Pouterin, a novel potential cytotoxic lectin-like protein with apoptosis-inducing activity in tumorigenic mammalian cells.
In this study, the cytotoxicity of pouterin in tumorigenic and non-tumorigenic mammalian cell lines was investigated. We found that HeLa, Hep-2 and HT-29 tumor cells were highly sensitive to pouterin cytotoxicity in a dose-dependent manner, whereas non-tumorigenic Vero cells and human lymphocytes were relatively resistant to the protein. Among the tumor cell lines, HeLa cells showed the highest susceptibility to pouterin cytotoxicity, exhibiting a time-dependent increase in LDH leakage and an IC(50) value of 5mug/mL. Morphological alterations such as rounding, cell shrinkage and chromatin condensation, consistent with apoptotic cell death were observed. Apoptosis induction was demonstrated by DNA fragmentation as detected by terminal dUTP nick-end labeling (TUNEL). Furthermore, HeLa cells incubated with pouterin showed disruption of the actin cytoskeleton. Western blot analysis revealed that pouterin caused increased expression of p21, thus indicating cell cycle arrest. Subsequent studies provided evidence that apoptosis may be partially explained in the activation of the tumor necrosis factor receptor 1 (TNFR1) signaling. Interestingly, a time-dependent decrease of the expression of p65 nuclear factor kappa B (NFkappaB) subunit, concomitant with a downregulation of the inhibitor of apoptosis protein 1 (IAP1) was observed, suggesting that TNFR-mediated apoptosis is the predominant pathway induced by pouterin in HeLa cells.
Boleti
Ana Paula de A
AP
Departamento de Bioquímica/IB, Universidade Estadual de Campinas, Campinas, SP, Brazil.
Ventura
Cláudio A
CA
Justo
Giselle Z
GZ
Silva
Rodrigo A
RA
de Sousa
Ana Carolina T
AC
Ferreira
Carmen V
CV
Yano
Tomomasa
T
Macedo
Maria Lígia R
ML
D008841:Actin Cytoskeleton / Q000187:drug effects / Q000648:ultrastructure; D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000633:toxicity*; D017209:Apoptosis; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000187:drug effects / Q000648:ultrastructure; D002522:Chlorocebus aethiops; D003603:Cytotoxins / Q000633:toxicity*; D053938:DNA Fragmentation; D006367:HeLa Cells; D006801:Humans; D008214:Lymphocytes / Q000187:drug effects / Q000648:ultrastructure; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D037121:Plant Lectins / Q000633:toxicity*; D032132:Pouteria / Q000737:chemistry; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D015854:Up-Regulation; D014709:Vero Cells
D000972:Antineoplastic Agents, Phytogenic; D003603:Cytotoxins; D016328:NF-kappa B; D037121:Plant Lectins; D047888:Receptors, Tumor Necrosis Factor, Type I