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Prevention and treatment of dermatological diseases: optimizing the protective action and efficacy of phenylpropanoid glycosides delivered through the skin by inclusion in biodegradable nanovesicles
No full textThe skin, the human body’s largest organ, serves critical roles in defense and physiological regulation. Maintaining its integrity is essential, yet it is constantly challenged by environmental factors such as ultraviolet radiation (UVR), extreme temperatures, and chemical pollutants. As a "complete carcinogen," UVR has the unique capacity to both initiate and promote tumor development, making it a significant contributor to skin cancer, with melanoma being the most aggressive and lethal form. The global incidence of melanoma continues to rise. Although targeted therapies and immunotherapies have advanced, clinical outcomes remain suboptimal due to tumor resistance and adverse effects, underscoring the need for innovative treatment strategies. Natural antioxidants, including Phenylpropanoid Glycosides (PPGs) and Hyperforin (HPF), show promising antitumor potential among emerging thera peutic agents. PPGs, a group of over 200 plant-derived polyphenols, and HPF, an acylphloroglucinol extracted from Hypericum perforatum, exhibit comple mentary pharmacological properties, such as antioxidant, anti-inflammatory, and antitumor activities. This thesis examines the dual role of the PPGs, particularly Echinacoside and Forsythoside B, in preventing skin tumors and treating melanoma and other skin cancers. To explore the protective properties of PPGs against UVR, normal hu man epidermal melanocytes (NHEM) and normal human dermal fibroblasts (NHDF) were pre-treated with these compounds. Solar UV radiation was then simulated by exposing cultured cells to a UV lamp. The protective effects of PPGs pre-treatment were analyzed by assessing cell viability and the activation of multiple stress signaling pathways. Our findings suggest that PPGs exert a mild UV protective effect in normal cells, although statistical significance was not consistently observed. To improve their stability and bioavailability under UV and temperature exposure, PPGs were nanoencapsulated. In collaboration with the company “Nanomnia”, a natural, biodegradable polymer-based process was employed, eliminating microplastics and enhancing targeted delivery and controlled release. This approach can also mitigate the environmental impact of conven tional sunscreen components, which are associated with marine ecosystem disruption. Preliminary studies revealed a mild UV protective effect in normal cells from encapsulated PPGs. Particular attention is given to the antitumor properties of Echinacoside and Forsythoside B against BRAF-mutated melanoma cell line (FO-1) and a human skin tumor cell line (A-431). The results reveal that these compounds selectively induce cytotoxicity in malignant cells without affecting normal f ibroblasts, keratinocytes, or melanocytes, even at concentrations four times higher than their EC50 values in cancer cells. Within 48 h of in vitro administration, PPGs promote apoptosis by cleaving Poly (ADP-ribose) Polymerase-1 (PARP-1), activating Ataxia-Telangiectasia Mutated (ATM) kinase, and increasing phosphorylated H2A histone family member X (γH2AX) levels, indicating DNA damage. Additionally, the phosphorylation of p38 mitogen-activated protein kinases (MAPK) suggests that stress response signaling pathways are involved. These findings demonstrate a slight protective effect on normal skin cells following UV stress, as well as the selective cytotoxicity and apoptotic potential of Echinacoside and Forsythoside B in skin cancer cell lines, highlighting their therapeutic promise in melanoma and other skin cancer. Encapsulated PPGs were evaluated to assess their protective activity on NHDF exposed to UV-A radiation. Preliminary results indicate that lower concentrations of encapsulated PPGs revealed a remarkable protective response through morphological analysis. However, quantitative analysis need to be carried out. Furthermore, Hyperforin (HPF), an acylphloroglucinol derived from Hy pericum perforatum, exhibits a variety of pharmacological activities, including antidepressant, anti-inflammatory, antimicrobial, and antitumor effects. Our f indings demonstrate that HPF exerts potent antitumor activity against BRAF-mutated melanoma cell lines (A375, FO-1, SK-Mel-28), including inhibition of proliferation, motility, colony formation, and induction of apop tosis. Its cytotoxic mechanisms involve lipid peroxidation and disruption of iron homeostasis through downregulation of glutathione peroxidase-4 (GPX 4), cystine transporter SLC7A11, and ferritin, thereby increasing cellular susceptibility to oxidative damage. Concurrently, HPF upregulates heme oxygenase-1 (HO-1), which promotes the release of free iron. Cytofluorimetric analysis confirmed an increase in lipid peroxidation, as evidenced by enhanced BODIPY-C11 fluorescence. HPF also induces autophagy, as indicated by elevated LC3B expression. Moreover, HPF exerts cytostatic effects by modulating key cell cycle regulators, including downregulation of cyclins D1 and A2, CDK4, and phos phorylated Rb, and upregulation of P21/waf1 and activated P53. Apoptotic effects are further enhanced by reductions in Bcl-2 and Bcl-xL levels and increased cleaved PARP-1. Furthermore, HPF alters the metabolism of melanoma cells inhibiting mitochondrial enzymes expression and compro mising the mitochondrial membrane potential. Although HPF modulates TRPC6 channels and elevates cytosolic Ca2+ and Zn2+ levels, its cytostatic effects appear largely independent of these pathways. Furthermore, HPF suppresses survival signaling by reducing the activity of transcription factors nuclear factor-kappa B (NF-κB) P65 and Signal Transducer and Activator of Transcription 3 (STAT3). HPF’s antimetastatic properties further reinforce its therapeutic potential. It downregulates many invasion markers, consistent with observed reductions in cell migration and colony formation. Collectively, these findings underscore HPF’s pleiotropic antitumor properties, includ ing cytostatic, cytotoxic, and antimetastatic effects, thereby supporting its potential as a therapeutic agent in melanoma management. These findings collectively highlight the therapeutic potential of PPGs and HPF. While PPGs demonstrate a slight UV-protective effect on normal f ibroblasts and melanocytes and a selective cytotoxic effect on malignant skin cells, HPF exerts broader cytostatic, cytotoxic, and antimetastatic activities on melanoma cell lines, providing a strong foundation for future translational research and therapeutic applications
Hyperforin enhances heme oxygenase-1 expression triggering lipid peroxidation in BRAF-mutated melanoma cells and hampers the expression of pro-metastatic markers
Get PDFHyperforin (HPF) is an acylphloroglucinol compound found abundantly in Hypericum perforatum extract which exhibits antidepressant, anti-inflammatory, antimicrobial, and antitumor activities. Our recent study revealed a potent antimelanoma effect of HPF, which hinders melanoma cell proliferation, motility, colony formation, and induces apoptosis. Furthermore, we have identified glutathione peroxidase-4 (GPX-4), a key enzyme involved in cellular protection against iron-induced lipid peroxidation, as one of the molecular targets of HPF. Thus, in three BRAF-mutated melanoma cell lines, we investigated whether iron unbalance and lipid peroxidation may be a part of the molecular mechanisms underlying the antimelanoma activity of HPF. Initially, we focused on heme oxygenase-1 (HO-1), which catalyzes the heme group into CO, biliverdin, and free iron, and observed that HPF treatment triggered the expression of this inducible enzyme. In order to investigate the mechanism involved in HO-1 induction, we verified that HPF downregulates the BTB and CNC homology 1 (BACH-1) transcription factor, an inhibitor of the heme oxygenase 1 (HMOX-1) gene transcription. Remarkably, we observed a partial recovery of cell viability and an increase in the expression of the phosphorylated and active form of retinoblastoma protein when we suppressed the HMOX-1 gene using HMOX-1 siRNA while HPF was present. This suggests that the HO-1 pathway is involved in the cytostatic effect of HPF in melanoma cells. To explore whether lipid peroxidation is induced, we conducted cytofluorimetric analysis and observed a significant increase in the fluorescence of the BODIPY C-11 probe 48 h after HPF administration in all tested melanoma cell lines. To discover the mechanism by which HPF triggers lipid peroxidation, along with the induction of HO-1, we examined the expression of additional proteins associated with iron homeostasis and lipid peroxidation. After HPF administration, we confirmed the downregulation of GPX-4 and observed low expression levels of SLC7A11, a cystine transporter crucial for the glutathione production, and ferritin, able to sequester free iron. A decreased expression level of these proteins can sensitize cells to lipid peroxidation. On the other hand, HPF treatment resulted in increased expression levels of transferrin, which facilitates iron uptake, and LC3B proteins, a molecular marker of autophagy induction. Indeed, ferritin and GPX-4 have been reported to be digested during autophagy. Altogether, these findings suggest that HPF induced lipid peroxidation likely through iron overloading and decreasing the expression of proteins that protect cells from lipid peroxidation. Finally, we examined the expression levels of proteins associated with melanoma cell invasion and metastatic potential. We observed the decreased expression of CD133, octamer-4, tyrosine-kinase receptor AXL, urokinase plasminogen activator receptor, and metalloproteinase-2 following HPF treatment. These findings provide further support for our previous observations, demonstrating the inhibitory effects of HPF on cell motility and colony formation in soft agar, which are both metastasis-related processes in tumor cells
BRAF-Mutated Melanoma Cell Lines Develop Distinct Molecular Signatures After Prolonged Exposure to AZ628 or Dabrafenib: Potential Benefits of the Antiretroviral Treatments Cabotegravir or Doravirine on BRAF-Inhibitor-Resistant Cells
Get PDFMelanoma is an aggressive cancer characterized by rapid growth, early metastasis, and poor prognosis, with resistance to current therapies being a significant issue. BRAF mutations drive uncontrolled cell division by activating the MAPK pathway. In this study, A375 and FO-1, BRAF-mutated melanoma cell lines, were treated for 4–5 months with RAF inhibitor dabrafenib or AZ628, leading to drug resistance over time. The resistant cells showed altered molecular signatures, with differences in cell cycle regulation and the propensity of cell death. Dabrafenib-resistant cells maintained high proliferative activity, while AZ628-resistant cells, especially A375 cells, exhibited slow-cycling, and a senescent-like phenotype with high susceptibility to ferroptosis, a form of cell death driven by iron. Antiretroviral drugs doravirine and cabotegravir, known for their effects on human endogenous retroviruses, were tested for their impact on these resistant melanoma cells. Both drugs reduced cell viability and colony formation in resistant cell lines. Doravirine was particularly effective in reactivating apoptosis and reducing cell growth in highly proliferative resistant cells by increasing tumor-suppressor proteins p16Ink4a and p27Kip1. These findings suggest that antiretroviral drugs can influence apoptosis and cell proliferation in RAF-inhibitor-resistant melanoma cells, offering potential therapeutic strategies for overcoming drug resistance
Salviolone from Salvia miltiorrhiza Roots Impairs Cell Cycle Progression, Colony Formation, and Metalloproteinase-2 Activity in A375 Melanoma Cells: Involvement of P21(Cip1/Waf1) Expression and STAT3 Phosphorylation
Get PDFMelanoma is a highly malignant solid tumor characterized by an elevated growth and propagation rate. Since, often, melanoma treatment cannot prevent recurrences and the appearance of metastasis, new anti-melanoma agents need to be discovered. Salvia miltiorrhiza roots are a source of diterpenoid derivatives, natural compounds with several biological activities, including antiproliferative and anticancer effects. Seven diterpenoid derivatives were purified from S. miltiorrhiza roots and identified by NMR and MS analysis. Tanshinone IIA and cryptotanshinone were detected as the main components of S. miltiorrhiza root ethanol extract. Although their antitumor activity is already known, they have been confirmed to induce a reduction in A375 and MeWo melanoma cell growth. Likewise, salviolone has been shown to impair the viability of melanoma cells without affecting the growth of normal melanocytes. The underlying anticancer activity of salviolone has been investigated and compared to that of cryptotanshinone in A375 cells, showing an increased P21 protein expression in a P53-dependent manner. In that way, salviolone, even more than cryptotanshinone, displays a multitarget effect on cell-cycle-related proteins. Besides, it modulates the phosphorylation level of the signal transducer and activator of transcription (STAT)3. Unexpectedly, salviolone and cryptotanshinone induce sustained activation of the extracellular signal-regulated kinases (ERK)1/2 and the protein kinase B (Akt). However, the blockage of ERK1/2 or Akt activities suggests that kinase activation does not hinder their ability to inhibit A375 cell growth. Finally, salviolone and cryptotanshinone inhibit to a comparable extent some crucial malignancy features of A375 melanoma cells, such as colony formation in soft agar and metalloproteinase-2 activity. In conclusion, it has been shown for the first time that salviolone, harboring a different molecular structure than tanshinone IIA and cryptotanshinone, exhibits a pleiotropic effect against melanoma by hampering cell cycle progression, STAT3 signaling, and malignant phenotype of A375 melanoma cells
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Lamivudine, Doravirine, and Cabotegravir downregulate the expression of Human Endogenous Retroviruses (HERVs), inhibit cell growth, and reduce invasive capability in melanoma cell lines
Get PDFThis study explores the impact of antiretroviral administration on the expression of human endogenous retroviruses (HERVs), cell growth, and invasive capability of human melanoma cell lines in culture. We investigated three antiretrovirals—lamivudine, doravirine, and cabotegravir—in A375, FO-1, and SK-Mel-28, BRAF-mutated, and in MeWo, P53-mutated, melanoma cell lines. The findings indicate a general capability of these drugs to downregulate the expression of HERV-K Pol and Env genes and hinder cell viability, mobility, and colony formation capacity of melanoma cells. The antiretroviral drugs also demonstrate selectivity against malignant cells, sparing normal human epithelial melanocytes. The study reveals that the integrase inhibitor cabotegravir is particularly effective in inhibiting cell growth and invasion across different cell lines in comparison with lamivudine and doravirine, which are inhibitors of the viral reverse transcriptase enzyme. The investigation further delves into the molecular mechanisms underlying the observed effects, highlighting the potential induction of ferroptosis, apoptosis, and alterations in cell cycle regulatory proteins. Our findings showed cytostatic effects principally revealed in A375, and SK-Mel-28 cell lines through a downregulation of retinoblastoma protein phosphorylation and/or cyclin D1 expression. Signs of ferroptosis were detected in both A375 cells and FO-1 cells by a decrease in glutathione peroxidase 4 and ferritin expression, as well as by an increase in transferrin protein levels. Apoptosis was also detected in FO-1 and SK-Mel-28, but only with cabotegravir treatment. Moreover, we explored the expression and activity of the stimulator of interferon genes (STING) protein and its correlation with programmed death-ligand 1 (PD-L1) expression. Both the STING activity and PD-L1 expression were decreased, suggesting that the antiretroviral treatments may counteract the detrimental effects of PD-L1 expression activation through the STING/interferon pathway triggered by HERV-K. Finally, this study underscores the potential therapeutic significance of cabotegravir in melanoma treatment. The findings also raise the prospect of using antiretroviral drugs to downregulate PD-L1 expression, potentially enhancing the therapeutic responses of immune checkpoint inhibitors
Hyperforin Elicits Cytostatic/Cytotoxic Activity in Human Melanoma Cell Lines, Inhibiting Pro-Survival NF-κB, STAT3, AP1 Transcription Factors and the Expression of Functional Proteins Involved in Mitochondrial and Cytosolic Metabolism
Get PDFHyperforin (HPF), the main component responsible for the antidepressant action of Hypericum perforatum, displays additional beneficial properties including anti-inflammatory, antimicrobic, and antitumor activities. Among its antitumor effects, HPF activity on melanoma is poorly documented. Melanoma, especially BRAF-mutated melanoma, is still a high-mortality tumor type and the currently available therapies do not provide solutions. We investigated HPF’s antimelanoma effectiveness in A375, FO1 and SK-Mel-28 human BRAF-mutated cell lines. Cell viability assays documented that all melanoma cells were affected by low HPF concentrations (EC50% 2–4 µM) in a time-dependent manner. A Br-deoxy-uridine incorporation assay attested a significant reduction of cell proliferation accompanied by decreased expression of cyclin D1 and A2, CDK4 and of the Rb protein phosphorylation, as assessed by immunoblots. In addition, the expression of P21/waf1 and the activated form of P53 were increased in A375 and SK-Mel-28 cells. Furthermore, HPF exerts cytotoxic effects. Apoptosis is induced 24 h after HPF administration, documented by an increase of cleaved-PARP1 and a decrease of both Bcl2 and Bcl-xL expression levels. Autophagy is induced, attested by an augmented LC3B expression and augmentation of the activated form of AMPK. Moreover, HPF lowers GPX4 enzyme expression, suggesting ferroptosis induction. HPF has been reported to activate the TRPC6 Ca++ channel and/or Ca++ and Zn++ release from mitochondria stores, increasing cytosolic Ca++ and Zn++ concentrations. Our data highlighted that HPF affects many cell-signaling pathways, including signaling induced by Ca++, such as FRA1, pcJun and pCREB, the expression or activity of which are increased shortly after treatment. However, the blockage of the TRPC6 Ca++ channel or the use of Ca++ and Zn++ chelators do not hinder HPF cytostatic/cytotoxic activity, suggesting that damages induced in melanoma cells may pass through other pathways. Remarkably, 24 h after HPF treatment, the expression of activated forms of the transcription factors NF-κB P65 subunit and STAT3 are significantly lowered. Several cytosolic (PGM2, LDHA and pPKM2) and mitochondrial (UQCRC1, COX4 and ATP5B) enzymes are downregulated by HPF treatment, suggesting a generalized reduction of vital functions in melanoma cells. In line with these results is the recognized ability of HPF to affect mitochondrial membrane potential by acting as a protonophore. Finally, HPF can hinder both melanoma cell migration and colony formation in soft agar. In conclusion, we provide evidence of the pleiotropic antitumor effects induced by HPF in melanoma cells
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