Metformin plus PIAF combination chemotherapy for hepatocellular carcinoma
Objectives: Metformin, the most used oral antidiabetic drug for the treatment of type 2 diabetus mellitus, has proved encouraging results when used in the treatment of various types of cancer such as triple-negative breast cancer. Despite compelling evidence of a role of metformin as an anticancer d...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
2012
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| Цитувати: | Metformin plus PIAF combination chemotherapy for hepatocellular carcinoma / B. Petrushev, C. Tomuleasa, O. Soritau, M. Aldea, T. Pop, S. Susman, G. Kacso, I. Berindan, A. Irimie, V. Cristea // Experimental Oncology. — 2012. — Т. 34, № 1. — С. 17-24. — Бібліогр.: 35 назв. — англ. |
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irk-123456789-1386852018-06-20T03:05:32Z Metformin plus PIAF combination chemotherapy for hepatocellular carcinoma Petrushev, B. Tomuleasa, C. Soritau, O. Aldea, M. Pop, T. Susman, S. Kacso, G. Berindan, I. Irimie, A. Cristea, V. Original contributions Objectives: Metformin, the most used oral antidiabetic drug for the treatment of type 2 diabetus mellitus, has proved encouraging results when used in the treatment of various types of cancer such as triple-negative breast cancer. Despite compelling evidence of a role of metformin as an anticancer drug, the mechanisms by which metformin exerts its oncostatic actions are not fully understood yet. Therefore, we tried to bring new insights by analyzing the anti-neoplastic effect of metformin for hepatocellular carcinoma-derived stem-like cells treated with conventional combination chemotherapy. Methods: Cancer stem-like cells previusly isolated from a hepatocellular carcinoma biopsy were treated with metformin, PIAF chemotherapy regimen and the combination of these two protocols. Measurements of lipid peroxidation, reduced glutathione, fluorescein diacetate and proliferation rates were determined, apart from the autophagy assay and apoptosis determination by chip flow cytometry. Results: Metformin alone and especially metformin in association with PIAF increases oxidative stress within the cells by increasing the levels of lipid peroxids as well as decreasing the levels of reduced glutathione. The MTT cell proliferation assay showed decreased proliferation rates for the arm treated with metformin and with the combination of drugs in comparison with the control arm, proving high correlation with the oxidative stress results. The autophagy assay and determination of apoptosis by chip flow cytometry confirmed the results obtained in the previous assays. Conclusion: Metformin could be used in chemotherapy treatments to induce reactive oxygen species and increase the cytostatics effects within the tumor cell. Still, further experiments must be carried out on murine models before we can move on and use this drugs in the adjuvant setting for unresectable primary liver cancer. 2012 Article Metformin plus PIAF combination chemotherapy for hepatocellular carcinoma / B. Petrushev, C. Tomuleasa, O. Soritau, M. Aldea, T. Pop, S. Susman, G. Kacso, I. Berindan, A. Irimie, V. Cristea // Experimental Oncology. — 2012. — Т. 34, № 1. — С. 17-24. — Бібліогр.: 35 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/138685 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Original contributions Original contributions |
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Original contributions Original contributions Petrushev, B. Tomuleasa, C. Soritau, O. Aldea, M. Pop, T. Susman, S. Kacso, G. Berindan, I. Irimie, A. Cristea, V. Metformin plus PIAF combination chemotherapy for hepatocellular carcinoma Experimental Oncology |
| description |
Objectives: Metformin, the most used oral antidiabetic drug for the treatment of type 2 diabetus mellitus, has proved encouraging results when used in the treatment of various types of cancer such as triple-negative breast cancer. Despite compelling evidence of a role of metformin as an anticancer drug, the mechanisms by which metformin exerts its oncostatic actions are not fully understood yet. Therefore, we tried to bring new insights by analyzing the anti-neoplastic effect of metformin for hepatocellular carcinoma-derived stem-like cells treated with conventional combination chemotherapy. Methods: Cancer stem-like cells previusly isolated from a hepatocellular carcinoma biopsy were treated with metformin, PIAF chemotherapy regimen and the combination of these two protocols. Measurements of lipid peroxidation, reduced glutathione, fluorescein diacetate and proliferation rates were determined, apart from the autophagy assay and apoptosis determination by chip flow cytometry. Results: Metformin alone and especially metformin in association with PIAF increases oxidative stress within the cells by increasing the levels of lipid peroxids as well as decreasing the levels of reduced glutathione. The MTT cell proliferation assay showed decreased proliferation rates for the arm treated with metformin and with the combination of drugs in comparison with the control arm, proving high correlation with the oxidative stress results. The autophagy assay and determination of apoptosis by chip flow cytometry confirmed the results obtained in the previous assays. Conclusion: Metformin could be used in chemotherapy treatments to induce reactive oxygen species and increase the cytostatics effects within the tumor cell. Still, further experiments must be carried out on murine models before we can move on and use this drugs in the adjuvant setting for unresectable primary liver cancer. |
| format |
Article |
| author |
Petrushev, B. Tomuleasa, C. Soritau, O. Aldea, M. Pop, T. Susman, S. Kacso, G. Berindan, I. Irimie, A. Cristea, V. |
| author_facet |
Petrushev, B. Tomuleasa, C. Soritau, O. Aldea, M. Pop, T. Susman, S. Kacso, G. Berindan, I. Irimie, A. Cristea, V. |
| author_sort |
Petrushev, B. |
| title |
Metformin plus PIAF combination chemotherapy for hepatocellular carcinoma |
| title_short |
Metformin plus PIAF combination chemotherapy for hepatocellular carcinoma |
| title_full |
Metformin plus PIAF combination chemotherapy for hepatocellular carcinoma |
| title_fullStr |
Metformin plus PIAF combination chemotherapy for hepatocellular carcinoma |
| title_full_unstemmed |
Metformin plus PIAF combination chemotherapy for hepatocellular carcinoma |
| title_sort |
metformin plus piaf combination chemotherapy for hepatocellular carcinoma |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2012 |
| topic_facet |
Original contributions |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/138685 |
| citation_txt |
Metformin plus PIAF combination chemotherapy for hepatocellular carcinoma / B. Petrushev, C. Tomuleasa, O. Soritau, M. Aldea, T. Pop, S. Susman, G. Kacso, I. Berindan, A. Irimie, V. Cristea // Experimental Oncology. — 2012. — Т. 34, № 1. — С. 17-24. — Бібліогр.: 35 назв. — англ. |
| series |
Experimental Oncology |
| work_keys_str_mv |
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| first_indexed |
2025-07-10T06:21:54Z |
| last_indexed |
2025-07-10T06:21:54Z |
| _version_ |
1837239915654414336 |
| fulltext |
Experimental Oncology ��� ������ ���� ��arc����� ������ ���� ��arc�� ��arc�� ��
METFORMIN PLUS PIAF COMBINATION CHEMOTHERAPY
FOR HEPATOCELLULAR CARCINOMA
B. Petrushev1,•, C. Tomuleasa2,3,*,•, O. Soritau3, M. Aldea1, T. Pop1,4,
S. Susman1, G. Kacso1,5, I. Berindan1,6, A. Irimie1,7, V. Cristea1,4
1Department of Medicine — Iuliu Hatieganu University of Medicine and Pharmacy, Cluj Napoca
2Department of Medicine, Division of Gastroenterology and Hepatology, The Johns Hopkins University
School of Medicine, Baltimore, MD, United States of America
3Department of Immunology, Ion Chiricuta Comprehensive Cancer Center, Cluj Napoca, Romania
4Octavian Fodor Regional Institute of Gastroenterology and Hepatology, Cluj Napoca, Romania
5Department of Medical Oncology, Ion Chiricuta Comprehensive Cancer Center, Cluj Napoca, Romania
6Department of Genetics, Ion Chiricuta Comprehensive Cancer Center, Cluj Napoca, Romania
7Department of Surgery, Ion Chiricuta Comprehensive Cancer Center, Cluj Napoca, Romania
Objectives: Metformin, the most used oral antidiabetic drug for the treatment of type 2 diabetus mellitus, has proved encouraging
results when used in the treatment of various types of cancer such as triple-negative breast cancer. Despite compelling evidence
of a role of metformin as an anticancer drug, the mechanisms by which metformin exerts its oncostatic actions are not fully under-
stood yet. Therefore, we tried to bring new insights by analyzing the anti-neoplastic effect of metformin for hepatocellular carci-
noma-derived stem-like cells treated with conventional combination chemotherapy. Methods: Cancer stem-like cells previusly
isolated from a hepatocellular carcinoma biopsy were treated with metformin, PIAF chemotherapy regimen and the combination
of these two protocols. Measurements of lipid peroxidation, reduced glutathione, fluorescein diacetate and proliferation rates were
determined, apart from the autophagy assay and apoptosis determination by chip flow cytometry. Results: Metformin alone and
especially metformin in association with PIAF increases oxidative stress within the cells by increasing the levels of lipid peroxids
as well as decreasing the levels of reduced glutathione. The MTT cell proliferation assay showed decreased prolife ration rates for
the arm treated with metformin and with the combination of drugs in comparison with the control arm, proving high correlation
with the oxidative stress results. The autophagy assay and determination of apoptosis by chip flow cytometry confirmed the results
obtained in the previous assays. Conclusion: Metformin could be used in chemotherapy treatments to induce reactive oxygen species
and increase the cytostatics effects within the tumor cell. Still, further experiments must be carried out on murine models before
we can move on and use this drugs in the adjuvant setting for unresectable primary liver cancer.
Key Words: metformin, chemotherapy, hepatocellular carcinoma, stem-like cells.
In ancient times� �epatology was inexistent� and
liver was a source of legends and spirituality. Two
t�ousand years BC t�e liver was t�oug�t to bear t�e
soul and priests used �epatoscopy in animals as a tool
for divine connection in order to predict t�e future and
t�e outcome of a certain military endeavor [�� �]. One
of t�e most famous legends was written by Hesiod and
describes Promet�eus stealing fire from Zeus� before
being puni�sed by t�e king of t�e gods to be c�ained
to a rock and �aving �is liver eaten by an eagle. But
t�e liver regenerated and regained its normal size
overnig�t [�� �]. Over time� t�e progress in t�e field
of �epatology and liver surgery was mostly for onco-
logic diseases� becoming more and more accurate and
sop�istiucated and years passed. An important step
for t�e improved outcome is t�e understanding t�at
liver cancer must be treated in specialized� interdis-
ciplinary centers w�ere a close relations�ip between
surgery� internal medicine and basic researc� �ave
t�e common goal to diagnose and cure t�is infermity.
Liver cancer is t�e 6t� most common cancer world-
wide and t�e t�ird most common cause of cancer
mortality on t�e planet� wit� more t�an �alf a million
deat�s annually� indicating t�at t�is pat�ology is not
only common� but also deadly. T�e �ig�es incidence
rates are in sub-Sa�aran Africa and eastern Asia� wit�
C�ina accounting for more t�an 5�% of cases [5]. He-
patocellular carcinoma �HCC� incidence is �ig� among
young adults and varies among different populations
and races� suggesting t�at t�is neoplasia is caused
by seve ral etiologal factors� t�e synergy of w�om �as
been s�own to be signifficant in �epatocarcinogenesis.
Risk factors include �epatitis B virus and �epatitis C vi-
rus infection� alco�ol consumption� smoking� aflatoxin
E exposure� �ormonal intake� occupational exposures
and c�ronic medical conditions suc� as metabolic
syndrome� diabetes mellitus� obesity or even t�yroid
diseases [6]. �ost cases of �epatocellular carcinoma
are diagnosed in an advanced stage because of t�e
relatively scarce symptoms. T�e patients survival
depends not only on tumor c�araceristics� but also
on t�e extent of t�e undelying disease. Fur�ermore�
t�e t�erapeutic agents available for HCC and t�e indi-
cations for aggresive t�erapy expand and an increase
of an interdisciplinary approac� between surgery� ra-
Received: December 8, 2011.
*Correspondence: E-mail: ciprian.tomuleasa@gmail.com
Authors marked with (•) contributed equally to this paper.
Abbreviations used: CSC — cancer stem-like cells; HCC — hepatocel-
lular carcinoma; PIAF — Cisplatin/Interferon α-2b/ Doxorubicin/5-
Fluorouracil, with Capecitabine replacing 5-Fluorouracil.
Exp Oncol ����
��� �� �����
ORIGINAL CONTRIBUTIONS
�8 Experimental Oncology ��� ������ ���� ��arc��
diot�erapy and c�emot�erapy is expected to c�allenge
our understanding of prognostic factors for HCC [�].
Encouraging results �ave recently emerged from
data publis�ed on breast cancer and point towards
metformin� an oral-antidiabetic used for decades in t�e
treatment of type � diabetes mellitus� as an efficient
anticancer drug [8]. �etformin �as already been
investigated by our team for t�e treatment of WHO
grade III and IV malignant gliomas in t�e laboratory
settings [9]� results being confirmed by retrospec-
tive epidemiological studies t�at reported a decrease
in cancer risk and also a better response to c�emo-
t�erapy of diabetic patients wit� breast cancer treated
wit� metformin [��].
In t�e current researc� paper� we studied t�e effect
of metformin plus PIAF �Cisplatin/Interferon α-�b/
Doxorubicin/5-Fluorouracil� wit� Capecitabine repla-
cing 5-Fluorouracil� wit� combination c�emot�erapy
on malignant liver stem-like cells in order to furt�er
investigate t�e basic mec�anisms by w�ic� t�is as-
sociation of drugs exerts its action on t�e cancer cell.
T�e aim of t�e current paper is to investigate t�e cor-
relation between an old drug and t�e best standard-
of-care for unresectable �epatocellular carcinoma�
wit� t�e final goal of improving t�e t�erapeutic index
of suc� patients.
MATERIALS AND METHODS
Reactives. �etformin ����-dimet�ylbiguanide
�ydroc�loride� was purc�esed from Wurwag P�arma
�Buc�arest� Romania� and used in a final concentra-
tion of �� m� diluted in p�osp�ate buffer solution
�PBS�. It was added prior to a slig�tly modiffied PIAF
regimen� wit� capecitabine replacing 5-fluoroura-
cil� as according to t�e results of von Delius et al
[��]. Doxorubicin was used at �.5 μg/mL� cisplatin
at �.�5 μg/mL� capecitabine at �� μg/mL and inter-
feron α-�b at � μg/mL� corresponding to t�e concen-
trations used in t�e preclinical models t�at were later
on followed by clinical trials [��]. N-Acetyl-L-Cysteine
�NAC� was obtained from Organika �Buc�arest� Ro-
mania� and used a final concentration of �5 μg/ml�
in order to fully assess t�e role of increased oxidative
stress in t�e arm wit� metformin plus PIAF combina-
tion c�emot�erapy.
Cell culture. Cancer stem-like cells isolated
as previusly described [��] �CSC cell line�� were cul-
tured along wit� normal liver stem cells �LIV cell line�
and t�e non-stem tumor cell line HepG� �bot� kindly
provided by t�e Assistant Professor Sergiu Susman�
�D� P�D at Department of Histology� Iuliu Hatieganu
University of �edicine and P�armacy from Cluj Na-
poca� Romania� and maintained in Ham’s F-�� and
Dulbecco’s �odified Essential �edium at �:� ratio�
supplemented wit� ��% fetal calf serum �FCS��
��� U/mL penicillin and ��� μg/mL streptomycin �all
from Sigma Aldric�� St Louis� �O� USA� in a ��°C �u-
midified incubator wit� a mixture of 95% air and 5%
carbon dioxide. All experiments were performed
on exponentially growing cells� wit� a doubling time of
ap proximately �� to �6 �. T�ese cells were previusly
reported to express t�e stem cell specific markers
albumin� α�-antitrypsin� α-fetoprotein� cytokera-
tin-�8� telomerase� CD9� and CD��� [��� ��].
Proliferation assay. Cell survival was assessed
using t�e �TT assay. For �-���5-dimet�ylt�iazol-
�-yl�-��5-dip�enyltetrazolium bromide ��TT� as-
says� cancer cells in monolayer culture were cul-
tivated at subconfluence in D�E�:F-�� media
supplemented wit� �5% FCS� ��� U/mL penicillin
and ��� μg/mL streptomycin complete media before
being was�ed twice wit� p�osp�ate buffer solution
�PBS�. Cells were t�en incubated wit� trypsin-EDTA�
resuspended in culture medium wit� FCS� counted and
plated in ��� μL media at �5 x ��� cells/well in 96-well
microtiter plates. After �� �� cells were was�ed and
treated wit� eit�er metformin or N-Acetyl-L-Cysteine�
before drugs being added. T�e control arms were
compared wit� t�e corresponding conventional cyto-
statics at identical concentrations. Absorbance of t�e
�TT was measured at �9� nm using a BioTek Synergy
� fluorescence microplate reader �Winooski� VT� USA�.
Lipid peroxidation assay. For bot� t�e determi-
nation of lipid peroxidation and reduced glutat�ion�
cells were seeded in �5 ml flasks at a density of � mil-
ions cells/flask. At �� and �8 � after treatments� cells
were detac�ed wit� trypsin and centrifugated 5 min�
�5�� rpm� at 9°C. T�e supernatant was removed and
t�e pellet was suspended in � ml lysis solution. T�en
eac� probe was sonicated to break t�e cellular mem-
branes. Peroxidase activity was determined by using
t�e indirect met�od t�at measures t�e rate f disappea-
rance of NADPH� adapted to laboratory conditions. T�e
reaction mixture consists of � m� GSH� �.�� U/ml glu-
tat�ione reducatse and �.�5 m� NADPH in 5� m� pBS
at a pH of �. Appropiate volumes of samples were
added for a final final reaction mixture incubated
at ��°C for 5 min. T�e assay was initially performed
wit� �� m� t-butyl �ydroperoxide solution� before
a decrease in absorbance at ��� nm for anot�er � min.
Lipid peroxidation activity was expressed as nmols
NAPDH consumed/min/mg lipids and were calculated
using 6.� x ���6 molar absorbtivity for NAPDH� as ac-
cording to Korde et al. [�5].
Reduced glutathione assay. Reduced GSH is an in-
tracellular antioxidant and is known to maintain cellular
redox balance. We t�erefore measured intracellular
GSH levels. Cell lysates were prepared as described
above and analyzed for GSH levels t�roug� a fluori-
metric met�od due to its property to make a fluorescent
reaction wit� o-p�talalde�yde. Cells are mixed wit� acid
tric�loracetic �TCA� ��% and after �� min centrifuged. T�e
supernatant was separated and �.� ml p�osp�ate buffer
wit� pH 8 and � ml of o-parafolmalde�yde were added.
After �5 min t�e intensity of emission at ��� nm on an ex-
citation of �5� nm was measured.
Glutat�ione concentration was measured using
a calibration curve realized wit� known concentrations
of glutat�ione obtained in t�e same way. Concentration
values are expressed in nmol/ml.
Experimental Oncology ��� ������ ���� ��arc����� ������ ���� ��arc�� ��arc�� �9
Dicloro-Fluorescein diacetate viability assay.
96 wells plates were prepared in t�e same way as de-
scribed in proliferation �TT assay� seeded wit� cells
and incubated for seven days. For t�e viability test
wit� fluorescein diacetate �FDA-an indicator of cell
viability�� cell monolayers were was�ed twice wit� PBS
supplemented wit� �g�+ and Ca �+ and stained for
5 min wit� FDA in t�e dark� at ��°C at a final concentra-
tion of ��� �. T�e wells were was�ed twice wit� PBS
supplemented wit� �g�+ and Ca �+ and fluorescence
intensity �FI� and were measured at �88 nm using
a BioTek Synergy � fluorescence microplate reader. All
t�e experiments were performed in triplicate.
Apoptosis quantification by chip flow cytometry.
T�e cells were treated as described above and cultured
for anot�er �� �. After incubation� t�e cells were trypsin-
ized� collected� and stained wit� Anexinn V-Cy5 Apopto-
sis Detection Kit �BioVision� for �� min in t�e dark. Calcein
A� was used as a viability marker� t�e cells were incu-
bated wit� � μ� Calcein A� for �� min at ��°C� was�ed
and quantified by on-c�ip flow cytometry according
to t�e manufacturer instructions �Agilent Tec�nologies�.
T�e number of apoptotic cells was assessed wit� Agilent
Lab-on-a-c�ip Bioanalyzer ���� �Agilent Tec�nologies��
as percent of apoptotic cells in live cells.
Autophagy assay. T�e autop�agy assay was
carried out using fluorescence microscopy and �ig�-
content imaging and analysis using cells previously fixed
in �.�% paraformalde�yde in PBS and permeabilized
wit� �.�% Triton X-��� diluted in PBS. T�en� t�e cells
were treated wit� ������ μ� c�loroquine for �6 ��
before t�e LC�B rabbit polyclonal antibody was diluted
in blocking buffer in order to prepare �.5 μg/ml working
solution. �.�% formalde�yde in PBS was added and
incubated for �5 min at room temperature before t�e
fixative was removed and cells was�ed t�ree times.
T�e next step was to add �.�% Triton X-��� in PBS and
cells were incubated for �5 min at room temperature.
T�e permeabilization buffer was removed� t�e primary
antibody added to t�e cells and incubated for � � be-
fore being once again removed and cells was�ed t�ree
times. Cells were t�en incubated wit� an anti-rabbit sec-
ondary antibody for �5 min� was�ed t�ree times once
more and additional DNA counterstaining was carried
out. Images were taken using a Zeiss Axiovert fluores-
cence microscope and autop�agosomes looked like
vesicular structures located in t�e perinuclear region.
Data analysis. Statistical significance values were
obtained by a one-way analysis of variance �ANOVA�
wit� 95% confidence level using Grap�Pad Prism
5 statistics program �La Jolla� CA� USA�. Data were
analyzed using one-way ANOVA wit� t�e Bonferroni
multiple comparison test �Kruskal-Wallis as nonpara-
metric�. Statistical significance was set at p < �.�5 and
all experiments were performed in triplicate.
RESULTS
Cell proliferation assay. �etformin doesn’t seem
to in�ibit �epatocellular carcinoma-derived cancer stem-
like cell proliferation bot� at �� � and after �8 � �Fig. ��
a and �� b�. Nevert�eless� t�e results are better w�en
metformin plus PIAF combination c�emot�erapy was
used and statistically signifficant data were obained�
as seen in Table � �at �� �� and Table � �at �8 ��. T�is
is especially important because �epatocarcinoma stem-
like cells �ave proven to be resistant to conventional
treatment wit� cisplatin/doxorubicin/capecitabine.
a
b
Contro
l
Metfo
rm
in
PIAF
Metfo
rm
in+PIAF
NAC
Metfo
rm
in+NAC
PIAF+NAC
Metfo
rm
in+NAC+PIAF
2.0
1.5
1.0
0.5
0.0
MTT 24h
Contro
l
Metfo
rm
in
PIAF
Metfo
rm
in+PIAF
NAC
Metfo
rm
in+NAC
PIAF+NAC
Metfo
rm
in+NAC+PIAF
1.5
1.0
0.5
0.0
MTT 48h
Fig. 1. T�e �TT cell proliferation assay s�ows t�at t�e combina-
tion of metformin plus PIAF is t�e best combination c�emot�erapy
option possible for �epatocellular carcinoma� bot� at �� � �� a�
and �8 � �� b�. Data is statistically significant �95% confidence
interval�� as seen in Table � �data at �� �� and Table � �data at �8 ��
Table 1. MTT assay at 24 h
Statistically significant groups 95% CI of difference
Control vs Metformin 0.4726 to 1.058
Control vs Metformin + PIAF 0.5284 to 1.114
Control vs NAC + Metformin + PIAF 0.3860 to 0.9714
Control vs NAC + Metformin + PIAF 0.4878 to 1.073
Metformin vs PIAF -1.293 to -0.6168
Metformin vs NAC -1.267 to -0.5906
Metformin vs NAC + PIAF -1.124 to -0.4476
PIAF vs Metformin + PIAF 0.6726 to 1.349
PIAF vs NAC + Metformin + PIAF 0.5302 to 1.206
PIAF vs NAC + Metformin + PIAF 0.6320 to 1.308
Metformin + PIAF vs NAC -1.322 to -0.6464
Metformin + PIAF vs NAC + PIAF -1.179 to -0.5034
NAC vs NAC + Metformin + PIAF 0.5040 to 1.180
NAC vs NAC + Metformin + PIAF 0.6058 to 1.282
NAC + Metformin + PIAF vs NAC + PIAF -1.037 to -0.3610
NAC + PIAF vs NAC + Metformin + PIAF 0.4628 to 1.139
Determination of lipid peroxidation. �etformin
increases t�e values of lipid peroxidation� bot� w�en
used alone or w�en combined wit� conventional c�e-
�� Experimental Oncology ��� ������ ���� ��arc��
mot�erapy drugs at �� and �8 �� as seen in Fig. �� a and
�� b. T�e most increased values of oxidative stress cor-
respond to t�e lowest proliferation rates obtained wit� t�e
treatment metformin plus PIAF c�emot�erapy.
Table 2. MTT assay at 48 h
Statistically significant groups 95% CI of difference
Control vs Metformin 0.3236 to 0.5920
Control vs Metformin + PIAF 0.3742 to 0.6426
Control vs NAC + Metformin + PIAF 0.3206 to 0.5890
Control vs NAC + PIAF -0.2736 to -0.005188
Control vs NAC + Metformin + PIAF 0.3686 to 0.6370
Metformin vs PIAF -0.6104 to -0.3060
Metformin vs NAC -0.6648 to -0.3604
Metformin vs NAC + PIAF -0.7494 to -0.4450
PIAF vs Metformin + PIAF 0.3566 to 0.6610
PIAF vs NAC + Metformin + PIAF 0.3030 to 0.6074
PIAF vs NAC + Metformin + PIAF 0.3510 to 0.6554
Metformin + PIAF vs NAC -0.7154 to -0.4110
Metformin + PIAF vs NAC + PIAF -0.8000 to -0.4956
NAC vs NAC + Metformin + PIAF 0.3574 to 0.6618
NAC vs NAC + Metformin + PIAF 0.4054 to 0.7098
NAC + Metformin + PIAF vs NAC + PIAF -0.7464 to -0.4420
NAC + PIAF vs NAC + Metformin + PIAF 0.4900 to 0.7944
a
b
Contro
l
Metfo
rm
in
PIAF
Metfo
rm
in+PIAF
NAC
Metfo
rm
in+NAC
PIAF+NAC
Metfo
rm
in+NAC+PIAF
1.0
0.8
0.6
0.4
0.2
0.0
Lipid peroxidation 24h
Contro
l
Metfo
rm
in
PIAF
Metfo
rm
in+PIAF
NAC
Metfo
rm
in+NAC
PIAF+NAC
Metfo
rm
in+NAC+PIAF
1.0
0.8
0.6
0.4
0.2
0.0
Lipid peroxidation 48h
Fig. 2. Lipid peroxidation values at �� � �a� and �8 � �b�
Determination of reduced glutathione. T�e
initial �ypot�esis t�at oxidative stress plays a key role
in t�e action of cytostatic drugs plus metformin in liver
cancer cell in�ibition is confirmed bot� by t�e data ob-
tained from t�e determination of lipid peroxidation and
reduced glutat�ione. Suc�� oxidative stress is most
increased w�en using t�is combination c�emot�erapy�
t�at is also corresponding to t�e lowest proliferation
rates at �� � �Fig. �� a� and �8 � �Fig. �� b�.
a
b
Contro
l
Metfo
rm
in
PIAF
Metfo
rm
in+PIAF
NAC
NAC+Metfo
rm
in
NAC+PIAF
NAC+Metfo
rm
in+PIAF
6
4
2
0
Reduced glutathione 24h
Contro
l
Metfo
rm
in
PIAF
Metfo
rm
in+PIAF
NAC
NAC+Metfo
rm
in
NAC+PIAF
NAC+Metfo
rm
in+PIAF
4
3
2
1
0
Reduced glutathione 48h
Fig. 3. Determination of reduced glutat�ione at �� � �a� and �8 � �b�.
Dicloro-FDA assay. T�e FDA assay confirms our
previous results� confirming increased oxidative stress
in cells t�at were treated wit� metformin plus cytostatics�
in comparison wit� cells treated eit�er wit� metformin
or PIAF� as illustrated in Fig. �. Data are s�own in Table �.
Contro
l
Metfo
rm
in
PIAF
Metfo
rm
in+PIAF
4000
3000
2000
1000
0
FDA Assay
OD
Fig. 4. Determination of increased oxidative stress using t�e
FDA assay
Apoptosis assay. After t�e incubation and
staining wit� Anexinn V-Cy5 Apoptosis Detection Kit
ad using calcein A� as a viability marker� t�e Agilent
Lab-on-a-c�ip Bioanalyzer ���� tec�nology allowed
us to assess t�e percentage of cells in apoptosis
Experimental Oncology ��� ������ ���� ��arc����� ������ ���� ��arc�� ��arc�� ��
as furt�er seen in Fig. 5. All t�e determinations were
carried out in triplicate� according to data provided
in Table �.
Table 3. FDA assay
Statistically significant groups 95% CI of difference
Control vs PIAF -632.2 to -267.8
Control vs Metformin + PIAF -871.2 to -506.8
Metformin vs PIAF -545.6 to -181.2
Metformin vs Metformin + PIAF -784.6 to -420.2
PIAF vs Metformin + PIAF -421.2 to -56.77
Contro
l
Metfo
rm
in
PIAF
Metfo
rm
in+PIAF
50
40
30
20
10
0
Apoptosis assay
OD
Fig. 5. Apoptosis assay compares in triplicate all t�e � arms
of our experiment� proving an increased apoptosis via oxidative
stress in cells treated wit� metformin plus temozolomide. T�e
data presented by using Agilent Lab-on-a-c�ip Bioanalyzer
���� is seen in t�e image presented bellow
Table 4. Apoptosis assay
Statistically significant groups 95% CI of difference
Control vs Metformin + PIAF -41.52 to -33.35
Metformin vs Metformin + PIAF -41.62 to -33.45
PIAF vs Metformin + PIAF -40.02 to -31.85
Autophagy assay. Autop�agy is confirmed
by Fig. 6 �a� b�� in comparison wit� t�e control —
as seen in Fig. 6 �c� d�. In t�e case of metformin
plus PIAF combination c�emot�erapy� vesicular-like
structures are seen in t�e perinuclear region of t�e
cells after incubation wit� t�e secondary anti-rabbit
monoclonal antibody and counterstaining of t�e
cell nucleus wit� DAPI �diamindino-�-p�enylindole
dye�. T�us� our initial �ypot�esis ans previous data
obtained are confirmed.
DISCUSSION
�ost cases of HCC are diagnosed in an advanced
stage because of t�e relatively scarce symptoms.
T�us� t�e oncologist must rely liver imaging in order
to proper assess t�e exact number and size of liver le-
sions� t�e location relative to t�e surrounding vessels�
t�e nature or origin of t�e tumor and most important�
t�e presence of extra�epatic spread. To t�e present
day� t�ere is no internationally-accepted consensus re-
garding t�e best strategy for t�e imaging of liver tumors
and it depends mostly on t�e request of t�e clinician�
t�e condition of t�e patient and t�e equipment avai-
lable in t�e �ospital. �ost centers use ultrasound �US��
computed tomograp�y �CT� and magentic resonance
a
c
b
d
Fig. 6. Autop�agy assay confirms t�e presence of perinuclear autop�agososmes after staining wit� t�e secondary antibody �a�
and counterstaining wit� DAPI of t�e nucleus �b�. Controls are seen in figures c and d
�� Experimental Oncology ��� ������ ���� ��arc��
imaging ��RI�� but some ot�ers tec�niques are avail-
able. T�ese include positron emission tomograpgy
�PET�� a CT during an arterial portograp�y �CTAP�
or during arteriograp�y �CTHA� and even laparoscopy
wit� intraoperative ultrasound [�6��8].
CT-based scanning is a more sensitive examina-
tion for t�e detection of focal liver lesions. If it’s still
not clear enog�� �RI �as an in�erent �ig� soft tussue
contrast because tissue-specific electromagnetic
paramaters suc� as t�e T� and T� relaxation time ef-
fect dominate t�e signal. So� �RI uses t�e many types
of sequences t�at investigate a different tissue for
eac� differential diagnosis between �ealt�y and pat�o-
logical liver parenc�yma. By using different contrast
mec�anisms� t�e �RI can provide bot� anatomical
and functional information� t�e c�emical s�ift imag-
ing being an example w�ere information is provided
wit� regard to t�e intracellular fat content of t�e liver
parenc�yma or a focal liver lesion [�9� ��].
Staging of t�e malignant liver mass is aimed
to stratify patients into groups wit� similar prognoses�
in order to �elp c�oose t�e best treatment� aid in pa-
tient counseling� allow comparisons of t�e outcome
of different t�erapy protocols and last but certainly not
least facilitate a good selection or randomization for
reserac� protocols. In t�e cse of HCC� t�e most widely
used in Europe and t�e US is t�e Barcelona Clinic Liver
Cancer �BCLC� system� proposed in �999 due to its
efficacy in bot� predicting prognosis and as a guide
to select t�e most appropiate t�erapy [��]. Even
if t�e BCLC staging was proven to be an efficient tool
in prognostics� t�e treatment algoritm is mostly based
on t�e Spanis� experience and is overly conserva-
tive wit� respect to t�e use of surgery. Patients wit�
large tumors are excluded from surgical resection
even if it �as been demonstrated a 5-year survival
of �5 to �9% after resection� wit� radiofrequency ab-
lation and et�anol injection recommended for t�ose
wit� multifocal disease t�at respect t�e �ilan criteria
[��]. T�e AJCC/UICC 6t� edition TN� staging system
is based on a study of t�e International Cooperative
Study Group on Hepatocellular Carcinoma� a analysis
of 59� cases from t�e US� Japan and France� w�o
all underwent surgical resection [��� ��]. T�e major
streng�t� is t�e use of centralized pat�ological review�
focusing on tumor multifocality� size and presence
of microvascular or major vascular c�aracteristics
of prognostic significance. It also brings toget�er t�e
data of liver fibrosis and cirr�osis based on t�e Is�ak
�istological grading� but t�e most important limitation
is t�at it was developed based only on resected tissue
and it’s applicability to patients undergoing ot�er non-
sugical locoregional treatments is questionable [�5].
But still� even t�oug� we take into consideration
t�e most permissive staging possible� most of t�e
patients diagnosed wit� a HCC are not eligible for
surgical resection. T�ese patients are considered
to be incurable and in order to ac�ieve a good quality
of life t�roug� increased toxicity for t�e cancer cells
and decresed toxicity for t�e �ealt�y tissue� modern
�epatology must develop a differet approac�� based
on recently publis�ed data. T�is very concept in used
in t�e current study. By experimenting on cancer cells
previously proven to �ave stem-like c�aracteristics�
our preliminary in vitro results aim to target t�e small
but very aggressive subpopulation t�oug�t to be re-
sponsible for clinical relapse and patient deat�.
Encouraging results �ave emerged from recent
data publis�ed on breast cancer and point towards
metformin� an oral-antidiabetic used for decades in t�e
treatment of type � diabetes mellitus� as an efficient
anticancer drug. �etformin �as been investigated
by our team for t�e treatment of WHO grade III and
IV malignant gliomas in t�e laboratory setting and t�e
results were furt�er confirmed by retrospective epide-
miological studies t�at reported a decrease in cancer
risk and also a better response to c�emot�erapy
of diabetic patients wit� breast cancer treated wit�
metformin [�6� �].
Still� despite compelling evidence of a role
of metformin as an anticancer drug� t�e mec�anisms
by w�ic� metformin exerts its oncostatic actions are
yet not fully understood. In t�e current researc� paper�
we studied t�e effect of metformin plus PIAF combi-
nation c�emot�erapy on HCC-derived stem-like cells
in order to furt�er investigate t�e basic mec�anisms
by w�ic� t�is association of drugs exerts its action
on t�e cancer cell. T�is study demonstrates for t�e
first time t�at t�e association between metformin and
conventional is linked to t�e generation of oxidative
stress� en�ancing its anti-neoplastic action. Recent
evidence s�ows t�at metformin decreases t�e reactive
oxigen species �ROS� in �ealt�y cells because of its
antioxidant and free radical scavenging abilities. Suc�
is t�e case of normal rat pancreatic islets� w�ere
metformin �as been proven to reduce t�e oxida-
tive stress induced by c�ronic exposure to �ig�
free fatty acids and restore t�e antioxidant status
in type � diabetic patients. Our results confirm t�is
experiment and furt�ermore� even if metformin �as
demonstrated powerful anti-oxidant properties in nor-
mal cells� in cancer stem-like ones isolated from a liver
tumor it stimulates t�e production of oxidative stress
bot� alone or in combination wit� c�emot�erapy. In-
creased values of oxidative stress are correlated wit�
decreased proliferation rates� t�us contradicting t�e
observation t�at oxidative stress contributes to drug
resistance.
Cellular redox �omeostasis is t�us maintained
by a fine balance between antioxidants and pro-
oxidants. Glutat�ione �GSH� is a critical intracellular
antioxidant responsible for maintaining redox balance
t�at can be oxidized to form a more complex structure
�GSSG�� wit� t�e ratio GSH/GSSG being a excellent
indicator of oxidative stress in t�e cells [��]. Decreased
GSH levels will indicate t�e s�ift of redox equilibrium
towards a pro-oxidant state. Our results s�ow t�at t�e
association between metformin and temozolomide �as
low values of reduced glutat�ione� w�ic� correlates
wit� t�e increase production of lipid peroxids.
Experimental Oncology ��� ������ ���� ��arc����� ������ ���� ��arc�� ��arc�� ��
It is well known t�at most of t�e liver pat�ology� may
it be non-alco�olic fatty liver disease �NAFLD�� non-
alco�olic steato�epatitis �NASH� syndrome� cirr�osis
and even �epatocellular carcinoma develop partially
due to an underlying constellation of closely related
risk factors known as metabolic syndrome or syn-
drome X [�8���]. Wit� a rapidly growing prevalence
in t�e Western world� t�is syndrome is c�aracterized
by obesity� insulin resistance� �yperinsulinemia� �y-
perglycaemia� dyslipidemia and �ypertension. As t�e
first step in disease appearance and development
is t�e accumulation of triglycerides in t�e �epatocyte
as a result of perip�eric insulin resistance. In time�
t�is will result in increased oxidative stress wit�in t�e
liver cell due to excessive production of ROS by t�e
mitoc�ondria and t�e cytoc�rome P-�5� system
and afterwards� an increse in lipid peroxidation�
pro-inflammatory cytokine and Fas ligand induction
promotes t�e progression from steatosis to NASH�
fibrosis and finally cirr�osis [����5]. W�en cirr�osis
appears� t�e natural �istory of t�e liver parenc�yma
most often evolves to eit�er �epatocellular carcinoma
or c�olnagiocarcinoma. Our data is supported by t�is
circuit of pat�ological c�anges of t�e �epatic tissue
and it is only natural t�at a drugs known for its action
against various features and risk factors of metabolic
syndrome via oxidative stress to en�ance t�e effect
of various cytostsic drugs and increase toxicity in t�e
malignant cell w�ile decreasing t�e same toxic effects
in t�e �ealt�y tissue.
CONCLUSIONS
Hepatocellular carcinomas are �eterogeneous
tumors wit� an inpredicable and most often let�al
clinical course. As t�e molecular approac�� previusly
t�oug�t to c�ange t�e management of a patient di-
agnosed wit� t�is dreadfull disease� �as proven most
often less effective t�an t�e classic approac�� our
researc� team turned to a drug used in t�e clinic for
decades and combines t�is oral antidiabetic wit� best
supportive care for advanced stage HCC. Our resuls
confirm previusly publis�ed papers t�at report t�e
sensitizing effect of metformin to tamoxifen t�erapy for
women wit� HER�/neu + breast cancer. Still� furt�er
data on animal models and retrospective analysis are
needed before we make a step forward in t�e field�
to p�ase I clinical trials� and start prescribing metfor-
min along wit� PIAF combination c�emot�erapy for
�epatocellular carcinoma tteatment.
ACKNOWLEDGEMENTS AND FUNDING
T�is researc� �as never been publis�ed before and
was financially supported by a researc� grant of t�e
Iuliu Hatieganu University of �edicine and P�armacy�
granted to Bobe Petrus�ev — �D. All t�e experiments
were carried out bot� at t�e Department of Cancer
Immunology� Ion C�iricuta Compre�ensive Cancer
Center in Cluj Napoca and at t�e G.I. Core Center
at Jo�ns Hopkins University� under t�e supervision
of Ciprian Tomuleasa� �D �Postdoctoral Researc�er
at T�e Jo�ns Hopkins University in Baltimore� �D�
USA� and Professor Victor Cristea� �D� P�D �Head
of Department and Attending p�ysician in Clinical
Gastroenterology and Hepatology at t�e Octavian
Fodor Regional Institute of Gastroenterology and
Hepato logy� Cluj Napoca� Romania�.
CONFLICTS OF INTEREST
None declared.
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