In Vitro Ultramorphological Assessment of Apoptosis on CEMss Induced by Linoleic Acid‐Rich Fraction from Typhonium flagelliforme Tuber

Abstract

The plant Typhonium flagelliforme, commonly known as “rodent tuber” in Malaysia, is often used as a health supplement and traditional remedy for alternative cancer therapies, including leukemia. This study aimed to evaluate in vitro anti-leukemic activity of dichloromethane extract/fraction number 7 (DCM/F7) from T. flagelliforme tuber on human T4 lymphoblastoid (CEMss) cell line. The DCM extract of tuber has been fractionated by column chromatography. The obtained fractions were evaluated for its cytotoxicity toward CEMss cells as well as human primary blood lymphocytes (PBLs). Assessment of apoptosis produced by the most active fraction was evaluated by various microscopic techniques and further confirmation of apoptosis was done by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Phytochemical screening was done by gas chromatography-mass spectrometry (GC-MS). The results shows that 7 out of 12 fractions showed significant cytotoxicity against the selected cell line CEMss, in which fractions DCM/F7, DCM/F11 and DCM/F12 showed exceptional activity with 3, 5 and 6.2 μg ml−1, respectively. Further studies in the non-cancerous PBL exhibited significant selectivity of DCM/F7 compared to other fractions. Cytological observations showed chromatin condensation, cell shrinkage, abnormalities of cristae, membrane blebbing, cytoplasmic extrusions and formation of apoptotic bodies as confirmed collectively by double-staining of acridine orange (AO)/propidium iodide (PI), SEM and TEM. In addition, DCM/F7 has increased the cellular DNA breaks on treated cells. GC-MS revealed that DCM/F7 contains linoleic acid, hexadecanoic acid and 9-hexadecanoic acid. The present results indicate that T. flagelliforme possess a valuable anti-leukemic effect and was able to produce distinctive morphological features of cell death that corresponds to apoptosis.

Typhonium flagelliforme induces apoptosis in CEMss cells via activation of caspase-9, PARP cleavage and cytochrome c release: Its activation coupled with G0/G1 phase cell cycle arrest

“…Our laboratory had previously demonstrated that a linoleic acid rich DCM/F7 fraction obtained from Typhonium flagelliforme tubers possess anti-leukemic effect, which showed distinctive morphological and ultramorphological features of cell death relating to apoptosis. Furthermore, it had been proven from this investigation that the cyctotoxic behavior of this DCM/F7 fraction is towards CEMss cells was selective (Mohan et al, 2010). In the current study, we established the probable mechanism of cell death induced by this fraction from Typhonium flagelliforme tuber, Fig.…”

“…Even though, several anti-cancer activities have been reported, yet the mechanism which included apoptosis, attributed by the presence of chemical constituents in TF remains unclear. The current research is a continuation of our previous work done and reported elsewhere (Mohan et al, 2010). In this present study, we aimed to establish the possible pathway of apoptosis induced in CEMss cells by DCM/F7 fraction obtained from Typhonium flagelliforme tuber.…”

“…Our previous study had clearly shown that 51.2% of the DCM/F7 is linoleic acid, followed by the presence of unsaturated aliphatic compounds (Mohan et al, 2010). Linoleic acid has been reported previously to be a well-known apoptotic agent (Fernanda Cury-Boaventura et al, 2006;Ip et al, 2000).…”

“…The results from our previous study (Mohan et al, 2010) indicated that the linoleic acid rich fraction (DCM/F7) showed selective anti-proliferative effect (IC 50 ) and apoptosis against CEMss. Therefore, we employed the same fraction for the current study.…”

“…In addition, DCM/F7 has increased cellular apoptotic DNA breaks in treated cells. GC-MS analysis further revealed that the DCM/F7 contains 51.2% of linoleic acid (Mohan et al, 2010). Even though, several anti-cancer activities have been reported, yet the mechanism which included apoptosis, attributed by the presence of chemical constituents in TF remains unclear.…”