ANTI-LEUKEMIC EFFECTS OF TYPHONIUM FLAGELLIFORME ON HUMAN LYMPHOBLASTOID CELLS (CEMss) AND MURINE LEUKEMIC (WEHI-3) MODEL

ANTI-LEUKEMIC EFFECTS OF TYPHONIUM FLAGELLIFORME ON HUMAN
LYMPHOBLASTOID CELLS (CEMss) AND MURINE LEUKEMIC (WEHI-3) MODEL

By
MURALI MOHAN SYAM MOHAN

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in
Fulfilment of the Requirements for the Degree of Doctor of Philosophy

October 2010

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Abstract of thesis presented to the senate of Universiti Putra Malaysia in fulfilment of the
requirement for the degree of Doctor of Philosophy

ANTI-LEUKEMIC EFFECTS OF TYPHONIUM FLAGELLIFORME ON HUMAN
LYMPHOBLASTOID CELLS (CEMss) AND MURINE LEUKEMIC (WEHI-3) MODEL

By

MURALI MOHAN SYAM MOHAN

October 2010

Chairman: Ahmad Bustamam Abdul, PhD

Faculty: Institute of Bioscience

To date, there has been no literature reported on the mechanism of Typhonium flagelliforme
and its effects on leukemia. Hence, the anti-leukemia effect of Typhonium flagelliforme was
investigated in vitro and in vivo leukemic model. Extraction and fractionation using organic
solvents were applied to obtain fractions from T. flagelliforme and subsequently, chemical
analysis was done using GC-MS. In vitro cytotoxic effects of extracts and fractions were
tested in several human cancer cell lines including leukemia (CEMss cells) using MTT assay.
Various microscopy techniques were used to study morphological changes occurring during
treatment. The Annexin V assay, TUNEL assay, cell cycle analysis and DNA laddering were
employed to detect apoptosis. Colourimetric assays for caspase-3 and 9, immunoblot
analysis for cytochrome c, BcL-2, PARP, FasL and β-actin were analysed. The in vivo model
of leukemia was induced in male BALB/c mice using WEHI-3 cells. The DCM extract of the
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plant tuber was used for treatment at various doses. Amongst 8 plant extracts investigated,
the dichloromethane (DCM) extracts of T. flagelliforme tuber demonstrated low and significant
anti proliferative effect against both CEMss (6.5±0.4 µg/ml) and WEHI-3 cells (24.0±5.2
µg/ml) (p<0.05). Further fractionation of the DCM tuber extract resulted into 12 fractions.
Seven of these 12 fractions showed significant cytotoxicity against CEMss, in which the
DCM/F7, DCM/F11 and DCM/F12 fractions showed highest anti-cancer activities of 3.0, 5.0
and 6.2 µg/ml respectively. Further studies of these fractions towards non cancerous
Peripheral Blood Lymphocytes (PBL) exhibited significant selectivity of DCM/F7 compared to
other fractions. Phytochemical analysis using GC-MS revealed that the DCM/F7 fraction
contains linoleic acid (51.20%), n-hexadecanoic acid (17.89%), 9-hexadecanoic acid (6.99%)
and Stigmasta-5,22-dien-3-ol (6.06%). Cytological observations exhibited chromatin
condensation, cell shrinkage, abnormalities of cristae, membrane blebbing, cytoplasmic
extrusions and formation of apoptotic bodies, further confirmed using AO/PI, SEM and TEM
analysis. The Annexin V and TUNEL assay revealed apoptotic induction in CEMss cells
exposed to the DCM/F7 in a time-dependent manner, whilst DNA fragmentation of CEMss
cells were detected using 1.0% agarose gel electrophoresis. The DCM/F7 fraction
significantly (p<0.05) stimulated both caspases 3 and 9 activities. The immunoblot results
revealed that DCM/F7 caused the release of mitochondrial cytochrome c and cleaved 116
kDa PARP into 85 kDa fragments. The Bcl-2 protein was found to decrease during treatment.
Meanwhile, FasL did not exhibit up or down regulation on treatment. Cell cycle analysis
revealed that there is significant (p<0.05) G1 phase arrest in a time-depended manner. The
DCM extract of T. flagelliforme tuber in vivo markedly inhibited the proliferation of WEHI-3 in
male BALB/c mice as evidenced by reduction in the percentage of immature monocytes as
well as granulocytes, liver weight, spleen weight and histopathological profiles of H&E stained
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spleen tissue. The DCM tuber extract of T. flagelliforme significantly decreased the spleen
tumor size, which had dose-dependent effects. Sections of spleen tissue of the BALB/c mice
treated with the extract. Treatment at 800 mg/kg dose showed evidence of apoptosis in
comparison to the control groups. Collectively, results presented in this study demonstrate
that T. flagelliforme, a local herbal medicinal plant in Malaysia inhibited the proliferation of
leukemia in vitro selectively, leading to the programmed cell death, which was later confirmed
to lead through mitochondrial pathways. Moreover, in vivo study on an orthotopic BALB/c
mice model clearly shows that, T. flagelliforme tuber extract has inhibited the proliferation of
leukemia via the induction of apoptosis.

Publication from this thesis

1. Syam, M., Ahmad, B., Siddig, I., Adel S, A.Z., Mohammed, A., Rasedee, A. and Manal
Mohamed, E., 2010. In vitro ultramorphological assessment of apoptosis on CEMss induced by
linoleic acid-rich fraction from typhonium flagelliforme tuber. Evidence-Based Complementary
and Alternative Medicine 2011.
www.hindawi.com/journals/ecam/2011/421894/abs/
2. Mohan, S., Bustamam, A., Ibrahim, S., Mohamed, M., Beng, N. and Isa, N., 2010. 563

Typhonium flagelliforme induces apoptosis through mitochondrial pathway. EJC Supplements
8, 144-144.
www.ejcancersupplements.info/article/S1359...1/abstract
3. Mohan, S., Abdul, A.B., Abdelwahab, S.I., Al-Zubairi, A.S., Sukari, M.A., Abdullah, R.,

Elhassan Taha, M.M., Ibrahim, M.Y. and Syam, S., 2010. Typhonium flagelliforme induces
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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. Journal of ethnopharmacology 131,
592-600.
4. Mohan, S., Bustamam, A., Ibrahim, S., Al-Zubairi, A.S. and Aspollah, M., 2008. Anticancerous

effect of Typhonium flagelliforme on human T4-lymphoblastoid cell line CEM-ss. Journal of
Pharmacology and Toxicology 3, 449-456.
linkinghub.elsevier.com/retrieve/pii/S0378874110005222
5. Mohan, S., Abdul, A.B., Wahab, S.I.A., Al-Zubairi, A.S., Elhassan, M.M. and Yousif, M.,

2008. Antibacterial and antioxidant activities of Typhonium flagelliforme (Lodd.) blume tuber.
American Journal of Biochemistry and Biotechnology 4, 402-407.
6. Syam Mohan, Ahmad Bustamam, Siddig Ibrahim, Adel S. Al-Zubairi, Mohamed Aspollah,

Rasedee Abdullah, Manal Mohamed Elhassan Ng Kuan Beng and Nurbaity Mohd Isa.
Typhonium flagelliforme inhibits the proliferation of murine leukemia WEHI-3 cells in vitro
and induces apoptosis in vivo. Leukemia Research. 34 (2010) 1483–1492;
doi:10.1016/j.leukres.2010.04.023)

Website

http://publicationslist.org/syammohanm

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