Fabrícia Dietrich

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.

Characterization of Ectonucleotidases in Human Medulloblastoma Cell Lines: ecto-5′NT/CD73 in Metastasis as Potential Prognostic Factor

Medulloblastoma (MB) is the most common malignant brain tumor in children and occurs mainly in the cerebellum. Important intracellular signaling molecules, such those present in the Sonic Hedgehog and Wnt pathways, are involved in its development and can also be employed to determine tumor grade and prognosis. Ectonucleotidases, particularly ecto-5′NT/CD73, are important enzymes in the malignant process of different tumor types regulating extracellular ATP and adenosine levels. Here, we investigated the activity of ectonucleotidases in three malignant human cell lines: Daoy and ONS76, being representative of primary MB, and the D283 cell line, derived from a metastatic MB. All cell lines secreted ATP into the extracellular medium while hydrolyze poorly this nucleotide, which is in agreement with the low expression and activity of pyrophosphate/phosphodiesterase, NTPDases and alkaline phosphatase. The analysis of AMP hydrolysis showed that Daoy and ONS76 completely hydrolyzed AMP, with parallel adenosine production (Daoy) and inosine accumulation (ONS76). On the other hand, D283 cell line did not hydrolyze AMP. Moreover, primary MB tumor cells, Daoy and ONS76 express the ecto-5′NT/CD73 while D283 representative of a metastatic tumor, revealed poor expression of this enzyme, while the ecto-adenosine deaminase showed higher expression in D283 compared to Daoy and ONS76 cells. Nuclear beta-catenin has been suggested as a marker for MB prognosis. Further it can promotes expression of ecto-5′NT/CD73 and suppression of adenosine deaminase. It was observed that Daoy and ONS76 showed greater nuclear beta-catenin immunoreactivity than D283, which presented mainly cytoplasmic immunoreactivity. In summary, the absence of ecto-5′NT/CD73 in the D283 cell line, a metastatic MB phenotype, suggests that high expression levels of this ectonucleotidase could be correlated with a poor prognosis in patients with MB.

Catʼs Claw Oxindole Alkaloid Isomerization Induced by Cell Incubation and Cytotoxic Activity against T24 and RT4 Human Bladder Cancer Cell Lines

The antitumor activity of Uncaria tomentosa, a native vine from the Amazonian rainforest, has been ascribed to pentacyclic oxindole alkaloids occurring in its bark. Former studies have shown that this activity, as well as its intensity, depends on whether cats claw alkaloids occur as original compounds or isomerized derivatives. This work addresses this aspect, using T24 and RT4 human bladder cancer cell lines for that purpose. Bark samples were extracted by dynamic maceration, prepurified with cross-linked polyvinylpyrrolidone and properly fractioned by an ion exchange process to obtain an oxindole alkaloid purified fraction. Alkaloid isomerization was induced by heating it under reflux at 85 °C. Samples collected after 5, 15, and 45 min of heating were analyzed by HPLC-PDA, freeze-dried at once, and separately assayed using the non-isomerized purified fraction for comparison purposes. The latter showed significant and dose-dependent cytotoxic activity against both T24 and RT4 cancer cell lines (IC50: 164.13 and 137.23 µg/mL, respectively). However, results for both cell lines were equivalent to those observed for isomerized samples (p > 0.05). The alkaloid isomerization induced by the incubation conditions (buffered medium pH 7.4 and temperature 37 °C) helps to explain the similar results obtained from non-isomerized and isomerized samples. Mitraphylline, speciophylline, uncarine F, and, to a lesser degree, pteropodine were more susceptible to isomerization under the incubation conditions. Thus, the alkaloid profile of all fractions and their cytotoxic activities against T24 and RT4 human bladder cancer cell lines are determined to a large extent by the incubation conditions.

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.

Genotoxicity and cytotoxicity of oxindole alkaloids from Uncaria tomentosa (cat's claw): Chemotype relevance.

ETHNOPHARMACOLOGICAL RELEVANCE Uncaria tomentosa (Willdenow ex Roemer & Schultes) DC. (Rubiaceae) or cats claw is a climber vine from the South American rainforest used in folk medicine for cancer treatment. Its antitumor activity has been mostly ascribed to pentacyclic oxindole alkaloids (POA) from stem bark and leaves while the activity of tetracyclic oxindole alkaloids (TOA) remains unknown. In recent times, the occurrence of three chemotypes based on its oxindole alkaloid profile was noticed in U. tomentosa, namely, chemotype I (POA cis D/E ring junction); chemotype II (POA trans D/E ring junction) or chemotype III (TOA). Consequently, the relationship between the chemotype and cytotoxic and genotoxic activities deserves attention. AIM OF THE STUDY To evaluate the influence of cats claw chemotypes on genotoxicity and cytotoxicity against non malignant and malignant human cell line models. MATERIAL AND METHODS Four authentic stem bark cats claw samples (SI-SIV) and two leaf samples (LII and LIII) were analyzed by HPLC-PDA, properly extracted and fractioned by ion-exchange to obtain oxindole alkaloid purified fractions (OAPFs). The freeze-dried fractions were assayed for genotoxicity and cytotoxicity against human leukocytes (non malignant cell line) by the micronuclei frequency method and the alkaline comet DNA assay, and the trypan blue method, respectively. Moreover, the cytotoxicity of each OAPF was evaluated against a human bladder cancer cell line (T24) and human glioblastoma cell line (U-251-MG) by MTT method (malignant cell lines). Additionally, the isomerization of oxindole alkaloids throughout the course of cell incubation was monitored by HPLC-PDA. RESULTS Based on HPLC-PDA analyses, sample SI was characterized as chemotype I, while samples SII and LII were characterized as chemotype II, and samples SIII, SIV and LIII as chemotype III. The chemotypes showed comparable cytotoxic activity toward malignant cell lines (T24 and U-251-MG) unlike human leukocytes (non malignant cell line), where this activity was clearly distinct. Chemotype II (POA trans D/E ring junction) showed a higher selectivity index (SI) against malignant cells (SI=1.11-3.04) than chemotype I (SI=0.10-0.19) and III (SI=0.21-0.57). No important genotoxic potential was found by micronuclei frequency and alkaline comet DNA assays. Despite the isomerization of oxindole alkaloids during the cell incubation, the chemotype of the cats claw samples remained unchanged. CONCLUSION Cats claw chemotypes showed different selectivity against human malignant cells, so that the correct identification of each chemotype seems to be important to better understand its antitumor potential.

The quinovic acid glycosides purified fraction from Uncaria tomentosa protects against hemorrhagic cystitis induced by cyclophosphamide in mice

Uncaria tomentosa is widely used in folk medicine for the treatment of numerous diseases, such as urinary tract disease. Hemorrhagic cystitis (HE) is an inflammatory condition of the bladder associated with the use of anticancer drugs such as cyclophosphamide (CYP). Sodium 2-mercaptoethanesulfonate (Mesna) has been used to prevent the occurrence of HE, although this compound is not effective in established lesions. It has been demonstrated that the purinergic system is involved in several pathophysiological events. Among purinergic receptors, P2X7 deserves attention because it is involved in HE induced by CYP and, therefore, can be considered a therapeutic target. The objective of this study was to investigate the potential therapeutic effect of the quinovic acid glycosides purified fraction (QAPF) from U. tomentosa in the mouse model of CYP-induced HE. Pretreatment with QAPF not only had a protective effect on HE-induced urothelial damage (edema, hemorrhage and bladder wet weight) but was also able to control visceral pain, decrease IL-1β levels and down-regulates P2X7 receptors, most likely by inhibit the neutrophils migration to the bladder. This research clearly demonstrates the promising anti-inflammatory properties of QAPF, supporting its use as complementary therapy. QAPF represents a promising therapeutic option for this pathological condition.

Ecto-5′-nucleotidase/CD73 contributes to the radiosensitivity of T24 human bladder cancer cell line

PurposeTrimodal therapy is a reasonable bladder-preserving option to radical cystectomy. However, many tumors are radioresistive. In this sense, the identification of new prognostic and predictive biomarkers that allow the selection of patients with better responses to radiation therapy would improve outcomes. With the aim of using ecto-5′-nucleotidase/CD73 as a predictive biomarker, the role of this enzyme in the context of radiotherapy in T24 human bladder cancer cell line was investigated.MethodsT24 cell line was exposure to a single dose of radiation (4 Gray) and trypan blue assay (pharmacological assays of viability/cumulative population doubling), flow cytometry (cell cycle/cell death/active caspase-3/ecto-5′-nucleotidase/CD73 protein staining), DAPI staining (nuclear morphometric assay), RT-PCR and real-time PCR, malachite green method (ectonucleotidase enzymatic assay), and HPLC (analysis of AMP metabolism) were carried out. T24 cell line in which ecto-5′-nucleotidase/CD73 has been completely silenced (5′KO) was also used.ResultsThe exposure of T24 cell line to a single dose (4 Gray) of radiation-induced cell death and triggered a transitory increase in ecto-5′-nucleotidase/CD73 expression, increased ectonucleotidase activity, and led to adenosine and inosine accumulation in the extracellular medium. Pharmacological inhibition or knocking out ecto-5′-nucleotidase/CD73 rescued cells’ proliferative capacity, reducing their sensitivity to radiation.ConclusionOur findings show that the induction of ecto-5′-nucleotidase/CD73 by radiation contributes to the radiosensitivity of T24 cell line.

Interference of ursolic acid treatment with glioma growth: An in vitro and in vivo study

Glioblastoma multiforme is the most devastating tumor in the brain. Ursolic acid (UA) is found in a variety of plants, and exhibits several pharmacological activities. In this study, we investigated the effects of UA in vitro, clarifying the mechanisms that mediate its toxicity and the long-lasting actions of UA in C6 glioma cells. We also evaluated the antitumor activity of UA in an in vivo orthotopic glioma model. Cell numbers were assessed using the Trypan blue exclusion test, and the cell cycle was characterized by flow cytometry using propidium iodide staining. Apoptosis was analyzed using an Annexin V kit and by examining caspase-3. Akt immunocontent was verified by Western blot and the long-lasting actions of UA were measured by cumulative population doubling (CPD). In vivo experiments were performed in rats to measure the effects on tumor size, malignant features and toxicological parameters. In vitro results showed that UA decreased glioma cell numbers, increased the sub-G1 fraction and induced apoptotic death, accompanied by increased active caspase-3 protein levels. Akt phosphorylation/activation in cells was also diminished by UA. With regard to CPD, cell proliferation was almost completely restored upon single UA treatments, but when the UA was added again, the majority of cells died, demonstrating the importance of re-treatment cycles with chemotherapeutic agents for abolishing tumor growth. In vivo, ursolic acid slightly reduced glioma tumor size but did not decrease malignant features. Ursolic acid may be a potential candidate as an adjuvant for glioblastoma therapy.