-TE, **p<0.001 vs NPZ-H2O, NPZ-TE and NPZ-DM.

IN VIVO STUDIES

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• Fig. 16. Effect of NPZ-DM treatment on UVB-induced infiltration of

leukocytes in irradiated (I) and nonirradiated (NI) mice. Values are expressed as mean ± SD (n = 4).

• Fig. 17. Effect of NPZ-DM treatment on the production of proinflammatory cytokines in irradiated (I)

and nonirradiated (NI) mice. Values are expressed as mean ± SD (n = 4).

• Acute UVB exposure increased MPO activity in the control group (I-C), which demonstrates the effectiveness of this assay and the apparent infiltration of leucocytes

into the inflamed skin. In addition, treating mice with NPZ-DM reduced this activity even not significantly suggesting the inhibition of UVB-induced infiltration of leukocytes by NPZ-DM treatment.

• Acute UVB exposure led to a higher production of both TNF-α and IL-1β in the skin of mice as compared to nonirradiated control mice (NI-C). In contrast, topical

application of NPZ-DM resulted in significantly lower production of TNF-α and IL-1β in the UVB irradiated mice (I-NPZ-DM) compared to irradiated control mice (I-C).

CONCLUSIONS

This study showed that all tested parameters were suitable either for NPZ-TE or NPZ-DM due to the synergistic combination of surfactant and ethanol or DMSO,respectively

In vivo studies demonstrated that NPZ-DM exposure was capable of preventing UVB-induced inflammation and blocking mediators of inflammation in mouse skin.

Future work will mainly cover the assessment of long-term stability of both formulations, as well as in vitro (3D skin models) and in vivo (mouse models) studies concerning the toxicity and therapeutic effect of 1-NPZ on skin cancer

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FINAL REMARKS

Both NPZ-TE and NPZ-DM formulations should be appropriate for therapeutic use This study identified for the first time 1-NPZ as a promising chemotherapeutic agent for the management of melanoma skin cancer

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ACKNOWLEDGMENTS

SUPERVISORS:



Professora DoutoraAndreiaAscenso (NanoBB,iMed.UL)



Doutora Helena Oliveira (CESAM, Universidade deAveiro)

COLLABORATORS:



Doutor Miguel Oliveira (CESAM,Universidade deAveiro)



Doutora Sandra Simões (NanoBB,iMed.UL)



Doutora Manuela Carvalheiro (NanoBB,iMed.UL)



Doutora Manuela Gaspar (NanoBB,iMed.UL)



Drª. Carla Euletério (NanoBB,iMed.UL)



Drª. Joana Marto (NanoBB,iMed.UL)

THANKYOUTOALL COLLABORATORS AND FORYOUR ATTENTION!

www.health-love-wealth.com

Mol Neurobiol DOI 10.1007/s12035-014-9068-z