Significant attained information from ethnopharmacological reports for our study is the Petiveria alliacea's
antitumor and immunomodulatory reported activities. To date at a molecular level, there is a lack of scientific evidence to explain such activities. For example, a methanolic extract was unable to induce cytotoxicity on Hep G2 cells. Nonetheless, no specific reason was given for the lack activity in this case [25
]. Several compounds isolated from Petiveria alliacea
, such as astilbin and dibenzyl trisulphide have been demonstrated to induce apoptosis or influence cell cycle or affect actin dynamics [20
]. The present study demonstrates that Petiveria alliacea's
F4 fraction contains substances capable of inducing G2 arrest in a dose and time dependent manner (Fig. ). The ability of F4 fraction to change cell morphology and induce G2 arrest was further investigated. Previous reports demonstrate that dibenzyl trisulphide (DTS), one of the sulfur compounds found in Petiveria alliacea
, might be responsible for this dual activity [20
]. DTS has been previously reported to exhibit potent immunomodulatory function, capable of increasing murine thymic weight along with up-regulation of parameters associated with the reticuloendothelial system, a system essential for molecules involved in immunomodulatory functions [23
]. Mice exposed to lethal dose of E. coli
were protected from death probably because an increase in phagocytic activity [27
]. DTS has also been reported having anti-fungal activity in vitro
], as well as insecticidal, acaricidal and insect repellent activities in vivo
DTS causes reversible microtubule disassembly, which may be due to attenuation of the tyrosyl residues dephosphorylation of the MAP kinases (erk1/erk2) [20
]. Along with the fact that MAP kinases are involved in development and apoptotic responses, this event suggests a molecular linkage between these two observations. Mixed-lineage kinase 3 (MLK-3, a kinase of the family controlling MAP kinases activity) inhibition, can cause mitotic arrest by a mechanism involving disruption of microtubule formation and spindle pole assembly [30
]. The latter data indicates that Petiveria alliacea
F4 fraction might inhibit MLK3.
Presence of apoptotic cells after treatment with F4 fraction clearly suggests that cell cycle arrest induces cell death (Fig. ). The F4 fraction from Petiveria alliacea did not cause mitochondrial membrane depolarization, suggesting that cell death is caused by mitochondrial independent mechanisms (Fig. and ). Differentiation of cell death mechanisms, such as necrosis or apoptosis, become necessary since an inflammatory response after tissue injury might be different. The induction of an immune response in situ could be the consequence of equilibrium between apoptosis and subsequent necrotic death.
The types of compounds tentatively found in Petiveria alliacea's F4 fraction are sulfur compounds, flavonoids, flavonoid glycosides, coumarin, a monomethylated cyclo hexitol and a fatty acid. The sulfur compounds reported for Petiveria alliacea
and probably present in F4 fraction are: thiobenzaldehyde S-oxide, dibenzyl sulfide, S-(2-hydroxiethyl)-phenylmetanethiosulfinate, glutamyl-S-benzyl cysteine and dibenzyltrisulfide (Fig. y ). It is likely that these compounds are produced by petiverins (benzyl sulfoxides) degradation during the plant extraction process [31
], and are associated with antitumor activity. Dibenzyl trisulfide, an immunomodulatory compound isolated from Petiveria
], is likely to be present in our fraction. Therefore, could be one of the compounds responsible for the biological activity present in F4 fraction. Pinitol, a monomethylated cyclohexitol reported in Petiveria alliacea
and possibly present in our fraction, has been reported to exhibit anti-inflammatory properties [32
], possibly acting on dendritic cells [33
]. Myricitrin, a flavonoid glycoside probably present in F4 fraction, has been reported to have analgesic, anti-inflammatory and antinociceptive properties [34
]. Coumarin, another compound possibly found in the F4 fraction is reported to exhibit anti-tumor activity in prostate cancer models [35
], and anti-inflammatory activities [36
]. Other compounds possibly present in F4 fraction, includes senfol (1,2 diisothiocyanato ethane), 3,5 diphenyltritiolan, 4 ethyl petiveral, 5-O-methyl leridol and lignoceric acid have no literature reports related to anti-tumoral activity.
Down-regulation of cytoskeleton proteins detected by mass spectrometric analysis is consistent with the cytoskeleton disruption observed by fluorescent microscopy. Moreover, changes in the concentration of proteins involved in translation and transduction processes, as well as those involved in cellular metabolism, could explain the decrease of tumor cells clonogenic ability, as well as the anti-tumor activity of Petiveria alliacea
. Currently, we are evaluating the coding genes for these proteins in order to determine if the changes are at the transcriptional level or whether the proteomic results are a consequence of differential management of the existing proteins in the tumor cells. The mechanism by which tumor cells undergo death should be determined. Our results indicate that there is DNA fragmentation; however, it is possible that oxidative stress, metabolic changes, necrosis or senescence are also ways by which tumor cells may undergo death. In fact, necrotic death can provide the necessary danger signals to induce dendritic cells activation, giving anti-tumoral protective immune response [37
]; although other mechanisms can be implied in this antigen transfer [38
]. Induction of an effective immune response is unknown, but possibly Petiveria alliacea
F4 fraction, can act as Sho-Saiko-to, or Juzen-taiho-to [40
], inducing reduction of primary tumors, metastasis, and generating a specific CD8+ CTL responses. Mechanisms implied in the process are unknown. However, it is critical to understand and elucidate the molecular mechanisms before the plant fraction can be used in the design of effective cancer drug therapeutics.