Sulforaphane Combined with Vitamin D Induces Cytotoxicity Mediated by Oxidative Stress, DNA Damage, Autophagy, and JNK/MAPK Pathway Modulation in Human Prostate Tumor Cells.

Prostate cancer ranks second in incidence worldwide. To date, there are no available therapies to effectively treat advanced and metastatic prostate cancer. Sulforaphane and vitamin D alone are promising anticancer agents in vitro and in vivo, but their low bioavailability has limited their effects in clinical trials. The present study examined whether sulforaphane combined with vitamin D at clinically relevant concentrations improved the cytotoxicity of the compounds alone towards DU145 and PC-3 human prostate tumor cells. To assess the anticancer activity of this combination, we analyzed cell viability (MTT assay), oxidative stress (CM-H2DCFDA), autophagy (fluorescence), DNA damage (comet assay), and protein expression (Western blot). The sulforaphane-vitamin D combination (i) decreased cell viability, induced oxidative stress, DNA damage, and autophagy, upregulated BAX, CASP8, CASP3, JNK, and NRF2 expression, and downregulated BCL2 expression in DU145 cells; and (ii) decreased cell viability, increased autophagy and oxidative stress, upregulated BAX and NRF2 expression, and downregulated JNK, CASP8, and BCL2 expression in PC-3 cells. Therefore, sulforaphane and vitamin D in combination have a potential application in prostate cancer therapy, and act to modulate the JNK/MAPK signaling pathway.

Diallyl Disulfide Induces Chemosensitization to Sorafenib, Autophagy, and Cell Cycle Arrest and Inhibits Invasion in Hepatocellular Carcinoma.

Hepatocellular carcinoma is the seventh most common type of cancer in the world, with limited treatment options. A promising strategy to treat cancer is to associate chemotherapeutics and plant bioactive compounds. Here, we examined whether diallyl disulfide (DADS; 50-200 µM) and sorafenib (SORA; 8 µM), either alone or in combination, were toxic to hepatocellular carcinoma cells (HepG2) in vitro. We assessed whether DADS and/or SORA induced cell death (LIVE/DEAD assay and autophagy) and cell cycle changes (flow cytometry), altered expression of key genes and proteins (RT-qPCR and Western blot), and modulated tumorigenesis signatures, such as proliferation (clonogenic assay), migration (wound healing), and invasion (inserts). The DADS + SORA combination elicited autophagic cell death by upregulating LC3 and NRF2 expression and downregulating FOS and TNF expression; induced the accumulation of cells in the G1 phase which thereby upregulated the CHEK2 expression; and inhibited invasion by downregulating the MMP2 expression. Predictive analysis indicated the participation of the MAPK pathway in the reported results. The DADS + SORA combination suppressed both cell invasion and clonogenic survival, which indicated that it dampened tumor growth, proliferation, invasion, and metastatic potential. Therefore, the DADS + SORA combination is a promising therapy to develop new clinical protocols.

Synephrine and caffeine combination promotes cytotoxicity, DNA damage and transcriptional modulation of apoptosis-related genes in human HepG2 cells.

The abusive consumption of thermogenic supplements occurs worldwide and deserves special attention due to their use to stimulate weight loss and prevent obesity. Thermogenic formulations usually contain Synephrine (SN) and Caffeine (CAF), stimulating compounds extracted from natural sources, but no genetic toxicology studies have predicted this hazardous combination potential. This study examined the toxicogenomic responses induced by SN and CAF, either alone or in combination, in the human hepatic cell line HepG2 in vitro. SN (0.03-30 µM) and CAF (0.6-600 µM) alone did neither decrease cell viability nor induce DNA damage, as assessed using the MTT and comet assays, respectively. SN (3 µM) and CAF (30-600 µM) were combined at concentrations similar to those found in commercial dietary supplements. SN/CAF at 3:90 and 3:600 µM ratios significantly decreased cell viability and increased DNA damage levels in HepG2 cells. CAF (600 µM) and the SN/CAF association at 3:60, 3:90, and 3:600 µM ratios promoted cell death by apoptosis, as demonstrated by flow cytometry. Similar results were observed in gene expression (RT-qPCR): SN/CAF up-regulated the expression of apoptosis- (BCL-2 and CASP9) and DNA repair-related (XPC) genes. SN/CAF at 3:90 µM also downregulated the expression of cell cycle control (CDKN1A) genes. In conclusion, the SN/CAF combination reduces cell viability by inducing apoptosis, damages DNA, and modulates the transcriptional expression of apoptosis-, cell cycle-, and DNA repair-related genes in human hepatic (HepG2) cells in vitro. These effects can be worrisome to consumers of thermogenic supplements.

p-synephrine induces transcriptional changes via the cAMP/PKA pathway but not cytotoxicity or mutagenicity in human gastrointestinal cells.

p-Synephrine (SN) is an alkaloid added to thermogenic formulations for weight loss that is predominantly absorbed in the human gastrointestinal tract (GI). As the adverse effects of SN on GI cells remain unclear, the aim of present study was to examine whether SN affected cell viability, cell cycle kinetics, genomic stability, redox status, and expression of cAMP/PKA pathway genes related to metabolism/energy homeostasis in stomach mucosa (MNP01) and colon adenocarcinoma (Caco-2) human cells. p-Synephrine at 25-5000 µM was not cytotoxic to both cell lines. At 2-200 µM, SN increased the formation of reactive oxygen species (ROS) but also enhanced levels of antioxidant defense molecules glutathione (GSH) and catalase (CAT) activity, which may account for the absence of cytotoxicity/mutagenicity in both cell lines. SN induced expression of the cAMP/PKA pathway genes ADCY3 and MAPK1 in MNP01 cells and MAPK1, GNAS, PRKACA, and PRKAR2A in Caco-2 cells, as well as modulated the transcription of genes related to cell proliferation (JUN; AKT1) and inflammation (RELA; TNF) in both cell lines. Therefore, the improved antioxidant state mitigated pro-oxidative effects attributed to SN. Evidence indicates that SN does not appear to exhibit adverse potential but modulated the cAMP/PKA pathway in human GI cell lines.

Bothrops moojeni L-amino acid oxidase induces apoptosis and epigenetic modulation on Bcr-Abl+ cells.

BACKGROUND: Resistance to apoptosis in chronic myeloid leukemia (CML) is associated with constitutive tyrosine kinase activity of the Bcr-Abl oncoprotein. The deregulated expression of apoptosis-related genes and alteration in epigenetic machinery may also contribute to apoptosis resistance in CML. Tyrosine kinase inhibitors target the Bcr-Abl oncoprotein and are used in CML treatment. The resistance of CML patients to tyrosine kinase inhibitors has guided the search for new compounds that may induce apoptosis in Bcr-Abl+ leukemic cells and improve the disease treatment. METHODS: In the present study, we investigated whether the L-amino acid oxidase isolated from Bothrops moojeni snake venom (BmooLAAO-I) (i) was cytotoxic to Bcr-Abl+ cell lines (HL-60.Bcr-Abl, K562-S, and K562-R), HL-60 (acute promyelocytic leukemia) cells, the non-tumor cell line HEK-293, and peripheral blood mononuclear cells (PBMC); and (ii) affected epigenetic mechanisms, including DNA methylation and microRNAs expression in vitro. RESULTS: BmooLAAO-I induced ROS production, apoptosis, and differential DNA methylation pattern of regulatory apoptosis genes. The toxin upregulated expression of the pro-apoptotic genes BID and FADD and downregulated DFFA expression in leukemic cell lines, as well as increased miR-16 expression - whose major predicted target is the anti-apoptotic gene BCL2 - in Bcr-Abl+ cells. CONCLUSION: BmooLAAO-I exerts selective antitumor action mediated by H2O2 release and induces apoptosis, and alterations in epigenetic mechanisms. These results support future investigations on the effect of BmooLAAO-I on in vivo models to determine its potential in CML therapy.

Caffeic acid and chlorogenic acid cytotoxicity, genotoxicity and impact on global DNA methylation in human leukemic cell lines.

Dietary phenolic compounds such as caffeic and chlorogenic acid exert an antiproliferative effect and modulate the gene-specific DNA methylation status in human breast tumor cells, but it remains unclear whether they interfere with global DNA methylation in human leukemia cells. We examined whether caffeic and chlorogenic acid (1-250 µM) exert antitumor action in human promyelocytic leukemia cells (HL-60) and human acute T-cell leukemia cells (Jurkat). Caffeic and chlorogenic acid did not reduce cell viability in the two cell lines, as assessed using the neutral red uptake and MTT assays. These phenolic acids (1-100 µM) neither induced DNA damage (comet assay) nor increased the micronuclei frequency (micronucleus assay) in HL-60 and Jurkat cells, indicating that they were not genotoxic or mutagenic. Analysis of global DNA methylation levels using a 5-mC DNA ELISA kit revealed that chlorogenic acid at a non-cytotoxic concentration (100 µM) induced global DNA hypomethylation in Jurkat cells, but not in HL-60 cells, suggesting that it exerts a cell-specific effect. Caffeic acid did not change global DNA methylation. As other phenolic compounds, chlorogenic acid probably modulates DNA methylation by targeting DNA methyltransferases. The hypomethylating action of chlorogenic acid can be beneficial against hematological malignances whose pathogenic processes involve impairment of DNA methylation.

Effects of sulforaphane on the oxidative response, apoptosis, and the transcriptional profile of human stomach mucosa cells in vitro.

Oxidative stress is a critical factor in the pathogenesis of several gastrointestinal diseases. Sulforaphane (SFN), a bioactive compound found in cruciferous vegetables, activates the redox-sensitive nuclear erythroid 2-related factor 2 (NRF2). In addition to its protective role, SFN exerts cytotoxic effects on cancer cells. However, there is a lack of information concerning the toxicity of SFN in normal cells. We investigated the effects of SFN on cell viability, antioxidant defenses, and gene expression in human stomach mucosa cells (MNP01). SFN reduced ROS formation and protected the cells against induced oxidative stress but high concentrations increased apoptosis. An intermediate SFN concentration (8 µM) was chosen for RNA sequencing studies. We observed upregulation of genes of the NRF2 (antioxidant) pathway, the DNA damage response, and apoptosis signaling; whereas SFN downregulated cell cycle and DNA repair pathway genes. SFN may be cytoprotective at low concentrations and cytotoxic at high concentrations.

Phospholipids modifications in human hepatoma cell lines (HepG2) exposed to silver and iron oxide nanoparticles.

Metallic nanoparticles such as silver (Ag NPs) and iron oxide (Fe3O4 NPs) nanoparticles are high production volume materials due to their applications in various consumer products, and in nanomedicine. However, their inherent toxicities to human cells remain a challenge. The present study was aimed at combining lipidomics data with common phenotypically-based toxicological assays to gain better understanding into cellular response to Ag NPs and Fe3O4 NPs exposure. HepG2 cells were exposed to different concentrations (3.125, 6.25, 12.5, 25, 50 and 100 µg/ml) of the nanoparticles for 24 h, after which they were assayed for toxic effects using toxicological assays like cytotoxicity, mutagenicity, apoptosis and oxidative stress. The cell membrane phospholipid profile of the cells was also performed using shotgun tandem mass spectrometry. The results showed that nanoparticles exposure resulted in concentration-dependent cytotoxicity as well as reduced cytokinesis-block proliferation index (CBPI). Also, there was an increase in the production of ROS and superoxide anions in exposed cells compared to the negative control. The lipidomics data revealed that nanoparticles exposure caused a modulation of the phospholipidome of the cells. A total of 155 lipid species were identified, out of which the fold changes of 23 were significant. The high number of differentially changed phosphatidylcholine species could be an indication that inflammation is one of the major mechanisms of toxicity of the nanoparticles to the cells.

Cytotoxicity, mutagenicity, oxidative stress and mitochondrial impairment in human hepatoma (HepG2) cells exposed to copper oxide, copper-iron oxide and carbon nanoparticles.

The increasing application of nanomaterials in various fields such as drug delivery, cosmetics, disease detection, cancer treatment, food preservation etc. has resulted in high levels of engineered nanoparticles in the environment, thus leading to higher possibility of direct or indirect interactions between these particles and biological systems. In this study, the toxic effects of three commercially available nanomaterials; copper oxide nanoparticles, copper-iron oxide nanopowders and carbon nanopowders were determined in the human hepatoma HepG2 cells using various toxicological assays which are indicative of cytotoxicity (MTT and neutral red assays), mutagenicity (cytokinesis-block micronucleus assay), oxidative stress (total reactive oxygen species and superoxide anion production) and mitochondrial impairment (cellular oxygen consumption). There was increased cytotoxicity, mutagenicity, and mitochondrial impairment in the cells treated with higher concentrations of the nanomaterials, especially the copper oxide nanoparticles. The fold production of reactive oxygen species was similar at the concentrations tested in this study but longer exposure duration resulted in production of more superoxide anions. The results of this study showed that copper oxide nanoparticles are highly toxic to the human HepG2 cells, thus implying that the liver is a target organ in human for copper oxide nanoparticles toxicity.

Transcriptome and DNA methylation changes modulated by sulforaphane induce cell cycle arrest, apoptosis, DNA damage, and suppression of proliferation in human liver cancer cells.

Abnormal epigenetic alterations are one of the keystones of cancer development. Epigenetic targeting drugs have become a promising and effective cancer therapy strategy. However, due to the high toxicity and unclear mechanisms of action of these drugs, natural compounds that cause epigenetic modulation have also been studied. Sulforaphane (SFN) is a promising bioactive compound for epigenetic targeting therapy. In this study, we investigate the effects of SFN on gene expression and DNA methylation in human hepatocellular carcinoma cells (HepG2). Using high throughput technologies in combination with cell-based assays, we find SFN is a potent anticancer agent, as it induces DNA damage, mitotic spindle abnormalities followed by apoptosis and proliferation inhibition in HepG2 cells. Our results show the upregulation of DNA damage response and cell cycle checkpoint genes. Also, we find the downregulation of cellular pathways frequently overexpressed in human cancer. As expected, SFN exerts epigenetic modulation effects by inhibiting histone deacetylases (HDACs). SFN might affect the activity of oncogenic transcription factors through methylation of its binding sites motifs. Our findings offer insights into SFN chemopreventive molecular effects in HepG2 cells and highlight SFN as a valuable natural approach to cancer therapy for future investigation.

Bothrops moojeni L-amino acid oxidase induces apoptosis and epigenetic modulation on Bcr-Abl+ cells

Resistance to apoptosis in chronic myeloid leukemia (CML) is associated with constitutive tyrosine kinase activity of the Bcr-Abl oncoprotein. The deregulated expression of apoptosis-related genes and alteration in epigenetic machinery may also contribute to apoptosis resistance in CML. Tyrosine kinase inhibitors target the Bcr-Abl oncoprotein and are used in CML treatment. The resistance of CML patients to tyrosine kinase inhibitors has guided the search for new compounds that may induce apoptosis in Bcr-Abl+ leukemic cells and improve the disease treatment. Methods: In the present study, we investigated whether the L-amino acid oxidase isolated from Bothrops moojeni snake venom (BmooLAAO-I) (i) was cytotoxic to Bcr-Abl+ cell lines (HL-60.Bcr-Abl, K562-S, and K562-R), HL-60 (acute promyelocytic leukemia) cells, the non-tumor cell line HEK-293, and peripheral blood mononuclear cells (PBMC); and (ii) affected epigenetic mechanisms, including DNA methylation and microRNAs expression in vitro. Results: BmooLAAO-I induced ROS production, apoptosis, and differential DNA methylation pattern of regulatory apoptosis genes. The toxin upregulated expression of the pro-apoptotic genes BID and FADD and downregulated DFFA expression in leukemic cell lines, as well as increased miR-16 expression - whose major predicted target is the anti-apoptotic gene BCL2 - in Bcr-Abl+ cells. Conclusion: BmooLAAO-I exerts selective antitumor action mediated by H2O2 release and induces apoptosis, and alterations in epigenetic mechanisms. These results support future investigations on the effect of BmooLAAO-I on in vivo models to determine its potential in CML therapy.(AU)

BjussuLAAO-II induces cytotoxicity and alters DNA methylation of cell-cycle genes in monocultured/co-cultured HepG2 cells.

BACKGROUND: The use of animal venoms and their toxins as material sources for biotechnological applications has received much attention from the pharmaceutical industry. L-amino acid oxidases from snake venoms (SV-LAAOs) have demonstrated innumerous biological effects and pharmacological potential against different cancer types. Hepatocellular carcinoma has increased worldwide, and the aberrant DNA methylation of liver cells is a common mechanism to promote hepatic tumorigenesis. Moreover, tumor microenvironment plays a major role in neoplastic transformation. To elucidate the molecular mechanisms responsible for the cytotoxic effects of SV-LAAO in human cancer cells, this study aimed to evaluate the cytotoxicity and the alterations in DNA methylation profiler in the promoter regions of cell-cycle genes induced by BjussuLAAO-II, an LAAO from Bothrops jaracussu venom, in human hepatocellular carcinoma (HepG2) cells in monoculture and co-culture with endothelial (HUVEC) cells. METHODS: BjussuLAAO-II concentrations were 0.25, 0.50, 1.00 and 5.00 µg/mL. Cell viability was assessed by MTT assay and DNA methylation of the promoter regions of 22 cell-cycle genes by EpiTect Methyl II PCR array. RESULTS: BjussuLAAO-II decreased the cell viability of HepG2 cells in monoculture at all concentrations tested. In co-culture, 1.00 and 5.00 µg/mL induced cytotoxicity (p < 0.05). BjussuLAAO-II increased the methylation of CCND1 and decreased the methylation of CDKN1A in monoculture and GADD45A in both cell-culture models (p < 0.05). CONCLUSION: Data showed BjussuLAAO-II induced cytotoxicity and altered DNA methylation of the promoter regions of cell-cycle genes in HepG2 cells in monoculture and co-culture models. We suggested the analysis of DNA methylation profile of GADD45A as a potential biomarker of the cell cycle effects of BjussuLAAO-II in cancer cells. The tumor microenvironment should be considered to comprise part of biotechnological strategies during the development of snake-toxin-based novel drugs.

Analysis of the cytotoxic, genotoxic, mutagenic, and pro-oxidant effect of synephrine, a component of thermogenic supplements, in human hepatic cells in vitro.

Thermogenic supplements containing synephrine (SN) are widely used to weight loss. SN is a proto-alkaloid naturally found in the bark of immature fruits of Citrus aurantium (bitter orange) that has been added to thermogenic supplements due to its chemical and pharmacological similarity with adrenergic amines, such as ephedrine and amphetamines. Although orally ingested SN is mainly metabolized in the liver, it remains unclear whether it affects the redox status and genetic material of human hepatic cells. The present study aims to examine whether SN affects cell viability, cell cycle, redox balance, genomic stability, and expression of the DNA damage response (DDR)-related genes ATM, ATR, CHEK1, CHECK2, TP53, and SIRT1 in HepG2 cells - used as in vitro hepatocyte model. SN induced overproduction of intracellular reactive oxygen species (ROS) after 6 h of treatment with the three concentrations tested (2, 20 and 200 µM). After 24 h of treatment, SN at 200 µM induced intracellular ROS overproduction and exerted cytostatic effects, while SN at 20 and 200 µM increased the levels of GPx and GSH. SN was not cytotoxic (2-5000 µM), genotoxic, and mutagenic and did not alter the expression of DDR-related genes (2-200 µM), indicating that the fast/specific SN metabolization and upregulation of antioxidant defense components to detoxify intracellular ROS were sufficient to prevent intracellular damage in HepG2 cells. In conclusion, SN showed no cytotoxic, genotoxic, and mutagenic potential at relevant concentrations for thermogenic users in human hepatic cells in vitro, although, it plays pro-oxidative action, and cytostatic effects. Taken together, our results suggest that other investigations about the hazard absence of this thermogenic compound should be performed.

BjussuLAAO-II induces cytotoxicity and alters DNA methylation of cell-cycle genes in monocultured/co-cultured HepG2 cells

The use of animal venoms and their toxins as material sources for biotechnological applications has received much attention from the pharmaceutical industry. L-amino acid oxidases from snake venoms (SV-LAAOs) have demonstrated innumerous biological effects and pharmacological potential against different cancer types. Hepatocellular carcinoma has increased worldwide, and the aberrant DNA methylation of liver cells is a common mechanism to promote hepatic tumorigenesis. Moreover, tumor microenvironment plays a major role in neoplastic transformation. To elucidate the molecular mechanisms responsible for the cytotoxic effects of SV-LAAO in human cancer cells, this study aimed to evaluate the cytotoxicity and the alterations in DNA methylation profiler in the promoter regions of cell-cycle genes induced by BjussuLAAO-II, an LAAO from Bothrops jaracussu venom, in human hepatocellular carcinoma (HepG2) cells in monoculture and co-culture with endothelial (HUVEC) cells. Methods: BjussuLAAO-II concentrations were 0.25, 0.50, 1.00 and 5.00 μg/mL. Cell viability was assessed by MTT assay and DNA methylation of the promoter regions of 22 cell-cycle genes by EpiTect Methyl II PCR array. Results: BjussuLAAO-II decreased the cell viability of HepG2 cells in monoculture at all concentrations tested. In co-culture, 1.00 and 5.00 μg/mL induced cytotoxicity (p < 0.05). BjussuLAAO-II increased the methylation of CCND1 and decreased the methylation of CDKN1A in monoculture and GADD45A in both cell-culture models (p < 0.05). Conclusion: Data showed BjussuLAAO-II induced cytotoxicity and altered DNA methylation of the promoter regions of cell-cycle genes in HepG2 cells in monoculture and co-culture models. We suggested the analysis of DNA methylation profile of GADD45A as a potential biomarker of the cell cycle effects of BjussuLAAO-II in cancer cells. The tumor microenvironment should be considered to comprise part of biotechnological strategies during the development of snake-toxin-based novel drugs.(AU)

Cell migration inhibition activity of a non-RGD disintegrin from Crotalus durissus collilineatus venom.

BACKGROUND: In recent decades, snake venom disintegrins have received special attention due to their potential use in anticancer therapy. Disintegrins are small and cysteine-rich proteins present in snake venoms and can interact with specific integrins to inhibit their activities in cell-cell and cell-ECM interactions. These molecules, known to inhibit platelet aggregation, are also capable of interacting with certain cancer-related integrins, and may interfere in important processes involved in carcinogenesis. Therefore, disintegrin from Crotalus durissus collilineatus venom was isolated, structurally characterized and evaluated for its toxicity and ability to interfere with cell proliferation and migration in MDA-MB-231, a human breast cancer cell line. METHODS: Based on previous studies, disintegrin was isolated by FPLC, through two chromatographic steps, both on reversed phase C-18 columns. The isolated disintegrin was structurally characterized by Tris-Tricine-SDS-PAGE, mass spectrometry and N-terminal sequencing. For the functional assays, MTT and wound-healing assays were performed in order to investigate cytotoxicity and effect on cell migration in vitro, respectively. RESULTS: Disintegrin presented a molecular mass of 7287.4 Da and its amino acid sequence shared similarity with the disintegrin domain of P-II metalloproteases. Using functional assays, the disintegrin showed low cytotoxicity (15% and 17%, at 3 and 6 µg/mL, respectively) after 24 h of incubation and in the wound-healing assay, the disintegrin (3 µg/mL) was able to significantly inhibit cell migration (24%, p < 0.05), compared to negative control. CONCLUSION: Thus, our results demonstrate that non-RGD disintegrin from C. d. collilineatus induces low cytotoxicity and inhibits migration of human breast cancer cells. Therefore, it may be a very useful molecular tool for understanding ECM-cell interaction cancer-related mechanisms involved in an important integrin family that highlights molecular aspects of tumorigenesis. Also, non-RGD disintegrin has potential to serve as an agent in anticancer therapy or adjuvant component combined with other anticancer drugs.

Cell migration inhibition activity of a non-RGD disintegrin from Crotalus durissus collilineatus venom

In recent decades, snake venom disintegrins have received special attention due to their potential use in anticancer therapy. Disintegrins are small and cysteine-rich proteins present in snake venoms and can interact with specific integrins to inhibit their activities in cell-cell and cell-ECM interactions. These molecules, known to inhibit platelet aggregation, are also capable of interacting with certain cancer-related integrins, and may interfere in important processes involved in carcinogenesis. Therefore, disintegrin from Crotalus durissus collilineatus venom was isolated, structurally characterized and evaluated for its toxicity and ability to interfere with cell proliferation and migration in MDA-MB-231, a human breast cancer cell line. Methods: Based on previous studies, disintegrin was isolated by FPLC, through two chromatographic steps, both on reversed phase C-18 columns. The isolated disintegrin was structurally characterized by Tris-TricineSDS-PAGE, mass spectrometry and N-terminal sequencing. For the functional assays, MTT and wound-healing assays were performed in order to investigate cytotoxicity and effect on cell migration in vitro, respectively. Results: Disintegrin presented a molecular mass of 7287.4 Da and its amino acid sequence shared similarity with the disintegrin domain of P-II metalloproteases. Using functional assays, the disintegrin showed low cytotoxicity (15% and 17%, at 3 and 6 µg/mL, respectively) after 24 h of incubation and in the wound-healing assay, the disintegrin (3 µg/mL) was able to significantly inhibit cell migration (24%, p < 0.05), compared to negative control. Conclusion: Thus, our results demonstrate that non-RGD disintegrin from C. d. collilineatus induces low cytotoxicity and inhibits migration of human breast cancer cells. Therefore, it may be a very useful molecular tool for understanding ECM-cell interaction cancer-related mechanisms involved in an important integrin family that highlights molecular aspects of tumorigenesis. Also, non-RGD disintegrin has potential to serve as an agent in anticancer therapy or adjuvant component combined with other anticancer drugs.(AU)