title: Piper nigrum ethanolic extract rich in piperamides causes ROS overproduction, oxidative damage in DNA leading to cell cycle arrest and apoptosis in cancer cells.
abstract: BACKGROUND
  Ayurvedic and Chinese traditional medicine and tribal people use herbal preparations containing Piper nigrum fruits for the treatment of many health disorders like inflammation, fever, asthma and cancer. In Brazil, traditional maroon culture associates the spice Piper nigrum to health recovery and inflammation attenuation.
  
  
  OBJECTIVE
  The aim of the current work was to evaluate the relationship between reactive oxygen species (ROS) overproduction, DNA fragmentation, cell cycle arrest and apoptosis induced by Piper nigrum ethanolic extract and its antitumor activity.
  
  
  METHODS
  The plant was macerated in ethanol. Extract constitution was assessed by TLC, UV-vis and ESI-IT-MS/MS spectrometry. The cytotoxicity, proliferation and intracellular ROS generation was evaluated in MCF-7 cells. DNA damage effects were evaluated through intercalation into CT-DNA, plasmid DNA cleavage and oxidative damage in CT-DNA. Tumor growth inhibition, survival time increase, apoptosis, cell cycle arrest and oxidative stress were assessed in Ehrlich ascites carcinoma-bearing mice.
  
  
  RESULTS
  Extraction yielded 64mg/g (36% piperine and 4.2% piperyline). Treatments caused DNA damage and reduced cell viability (EC50=27.1±2.0 and 80.5±6.6µg/ml in MCF-7 and HT-29 cells, respectively), inhibiting cell proliferation by 57% and increased ROS generation in MCF-7 cells (65%). Ehrlich carcinoma was inhibited by the extract, which caused reduction of tumor growth (60%), elevated survival time (76%), cell cycle arrest and induced apoptosis. The treatment with extract increased Bax and p53 and inhibited Bcl-xL and cyclin A expression. It also induced an oxidative stress in vivo verified as enhanced lipid peroxidation and carbonyl proteins content and increased activities of glutathione reductase, superoxide dismutase and catalase. GSH concentration was decreased in tumor tissue from mice.
  
  
  CONCLUSIONS
  The ethanolic extract has cytotoxic and antiproliferative effect on MCF-7 cells and antitumor effect in vivo probably due to ROS overproduction that induced oxidative stress affecting key proteins involved in cell cycle arrest at G1/S and triggering apoptosis. Finally, the overall data from this study are well in line with the traditional claims for the antitumor effect of Piper nigrum fruits.
authors.affiliation: Laboratório de Bioquímica Experimental, Departamento de Bioquímica, Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil.
authors.forename: Valdelúcia Maria Alves
authors.initials: VM
authors.lastname: de Souza Grinevicius
authors.affiliation: Programa de Pós-Graduação em Ciências da Saúde da Universidade do Sul de Santa Catarina (UNISUL), SC, Brazil.
authors.forename: Maicon Roberto
authors.initials: MR
authors.lastname: Kviecinski
authors.affiliation: Laboratório de Bioquímica Experimental, Departamento de Bioquímica, Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil.
authors.forename: Nádia Sandrini Ramos
authors.initials: NS
authors.lastname: Santos Mota
authors.affiliation: Laboratório de Bioquímica Experimental, Departamento de Bioquímica, Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil.
authors.forename: Fabiana
authors.initials: F
authors.lastname: Ourique
authors.affiliation: Laboratório de Bioquímica Experimental, Departamento de Bioquímica, Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil.
authors.forename: Luiza Sheyla Evenni
authors.initials: LS
authors.lastname: Porfirio Will Castro
authors.affiliation: Laboratório de Bioquímica Experimental, Departamento de Bioquímica, Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil.
authors.forename: Rafaela Rafognato
authors.initials: RR
authors.lastname: Andreguetti
authors.affiliation: Laboratório de Bioquímica Experimental, Departamento de Bioquímica, Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil.
authors.forename: João Francisco
authors.initials: JF
authors.lastname: Gomes Correia
authors.affiliation: Departamento de Ecologia e Zoologia,Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil.
authors.forename: Danilo Wilhem
authors.initials: DW
authors.lastname: Filho
authors.affiliation: NITBIO, Universidade Federal de Santa Catarina, Araranguá, SC, Brazil.
authors.forename: Claus Tröger
authors.initials: CT
authors.lastname: Pich
authors.affiliation: Laboratório de Bioquímica Experimental, Departamento de Bioquímica, Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil. Electronic address: rozangela.pedrosa@ufsc.br.
authors.forename: Rozangela Curi
authors.initials: RC
authors.lastname: Pedrosa
mesh_terms: D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000302:isolation & purification / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000378:metabolism; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002286:Carcinoma, Ehrlich Tumor / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D059447:Cell Cycle Checkpoints / Q000187:drug effects*; D018797:Cell Cycle Proteins / Q000378:metabolism; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D000431:Ethanol / Q000737:chemistry*; D005260:Female; D019073:HT29 Cells; D006801:Humans; D015227:Lipid Peroxidation / Q000187:drug effects; D061986:MCF-7 Cells; D008297:Male; D008807:Mice, Inbred BALB C; D016877:Oxidants / Q000302:isolation & purification / Q000494:pharmacology*; D018384:Oxidative Stress / Q000187:drug effects*; D008517:Phytotherapy; D029222:Piper nigrum / Q000737:chemistry*; D010880:Piperidines / Q000302:isolation & purification / Q000494:pharmacology*; D010936:Plant Extracts / Q000302:isolation & purification / Q000494:pharmacology*; D010946:Plants, Medicinal; D050050:Protein Carbonylation / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012997:Solvents / Q000737:chemistry*; D013997:Time Factors; D047368:Tumor Burden / Q000187:drug effects; D015854:Up-Regulation
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D051017:Apoptosis Regulatory Proteins; D014408:Biomarkers, Tumor; D018797:Cell Cycle Proteins; D016877:Oxidants; D010880:Piperidines; D010936:Plant Extracts; D017382:Reactive Oxygen Species; D012997:Solvents; D000431:Ethanol
keywords: Antitumor activity; Apoptosis; Cell cycle arrest; DNA damage; Doxorubicin (PubChem CID: 31703); Piper nigrum; Piperine (PubChem CID: 638024); Piperyline (PubChem CID: 636537); ROS overproduction