Natalia Girola

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.

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.

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.

A novel microtubule de-stabilizing complementarity-determining region C36L1 peptide displays antitumor activity against melanoma in vitro and in vivo

Short peptide sequences from complementarity-determining regions (CDRs) of different immunoglobulins may exert anti-infective, immunomodulatory and antitumor activities regardless of the specificity of the original monoclonal antibody (mAb). In this sense, they resemble early molecules of innate immunity. C36L1 was identified as a bioactive light-chain CDR1 peptide by screening 19 conserved CDR sequences targeting murine B16F10-Nex2 melanoma. The 17-amino acid peptide is readily taken up by melanoma cells and acts on microtubules causing depolymerization, stress of the endoplasmic reticulum and intrinsic apoptosis. At low concentrations, C36L1 inhibited migration, invasion and proliferation of B16F10-Nex2 cells with cell cycle arrest at G2/M phase, by regulating the PI3K/Akt signaling axis involving Rho-GTPase and PTEN mediation. Peritumor injection of the peptide delayed growth of subcutaneously grafted melanoma cells. Intraperitoneal administration of C36L1 induced a significant immune-response dependent anti-tumor protection in a syngeneic metastatic melanoma model. Dendritic cells stimulated ex-vivo by the peptide and transferred to animals challenged with tumor cells were equally effective. The C36 VL CDR1 peptide is a promising microtubule-interacting drug that induces tumor cell death by apoptosis and inhibits metastases of highly aggressive melanoma cells.

A novel cell‐penetrating peptide derived from WT1 enhances p53 activity, induces cell senescence and displays antimelanoma activity in xeno‐ and syngeneic systems

The Wilms tumor protein 1 (WT1) transcription factor has been associated in malignant melanoma with cell survival and metastasis, thus emerging as a candidate for targeted therapy. A lysine–arginine rich peptide, WT1‐pTj, derived from the ZF domain of WT1 was evaluated as an antitumor agent against A2058 human melanoma cells and B16F10‐Nex2 syngeneic murine melanoma. Peptide WT1‐pTj quickly penetrated human melanoma cells and induced senescence, recognized by increased SA‐β‐galactosidase activity, enhanced transcriptional activity of p53, and induction of the cell cycle inhibitors p21 and p27. Moreover, the peptide bound to p53 and competed with WT1 protein for binding to p53. WT1‐pTj treatment led to sustained cell growth suppression, abrogation of clonogenicity and G2/M cell cycle arrest. Notably, in vivo studies showed that WT1‐pTj inhibited both the metastases and subcutaneous growth of murine melanoma in syngeneic mice, and prolonged the survival of nude mice challenged with human melanoma cells. The 27‐amino acid cell‐penetrating WT1‐derived peptide, depends on C3 and H16 for effective antimelanoma activity, inhibits proliferation of WT1‐expressing human tumor cell lines, and may have an effective role in the treatment of WT1‐expressing malignancies.

Melanoma: Perspectives of a Vaccine Based on Peptides

Anti-tumor peptides have been identified in internal sequences of immunoglobulins, mainly the complementarity determining regions (CDRs), and also as sequences of signaling proteins. The in vitro effects of these peptides against several tumor cell lines involved different receptors and mechanisms mainly affecting the cell invasion, proliferation, cell cycle and apoptosis. Peptides also exerted in vivo protection using a melanoma subcutaneous and metastatic model, but this effect depended on the immune response. More specifically, peptides activated dendritic cells (DCs) and these cells exerted a potent anti-tumor effect. Experiments with DCs activated ex-vivo and transferred to tumor-challenged animals showed a protection identical to that of peptides alone suggesting that the latter activate DCs in vivo. A vaccine based on peptides is encouraged by the protection exerted by a signaling protein-derived peptide that caused arrest of a 200-mm3 growing subcutaneous melanoma, and by Ig-CDR readily synthesized, purified peptides that displayed anti-metastatic melanoma activities.

Benzofuroxan derivatives N-Br and N-I induce intrinsic apoptosis in melanoma cells by regulating AKT/BIM signaling and display anti metastatic activity in vivo

BackgroundMalignant melanoma is an aggressive type of skin cancer, and despite recent advances in treatment, the survival rate of the metastatic form remains low. Nifuroxazide analogues are drugs based on the substitution of the nitrofuran group by benzofuroxan, in view of the pharmacophore similarity of the nitro group, improving bioavailability, with higher intrinsic activity and less toxicity. Benzofuroxan activity involves the intracellular production of free-radical species. In the present work, we evaluated the antitumor effects of different benzofuroxan derivatives in a murine melanoma model.MethodsB16F10-Nex2 melanoma cells were used to investigate the antitumor effects of Benzofuroxan derivatives in vitro and in a syngeneic melanoma model in C57Bl/6 mice. Cytotoxicity, morphological changes and reactive oxygen species (ROS) were assessed by a diphenyltetrasolium reagent, optical and fluorescence microscopy, respectively. Annexin-V binding and mitochondrial integrity were analyzed by flow cytometry. Western blotting and colorimetry identified cell signaling proteins.ResultsBenzofuroxan N-Br and N-I derivatives were active against murine and human tumor cell lines, exerting significant protection against metastatic melanoma in a syngeneic model. N-Br and N-I induce apoptosis in melanoma cells, evidenced by specific morphological changes, DNA condensation and degradation, and phosphatidylserine translocation in the plasma membrane. The intrinsic mitochondrial pathway in B16F10-Nex2 cells is suggested owing to reduced outer membrane potential in mitochondria, followed by caspase −9, −3 activation and cleavage of PARP. The cytotoxicity of N-Br and N-I in B16F10-Nex2 cells is mediated by the generation of ROS, inhibited by pre-incubation of the cells with N-acetylcysteine (NAC). The induction of ROS by N-Br and N-I resulted in the inhibition of AKT activation, an important molecule related to tumor cell survival, followed by upregulation of BIM.ConclusionWe conclude that N-Br and N-I are promising agents aiming at cancer treatment. They may be useful in melanoma therapy as inducers of intrinsic apoptosis and by exerting significant antitumor activity against metastatic melanoma, as presently shown in syngeneic mice.

Pyrostegia venusta heptane extract containing saturated aliphatic hydrocarbons induces apoptosis on B16F10-Nex2 melanoma cells and displays antitumor activity in vivo

Background: Pyrostegia venusta (Ker. Gawl.) Miers (Bignoniacea) is a medicinal plant from the Brazilian Cerrado used to treat leucoderma and common diseases of the respiratory system. Objective: To investigate the antitumor activity of P.venusta extracts against melanoma. Materials and Methods: The cytotoxic activity and tumor induced cell death of heptane extract (HE) from P. venusta flowers was evaluated against murine melanoma B16F10-Nex2 cells in vitro and in a syngeneic model in vivo. Results: We found that HE induced apoptosis in melanoma cells by disruption of the mitochondrial membrane potential, induction of reactive oxygen species and late apoptosis evidenced by plasma membrane blebbing, cell shrinkage, chromatin condensation and DNA fragmentation, exposure of phosphatidylserine on the cell surface and activation of caspase-2,-3,-8,-9. HE was also protective against singeneyc subcutaneous melanoma HE compounds were also able to induce cell cycle arrest at G2/M phases on tumor cells. On fractionation of HE in silica gel we isolated a cytotoxic fraction that contained a mixture of saturated hydrocarbons identified by 1H NMR and GC-MS analyses. Predominant species were octacosane (C28H58-36%) and triacontane (C30H62-13%), which individually showed significant cytotoxic activity against murine melanoma B16F10-Nex2 cells in vitro and a very promising antitumor protection against subcutaneous melanoma in vivo. Conclusion: The results suggest that the components of the heptane extract, mainly octasane and triacontane, which showed antitumor properties in experimental melanoma upon regional administration, might also be therapeutic in human cancer, such as in the mostly epidermal and slowly invasive melanomas, such as acral lentiginous melanoma, as an adjuvant treatment to surgical excision.

AC-1001 H3 CDR peptide induces apoptosis and signs of autophagy in vitro and exhibits antimetastatic activity in a syngeneic melanoma model

Antibody‐derived peptides modulate functions of the immune system and are a source of anti‐infective and antitumor substances. Recent studies have shown that they comprise amino acid sequences of immunoglobulin complementarity‐determining regions, but also fragments of constant regions. VH CDR3 of murine mAb AC‐1001 displays antimetastatic activities using B16F10‐Nex2 murine melanoma cells in a syngeneic model. The peptide was cytotoxic in vitro in murine and human melanoma cells inducing reactive oxygen species (ROS) and apoptosis by the intrinsic pathway. Signs of autophagy were also suggested by the increased expression of LC3/LC3II and Beclin 1 and by ultrastructural evidence. AC‐1001 H3 bound to both G‐ and F‐actin and inhibited tumor cell migration. These results are important evidence of the antitumor activity of Ig CDR‐derived peptides.

Circadian variation and in vitro cytotoxic activity evaluation of volatile compounds from leaves of Piper regnellii (Miq) C. DC. var. regnellii (C. DC.) Yunck (Piperaceae)

Abstract Aiming detection of circadian variation in the chemical composition of volatiles from Piper regnellii, the leaves were collected during four different periods (8, 12, 16 and 20 h) in the same day. After extraction by hydrodistillation and GC/MS analysis, no significant variation was observed for the main compounds: germacrene D (45.6 ± 1.5–51.4 ± 3.1%), α–chamigrene (8.9 ± 1.3–11.3 ± 2.7%) and β–caryophyllene (8.2 ± 0.9–9.5 ± 0.3%). Evaluation of in vitro cytotoxicity against several cancer and non-tumourigenic cells indicated promising activity, especially to HeLa (human cervical carcinoma) with IC50 ranging from 11 ± 3 to 17 ± 3 μg/mL. The obtained volatile oils were pooled and subjected to fractionation to afford pure β-caryophyllene, α-chamigrene and germacrene D, being this last compound the more active against HeLa cells with IC50 of 7 ± 1 μg/mL (34 ± 5 μM). Therefore, the predominance of germacrene D in all analysed oils could justify, at least in part, the activity observed for the volatile compounds from P. regnellii leaves.