Phloroglucinol derivatives from Hypericum species induce in vitro proliferation of cells involved in the wound healing process.

The genus Hypericum (Hypericaceae) is a recognized source of therapeutic agents, being some species widely used due to their wound healing properties. In a previous study, south Brazilian species H. caprifoliatum, H. carinatum, H. connatum, H. myrianthum and H. polyanthemum demonstrated potential to induce proliferation of keratinocytes. In the present study, the effect of phloroglucinol derivatives isolated from Hypericum on cell proliferation of human keratinocytes, fibroblasts and stem cells was investigated. The best results, determined by the MTT assay, were achieved with cariphenone B at concentrations of 0.01 and 0.1 µM (122.3% and 114%, respectively) on HaCaT cells. Uliginosin B was able to induce the proliferation of mesenchymal stem cells (129% at 10 µM) and MRC5 fibroblasts (152.5% at 5 µM). These findings confirm the capacity of phloroglucinol derivatives to induce the in vitro cellular proliferation and reinforce the importance of Hypericum species as potential sources of wound healing compounds.

Imatinib mesylate affects extracellular ATP catabolism and expression of NTPDases in a chronic myeloid leukemia cell line.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm, characterized by the occurrence of the t(9;22)(q34;q11) translocation. First-line therapy for CML consists of treatment with imatinib mesylate, which selectively inhibits the BCR-ABL protein by competing for its ATP-binding site. Adenine nucleotide signaling is modulated by the ectonucleotidases and this pathway is related to tumorigenic processes. Considering the relationship between ATP and cancer, we aimed to evaluate the influence of imatinib mesylate on the expressions and functions of the NTPDase and ecto-5'-nucleotidase (CD73) enzymes in imatinib-sensitive and -resistant K-562 cell lines. mRNA analysis showed that K-562 cells express all ENTPDs and NT5E. However, when treated with imatinib mesylate for 24 h, the expression of ENTPD1, -2, -3 and -5 increased, leading to a higher nucleotides hydrolysis rate. HPLC analysis identified increased ATP degradation in cells after 24 h of treatment, with consequent ADP and AMP formation, corroborating the increase in gene and protein expression of ectonucleotidases as observed in previous results. On the other hand, we observed that imatinib-resistant K-562 cells presented a decrease in nucleotide hydrolysis and expressions of ENTPD1 and -5. These results suggest an involvement of imatinib in modulating ectonucleotidases in CML that will need further investigation. Since these ectonucleotidases have important catalytic activities in the tumor microenvironment, their modulation in CML cells may represent an important therapeutic approach to regulate levels of extracellular adenine nucleotides.

Enhanced and Selective Antiproliferative Activity of Methotrexate-Functionalized-Nanocapsules to Human Breast Cancer Cells (MCF-7).

Methotrexate is a folic acid antagonist and its incorporation into nanoformulations is a promising strategy to increase the drug antiproliferative effect on human breast cancer cells by overexpressing folate receptors. To evaluate the efficiency and selectivity of nanoformulations containing methotrexate and its diethyl ester derivative, using two mechanisms of drug incorporation (encapsulation and surface functionalization) in the in vitro cellular uptake and antiproliferative activity in non-tumoral immortalized human keratinocytes (HaCaT) and in human breast carcinoma cells (MCF-7). Methotrexate and its diethyl ester derivative were incorporated into multiwall lipid-core nanocapsules with hydrodynamic diameters lower than 160 nm and higher drug incorporation efficiency. The nanoformulations were applied to semiconfluent HaCaT or MCF-7 cells. After 24 h, the nanocapsules were internalized into HaCaT and MCF-7 cells; however, no significant difference was observed between the nanoformulations in HaCaT (low expression of folate receptors), while they showed significantly higher cellular uptakes than the blank-nanoformulation in MCF-7, which was the highest uptakes observed for the drug functionalized-nanocapsules. No antiproliferative activity was observed in HaCaT culture, whereas drug-containing nanoformulations showed antiproliferative activity against MCF-7 cells. The effect was higher for drug-surface functionalized nanocapsules. In conclusion, methotrexate-functionalized-nanocapsules showed enhanced and selective antiproliferative activity to human breast cancer cells (MCF-7) being promising products for further in vivo pre-clinical evaluations.

Antineoplastic potential of the aqueous crude extract of Eugenia uniflora L. in human cervical cancer

Abstract Considering the high prevalence of human cervical cancer and the adverse effects of the available treatments, it is important to develop studies involving plants. Eugenia uniflora L. is a Brazilian native plant widely used in folk medicine and some biological effects have already been described. In this study, we investigated the biologicals effects of the aqueous crude extract of E. uniflora leaves in relation to the viability of human cervical cancer cells (SiHa), non-tumorigenic cells HaCaT and human lymphocytes. Our results demonstrated that different concentrations of E. uniflora's extract significantly inhibited the viability of the Siha cell line at 24, 48 and 72 hours of treatment, but did not induce significant changes in the HaCat cell line and human lymphocytes. Tumor cells had adhesion capacity, migration processes, ability of colony forming and the potential to recover its viability after treatment. withdrawal, significantly reduced. The nuclear morphology revealed chromatin condensation, and the flow cytometry showed predominantly cell death by apoptosis in the treated tumor cells. Therefore, the E. uniflora's extract may contribute for future studies aiming at new therapeutic perspectives for human cervical cancer.

Arginylglycylaspartic Acid-Surface-Functionalized Doxorubicin-Loaded Lipid-Core Nanocapsules as a Strategy to Target Alpha(V) Beta(3) Integrin Expressed on Tumor Cells.

Doxorubicin (Dox) clinical use is limited by dose-related cardiomyopathy, becoming more prevalent with increasing cumulative doses. Previously, we developed Dox-loaded lipid-core nanocapsules (Dox-LNC) and, in this study, we hypothesized that self-assembling and interfacial reactions could be used to obtain arginylglycylaspartic acid (RGD)-surface-functionalized-Dox-LNC, which could target tumoral cells overexpressing αvß3 integrin. Human breast adenocarcinoma cell line (MCF-7) and human glioblastoma astrocytoma (U87MG) expressing different levels of αvß3 integrin were studied. RGD-functionalized Dox-LNC were prepared with Dox at 100 and 500 mg·mL-1 (RGD-MCMN (Dox100) and RGD-MCMN (Dox500)). Blank formulation (RGD-MCMN) had z-average diameter of 162 ± 6 nm, polydispersity index of 0.11 ± 0.04, zeta potential of +13.2 ± 1.9 mV and (6.2 ± 1.1) × 1011 particles mL-1, while RGD-MCMN (Dox100) and RGD-MCMN (Dox500) showed respectively 146 ± 20 and 215 ± 25 nm, 0.10 ± 0.01 and 0.09 ± 0.03, +13.8 ± 2.3 and +16.4 ± 1.5 mV and (6.9 ± 0.6) × 1011 and (6.1 ± 1.0) × 1011 particles mL-1. RGD complexation was 7.73 × 104 molecules per nanocapsule and Dox loading were 1.51 × 104 and 7.64 × 104 molecules per nanocapsule, respectively. RGD-functionalized nanocapsules had an improved uptake capacity by U87MG cells. Pareto chart showed that the cell viability was mainly affected by the Dox concentration and the period of treatment in both MCF-7 and U87MG. The influence of RGD-functionalization on cell viability was a determinant factor exclusively to U87MG.

Data of PCL-b-P(MMA-DMAEMA)2 characterization and related assays.

The data presented here are related to the research paper entitled "PCL-b-P(MMA-co-DMAEMA)2 new triblock copolymer for novel pH-sensitive nanocapsules intended for drug delivery to tumors" by Franco et al. [1]. Characterization data of PCL-diol, macroinitiator Br-PCL-Br, homopolymers (PMMA and PDMAEMA) and copolymers (batch 1 and batch 2) analyzed by FTIR, SEC and NMR, as well as, characterization of PCL-NS formulation by laser diffraction and DLS analysis, initial nanocapsule formulations and 1C-NC and 2C-NC formulations, including hydrodynamic diameter at different pH media, and DMSO cytotoxicity.

Liquid formulation containing doxorubicin-loaded lipid-core nanocapsules: Cytotoxicity in human breast cancer cell line and in vitro uptake mechanism.

Cancer is a major public health problem in the world, being breast cancer the most frequent cancer affecting women. Despite advances in detection and treatment, mortality rates remain high. Therefore, new approaches for breast cancer treatments are necessary. In this study, our objective was to develop a liquid formulation containing doxorubicin-loaded lipid-core nanocapsules (DOX-LNC), to evaluate the in vitro antiproliferative activity and to determine the nanocapsules uptake by MCF-7 cells. Lipid-core nanocapsules (LNC), blank formulation, and DOX-LNC, proposed treatment, were prepared by self-assembling using the solvent displacement method. Hydrodynamic mean diameters (z-average) were respectively 191±31nm and 230±23nm presenting narrow size distributions. Drug content was 0.102±0.029mgmL-1 with an encapsulation efficiency higher than 90%. Formulations were applied to semiconfluent MCF-7 cells. After 24h, LNC showed no cytotoxicity, while DOX-LNC showed an IC50 of 4.49 micromolar. After 72h of incubation, DOX-LNC showed an IC50 of 1.60 micromolar demonstrating a sustained effect. The nanocapsules were internalized by endocytosis mediated by caveolin and by fluid phase endocytosis, which are active transport mechanisms. In conclusion, the liquid formulation containing DOX-LNC showed to be a promising product for the breast cancer treatment opening new avenues for further in vivo studies.

Bromelain-Functionalized Multiple-Wall Lipid-Core Nanocapsules: Formulation, Chemical Structure and Antiproliferative Effect Against Human Breast Cancer Cells (MCF-7).

PURPOSE: This study was conducted a promising approach to surface functionalization developed for lipid-core nanocapsules and the merit to pursue new strategies to treat solid tumors. METHODS: Bromelain-functionalized multiple-wall lipid-core nanocapsules (Bro-MLNC-Zn) were produced by self-assembling following three steps of interfacial reactions. Physicochemical and structural characteristics, in vitro proteolytic activity (casein substrate) and antiproliferative activity (breast cancer cells, MCF-7) were determined. RESULTS: Bro-MLNC-Zn had z-average diameter of 135 nm and zeta potential of +23 mV. The complex is formed by a Zn-N chemical bond and a chelate with hydroxyl and carboxyl groups. Bromelain complexed at the nanocapsule surface maintained its proteolytic activity and showed anti-proliferative effect against human breast cancer cells (MCF-7) (72.6 ± 1.2% at 1.250 µg mL-1 and 65.5 ± 5.5% at 0.625 µg mL-1). Comparing Bro-MLNC-Zn and bromelain solution, the former needed a dose 160-folds lower than the latter for a similar effect. Tripan blue dye assay corroborated the results. CONCLUSIONS: The surface functionalization approach produced an innovative formulation having a much higher anti-proliferative effect than the bromelain solution, even though both in vitro proteolytic activity were similar, opening up a great opportunity for further studies in nanomedicine.

Characterization of Phloroglucinol-enriched Fractions of Brazilian Hypericum Species and Evaluation of Their Effect on Human Keratinocytes Proliferation.

In this study, a phytochemical and biological investigation on five South Brazilian Hypericum species (Hypericum caprifoliatum, Hypericum carinatum, Hypericum connatum, Hypericum myrianthum, and Hypericum polyanthemum) was carried out. The phloroglucinol-enriched fractions (PEF) of the flowering aerial parts were analyzed by high-performance liquid chromatography for the content of uliginosin A (1), japonicin A (2), uliginosin B (3), hyperbrasilol B (4), and the three benzopyrans, that is, 6-isobutyryl-5,7-dimethoxy-2,2-dimethyl-benzopyran (HP1) (5), 7-hydroxy-6-isobutyryl-5-methoxy-2,2-dimethyl-benzopyran (HP2) (6), and 5-hydroxy-6-isobutyryl-7-methoxy-2,2-dimethyl-benzopyran (HP3) (7). After chemical characterization, the PEF were assayed for cell proliferation on human keratinocyte cell line by MTT. The H. carinatum and H. polyanthemum PEF demonstrated better results with an increase in cell proliferation (138.7% and 120.6%, respectively). The cell counting and Ki-67 assay with H. carinatum PEF confirmed the MTT results. The cell cycle distribution indicates an increase in the cells at S and G2/M phases, which is indicative of proliferation induction. In summary, the results indicate an induction of HaCaT proliferation by the treatment with H. carinatum PEF (at 10 and 15 µg/mL), suggesting a possible use as wound healing agent. Copyright © 2016 John Wiley & Sons, Ltd.

DPPIV/CD26: a tumor suppressor or a marker of malignancy?

Dipeptidyl peptidase IV (DPPIV/CD26) is a multifunctional protein with intrinsic peptidase activity that inactivates or degrades some bioactive peptides. It is the main cellular binding protein for ecto-adenosine deaminase and interacts with extracellular matrix proteins, besides participating in different signaling pathways. Due to these multiple functions, DPPIV/CD26 has been shown to be closely related to the tumor process. It has been reported that the progression of certain types of cancer is accompanied by a decrease in DPPIV/CD26 expression, and studies have shown that the malignant phenotype can be reverted when DPPIV/CD26 expression is induced in these cancer cells, characterizing this protein as a tumor suppressor. On the other hand, DPPIV/CD26 was described as a protein associated with invasion and metastatic spread, characterizing it as a marker of malignancy. Thus, this review explores the roles of DPPIV/CD26 expression in tumor progression in different types of cancer and demonstrates the importance of this protein as a promising therapeutic target and tumor biomarker.

Proliferative effect of plants used for wound healing in Rio Grande do Sul state, Brazil.

ETHNOPHARMACOLOGICAL RELEVANCE: Wounds are normally resolved in a few days, but chronic wounds represent a major burden because of economic and social factors. Thereby, the search for new agents is ongoing and natural products become a great target. Also, Brazil as a consumer of herbal medicines with rich social diversity is promising for ethnopharmacological studies. AIMS OF THE STUDY: The study aims to find the plants popularly used for wound healing purposes in Rio Grande do Sul state, and test the traditional knowledge through an in vitro screening. MATERIALS AND METHODS: Ethnobotanical studies from state of Rio Grande do Sul were analyzed to find the most used plants to treat wounds. The selected species were collected, identified and ethanolic and aqueous extracts were prepared. After, proliferative capacity was accessed by MTT assay in a keratinocyte cell line (HaCaT). RESULTS: The survey comprehended almost all state regions and led to 117 plant species from 85 genera, from which 14 were selected for in vitro testing. Aqueous extracts from Achyrocline satureioides DC Lam., Matricaria recutita L., Melia azedarach L. and Mirabilis jalapa L. demonstrated the ability to stimulate keratinocyte growth up to 120% in concentrations of 25 µg/mL and 50 µg/mL. The ethanolic extract of A. satureioides was able to stimulate keratinocyte and fibroblast proliferation on the lower concentration tested, 1 µg/mL, being the most promising species. CONCLUSIONS: The traditional knowledge collected from the ethnobotanical studies was accessed by in vitro investigation and extracts from Achyrocline satureioides, Matricaria recutita, Melia azedarach and Mirabilis jalapa can influence positively cell proliferation.

Differential Expression and Enzymatic Activity of DPPIV/CD26 Affects Migration Ability of Cervical Carcinoma Cells.

Dipeptidyl peptidase IV (DPPIV/CD26) is a transmembrane glycoprotein that inactivates or degrades some bioactive peptides and chemokines. For this reason, it regulates cell proliferation, migration and adhesion, showing its role in cancer processes. This enzyme is found mainly anchored onto the cell membrane, although it also has a soluble form, an enzymatically active isoform. In the present study, we investigated DPPIV/CD26 activity and expression in cervical cancer cell lines (SiHa, HeLa and C33A) and non-tumorigenic HaCaT cells. The effect of the DPPIV/CD26 inhibitor (sitagliptin phosphate) on cell migration and adhesion was also evaluated. Cervical cancer cells and keratinocytes exhibited DPPIV/CD26 enzymatic activity both membrane-bound and in soluble form. DPPIV/CD26 expression was observed in HaCaT, SiHa and C33A, while in HeLa cells it was almost undetectable. We observed higher migratory capacity of HeLa, when compared to SiHa. But in the presence of sitagliptin SiHa showed an increase in migration, indicating that, at least in part, cell migration is regulated by DPPIV/CD26 activity. Furthermore, in the presence of sitagliptin phosphate, SiHa and HeLa cells exhibited a significant reduction in adhesion. However this mechanism seems to be mediated independent of DPPIV/CD26. This study demonstrates, for the first time, the activity and expression of DPPIV/CD26 in cervical cancer cells and the effect of sitagliptin phosphate on cell migration and adhesion.

CD26 a cancer stem cell marker and therapeutic target.

Cancer stem cells (CSCs) comprise a tumor subpopulation responsible for tumor maintenance, resistance to chemotherapy, recurrence and metastasis. The identification of this cell group is very important, but there is still no consensus on its characterization. Several CSC markers have been described, like CD133, CD24, CD44 and ALDH1, but more research to identify new markers to facilitate the identification of CSC in a heterogeneous tumoral mass is required. Thus, this article describes the CD26 expression as a CSC marker and the role that it plays in different types of cancer. CD26 expression correlates with some characteristics of CSCs, like the formation of spheres in vitro, formation of new tumors, and resistance to chemotherapy. CD26 is therefore suggested as an auxiliary marker for CSC in different types of cancer, and as a potential therapeutic target.

Adenosine uptake is the major effector of extracellular ATP toxicity in human cervical cancer cells.

In cervical cancer, HPV infection and disruption of mechanisms involving cell growth, differentiation, and apoptosis are strictly linked with tumor progression and invasion. Tumor microenvironment is ATP and adenosine rich, suggesting a role for purinergic signaling in cancer cell growth and death. Here we investigate the effect of extracellular ATP on human cervical cancer cells. We find that extracellular ATP itself has a small cytotoxic effect, whereas adenosine formed from ATP degradation by ectonucleotidases is the main factor responsible for apoptosis induction. The level of P2 × 7 receptor seemed to define the main cytotoxic mechanism triggered by ATP, since ATP itself eliminated a small subpopulation of cells that express high P2 × 7 levels, probably through its activation. Corroborating these data, blockage or knockdown of P2 × 7 only slightly reduced ATP cytotoxicity. On the other hand, cell viability was almost totally recovered with dipyridamole, an adenosine transporter inhibitor. Moreover, ATP-induced apoptosis and signaling-p53 increase, AMPK activation, and PARP cleavage-as well as autophagy induction were also inhibited by dipyridamole. In addition, inhibition of adenosine conversion into AMP also blocked cell death, indicating that metabolization of intracellular adenosine originating from extracellular ATP is responsible for the main effects of the latter in human cervical cancer cells.

Controlled release of raloxifene by nanoencapsulation: effect on in vitro antiproliferative activity of human breast cancer cells.

Raloxifene hydrochloride (RH) is considered to be an antiproliferative agent of mammary tissue. The aim of this study was to investigate the effect of the encapsulation of RH in polymeric nanocapsules with anionic or cationic surface on its release profile and antiproliferative activity. They were prepared by interfacial deposition of preformed polymer, followed by wide physicochemical characterization. The in vitro RH release was assessed by the dialysis membrane method and the data analyzed by mathematical modeling. The antiproliferative effect on MCF-7 cell viability was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as well as by counting viable cells. They had high encapsulation efficiency, low polydispersity, and nanometric mean size. Nanocapsules prepared with Eudragit(®) RS100 and Eudragit(®) S100 presented positive and negative zeta potentials, respectively. Drug release studies demonstrated controlled release of RH from anionic nanocapsules, which could be explained due to a stronger interaction of the drug to these nanocapsules and the larger amount of entrapped drug. On the other hand, this control was not observed from cationic nanocapsules due to the larger amount of drug adsorbed onto their surface. MCF-7 cell viability studies and cell counting showed that RH-loaded Eudragit(®) RS100 nanocapsules promote the best antiproliferative activity after 24 hours of treatment, whereas the best activity was observed for RH-loaded Eudragit(®) S100 nanocapsules after 72 hours. Furthermore, the combined treatment of these formulations improved the antiproliferative effect during the entire treatment.

Ectonucleotidase expression profile and activity in human cervical cancer cell lines.

Cervical cancer is the third most frequent cancer in women worldwide. Adenine nucleotide signaling is modulated by the ectonucleotidases that act in sequence, forming an enzymatic cascade. Considering the relationship between the purinergic signaling and cancer, we studied the E-NTPDases, ecto-5'-nucleotidase, and E-NPPs in human cervical cancer cell lines and keratinocytes. We evaluated the expression profiles of these enzymes using RT-PCR and quantitative real-time PCR analysis. The activities of these enzymes were examined using ATP, ADP, AMP, and p-nitrophenyl-5'-thymidine monophosphate (p-Nph-5'-TMP) as substrate, in a colorimetric assay. The extracellular adenine nucleotide hydrolysis was estimated by HPLC analysis. The hydrolysis of all substrates exhibited a linear pattern and these activities were cation-dependent. An interesting difference in the degradation rate was observed between cervical cancer cell lines SiHa, HeLa, and C33A and normal imortalized keratinocytes, HaCaT cells. The mRNA of ecto-5'-nucleotidase, E-NTPDases 5 and 6 were detectable in all cell lines, and the dominant gene expressed was the Entpd 5 enzyme, in SiHa cell line (HPV16 positive). In accordance with this result, a higher hydrolysis activity for UDP and GDP nucleotides was observed in the supernatant of the SiHa cells. Both normal and cancer cells presented activity and mRNAs of members of the NPP family. Considering that these enzymes exert an important catalytic activity, controlling purinergic nucleotide concentrations in tumors, the presence of ectonucleotidases in cervical cancer cells can be important to regulate the levels of extracellular adenine nucleotides, limiting their effects.