Carlos R. Figueiredo

α-Pinene isolated from Schinus terebinthifolius Raddi (Anacardiaceae) induces apoptosis and confers antimetastatic protection in a melanoma model

Malignant melanoma is one the most aggressive types of cancer and its incidence has gradually increased in the last years, accounting for about 75% of skin cancer deaths. This poor prognosis results from the tumor resistance to conventional drugs mainly by deregulation of apoptotic pathways. The aim of this work was to investigate the cell death mechanism induced by α-pinene and its therapeutic application. Our results demonstrated that α-pinene was able to induce apoptosis evidenced by early disruption of the mitochondrial potential, production of reactive oxygen species, increase in caspase-3 activity, heterochromatin aggregation, DNA fragmentation and exposure of phosphatidyl serine on the cell surface. Most importantly, this molecule was very effective in the treatment of experimental metastatic melanoma reducing the number of lung tumor nodules. This is the first report on the apoptotic and antimetastatic activity of isolated α-pinene.

Jacaranone induces apoptosis in melanoma cells via ROS-mediated downregulation of Akt and p38 MAPK activation and displays antitumor activity in vivo.

Background Malignant melanoma is a deadly type of metastatic skin cancer with increased incidence over the past 30 years. Despite the advanced knowledge on the biology, immunobiology and molecular genetics of melanoma, the alternatives of treatment are limited with poor prognosis. On clinical trials, natural products and among them redox-active quinones have been tested in the attempt to control the growth of cancer cells. Recently, we isolated jacaranone from Pentacalia desiderabilis, a benzoquinone derivative that showed a broad antitumor activity and protective anti-melanoma effect in a syngeneic model. The purified substance is active at micromolar concentrations, is not hemolytic, and is not toxic in naïve mice. Methodology/Principal Findings The jacaranone antitumor activity was shown against several human cancer cell lines in vitro. Moreover, the induction of apoptosis in murine melanoma cells and jacaranone antitumor activity in vivo, in a melanoma experimental model, were also shown. Jacaranone renders antiproliferative and proapoptotic responses in tumor cells, by acting on Akt and p38 MAPK signaling pathways through generation of reactive oxygen species (ROS). The free radical scavenger N-acetyl-cysteine (NAC) was able to completely suppress cell death induced by jacaranone as it blocked Akt downregulation, p38 MAPK activation as well as upregulation of proapoptotic Bax. Notably, treatment of melanoma growing subcutaneously in mice with jacaranone significantly extended the mean survival times in a dose-dependent manner. Conclusions/Significance The results provide evidence for the mechanisms of action of jacaranone and emphasize the potential use of this quinone for the treatment of melanoma.

Essential oils from Schinus terebinthifolius leaves – chemical composition and in vitro cytotoxicity evaluation

Context: In folk medicine, Schinus terebinthifolius Raddi (Anacardiaceae), has been used as a remedy for ulcers, respiratory problems, wounds, rheumatism, gout, diarrhea, skin ailments and arthritis, as well as to treat tumors and leprosy. Objective: To investigate the chemical composition and cytotoxicity of essential oil from leaves of S. terebinthifolius as well as the identification of active compounds from this oil. Material and methods: Essential oil from S. terebinthifolius leaves, obtained by hydrodistillation using a Clevenger-type apparatus, was characterized in terms of its chemical composition. Also, the crude oil was subjected to chromatographic separation procedures to afford an active fraction composed of α- and β-pinenes. These compounds, including hydrogenation (pinane) and epoxydation (α-pinene oxide) derivatives from α-pinene, were tested in vitro against murine melanoma cell line (B16F10-Nex2) and human melanoma (A2058), breast adenocarcinoma (MCF7), leukemia (human leukemia (HL-60) and cervical carcinoma (HeLa) cell lines. Results: Forty-nine constituents were identified in the oil (97.9% of the total), with germacrene D (23.7%), bicyclogermacrene (15.0%), β-pinene (9.1%) and β-longipinene (8.1%) as the main compounds. The crude essential oil showed cytotoxic effects in several cell lines, mainly on leukemia and human cervical carcinoma. Fractions composed mainly of α- and β-pinenes as well as those composed of individually pinenes showed effective activities against all tested cell lines. Aiming to determinate preliminary structure/activity relationships, α-pinene was subjected to epoxydation and hydrogenation procedures whose obtained α-pinene oxide showed an expressive depression in its cytotoxicity effect, similar as observed to pinane derivative. Discussion and conclusion: The obtained results indicated that the monoterpenes α- and β-pinenes could be responsible to the cytotoxic activity detected in the crude oil from leaves of S. terebinthifolius. In addition, it was possibly inferred that the presence of double bond in their structures, mainly at endocyclic position, is crucial to cytotoxic potential detected in these derivatives.

β-Actin-binding Complementarity-determining Region 2 of Variable Heavy Chain from Monoclonal Antibody C7 Induces Apoptosis in Several Human Tumor Cells and Is Protective against Metastatic Melanoma

Background: Immunoglobulin complementarity-determining region (CDR) peptides frequently display antitumor activities. Results: Monoclonal antibody C7 CDR-H2 binds to β-actin and induces polymerization, F-actin stabilization, and tumor cell death. Conclusion: Alterations of actin dynamics trigger reactive oxygen species and tumor cell apoptosis. Significance: The in vitro apoptotic effect and in vivo antimetastatic activity of peptide C7H2 makes it a candidate to be developed as an anticancer drug. Complementarity-determining regions (CDRs) from monoclonal antibodies tested as synthetic peptides display anti-infective and antitumor activities, independent of the specificity of the native antibody. Previously, we have shown that the synthetic peptide C7H2, based on the heavy chain CDR 2 from monoclonal antibody C7, a mAb directed to a mannoprotein of Candida albicans, significantly reduced B16F10 melanoma growth and lung colony formation by triggering tumor apoptosis. The mechanism, however, by which C7H2 induced apoptosis in tumor cells remained unknown. Here, we demonstrate that C7H2 interacts with components of the tumor cells cytoskeleton, being rapidly internalized after binding to the tumor cell surface. Mass spectrometry analysis and in vitro validation revealed that β-actin is the receptor of C7H2 in the tumor cells. C7H2 induces β-actin polymerization and F-actin stabilization, linked with abundant generation of superoxide anions and apoptosis. Major phenotypes following peptide binding were chromatin condensation, DNA fragmentation, annexin V binding, lamin disruption, caspase 8 and 3 activation, and organelle alterations. Finally, we evaluated the cytotoxic efficacy of C7H2 in a panel of human tumor cell lines. All tumor cell lines studied were equally susceptible to C7H2 in vitro. The C7H2 amide without further derivatization significantly reduced lung metastasis of mice endovenously challenged with B16F10-Nex2 melanoma cells. No significant cytotoxicity was observed toward nontumorigenic cell lines on short incubation in vitro or in naïve mice injected with a high dose of the peptide. We believe that C7H2 is a promising peptide to be developed as an anticancer drug.

Chemical composition and cytotoxicity evaluation of essential oil from leaves of Casearia sylvestris, its main compound α-zingiberene and derivatives.

Casearia sylvestris (Salicaceae), popularly known as “guaçatonga”, is a plant widely used in folk medicine to treat various diseases, including cancer. The present work deals with the chemical composition as well as the cytotoxic evaluation of its essential oil, its main constituent and derivatives. Thus, the crude essential oil from leaves of C. sylvestris was obtained using a Clevenger type apparatus and analyzed by GC/MS. This analysis afforded the identification of 23 substances, 13 of which corresponded to 98.73% of the total oil composition, with sesquiterpene α-zingiberene accounting for 50% of the oil. The essential oil was evaluated for cytotoxic activity against several tumor cell lines, giving IC50 values ranging from 12 to 153 μg/mL. Pure α-zingiberene, isolated from essential oil, was also evaluated against the tumor cell lines showing activity for HeLa, U-87, Siha and HL60 cell lines, but with IC50 values higher than those determined for the crude essential oil. Aiming to evaluate the effect of the double bonds of α-zingiberene on the cytotoxic activity, partially hydrogenated α-zingiberene (PHZ) and fully hydrogenated α-zingiberene (THZ) derivatives were obtained. For the partially hydrogenated derivative only cytotoxic activity to the B16F10-Nex2 cell line (IC50 65μg/mL) was detected, while totally hydrogenated derivative showed cytotoxic activity for almost all cell lines, with B16F10-Nex2 and MCF-7 as exceptions and with IC50 values ranging from 34 to 65 μg/mL. These results indicate that cytotoxic activity is related with the state of oxidation of compound.

Mastoparan induces apoptosis in B16F10-Nex2 melanoma cells via the intrinsic mitochondrial pathway and displays antitumor activity in vivo

Mastoparan is an α-helical and amphipathic tetradecapeptide obtained from the venom of the wasp Vespula lewisii. This peptide exhibits a wide variety of biological effects, including antimicrobial activity, increased histamine release from mast cells, induction of a potent mitochondrial permeability transition and tumor cell cytotoxicity. Here, the effects of mastoparan in malignant melanoma were studied using the murine model of B16F10-Nex2 cells. In vitro, mastoparan caused melanoma cell death by the mitochondrial apoptosis pathway, as evidenced by the Annexin V-FITC/PI assay, loss of mitochondrial membrane potential (ΔΨm), generation of reactive oxygen species, DNA degradation and cell death signaling. Most importantly, mastoparan reduced the growth of subcutaneous melanoma in syngeneic mice and increased their survival. The present results show that mastoparan induced caspase-dependent apoptosis in melanoma cells through the intrinsic mitochondrial pathway protecting the mice against tumor development.

A New Phage-Display Tumor-Homing Peptide Fused to Antiangiogenic Peptide Generates a Novel Bioactive Molecule with Antimelanoma Activity

Phage-display peptide libraries have been widely used to identify specific peptides targeting in vivo tumor cells and the tumor vasculature and playing an important role in the discovery of antitumor bioactive peptides. In the present work, we identified a new melanoma-homing peptide, (-CVNHPAFAC-), using a C7C phage-display library directed to the developing tumor in syngeneic mice. Phage were able to preferentially target melanoma in vivo, with an affinity about 50-fold greater than that with normal tissue, and the respective synthesized peptide displaced the corresponding phage from the tumor. A preferential binding to endothelial cells rather than to melanoma cells was seen in cell ELISA, suggesting that the peptide is directed to the melanoma vasculature. Furthermore, the peptide was able to bind to human sonic hedgehog, a protein involved in the development of many types of human cancers. Using a new peptide approach therapy, we coupled the cyclic peptide to another peptide, HTMYYHHYQHHL-NH2, a known antagonist of VEGFR-2 receptor, using the GYG linker. The full peptide CVNHPAFACGYGHTMYYHHYQHHL-NH2 was effective in delaying tumor growth (P < 0.05) and increasing animal survival when injected systemically, whereas a scramble-homing peptide containing the same antagonist did not have any effect. This is the first report on the synthesis of a tumor-homing peptide coupled to antiangiogenic peptide as a new anticancer therapeutics. Mol Cancer Res; 9(11); 1471–8. ©2011 AACR.

Chemical composition and in vitro cytotoxic effects of the essential oil from Nectandra leucantha leaves

Abstract Context: Nectandra (Lauraceae) species have been used in folk medicine as an antidiarrheal, analgesic, antifungal, etc., and have many pharmacological proprieties. Objective: Investigation of the chemical composition and cytotoxicity of essential oil from Nectandra leucantha Nees & Mart. leaves. This is the first study involving N. leucantha reported in the literature. Material and methods: The essential oil of N. leucantha leaves was obtained by hydrodistillation. Its chemical composition was determined using a combination of GC/FID, GC/MS, and determination of Kovats index (KI). In vitro cytotoxic activity was evaluated against six cancer cell lines – murine melanoma (B16F10-Nex2), human glioblastome (U-87), human cervical carcinoma (HeLa), human colon carcinoma (HCT), human breast adenocarcinoma (MCF7), and human cervical tumor (Siha) as well as against one non-tumorigenic cell line – human foreskin fibroblast (HFF). Results: Thirty-three compounds were identified primarily sesquiterpenes (81.41%), the main compounds being bicyclogermacrene (28.44%), germacrene A (7.34%), spathulenol (5.82%), and globulol (5.25%). Furthermore, monoterpenes were also found in the analyzed oil (12.84%), predominantly α- and β-pinenes (6.59 and 4.57%, respectively). The crude essential oil displayed significant cytotoxic activity against B16F10-Nex2 (IC50 33 ± 1 μg/mL) and U87 (IC50 75.95 ± 0.03 μg/mL) and HeLa (IC50 60 ± 12 μg/mL) cell lines. The main identified compound, bicyclogermacrene, displayed IC50 ranging from 3.1 ± 0.2 to 21 ± 6 μg/mL. Discussion and conclusion: The results indicate that the crude oils from leaves of N. leucantha displayed cytotoxic activity being bicyclogermacrene, the main compound identified in the crude oil responsible, at least in part, for this potential.

Cytotoxic effects of dillapiole on MDA-MB-231 cells involve the induction of apoptosis through the mitochondrial pathway by inducing an oxidative stress while altering the cytoskeleton network.

Breast cancer is the worlds leading cause of death among women. This situation imposes an urgent development of more selective and less toxic agents. The use of natural molecular fingerprints as sources for new bioactive chemical entities has proven to be a quite promising and efficient method. Here, we have demonstrated for the first time that dillapiole has broad cytotoxic effects against a variety tumor cells. For instance, we found that it can act as a pro-oxidant compound through the induction of reactive oxygen species (ROS) release in MDA-MB-231 cells. We also demonstrated that dillapiole exhibits anti-proliferative properties, arresting cells at the G0/G1 phase and its antimigration effects can be associated with the disruption of actin filaments, which in turn can prevent tumor cell proliferation. Molecular modeling studies corroborated the biological findings and suggested that dillapiole may present a good pharmacokinetic profile, mainly because its hydrophobic character, which can facilitate its diffusion through tumor cell membranes. All these findings support the fact that dillapiole is a promising anticancer agent.

Anti-tumor activities of peptides corresponding to conserved complementary determining regions from different immunoglobulins.

Short synthetic peptides corresponding to sequences of complementarity-determining regions (CDRs) from different immunoglobulin families have been shown to induce antimicrobial, antiviral and antitumor activities regardless of the specificity of the original monoclonal antibody (mAb). Presently, we studied the in vitro and in vivo antitumor activity of synthetic peptides derived from conserved CDR sequences of different immunoglobulins against human tumor cell lines and murine B16F10-Nex2 melanoma aiming at the discovery of candidate molecules for cancer therapy. Four light- and heavy-chain CDR peptide sequences from different antibodies (C36-L1, HA9-H2, 1-H2 and Mg16-H2) showed cytotoxic activity against murine melanoma and a panel of human tumor cell lineages in vitro. Importantly, they also exerted anti-metastatic activity using a syngeneic melanoma model in mice. Other peptides (D07-H3, MN20v1, MS2-H3) were also protective against metastatic melanoma, without showing significant cytotoxicity against tumor cells in vitro. In this case, we suggest that these peptides may act as immune adjuvants in vivo. As observed, peptides induced nitric oxide production in bone-marrow macrophages showing that innate immune cells can also be modulated by these CDR peptides. The present screening supports the search in immunoglobulins of rather frequent CDR sequences that are endowed with specific antitumor properties and may be candidates to be developed as anti-cancer drugs.