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Leukemia Research 34 (2010) 1483–1492

Contents lists available at ScienceDirect

Leukemia Research

journa l homepage: www.e lsev ier .com/ locate / leukres

yphonium flagelliforme inhibits the proliferation of murine leukemia WEHI-3ells in vitro and induces apoptosis in vivo

yam Mohana, Ahmad Bustamam Abdula,b,∗, Siddig Ibrahim Abdelwahaba, Adel S. Al-Zubairi a,c,ohamed Aspollah Sukarid, Rasedee Abdullahe, Manal Mohamed Elhassan Tahaa, Ng Kuan Benga,urbaity Mohd Isaa

UPM-MAKNA Cancer Research Laboratory, Institute of Bioscience, University Putra Malaysia, Serdang, MalaysiaDepartment of Biomedical Sciences, Faculty of Medicine and Health Sciences, University Putra Malaysia, Serdang, MalaysiaDepartment of Clinical Biochemistry, University of Sana’a, Sana’a, YemenDepartment of Chemistry, Faculty of Science, University Putra Malaysia, Serdang, MalaysiaDepartment of Pathology and Microbiology, Faculty of Veterinary Medicine, University Putra Malaysia, Serdang, Malaysia

r t i c l e i n f o

rticle history:eceived 8 February 2010eceived in revised form 23 April 2010ccepted 27 April 2010vailable online 1 June 2010

eywords:

a b s t r a c t

Typhonium flagelliforme (TF) is a tropical plant, traditionally used by the ethnic population of Malaysia forthe cure of various cancers. This plant had shown to induce antiproliferative effect as well as apoptosis incancer cells. However, there is no available information to address that TF affects murine leukemia cells invitro and in vivo. Here, we investigated in vitro and in vivo effects of TF on murine leukemia WEHI-3 cells.It was found that the growth of leukemia cells in vitro was inhibited by the various extracts of TF. Amongthese fractions, the dichloromethane (DCM) tuber extracts of TF showed the lowest IC50 (24.0 ± 5.2 �g/ml)and had demonstrated apoptogenic effect when observed under fluorescent microscope. We investigated

poptosisALB/c miceytotoxicityyphonium flagelliformeEHI-3 leukemia cells

the in vivo effects of DCM tuber extracts of TF on murine leukemia cells, and the results showed that thecounts of immature granulocytes and monocytes were significantly decreased in peripheral blood ofBALB/c leukemia mice after the oral administration of DCM tuber extracts of TF for 28 days with threedoses (200, 400 and 800 mg/kg). These results were confirmed by observing the spleen histopathology and

pleenin th

morphology of enlarged sthe cell death mechanism

. Introduction

Leukemia is a group of heterogeneous neoplastic disorder ofhite blood cells characterized by the uncontrolled proliferation

nd block in differentiation of hematopoietic cells [1]. In Malaysia,eukemia is the commonest cancer occurring in children. The crudencidence rate of pediatric malignancies in Malaysia was 77.4 per

illion children aged less than 15 years. Among cancer deaths in998, leukemia stands at the fourth place of commonest diagnoses2]. Treatment of leukemia is a multidisciplinary effort. The modal-ties of treatment include radiotherapy, chemotherapy, hormonalherapy, immune therapy and symptomatic and supportive ther-

py. A keen interest in anti-cancer therapy of herbal plants haseveloped recently. This interest in therapy with drugs of plantrigin is due to several reasons; namely, conventional medicinean be inefficient (e.g. side effects and ineffective therapy), abu-

∗ Corresponding author at: UPM-MAKNA Cancer Research Laboratory, Institutef Bioscience, University Putra Malaysia, 43400 UPM Serdang, Malaysia.el.: +60 12 6894693; fax: +60 38 9472101.

E-mail address: abustamam@putra.upm.edu.my (A.B. Abdul).

145-2126/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.oi:10.1016/j.leukres.2010.04.023

and liver in leukemia mice when compared with the control. Furthermore,e spleen tissue of treated mice was found via apoptosis.

© 2010 Elsevier Ltd. All rights reserved.

sive and/or incorrect use of synthetic drugs results in side effectsand other problems, a large percentage of the world’s populationdoes not have access to conventional pharmacological treatment,and folk medicine and ecological awareness suggest that “natural”products are harmless [3].

The plant Typhonium flagelliforme, commonly known as ‘rodenttuber’ in Malaysia, is often used as traditional remedy for alter-native cancer therapies, including leukemia by various ethnicpopulations [4]. This plant is widely distributed in soft, damp andshady habitats in South-East Asia reaching Northern Australia andSouth India [5]. TF is now commercially available as a healthcaresupplement to cure breast, lung, rectum, liver, prostate, leukemia,pancreas and cervical cancer [6]. There are few in vitro studies thathave established the anti-cancer properties of TF on different can-cer cell lines, including lung cancer NCI-H23 [7,8], breast cancerHS578T [5,7], liver cancer Hep-G2, breast cancer T-47D [7] andmurine P388 leukemia cells [9]. But till today there is no animal

study done to evaluate the anti-cancer pharmacological potency ofTF.

Nature is an attractive source of new therapeutic candidate asa tremendous chemical diversity is found in millions of species ofmicroorganisms, animals, marine organisms and most notably from

1484 S. Mohan et al. / Leukemia Research 34 (2010) 1483–1492

Fig. 1. Fluorescent micrograph of acridine orange and propidium iodide double-stained WEHI-3 cells. Cells were treated at IC50 of DCM tuber extract of Typhonium flagelliformeat time-dependent manner. Cells were cultured in RPMI 1640 media maintained at 37 ◦C and 5% CO2. (A) Untreated cells showed normal structure without prominent apoptosisand necrosis. (B) Early apoptosis features were seen after 24 h representing intercalated acridine orange (bright green) among the fragmented DNA. (C) Blebbing and nuclearmargination were noticed in 48 h treatment. (D) Late apoptosis was seen in 72 h incubated cells, whereby positive staining with orange color represents the hallmark of lateapoptosis. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Table 1Cytotoxic effect of Typhonium flagelliforme on the viability of WEHI-3 cancer cells.Cells were cultured in RPMI 1640 at 37 ◦C and 5% CO2.

Solvents IC50 ± S.D.

Leaves Tuber

Hexane 49.5 ± 2.7 82.0 ± 6.4Dichloromethane 67.0 ± 1.8 24.0 ± 5.2Ethyl acetate >100 75.0 ± 4.1Methanol >100 88.5 ± 4.3DMSO (0.1%, v/v) – –

FW*

psbo

Fig. 3. Percentage body weight changes of BALB/c mice treated with single doses ofTyphonium flagelliforme tuber DCM extract. Values are average of two mice. *Mice

ig. 2. Percentages of viable, early apoptotic and late apoptosis cells observed inEHI-3 cells in vitro after DCM tuber extract of Typhonium flagelliforme treatment.

Indicates P < 0.05.

lants [10,11]. Plants have been recognized as the most importantources which might have numerous therapeutic agents. They haveeen used for thousands of years to improve health and well beingf civilization as well as have been proven using modern scientific

died due to toxicity.

approaches to have both medicinal and nutritional values [12]. Theresults from these plants have revealed the potential of medicinalplants in the area of pharmacology [13]. Even though the medicinalplant TF have been used as a cure for leukemia, there is no availableinformation to address the effects of this plant on the leukemiacells in vitro and in vivo and also have no reports to demonstratethe cell death mechanism in mice. Therefore, in the present study,

we investigated the effects as well as mechanism of TF on WEHI-3leukemia in vitro and in vivo.

S. Mohan et al. / Leukemia Research 34 (2010) 1483–1492 1485

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ig. 4. Representative pictures of BALB/c mice. BALB/c mice after injection with Without DCM tuber extract of Typhonium flagelliforme for 4 weeks. Blood was collect

hanges (B) and weighed (C). *P < 0.05.

. Materials and methods

.1. Preparation of plant extract

Typhonium flagelliforme (Lodd.) Blume (Araceae) whole plant (leaves andubers) was collected from the state of Selangor, Malaysia. Authentication wasone at the faculty of science, University Putra Malaysia. The harvested plants

ere washed thoroughly with running tap water and then distilled water, fol-

owed by separation in to aerial parts as well as tubers before drying. All thelant materials were air dried and then oven dried at reduced temperature.he fully dried plants were powdered and weighed before cold maceration.he powdered leaves and tubers were extracted with different solvents inhe order of increasing polarity. The solvents used were hexane, DCM, ethyl

cells (1 × 106 cells/animal) in phosphate buffered saline (PBS) and treated with oranimals were sacrificed (A) for examination of spleen, which showed morphologic

acetate and methanol. The extraction was done for 7 days with occasionalshaking and the process was repeated three times. The combined extractswere filtered through Whatman® No. 41 filter paper (pore size 20–25 �m)and dried under vacuum using a rotary evaporator (Buchi, R-210, Switzerland)and then weighed to calculate the yield of the extracts and kept at 4 ◦C untilrequired.

2.2. Cells and animals

The murine monomyelocytic WEHI-3 leukemia cells were purchased from ATCC.The cell line was cultured in RPMI 1640 (PAA, Austria) containing 10% fetal bovineserum (FBS), 100 U/ml penicillin and 100 �g/ml streptomycin at 37 ◦C in a humid-ified atmosphere of 5% CO2. Cells were split every 3 days to maintain exponential

1 Research 34 (2010) 1483–1492

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Fig. 5. The effects of Typhonium flagelliforme DCM tuber extract and ATRA on thebody weight (A), percentage of immature monocytes in peripheral blood (B) and per-centage of immature granulocytes in peripheral blood (C) of BALB/c mice burdenedwith WEHI-3 leukemia cells. Data were mean ± S.D. (n = 10). *P < 0.05 as compared

486 S. Mohan et al. / Leukemia

rowth before experiments. Male BALB/c mice of 22–28 g in weight at the age ofweeks were obtained from commercial laboratory animal supplier (Biotanjung

nterprises, Malaysia). The mice were bred with clean water and nutrient food intandard conditions of constant temperature and humidity.

.3. In vitro cytotoxicity assay

The toxicity profiles of the plant extracts were assessed using the 3-4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) microcultureetrazolium viability assay [14]. The plant extracts were dissolved in DMSO to pre-are the stock solution and thereafter, the various concentrations of samples wererepared by dissolving the stock solution in media and were plated out in tripli-ates. The final concentration of DMSO was kept at 0.1% (v/v). Each plate includedntreated cell controls and a blank cell-free control. After 68 h of incubation, MTT5 mg/ml) was added to each well and the plates incubated for a further 4 h andhe media was removed. DMSO was later added into each well to solubilize the for-

azan crystals. The absorbance was read at wavelength of 595 nm using a microtitrelate reader (Tecan Sunrise Basic, Austria). The percentage cellular viability was cal-ulated with the appropriate controls taken into account. The concentration whichnhibited 50% of cellular growth (IC50 value) was determined. All experiments forach extract were carried out in triplicates. The inhibitory rate of cell prolifera-ion was calculated by the following formula: Growth inhibition = OD control − ODreated/OD control × 100. The cytotoxicity of sample on cancer cells was expresseds IC50 values (the drug concentration reducing the absorbance of treated cells by0% with respect to untreated cells).

.4. In vitro detection of apoptosis using propidium iodide and acridine orangeouble-staining

DCM tuber extract of TF induced cell death in WEHI-3 cells was quantified usingropidium iodide (PI) and acridine orange (AO) double-staining according to stan-ard procedures and examined under fluorescence microscope (Lieca attached with-Floro Software). Briefly, treatment was carried out in a 25 ml culture flask (Nunc).EHI-3 cells were plated at concentration of 1 × 106 cell/ml and treated with DCM

uber extract of TF at IC50 concentration. Flasks were incubated in atmosphere of% CO2 at 37 ◦C for 24, 48 and 72 h. The cells were then spin down at 1000 rpm for0 min. Supernatant was discarded and the cells were washed twice using phos-hate buffered saline (PBS) after centrifuging at 1000 rpm for 10 min to remove theemaining media. Ten microliters of fluorescent dyes containing acridine orangeAO, 10 �g/ml) and propidium iodide (PI, 10 �g/ml) were added into the cellular pel-et at equal volumes of each. Freshly stained cell suspension was dropped into a glasslide and covered by coverslip. Slides were observed under UV-fluorescence micro-cope within 30 min before the fluorescence color starts to fade. The percentagesf viable, early apoptotic, late apoptosis and secondary necrotic cells were deter-ined in more than 200 cells. Acridine orange (AO) and propidium iodide (PI) are

ntercalating nucleic acid specific fluorochromes which emit green and orange flu-rescence, respectively, when they are bound to DNA. Of the two, only AO can crosshe plasma membrane of viable and early apoptotic cells.

.5. In vivo studies

.5.1. Starting dose determination by maximum tolerated dose (MTD) analysisMale BALB/c mice at 8 weeks of age were acclimatized for 5 days during which

nimal were examined to confirm suitability for the study. Mice were kept in indi-idual cages and given free access to food and water. To establish the MTD, DCMuber extract of TF was dissolved in olive oil and administered to BALB/c mice atscalating doses (two mice per group) for a treatment period of 14 days by oralavage. Body weights of the mice were measured daily after the extract administra-ion. The percentage maximum weight loss (relative to initial starting weight) wasecorded, with a weight loss of greater than 15% considered as toxic [15]. The MTDs the dose one step lower than toxic dose. The MTD was selected for in vivo studyn BALB/c mice.

.5.2. Drug treatmentThe animals for the experiment were divided into seven groups (10 animals per

roup) of male BALB/c mice (groups 0–6). Group 0 was served as negative controlanimals without leukemia burden). The remaining groups of mice were injectedith WEHI-3 cells (1 × 106 cells/animal). Then group 1 was served as leukemia con-

rol. Group 2 was treated with olive oil (vehicle). Groups 3, 4 and 5 were treated withCM crude extract of TF tuber in olive oil (200, 400 and 800 mg/kg), respectively.roup 6 was treated with ATRA 5 mg/kg (positive control). All animals were admin-

stered with the oral dose (oral gavage) as mentioned above for every alternativeays up to 4 weeks before being weighed and sacrificed.

.5.3. Leukocyte counting in peripheral blood of treated miceBlood from treated BALB/c mice tail vein was collected after inoculation of

EHI-3 leukemia cells for 7, 14, 21 and 28 days. Immature monocytes and gran-locytes counting based on cell morphology were done by Giemsa staining withlood smears.

with the control. Where, group 0 is normal control, group 1 is Leukemia control,group 2 is Vehicle control, group 3 is 200 mg/kg treatment, group 4 is 400 mg/kgtreatment, group 5 is 800 mg/kg treatment and group 6 is ATRA 5 mg/kg.

S. Mohan et al. / Leukemia Research 34 (2010) 1483–1492 1487

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ig. 6. Representative pictures of immature blood cells present in peripheral bloegakaryocyte (C), myeloblast (D), neutrophilic metamyelocyte (E) and basophilic

.5.4. Tissues samples (liver and spleen)All animals from each group were weighed before blood was sampled. The liver

nd spleen samples were obtained, weighed individually and spleen was used foristopathology.

.5.5. HistopathologyThe spleen tissue was fixed in 10% formalin for at least 3 days to assure proper

xation. After which the tissue was processed using a tissue processor (Leica TP020-1-1, Germany). The processed tissue was embedded in paraffin wax (LeicaG1160 TP 1020, Germany) and was sectioned using microtome (Leica RM 2145).issue sections were flattened in a water bath (Leica HI1210, Germany) at 40 ◦C, and

hen loaded onto poly-l-lysine coated glass slides and stained with hematoxylin andosin.

.5.6. Detection of apoptosis using TUNEL assayFor further confirmation of the incidence of apoptosis, TUNEL assay was per-

ormed using DeadEndTM fluorometric TUNEL system (Promega, USA). The assay

leukemic Balb/C mice. Immature monocyte (A), eosinophilic band (B), immaturecyte (F).

was then conducted according to the manufacturer’s instructions. Briefly, the tis-sue sections were deparaffinized by immersing slides in fresh xylene. The tissuesections were fixed by immersing the slides in 4% methanol-free formaldehydesolution in PBS for 15 min at room temperature. 20 �g/ml proteinase K solu-tions were added to each slide to cover the tissue section for permeability.The tissue sections were covered with 100 �l of equilibration buffer followedby 50 �l of rTdT incubation buffer. The samples were stained by propidiumiodide solution and were analyzed under a confocal microscope using a standardfluorescent filter set to view the green fluorescence produced by fluorescein-12-dUTP.

2.6. Statistical analysis

The results were expressed as mean ± S.D. and the difference between groupswas analyzed by one-way ANOVA.

1488 S. Mohan et al. / Leukemia Research 34 (2010) 1483–1492

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ig. 7. DCM tuber extract of Typhonium flagelliforme affected the changes of we1 × 106 cells/animal) and treated with or without DCM tuber extract of Typhonind photographed (B). *P < 0.05.

. Results

.1. The effects of Typhonium flagelliforme on proliferation ofEHI-3 leukemia cells in vitro

The MTT is a standard colorimetric assay (an assay whicheasures changes in color) for measuring cellular growth. The max-

mum concentration used in this study was 100 �g/ml. Methanolxtracts of both leaves and tubers did not show any inhibitionelow this range. All the other extracts exhibited different IC50gainst the WEHI-3 cells, in which DCM extracts of tuber showedhe lowest IC50 with 24.0 ± 5.2 �g/ml (Table 1). The Americanational Cancer Institute guidelines set the limit of activity forrude extracts at a 50% inhibition (IC50) of proliferation of less than0 �g/ml after an exposure time of 72 h [16]. As a result, only theCM extract of tuber was selected for further studies.

.2. In vitro quantification of apoptosis using propidium iodidend acridine orange double-staining

Treated WEHI-3 cells were scored under fluorescence micro-cope in order to quantify viable, early apoptosis and late apoptoticells. We counted 200 cells arbitrarily and differentially, togetherith the untreated negative control. The study revealed that DCM

uber extract of TF triggered morphological features that relates

o apoptosis in a time-dependent manner (Fig. 1). Early apopto-is was obvious by intercalated AO within the fragmented DNA. Ineveral of such cases, the fluorescent bright green color could beeen in treated WEHI-3 cells only. In contrast, untreated cells werebserved with a green intact nuclear structure. At 24 h treatment

f liver tissues from BALB/c mice. The animals were injected with WEHI-3 cellsgelliforme for 4 weeks. Livers were individually collected and were weighed (A)

with DCM tuber extract of TF, blebbing and nuclear chromatin con-densation were noticed (moderate apoptosis). In addition, in thelate stages of apoptosis, changes such as the presence of reddish-orange color due to the binding of AO to denatured DNA wereobserved after 48 and 72 h treatment. Differential scoring of treatedWEHI-3 cells (200 cells population) showed that there is a statisti-cal significant (P < 0.05) difference in both early and late apoptosispositive cells, which indicates clearly that a time-dependent apop-togenic effect has occurred (Fig. 2).

3.3. Determination of maximum tolerated dose of DCM crudeextract of Typhonium flagelliforme tuber in BALB/c mice

Toxicity was assessed by measuring the body weight daily for14 consecutive days following single oral dose of DCM extract ofTF tuber as mentioned earlier. The percentage maximum weightloss (relative to initial starting weight) was recorded and a weightloss greater than 15% was considered to be toxic. MTD was thedose one step lower than the toxic dose. From the obtained results1000 mg/kg was selected as MTD as there was more than 15%weight loss observed at dose of 2000 mg/kg. Control as well as500 mg/kg dose treated animals showed up to 15% body weight gainat 14th day. Meanwhile, the highest dose (4000 mg/kg) receivedanimals died at day 8 (Fig. 3).

3.4. WEHI-3 induced leukemia model in BALB/c mice

Representative pictures of the animals after injection withWEHI-3 cells for 4 weeks that also showed the leukemia spleentumors are given in Fig. 4A. The size of spleen showed that DCM

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S. Mohan et al. / Leukemia

uber extract of TF decreased the spleen tumor size and had dose-ependent effects (Fig. 4B).

.5. Typhonium flagelliforme maintained the body weight andeduced the immature cells in peripheral blood of leukemic mice

The average body weight of animals with leukemia burdeneduced significantly at the end of experiment (Fig. 5A). At theame time, the treatments, especially the highest dose (800 mg/kg)aintained the rise in body weight and reached near to the bodyeight of normal mice. After burden with WEHI-3 leukemia cellsp to 28 days, the numbers of both peripheral granulocytes andonocytes in the TF DCM tuber extract treated leukemia BALB/cice were significantly lower than that of the control group with-

ut treatment (Fig. 5B and C). In addition, it can be observed thathe olive oil treatment (vehicle control) did not reduce the imma-ure cells, showing that it served as an inert vehicle in this study.part from that, the positive control used in the study (ATRAmg/kg) showed a marked decrease in cancer cells significantly

Fig. 6).

.6. Typhonium flagelliforme affects the weight andistopathology of liver and spleen tissues in BALB/c mice

Liver and spleen tissues were isolated from animals and werehotographed, weighed and histopathologically examined. The sizef spleen showed that DCM tuber extract of TF decreased sig-ificantly the spleen tumor size and had dose-dependent effectsFig. 4C). Even though there is a difference in spleen weight, the low-st dose (200 mg/kg) fails to show significant reduction in spleeneight. The liver size of leukemia mice also reduced significantly

fter treatment with DCM tuber extract of TF (Fig. 7A and B). Allhe treatment doses showed significant difference in liver weights compared to the control.

The histopathological examination indicated that, after treat-ent spleen demonstrated a pattern ranging from minimal

istopathological change to scanty small neoplastic cell nestsresent in the sinusoid (Fig. 8B) when compared to controlFig. 8A). The spleens, belonging to the treatment groups eitherisclosed markedly decreased numbers of neoplastic cells or theells were hard to find in the red pulp. The marked expan-ion of red pulp was seen in examined spleen tissues. Theeoplastic cells in examined spleen tissues presented large irreg-lar nuclei with clumped chromatin; prominent nucleoli andbundant clear and light eosinophilic cytoplasm were seenFig. 8C).

.7. Typhonium flagelliforme induces apoptosis in thepleenocytes of leukemic mice

Spleen tissue sections of BALB/c mice treated with DCM tuberxtract of TF and ATRA showed increased number of apoptotic cells,vidently with higher green fluorescence when viewed under aonfocal microscope. Treatment at 800 mg/kg (Fig. 9C) and ATRAmg/kg doses induced augmentation in apoptosis to the spleen

issues in comparison to leukemic control group (Fig. 9B), whichhowed no evidence of apoptosis. Normal spleen tissues howeveremonstrated very few apoptotic cells (Fig. 9A).

Fig. 10 shows the mean percentage of apoptosis in spleen tis-ues after treatment with DCM tuber extract of TF, ATRA and oliveil. There was a significant mean difference between the treatment

roups with P < 0.01 analyzed using one-way ANOVA. A post hocomparison test (one-way ANOVA) showed a significant differencen mean percentage of apoptosis between olive oil treatment group0%) with DCM tuber extract of TF 800 mg/kg (12.67%), and ATRA23.33%) treatment groups (P < 0.01). A significant mean difference

represent neoplastic cells and effects of DCM extract of Typhonium flagelliforme(800 mg/kg) on the histopathology of the spleen from leukemic BALB/c mice (C).

was also found between the highest dose treatment groups of DCMtuber extract of TF and ATRA 5 mg/kg doses as compared to the nor-

mal group of mice, the mean apoptotic percentage being 1.667%(P < 0.01). The statistical data suggest that the apoptotic effect ofDCM tuber extract of TF is in dose depended manner.

1490 S. Mohan et al. / Leukemia Research 34 (2010) 1483–1492

Fig. 9. TUNEL assay was used to detect the apoptosis on spleen tissue induced by DCM tuber extract of Typhonium flagelliforme. Micrograph of spleen tissue of normal BALB/cmice exhibiting less apoptotic cells (white arrows) (A), spleen tissue of BALB/c mice induced with leukemia without treatment, were cells observed to proliferate aggressively(B), significantly increased cells with apoptosis were observed on 800 mg/kg DCM tuber extract for treatment (C) and normal spleen tissue treated with 800 mg/kg DCM tuberextract does not showed the presence of apoptosis cells (D).

Fig. 10. The mean percentage of apoptotic cells in spleen tissue sectioning of BALB/c mice having leukemia burden after treatments with DCM tuber extract of Typhoniumflagelliforme, ATRA and olive oil. The findings showed that there was a significant mean difference between the treatments groups with P < 0.01 (analyzed using post hoccomparison test one-way ANOVA).

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. Discussion

At present, chemotherapy is considered as one of the mostfficient cancer treatment approach. Even though chemotherapyignificantly recover symptoms of patients with leukemia, onlynpretentious increase in survival rate can be achieved and henceatients turned to alternative medicines, including herbal thera-ies as a supportive care [17]. Nowadays there is growing attention

n the use of plant materials for the treatment of various cancersue to the fact that natural agents that restrain the proliferationf malignant cells by inducing apoptosis may represent a use-ul mechanistic approach to cancer chemotherapy and developffective therapeutic agents [18]. Although, TF had been showno promote antiproliferative effect in different in vitro cancer cellines including leukemia, the effect of this plant has not yet beenstablished in an animal model. So in the present study, we exam-ned the cytotoxicity of TF in WEHI-3 cells in vitro as well as inivo.

In the in vitro experiments, as shown in MTT results, the DCMxtract of tuber of this plant had stronger cytotoxicity than theest. It is well known that in apoptosis, the earliest recognizedorphological changes are compaction and segregation of the

uclear chromatin, with the result of chromatin margination andondensation of the cytoplasm. Progression of the condensations accompanied by convolution of the nuclear and cell outlinesollowed by breaking up of the nucleus into discrete fragmentsnd by budding of the cell as a whole to produce membrane-ounded apoptotic bodies. The microscopic examination carriedut in the current study using AO/PI confirms that there was a time-ependent apoptogenic effect on WEHI-3 cells in vitro. As such,his active extract has been used for the animal experiments tossess the anti-leukemic activity of TF. In the cancer pharmaco-ogical experiments, in vivo study plays an inevitable role. Because

uch of the research in this area is solely based on simplified in vitroystems that cannot take into account the complexity of biotrans-ormation processes. In addition, experimental animals provide thepportunity to further examine chemo-preventive properties inespect of their absorption, distribution and impact on individualrgans, and finally the kinetics of their metabolism and excretion19]. WEHI-3 cells, a murine monomyelocytic leukemia cells, wereriginally derived from the BALB/c mouse [20]. The in vivo ani-al model of leukemia through the injection of WEHI-3 cells i.p.

n mice is well established [21–24]. Moreover, the WEHI-3 cells inivo model have been recognized to be an ideal system for the studyf antileukemia activity such as ATRA, aclacinomycin A, IL-6, G-CSF23,25]. Murine host systems have been extensively applied in theeukemia research [26–28], may be due to common and assess-ble option in the pursuit of any model of human disease. Besides,hey are easily obtainable, relatively inexpensive to maintain, andheir immunology, genetics, and metabolism are well known andppreciated [29].

We selected the 200, 400 and 800 mg/kg doses for in vivo exper-ment based on results obtained from MTD studies. The maximumolerated dose (MTD) is defined as the highest dose of the test agenturing the chronic study that can be predicted not to alter thenimal’s normal longevity from effects other than carcinogenic-ty [30]. If the MTD has been chosen appropriately, there shoulde no adverse effect on survival, only a modest decrement in bodyeight gain and minimal overt signs of toxicity. The MTD has been

xceeded if there is increased mortality, severe body weight decre-ent, or marked signs of toxicity [31]. In this study, toxicity was

ssessed by measuring the body weight daily for 14 consecutiveays following single oral dose of DCM extract of TF tuber as men-ioned earlier. The result demonstrates that group which receives000 mg/kg showed the body weight loss more than 15% comparedo control by the day 10. Furthermore, both the animals died at day

rch 34 (2010) 1483–1492 1491

12. So the 1000 mg/kg dose has been selected as the MTD for the invivo experiments.

In the WEHI-3 cells orthotopic mice model, the BALB/c micewere injected i.p. with WEHI-3 cells 1 day prior to the treat-ment with DCM tuber extract of TF, and then the animals weresacrificed at 28th day. The WEHI-3 leukemia animal model wascharacterized by elevated peripheral monocytes and granulocyteswith immature morphology and by apparently enlarged and infil-trated spleens as compared with the normal counterpart. It wasrecommended to count different classes of immature leukocytesin peripheral blood as well as to determine the enlarged spleenweights to establish the efficacy criteria for the treatment on dif-ferentiation of leukemia BALB/c mice in vivo [22]. The availableresults in the present study are well correlated with these crite-ria. We also see the enlarged size of spleen and liver of leukemiacontrol group and DCM extract of TF decreased the size of spleenas well as liver and there was a significant difference betweenthe control and treatment groups. Moreover, treatments signifi-cantly prevented the loss of body weight (Fig. 3A). In support tothese findings, the histopathological examination indicated thatafter treatment, spleen demonstrated a pattern ranging from min-imal histopathological change to scanty small neoplastic cell nestspresent in the sinusoid. Reduction in the infiltration of immaturemyeloblastic cells into splenic red pulp also observed. Similar kindof results was observed previously in BALB/c mice leukemia model[23,26,32].

Treatment of tumors is directed not only on inhibition of cellproliferation, but also on induction of apoptosis of tumor cells. Moreand more attention is paid to the ability of drugs to induce apopto-sis in the process of evaluation of anti-tumor agents’ effectiveness.That is why we evaluated the ability of DCM extract of TF in theinduction of apoptosis in vivo. There was a significant mean dif-ference between the treatment groups in the apoptosis producedby control and treatment groups. In addition, the positive controlused in this study, ATRA demonstrated a higher proportion of apop-totic cells. ATRA is known to induce differentiation and apoptosisin human leukemia cells in vitro and in vivo [33].

So far, our results showed that, DCM tuber extracts of TF inhib-ited the cell proliferation of WEHI-3 cells in vitro and repressedthe spleen leukemia tumor growth in a WEHI-3 leukemia murinemodel in vivo. In addition, our study for the first time clearly showedthat TF has direct cytotoxicity in WEHI-3 leukemia cells and inhibitscell growth via inducing apoptosis. Therefore, more study is neededto further evaluate the molecular mechanisms, its pathways andthe active phytochemicals before being used as a potential thera-peutic agent for leukemia.

Conflict of interest statement

None.

Acknowledgements

This research was funded in part by the National Cancer Coun-cil (MAKNA), Malaysia. The authors also acknowledge additionalsupport from University Putra Malaysia (UPM), Serdang, Malaysia(Grant No. RUGS 91143).

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