Fabrício Figueiró

Indomethacin-loaded nanocapsules treatment reduces in vivo glioblastoma growth in a rat glioma model

Multimodal combinations of target agents with radiation and chemotherapy may enhance cancer treatment efficacy; however, despite these treatments, gliomas recur early due to their highly proliferative, infiltrative and invasive behaviors. Nanoparticles of biodegradable polymers for anticancer drug delivery have attracted intensive interest in recent years since they may provide a sustained, controlled and targeted delivery. In the present study, we investigated the effect of indomethacin-loaded nanocapsules in an experimental glioma model. The rats treated with indomethacin-loaded nanocapsules demonstrated a significant reduction in tumor size and half of these animals presented just cells with characteristics of a residual tumor, as shown by immunostaining for nestin. Pathological analyses showed that the treated gliomas presented a significant reduction in the mitotic index and other histological characteristics that indicate a less invasive/proliferative tumor. An important finding of the present study is that indomethacin carried by polymeric nanocapsules achieved higher intracerebral drug concentrations than those of indomethacin in solution. Furthermore, indomethacin achieved a greater concentration in the hemisphere where the glioma was implanted, compared with the contralateral healthy hemisphere. Indomethacin-loaded nanocapsule treatment did not cause characteristics of toxicity and increased the survival of animals. Thus, our results show that polymeric nanocapsules are able to increase the intratumoral bioavailability of indomethacin and reduce the growth of implanted gliomas. Data suggest that indomethacin-loaded nanocapsules could offer new and potentially highly effective strategies for the treatment of malignant gliomas.

Autophagy inhibition improves the efficacy of curcumin/temozolomide combination therapy in glioblastomas

Glioblastoma is a devastating primary brain tumor resistant to conventional therapies. In this study, we tested the efficacy of combining temozolomide with curcumin, a phytochemical known to inhibit glioblastoma growth, and investigated the mechanisms involved. The data showed that synergy between curcumin and temozolomide was not achieved due to redundant mechanisms that lead to activating protective autophagy both in vitro and in vivo. Autophagy preceded apoptosis, and blocking this response with autophagy inhibitors (3-methyl-adenine, ATG7 siRNA and chloroquine) rendered cells susceptible to temozolomide and curcumin alone or combinations by increasing apoptosis. While curcumin inhibited STAT3, NFκB and PI3K/Akt to affect survival, temozolomide-induced autophagy relied on the DNA damage response and repair components ATM and MSH6, as well as p38 and JNK1/2. However, the most interesting observation was that both temozolomide and curcumin required ERK1/2 to induce autophagy. Blocking this ERK1/2-mediated temozolomide and curcumin induced autophagy with resveratrol, a blood-brain barrier permeable drug, improved temozolomide/curcumin efficacy in brain-implanted tumors. Overall, the data presented demonstrate that autophagy impairs the efficacy of temozolomide/curcumin, and inhibiting this phenomenon could provide novel opportunities to improve brain tumor treatment.

Selective cytotoxicity of indomethacin and indomethacin ethyl ester-loaded nanocapsules against glioma cell lines: an in vitro study.

Gliomas are the most common and devastating tumors of the central nervous system. Several studies have suggested that nonsteroidal anti-inflammatory drugs (NSAIDs) are promising anticancer agents. Biodegradable nanoparticulate systems have received considerable attention as potential drug delivery vehicles. The aim of this study was to evaluate the effects of indomethacin-loaded nanocapsules and indomethacin ethyl ester-loaded nanocapsules on glioma cell lines. In addition, the effect of these formulations on normal neural tissue was also evaluated. In order to investigate this, glioma cell lines (U138-MG and C6) and hippocampal organotypic cultures were used. The main finding of the present study is that indomethacin-loaded nanocapsules formulation was more potent than a solution of indomethacin in decreasing the viability and cell proliferation of glioma lines. Indomethacin and indomethacin ethyl ester associated together in the same nanocapsule formulation caused a synergic effect decreasing glioma cell proliferation. In addition, when the glioma cells were exposed to 25 microM of indomethacin-loaded nanocapsules or indomethacin ethyl ester-loaded nanocapsules, a necrotic cell death was observed. Interestingly, 5 microM of indomethacin-loaded nanocapsules was able to cause an antiproliferative effect without promoting necrosis in glioma cells. Another important finding was that the cytotoxic effect induced by 25 microM or 50 microM of indomethacin-loaded nanocapsules or indomethacin ethyl ester-loaded nanocapsules, in glioma cells was not observed in the organotypic cultures, indicating selective cytotoxicity of those formulations for tumoral cells. Further investigations using in vivo glioma model should be helpful to confirm the distinct effects of indomethacin-loaded nanocapsules and indomethacin ethyl ester-loaded nanocapsules, in normal versus tumoral cells.

Involvement of purinergic system in the release of cytokines by macrophages exposed to glioma-conditioned medium.

Macrophages are involved in cancer progression. M1 macrophages have an antitumor effect, whereas M2 phenotype are associated with tumor growth. The progression of gliomas involves the participation of an inflammatory microenvironment. Adenosine triphosphate (ATP) can act as pro‐inflammatory signal, whereas adenosine has opposite properties. The biological effects of extracellular nucleotides/nucleosides mediated by purinergic receptors are controlled by ectonucleotidases. In the present work, we evaluated whether glioma‐conditioned medium (GL‐CM) modulates macrophage differentiation and the participation of ATP and adenosine in the release of pro‐and anti‐inflammatory cytokines by these cells. The results show that macrophages exposed to GL‐CM were modulated to an M2‐like phenotype. HPLC analysis of GL‐CM demonstrated the presence of significant amounts of ATP and its metabolites. Macrophages exposed to GL‐CM presented decreased ATP and AMP hydrolysis and increased IL‐10 and MCP‐1 secretion, effects that were diminished by P1 or P2 antagonists. GL‐CM did not alter the release of IL‐6 by macrophages, although treatment with ATP promoted an increase in the release of IL‐6, which was prevented by a P2X7 antagonist. In summary, we found that A2A and P2X7 activation is necessary for IL‐10, MCP‐1, and IL‐6 release by macrophages exposed to GL‐CM, which, in turn, modulates the macrophages to M2‐phenotype. The present study establishes a relationship between M2‐like polarization, cytokine release and purinergic receptor activation in macrophages exposed to GL‐CM. Therefore, the data presented herein contributes to advancing in the field of cancer‐related inflammation and point specific purinergic receptors as targets for modulation of the phenotype of glioma‐associated macrophages. J. Cell. Biochem. 116: 721–729, 2015.

Quinovic acid glycosides purified fraction from Uncaria tomentosa induces cell death by apoptosis in the T24 human bladder cancer cell line

Bladder cancer is the second most prevalent malignancy in the genitourinary tract and remains a therapeutic challenge. In the search for new treatments, researchers have attempted to find compounds with low toxicity. With this goal in mind, Uncaria tomentosa is noteworthy because the bark and root of this species are widely used in traditional medicine and in adjuvant therapy for the treatment of numerous diseases. The objective of this study was to investigate the antitumor effect of one purified bioactive fraction of U.tomentosa bark on cell proliferation in two human bladder cancer cell lines, T24 and RT4. Quinovic acid glycosides purified fraction (QAPF) of U.tomentosa decreased the growth and viability of both T24 and RT4 cell lines. In T24 cells, QAPF induced apoptosis by activating caspase-3 and NF-κB. Further study showed that this fraction does not induce cell cycle arrest and does not alter PTEN and ERK levels. In conclusion, we demonstrated that QAPF of U.tomentosa has a potent inhibitory effect on the growth of human bladder cancer cell lines by inducing apoptosis through modulation of NF-κB, and we suggest that QAPF may become a potential therapeutic agent for the prevention and/or treatment of this cancer.

Selective cytotoxicity and apoptosis induction in glioma cell lines by 5-oxygenated-6,7-methylenedioxycoumarins from Pterocaulon species

The coumarins 5-methoxy-6,7-methylenedioxycoumarin 1 5-(3-methyl-2-butenyloxy)-6,7-methylenedioxycoumarin 2 and 5-(2,3-dihydroxy-3-methylbutyloxy)-6,7-methylenedioxycoumarin 3 isolated from Pterocaulon species showed significant cytotoxicity against two glioma cells lines. Compound 1 presented IC(50) values of 34.6 μM and 31.6 μM against human (U138-MG) and rat (C6) glioma cells, respectively, and this compound was at least two times more potent than compounds 2 and 3. This result could be explained by the planar conformation adopted by 1 through a non-classical hydrogen bond between a hydrogen of the methoxy and the oxygen of the methylenedioxy groups. Another important finding was that the cytotoxic effect induced by 1 in glioma cells was not observed in organotypic cultures, indicating a selective cytotoxicity for tumor cells.

Quercetin derivative induces cell death in glioma cells by modulating NF-κB nuclear translocation and caspase-3 activation.

Treated glioblastoma multiforme (GBM) patients only survive 6 to 14months after diagnosis; therefore, the development of novel therapeutic strategies to treat gliomas remains critically necessary. Considering that phenolic compounds, like quercetin, have the potential to be used in the chemotreatment of gliomas and that some flavonoids exhibit the ability to cross the BBB, in the present study, we investigated the antitumor effect of flavonoids (including chalcones, flavones, flavanones and flavonols). Initially their activities were tested in C6 glioma cells screened using the MTT method, resulting in the selection of chalcone 2 whose feasibility was confirmed by a Trypan Blue exclusion assay in the low μM range on C6 glioma cells. Cell cycle and apoptotic death analyses on C6 glioma cells were also performed, and chalcone 2 increased the apoptosis of the cells but did not alter the cell cycle progression. In addition, treatments with these two compounds were not cytotoxic to hippocampal organotypic cultures, a model of healthy neural cells. Furthermore, the results indicated that 2 induced apoptosis by inhibition of NF-κB and activation of active caspase-3 in glioma cells, suggesting that it is a potential prototype to develop new treatments for GBM in the future.

Ecto-5'-Nucleotidase Overexpression Reduces Tumor Growth in a Xenograph Medulloblastoma Model.

Background Ecto-5’-nucleotidase/CD73 (ecto-5’-NT) participates in extracellular ATP catabolism by converting adenosine monophosphate (AMP) into adenosine. This enzyme affects the progression and invasiveness of different tumors. Furthermore, the expression of ecto-5’-NT has also been suggested as a favorable prognostic marker, attributing to this enzyme contradictory functions in cancer. Medulloblastoma (MB) is the most common brain tumor of the cerebellum and affects mainly children. Materials and Methods The effects of ecto-5’-NT overexpression on human MB tumor growth were studied in an in vivo model. Balb/c immunodeficient (nude) 6 to 14-week-old mice were used for dorsal subcutaneous xenograph tumor implant. Tumor development was evaluated by pathophysiological analysis. In addition, the expression patterns of adenosine receptors were verified. Results The human MB cell line D283, transfected with ecto-5’-NT (D283hCD73), revealed reduced tumor growth compared to the original cell line transfected with an empty vector. D283hCD73 generated tumors with a reduced proliferative index, lower vascularization, the presence of differentiated cells and increased active caspase-3 expression. Prominent A1 adenosine receptor expression rates were detected in MB cells overexpressing ecto-5’-NT. Conclusion This work suggests that ecto-5’-NT promotes reduced tumor growth to reduce cell proliferation and vascularization, promote higher differentiation rates and initiate apoptosis, supposedly by accumulating adenosine, which then acts through A1 adenosine receptors. Therefore, ecto-5’-NT might be considered an important prognostic marker, being associated with good prognosis and used as a potential target for therapy.

4-Methylcoumarins with cytotoxic activity against T24 and RT4 human bladder cancer cell lines

Bladder cancer is one of the most prevalent malignancies of the genitourinary tract, and approximately 25% of patients develop superficial cancers with invasive and metastatic pathology. Coumarins and their derivatives have antiproliferative activity and induce apoptosis in several cancer cell lines. Due to the potential therapeutic applications of these compounds, a series of 4-methylcoumarins were synthesized to investigate the antitumor cytotoxic effect in the T24 and RT4 human bladder cancer cell lines. The microwave-assisted synthesis of the coumarinsvia Pechmann condensation with modifications at position 7 was performed with excellent yields (74–100%). The 7,8-dihydroxy-4-methyl-2-oxo-2H-chromene-5-carboxylic acid derivative (3c) exhibited greater cytotoxicity against T24 cells, which represent a more malignant cell line compared to RT4. In the T24 cells, cell cycle analysis revealed a large number of cells in the sub-G1 phase after treatment with 3c, which is indicative of apoptosis. Apoptotic death was confirmed by annexin V staining assay. Based on the chemical structures of the compounds, it is suggested that the 5-carboxycoumarin ring group has a positive influence on the cytotoxic activity. These results indicate that these new compounds are promising for further chemical modulation to discover a new antitumor agent.

Kaempferol-loaded mucoadhesive nanoemulsion for intranasal administration reduces glioma growth in vitro

&NA; In order to search for new approaches to treat glioma, intranasal administration has been proposed as an alternative route to deliver drugs into the brain. Among the drug alternatives, kaempferol (KPF) has been reported to induce glioma cell death. This study aimed to prepare nanoemulsions containing KPF with and without chitosan to investigate their potential for brain delivery following intranasal administration, and to evaluate their antitumor activity against glioma cells. KPF‐loaded nanoemulsion (KPF‐NE) and KPF‐loaded mucoadhesive nanoemulsion (KPF‐MNE) were prepared by high‐pressure homogenization technique and were characterized for their globule size, zeta potential, drug content, pH, viscosity, mucoadhesive strength and morphology. KPF from KPF‐MNE showed significantly higher permeation across the mucosa in ex vivo diffusion studies. Histopathological examination suggests both nanoemulsions to be safe for the nasal mucosa and able to preserve KPF antioxidant capability. KPF‐MNE enhanced significantly the amount of drug into rats brain following intranasal administration (5‐ and 4.5‐fold higher than free drug and KPF‐NE, respectively). In addition, KPF‐MNE reduced C6 glioma cell viability through induction of apoptosis to a greater extent than either free KPF or KPF‐NE. The mucoadhesive nanoemulsion developed for intranasal administration may be a promising system for delivery to the brain, and KPF‐MNE is a candidate for further antiglioma trials. Graphical abstract Figure. No caption available.