Inhibitory effect of kinetin riboside in human heptamoa, HepG2

Jane Cheong,aDavid Goh,

aJean Wan Hong Yong,

bSwee Ngin Tan

b

and Eng Shi Ong*c

Received 20th August 2007, Accepted 1st October 2008

First published as an Advance Article on the web 25th November 2008

DOI: 10.1039/b712807j

Cytokinins ribosides such as kinetin riboside are a class of plant hormone that were first identified

as factors that promote cell division and have since been implicated in many other aspects of

plant growth and development. From the data obtained from cell cycle analysis with flow

cytometry, the in vitro growth inhibition of human heptamoa, HepG2 cells with kinetin riboside

was mediated by causing G2/M cell cycle arrest and cell death. At the same time, treatment with

various doses of kinetin riboside in HepG2 cells did not result in a population of cells positive for

the active caspase 3. Differentially expressed proteins in the mitochondria of HepG2 cells with cell

death induced by kinetin riboside were investigated. Without the use of stable isotope labeling,

the proposed method using LC/MSMS provided a rapid approach to study the differentially

expressed proteins in the mitochondria due to the cell death induced by kinetin riboside in HepG2

cells. The ability of kinetin riboside to induce cell death and attenuate G1 to S transition is

probably a consequence of its ability to interfere with several components in the mitochondria.

Hence, it was proposed that the cell death caused by kinetin riboside in HepG2 cells affected a

network of proteins involved in cell death and electron transport.

Introduction

Cytokinins, N6-substituted adenine derivatives, are a class of

plant hormones that were first identified as factors that

promoted cell division and have since been implicated in many

other aspects of plant growth and development including

shoot initiation and growth, apical dominance, senescence,

and photomorphogenic development.1,2 The effects of various

adenine analogues on the growth and differentiation of human

myeloid leukemia HL-60 cells were examined. Kinetin riboside

(Fig. 1A) was observed to be one of most potent cytokinins

investigated for growth inhibition and apoptosis.3 Kinetin

ribosides were shown to act on mouse and human tumor cells

such as M4 Beu human and B16 murine melanoma cells at low

concentration.4 Kinetin (free base and riboside), which was

assumed by many scientists to be a synthetic 3 cytokinin plant

growth hormone, has been detected for the first time in the

endosperm 4 liquid of fresh young coconut fruits (‘‘coconut

water’’) with a LC/MSMS method.5

The mitochondria have long been considered to play a

straightforward but critical role in the life of the cell and as

a key regulator of mammalian apoptotic cell death. They carry

out energy oxidative reactions that create the vast majority of

ATP necessary to support all cellular functions. Interruption

of this mitochondrial function in vivo leads to death. In the

Fig. 1 (A) Chemical structure of kinetin riboside, R: b-D-ribofur-

anosyl and (B) growth inhibitory effect of kinetin riboside on HepG2

human liver cancer cells. Cells were treated with different concentra-

tions of kinetin riboside for 2 days when the cell viability was

determined by the MTT assay. The growth inhibition was calculated

as percentage of inhibition compared with the control.

a Applied Science School, Temasek Polytechnic, 21 Tampinese Avenue 1,Singapore, 529757, Republic of Singapore

bNatural Sciences and Science Education Academic Group, NanyangTechnological University, 1 Nanyang Walk, Singapore, 637616,Singapore

cDepartment of Community, Occupational and Family Medicine,National University of Singapore, 16, Medical Drive, Singapore,117597. E-mail: cofoes@nus.edu.sg

This journal is �c The Royal Society of Chemistry 2009 Mol. BioSyst., 2009, 5, 91–98 | 91

PAPER www.rsc.org/molecularbiosystems | Molecular BioSystems

mitochondria two main pathways, the intrinsic and

extrinsic pathways, can lead to apoptosis. The extrinsic pathway

involves a death receptor protein and an adapter protein.

This in turn interacts with the cysteine aspartate protease

pro-caspase 8 and often pro-caspase 10. Activation of

caspase 8 culminates in the activation of other executioner

caspases such as caspase 3, 6 and 7. The intrinsic pathway

directly releases soluble proteins contained in the mito-

chondria inter-membrane space. These molecules include

cytochrome c, apoptosis-inducing factors (AIF), Smac/

DIABLO and endonuclease G (EndoG).6–9 Another apoptotic

gene, death associated protein 3, localized to the mitochondria

is involved in the process of mitochondrial fragmentation

during cell death.10 With proteomic analysis, mitochondrial

proteins that may hold the key to the mechanisms by

which copper-zinc superoxide dismutase (SOD1) mutants

cause mitochondrial dysfunction and neuronal death were

investigated.11

Proteomic analysis often involves the identification and

quantification of expressed protein components in cells, tissue

and organisms. It is a useful tool in investigating biological

events as it provides significant information about the relevant

gene products and how their levels and modifications change

in response to the effects of various internal and external

factors. The quantitative profiling of tryptic digest of proteins

in complex mixtures without isotope labeling using liquid

chromatography and mass spectrometry had been reported.

The expected and calculated protein ratios differed no more

than 16%.12 An approach using proteolytic digest with two

dimensional liquid chromatography nanospray mass spectro-

metry without the use of stable isotope was applied for the

study of differential protein expression in epidermal cell lines

grown in the presence or absence of epidermal growth factor.13

In our laboratory, a method using proteolytic digest with two-

dimensional liquid chromatography with tandem mass spec-

trometry without the use of stable isotope was used to identify

differentially expressed proteins in human liver cancer cell lines

(HepG2) and colon cancer cell lines, LoVo in response to the

standardized extract from Scutellariae radix14 and Scutellaria

barbata.15 At the same time, the method was applied for the

profiling of differentially expressed proteins of mouse liver in

the control group and groups treated with standardized

extract from Scutellariae radix.16 The proposed method was

able to identify changes at the molecular level and have

satisfactory level of reproducibility.14–17 Currently, other label

free protein quantitation with LC/MSMS from complex pro-

teomes had been reported.18–20

In this work, we will study the inhibitory effects of kinetin

riboside in human heptamoa, HepG2. The mitochondrial

proteins obtained from the control and treated cells were

digested with trypsin and the peptides were separated by

reversed phase liquid chromatography tandem mass spectro-

metry (LC/MSMS). For the current method, labeling of cells

with stable isotope would not used. The method developed

allowed the molecular mechanism due to the inhibitory effects

of the kinetin riboside to be investigated. The differentially

expressed proteins identified using the current method will be

used to correlate with cell viability assay, cell cycle analysis

and activation of caspase 3 with flow cytometry.

Experimental

Chemicals and reagents

Dulbecco’s modified Eagles medium (DMEM), penicillin,

streptomycin, trypsin–EDTA were obtained from Hyclone

(Logan, Utah, USA). Fetal bovine serum (FBS) was obtained

from Biological Industries (Israel). Dimethyl sulfoxide

(DMSO), methanol and acetonitrile of HPLC grade were

purchased from APS (NSW, Australia). Ultra pure water

was obtained from Millipore Alpha-Q water system (Bedford,

MA, USA). Sequencing grade modified trypsin was purchased

from Promega (Madison, WI). Formic acid and ammonium

acetate were purchased from Merck (Darmstadt, Germany).

Kinetin riboside (499.0% purity, MW: 347.33) was purchased

from Olchemim (Olomouc, Czech).

Cell cultures

All cell lines were obtained from ATCC. Human Hepatoma

HepG2 was maintained with Eagle’s minimum essential

medium with Earle’s BSS and 2 mM L-glutamine (EMEM)

that was modified to contain 1 mM sodium pyruvate, 0.1 mM

nonessential amino acids and 1.5 g l�1 sodium bicarbonate

and supplemented with 10% fetal bovine serum (FBS),

100 U ml�1 Penicillin, 100 mg ml�1 Stretomycin, incubated

at 37 1C, and 5% CO2 atmosphere. Initially, 10 mg of kinetin

riboside was dissolved in 10 ml of water to form stock

solution A.

Cell viability with MTT assay

HepG2 cells were seeded at a concentration of 5 � 104

cells ml�1 in a 96 well plate. After overnight incubation,

serial concentrations of kinetin riboside were added. Each

concentration was repeated three times. These cells were

incubated in a humidified atmosphere with 5% CO2 for

2 days. Then, 20 ml MTT (3-(4,5-dimethylthiazol-2-yl)-2,

5-diphenyl tetrazolium bromide) (Sigma) solution (4.14 mg ml�1)

was added to each well and incubated at 37 1C for 4 h.

The medium was removed and formazan was dissolved in

DMSO and the optical density was measured at 590 nm using

a Bio-assay reader (BioRad, USA). The growth inhibition was

determined using: growth inhibition = (control O.D. �sample O.D.)/control O.D.

Flow cytometry analysis

A total of 1 � 106 cells were plated on 75 cm2 tissue culture

flask and incubated for 24 h at 37 1C. Cells were treated with

extracts from kinetin riboside or control with medium for

another 48 h. Cells were then washed, pelleted, fixed with cold

70% ethanol for at least 30 min. Before analysis, the

70% ethanol was removed by spinning at 2000 rpm and washed

with phosphate saline buffer (PBS). The resulting solution

was incubated with 100 mg ml�1 Rnase A and 50 mg ml�1 of

propidium iodide at room temperature for 30 min. Samples

were immediately analyzed by flow cytometry (Becton

Dickinson, San Jose, CA). Cell cycle distribution was

determined using Modfit software (Verify Software House,

Topsham, ME).

92 | Mol. BioSyst., 2009, 5, 91–98 This journal is �c The Royal Society of Chemistry 2009

Flow cytometric analysis of active caspase 3

A total of 1 � 105 of HepG2 cells were plated on 25 cm2 tissue

culture flask and incubated for 24 h at 37 1C. Cells were

treated with kinetin riboside or control with medium for

another 48 h. The study of the activation of caspase 3 was

done using a kit available from BD Biosciences (Becton

Dickinson, San Jose, CA). Briefly, the cells were washed,

pelleted, thoroughly resuspended with 250 ml of BD Cytofix/

Cytoperm solution and incubated for 20 min at 4 1C. The cells

(1 � 106 cells) were then washed 2 times in a buffer containing

a cell permeabilizing agent such as saponin (BD Perm/Washt

buffer). Finally, the cells were thoroughly resuspend fixed/

permeabilized in 50 ml of BD Perm/Washt buffer containing

the anti active caspase 3 monoclonal antibodies mAb

(C92-605). The cells were allowed to incubate at room

temperature for 60 min in the dark. Following incubation,

cells were washed with BD Perm/Washt buffer, resuspended

in BD Perm/Washt buffer and analyzed by flow cytometry.

For positive control, HepG2 cells were treated with 1 mMstaurosporine and 2 mM of camptothecin for 48 h.

Mitochondrial protein extraction and protein analysis

A total of 1 � 105 cells were plated on a 75 cm2 tissue culture

flasks and incubated for 24 h at 37 1C under 5% CO2. Cells

were treated with kinetin riboside (8.33 and 16.67 mg l�1) or

control with medium for another 48 h. The mitochondria

proteins were isolated using a kit from Pierce (Rockford,

IL). Cells were trypsinized and pelleted at 2000 rpm for

5 min. The supernatant was discarded and 1 ml phosphate

buffered saline (PBS) was used to reconstitute the pellet. It was

centrifuged at 2000 rpm for another 5 min and the supernatant

was removed. Proteins were extracted with 800 ml reagent Aand vortexed at medium speed for 5 s. It was incubated on ice

for 2 min. 10 ml reagent B was added and vortexed at

maximum speed for 5 s. The tube was incubated on ice for

5 min and vortexed at maximum speed every minute. 800 mlReagent C was added and the tube was inverted several times

to mix. It was then centrifuged at 2500 rpm for 10 min at 4 1C.

The supernatant was pelleted at 9500 rpm for 15 min at 4 1C.

500 ml Reagent C was added to the pellet and centrifuged at

9500 rpm for another 5 min. The supernatant was discarded

and 0.5 ml of M-Per (Pierce, Rockford, IL) was added to

reconstitute the pellet. Protein concentration was assayed

using Bradford assay reagent (Pierce, Rockford, IL).

The proteins were reduced with DTT (3 ml of 1000 mM in

water). The mixture was incubated at 37 1C for 30 min. To

alkylate the protein, iodoacetamide (7 ml of 1000 mM in 0.1 M

KOH) was added and the mixture was incubated at room

temperature for an additional 30 min in the dark. DTT (13 mlof 1000 mM in water) was added to react with excess iodo-

acetamide. The reduced and alkylated proteins were digested

with sequencing grade trypsin (1 : 50) for 18 h.

For C-18 SPE, each 500 mg Strata (Phenomenex, USA)

C-18 SPE column was conditioned with 10 ml of methanol,

water and water with 0.1% acetic acid before loading the

enzymatically digested samples. The loaded SPE columns

were washed with 5 ml of water with 0.1% formic acid before

eluting with 0.5 ml acetonitrile. Excess solvent for each of the

fractions collected was evaporated in a speedvac. All fractions

were reconstituted in 100 ml of water with 0.1% acetic acid

prior to analysis by LC/MSMS.

Reversed phase HPLC/MSMS analysis for protein expression

For LC/MSMS experiments, an Agilent 1100 series (Waldbronn,

Germany) equipped with a quaternary gradient pump, auto-

sampler with sample cooler, column oven and diode array

detector was coupled with a Agilent LC/MSD Trap VL ion trap

mass spectrometer (Waldbronn, Germany). The gradient elution

involved a mobile phase consisting of (A) water with 0.1%

formic acid and (B) acetonitrile with 0.1% formic acid. The

initial condition was set at 5% of (B), gradient up to 40% in

70 min, up to 90% in the next 5 min and returning to initial

condition for 15 min. Oven temperature was set at 40 1C and

flow rate was set at 200 ml min�1. For all experiments, 30 mlof sample was injected. The column used for the separation

was a reversed phase C18 Jupiter, 150 � 2.0 mm, 5 m, 300 A

(Phenomenex, USA). The ESI-MS was acquired in the positive

ion mode. The scanning mass range was from 400 to 1500. The

heated capillary temperature was maintained at 350 1C, the

drying gas and nebulizer nitrogen gas flow rates were 10 l min�1

and 50 psi, respectively. Data were acquired using auto-

mated MSMS. The target was set at 30000, maximum accumu-

lation time: 300 ms, the number of average scans was 5 and

SmartSelectt was used.

Database searching procedure of MS/MS data for protein

identification

Mass data collected during a LC/MSMS run were submitted

to the search software Mascot (http://www.matrixscience.com).

Preliminary protein identifications were obtained by com-

paring experimental data to NCBInr database and Swiss-prot.

The taxonomy was set to Homo sapien (human), 1 missed

cleavage was allowed and carbamidomethyl was selected for

fixed modifications. Peptide charge was set at 2+ and 3+.

Searches were done with a tolerance on mass measurement of

1.0 Da in MS mode. From the searches obtained fromMascot,

only the first five hits were considered. For MS/MS ion search,

proteins with one peptide ion scoring higher than 45 or two

peptide ions scoring higher than 30 were considered an

unambiguous identification without manual inspection.

A sequence tag of several continuous amino acids (5–20

residues) and the peptide mass were generally sufficient to

identify the protein of a peptide. The sub-cellular location and

tissue specificity of the proteins identified was examined using

Swiss-prot. The raw data were inspected manually for con-

firmation prior to acceptance.

Results

Growth inhibition of kinetin ribosides on human liver cancer cell

lines

The effect of different doses of kinetin riboside on cell growth

was examined on HepG2 cell lines. Under the experimental

conditions used with 48 h treatment, kinetin riboside exhibited a

marked growth inhibitory effect on HepG2 in a dose-dependent

manner (Fig. 1B). The IC50 was approximately 8.33� 0.73 mg l�1

This journal is �c The Royal Society of Chemistry 2009 Mol. BioSyst., 2009, 5, 91–98 | 93

for HepG2. A significant cell shrinkage and change in cell shape

was observed in HepG2 cells treated with a higher concentration

of kinetin riboside.

The effects of kinetin riboside on cell proliferation may be

due to cell cycle regulation, we examined the effect on cell cycle

perturbation using flow cytometric analysis. It was observed

that kinetin ribosides at 1.67 mg l�1 did not induce a mark

increase sub-G0 phase in HepG2 cells. However, different

doses of kinetin riboside were observed to decrease the popu-

lation of G1 phase and an increased in the G2/M phase after

48 h. At the same time, a marked increase in sub-G0 phase

was observed in a dose dependent manner (Fig. 2). The results

were repeated on a different day and a similar trend was

observed. The results showed that kinetin riboside induced

in vitro growth inhibition of HepG2 cells was mediated by

causing G2/M cell cycle arrest and cell death.

Treatment with different doses of kinetin riboside for 48 h

did not result in induced capase-3 activity in HepG2 cells

(Fig. 3). From Fig. 3A, the untreated HepG2 cells were

primarily negative for active caspase 3. The different doses

of kinetin riboside did not result in any activation of active

caspase 3 (Fig. 3B, C, D and E). As a positive control, human

liver cells treated with 1 mM staurosporine that will result in

the activation of active caspase 3 was used.21 The determina-

tion of caspase 3/7 activities in HepG2 cells treated for 24 h

with butyric acid, carbonyl cyanide 4 (trifluoromethoxy)

phenylhydrazone and camptothecin was reported.22 By

comparing the results obtained with the positive control, the

different doses of kinetin ribosides did not result in a signi-

ficant activation of active caspase 3. For the positive control

(Fig. 3F), activation of caspase 3 can be seen in the changes of

the profile of the stained and unstained HepG2 cells. We

propose that HepG2 cells treated with different doses of

kinetin ribosides had resulted in a significant degree of DNA

fragmentation and cell death. At the same time, the various

doses used did not result in a population of cells positive for

the active caspase 3.

Effect of kinetin riboside on cell mitochondria protein expression

by HPLC/MSMS

Using reversed phase LC/MSMS, we next assessed the effect of

kinetin riboside on the expression of mitochondria proteins

that may be involved in the control of cellular proliferation.

The reproducibility of the proposed method was validated by

profiling the mitochondria proteins from 3 different untreated

HepG2 cells (Fig. 4A, B and C). By superimposing the

Fig. 2 Cell cycle analysis by flow cytometry. The HepG2 cells lines were

(A) treated withmedium for control, (B) treated with 1.67mg l�1 of kinetin

riboside (C) treated with 8.33 mg l�1 of kinetin riboside, (D) treated with

16.67 mg l�1 of kinetin riboside and (E) treated with 33.33 mg l�1

of kinetin riboside for 48 h. The significant increase in the sub G0 phase

in C, D and E is indicative of cell death.

Fig. 3 Flow cytometric analysis of active caspase 3 in HepG2 cells using

the anti-active caspase 3 mAb (clone C92-605). Stained and unstained

HepG2 cells were (A) left untreated (control), (B) treated with 1.67 mg l�1

of kinetin riboside, (C) treated with 8.33 mg l�1 of kinetin riboside, (D)

treated with 16.67 mg l�1 of kinetin riboside, (E) treated with 33.33 mg l�1

of kinetin riboside and (F) stained and unstainedHepG2 cells treated with

1 mM of staurosporine for 48 h (positive control). The untreated HepG2

cells were primarily negative for active caspase 3. The higher doses kinetin

riboside (4.0 and 24.0 mg l�1) did not result in a significant activation of

active caspase 3.

94 | Mol. BioSyst., 2009, 5, 91–98 This journal is �c The Royal Society of Chemistry 2009

chromatograms (TIC) from the all three different untreated

groups, it was found that good reproducibility was obtained

from cell lysates from 3 different flasks. The experiments were

repeated on a different day with a different analyst and a

similar reproducibility was observed. Using the current

LC/MSMS approach, however, higher degree of variation of

the chromatographic profile was observed from cell lysates

obtained from different days. The method variation on the

same day was estimated to be less than 20% and our data

showed the feasibility of profiling the tryptic digest of the

mitochondria proteins without the use of isotope labeling.

From the data obtained in Fig. 4, it was clear that by

superimposing the different chromatograms obtained, it was

possible to identify differentially expressed peptides from the

mitochondria proteins.

The chromatograms in Fig. 5A and B showed the tryptic

digest of two different cell lysates with reversed phase HPLC/

MSMS from the HepG2 cell lines for the control and cell line

treated with 16.67 mg l�1 of kinetin riboside, respectively. By

superimposing the chromatograms (TICs) from Fig. 5A and

B, a number of peptides that remained unchanged in the

control and treated group were obtained. It was proposed

that they were likely to be house keeping proteins as observed

in our earlier works.14–16 With the assistance of peptides from

house keeping proteins, the identification of peptides from

proteins where their expression had been modified can be

obtained. Hence, a list of mitochondria proteins (Table 1)

where the expression were changed by more than 2 fold after

treatment with 16.67 mg l�1 of kinetin riboside was identified

using tandem mass spectrometry. At the same time, we did

not observe significant changes in the mitochondria protein

expression for HepG2 cells treated with 8.33 mg l�1 of kinetin

riboside. The criteria was selected as it was observed that the

proposed method would not induce variation higher than

2 fold (peak reduction or increased by two times).

The current approach of identification of proteins was based

on our earlier works14–17 and other reports.11,13,23–28 The

potential for false positive identifications from large databases

through tandem mass spectra data had been discussed.28,29

Fig. 4 Chromatograms (TIC) from LCMSMS of three different cell

lysates of the mitochondria proteins from untreated Hep G2 cell line.

With the assistance of the software, by superimposing the chromato-

grams (TIC) from the all the different untreated groups it was

found that good reproducibility was obtained from cell lysates from

3 different flasks.

Fig. 5 Chromatograms (TIC) from LCMSMS of mitochondria

proteins from (A) HepG2 cell line, control with medium, (B) HepG2

cell line, treated with 16.67 mg l�1 of kinetin riboside for 48 h. The box

regions are where differential expressed peptides (up-regulated or

down-regulated more than two times) were observed. Peak 1–6 is as

labeled in Table 1.

This journal is �c The Royal Society of Chemistry 2009 Mol. BioSyst., 2009, 5, 91–98 | 95

Hence, for the searches obtained from Mascot, websites

such as Swiss-prot (http://us.expasy.org/sprot) and NCBInr

database (http://www.ncbi.nlm.nih.gov) were used to exa-

mine domains and motifs present in identified proteins. In

this work, the proteins identified must be present in the

mitochondria and the tissue specificity was examined using

Swiss-prot.

Discussion

Cytokinin ribosides such as kinetin riboside, isopentenyl-

adenosine and benzyl-aminopurine were found to be the

most potent for growth inhibition and apoptosis in human

myeloid leukemia cells (HL-60). Cytokinin ribosides greatly

reduced the intracellular ATP content and disturbed the

mitochondrial membrane potential and accumulation of

reactive oxygen species, whereas cytokinin did not.3 The

growth inhibition and induction of cell death of kinetin

ribosides in HepG2 in this study was consistent with other

works on other cell lines.3,4

Active caspase 3 is a marker for cells undergoing apopto-

sis.30,31 Caspases were originally identified as the mediators of

apoptosis where it was hypothesized that many of their

substrates were essential proteins whose destruction ensured

the inevitability of cell death. However, caspase-independent

cell death is observed in many systems indicating that cells still

die even if the executioner is absent.30,32–35 Cell death induced

by 5-hydroxyphenyl butanoate retinamide in human cancer

cell lines such as HCT116 and MCF7 did not result in

cleavages of caspase 3 and 8. However, cleavages of caspases

3 and 8 were evident in PLC/PRF/5 and CaSki cells.36

Activation of caspase 3 was observed within 6 h after treat-

ment of HL-60 cells with kinetin riboside.3 For HepG2 cells,

treatment with different doses of kinetin riboside had resulted

in cell death but not the activation of active caspase 3.

Mitochondria are an important part of the apoptotic

machinery. The mechanisms of action of chemically diverse

small molecules on specific mitochondria loci such as respira-

tory chain, DNA biogenesis, potassium channels, the Bcl-2

family proteins and the permeability transition pores were well

reported.9,37 Mitochondrial damage promotes apoptosis in

two ways. On the one hand, it leads to the release of apopto-

genic factors and on the other hand, it disrupts energy

production of the cell. The ability of kinetin riboside to induce

cell death and attenuate G1 to S transition is probably a

consequence of its ability to interfere with several components

in the mitochondria.

Genetic and biochemical studies have demonstrated that

Bcl-2 family proteins are central to the regulation of mito-

chondria membrane permeabilization. The Bcl-2 family

proteins localize or can be targeted to the mitochondria and

regulate the permeability of the outer membrane to various

apoptotic factors.8,9,35 The identification of the Bcl2-antagonist

of cell death protein with the current method was

consistent with all the data obtained from cell viability assay

and cell cycle analysis. It is located at the outer mitochondrial

membrane, upon phosphorylation, it locates to the cytoplasm.

The Bcl2-antagonist of cell death protein encoded by this

gene is a member of the Bcl-2 family. This protein positively

regulates cell apoptosis by forming heterodimers with

Bcl-xL and Bcl-2, and reversing their death repressor activity.

Proapoptotic activity of this protein is regulated through

its phosphorylation. Protein kinases AKT and MAP kinase,

as well as protein phosphatase calcineurin were found to be

involved in the regulation of this protein.38

Heat shock proteins (HSP) are the products of several

distinct gene families that are required for cell survival during

stress.39 The significances of chaperonin 10-mediated inhibi-

tion of ATP hydrolysis by chaperonin 60 was reported.40

Recent works have proposed the anti-apoptotic role of two

heat shock proteins, Hsp10 and Hsp60 in various cells. These

Table 1 Identification of mitochondria proteins in the HepG2 cell line found to be significantly different (more than 2 times) in the Control andTreated Group (16.67 mg l�1)

Accession Mass/Da Description Up/down regulated Proposed functions

1 P49448 61 434 Glutamate dehydrogenase[NAD (P)]

Down regulated Molecular function: electron transporter activity;glutamate dehydrogenase activity.Biological process: glutamate catabolism.

2 Q92934 18 392 Bcl2-antagonist of cell death Down regulated Molecular function: protein binding.Biological process: apoptotic program; andinduction of apoptosis

3 Q9UNM1 10 295 Chaperonin 10-related protein[Fragment]

Up regulated Molecular function: ATP binding, unfoldedprotein binding.Biological process: protein folding.

4 Q5KTR4 111 651 Flavoprotein oxidoreductase Up regulated Molecular function: oxidoreductase activity; zincion bindingBiological processs: electron transport; andmetabolism

5 P40939 83 000 Trifunctional enzyme alphasubunit, mitochondrial[Precursor]

Down regulated Molecular function: 3-hydroxylacyl-CoAdehydrogenase activity; acetyl-CoAC-acetyltransferase activity; and enoyl-CoAhydratase activity.Biological function: lipid metabolism, metabolismand fatty acid metabolism

6 Q5SZ02 19 762 Mitochondrial ribosomalprotein L24 [Fragment]

Down regulated Molecular function: structural constituent ofribosomeBiological process: protein biosynthesis.

96 | Mol. BioSyst., 2009, 5, 91–98 This journal is �c The Royal Society of Chemistry 2009

two proteins can be induced when cells are under stress. An

over-expression of Hsp10 and Hsp60 differentially modulated

the Bcl-2 family and in turn attenuate doxorubicin induced

apoptosis in primary cardiomyocytes.41 Over-expression of

Hsp10 by adenoviral infection decreased myocyte death

induced by hydrogen peroxide, sodium cyanide, simu-

lated ischemia and reoxygenation.42 The identification of

Chaperonin 10-related protein in HepG2 cells treated with

kinetin riboside was consistent with the proposition of cell

death. The up-regulation of Chaperonin 10-related pro-

tein and down-regulation of Bcl2-antagonist of cell death

suggested a possible link between the two proteins.

Electron transport is carried out in the mitochondrial inner

membrane through a series of membrane-embedded proteins

that communicate via several smaller molecules, the lipid-

soluble ubiquinone and the water soluble protein cytochrome c.6

In our current work, the growth inhibition of HepG2 cells with

kinetin riboside resulted in proteins such as glutamate dehy-

drogenase, flavoprotein oxidoreductase and trifunctional en-

zyme alpha subunit, mitochondrial [Precursor] that were

involved in electron transport and metabolism to be differen-

tially expressed. For trifunctional enzyme alpha subunit,

mitochondrial [Precursor], it encodes the alpha and beta

subunits of the mitochondrial trifunctional protein, respec-

tively. The heterocomplex contains 4 alpha and 4 beta sub-

units and catalyzes 3 steps in mitochondrial beta-oxidation of

fatty acids, including the long-chain 3-hydroxyl-CoA dehydro-

genase (LCHAD) step. The alpha subunit harbors the

3-hydroxyacyl-CoA dehydrogenase and enoyl-CoA hydratase

activities.38 Lastly, cell death induced by kinetin riboside had

affected the mitochondrial ribosomal protein L24 [Fragment]

that was involved in protein biosynthesis.

Conclusions

This paper reports our current data on the inhibitory effects of

kinetin ribosides in human cancer cell lines. Together with

other techniques, the current method allows us to study the

differentially expressed proteins in the mitochondria due to the

inhibitory effects of different doses of kinetin riboside in

human liver cancer cells, HepG2. The data obtained from cell

cycle analysis with flow cytometry provides some insights for

the proteins that may be differentially expressed in HepG2 cell

lines treated with kinetin riboside.13,14 In our earlier work,

we did not identify any differentially expressed proteins that

were associated with signal transduction, cell cycle and cell

death when significant changes in sub G0, G0/G1, S and G2/M

phases were not observed in cell cycle analysis with flow

cytometry.13

Without the use of stable isotope labeling, the proposed

method provided a rapid approach to study the molecular

mechanism due to the inhibitory effects of different doses

of kinetin riboside on HepG2 cell lines. The differentially

expressed proteins identified in the mitochondria were consis-

tent with what was obtained from cell cycle analysis with flow

cytometry. It provided further information on how the mito-

chondria play a critical role in the life of the cell and as a key

regulator of mammalian apoptotic cell death. The cell death

caused by kinetin riboside in HepG2 cells did not result in an

activation of active caspase 3 and affected a network of

proteins involved in cell death and electron transport.

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