Click here to load reader

metformin

  • View
    91

  • Download
    1

Embed Size (px)

Text of metformin

Experimental Oncology arc Exp Oncol ORIGINAL CONTRIBUTIONS

METFORMIN PLUS PIAF COMBINATION CHEMOTHERAPY FOR HEPATOCELLULAR CARCINOMAB. 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 1 Department of Medicine Iuliu Hatieganu University of Medicine and Pharmacy, Cluj Napoca 2 Department of Medicine, Division of Gastroenterology and Hepatology, The Johns Hopkins University School of Medicine, Baltimore, MD, United States of America 3 Department of Immunology, Ion Chiricuta Comprehensive Cancer Center, Cluj Napoca, Romania 4 Octavian Fodor Regional Institute of Gastroenterology and Hepatology, Cluj Napoca, Romania 5 Department of Medical Oncology, Ion Chiricuta Comprehensive Cancer Center, Cluj Napoca, Romania 6 Department of Genetics, Ion Chiricuta Comprehensive Cancer Center, Cluj Napoca, Romania 7 Department of Surgery, Ion Chiricuta Comprehensive Cancer Center, Cluj Napoca, RomaniaObjectives: 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. 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 tousand years BC te liver was tougt to bear te soul and priests used epatoscopy in animals as a tool for divine connection in order to predict te future and te outcome of a certain military endeavor [ ]. One of te most famous legends was written by Hesiod and describes Prometeus stealing fire from Zeus before being punised by te king of te gods to be cained to a rock and aving is liver eaten by an eagle. But te liver regenerated and regained its normal size overnigt [ ]. Over time te progress in te field of epatology and liver surgery was mostly for oncologic diseases becoming more and more accurate and sopistiucated and years passed. An important step for te improved outcome is te understanding tat liver cancer must be treated in specialized interdisciplinary centers were a close relationsip between surgery internal medicine and basic researc ave te common goal to diagnose and cure tis infermity.Received: December 8, 2011. *Correspondence: E-mail: [email protected] Authors marked with () contributed equally to this paper. Abbreviations used: CSC cancer stem-like cells; HCC hepatocellular carcinoma; PIAF Cisplatin/Interferon -2b/ Doxorubicin/5Fluorouracil, with Capecitabine replacing 5-Fluorouracil.

Liver cancer is te 6t most common cancer worldwide and te tird most common cause of cancer mortality on te planet wit more tan alf a million deats annually indicating tat tis patology is not only common but also deadly. Te iges incidence rates are in sub-Saaran Africa and eastern Asia wit Cina accounting for more tan 5% of cases [5]. Hepatocellular carcinoma HCC incidence is ig among young adults and varies among different populations and races suggesting tat tis neoplasia is caused by several etiologal factors te synergy of wom as been sown to be signifficant in epatocarcinogenesis. Risk factors include epatitis B virus and epatitis C virus infection alcool consumption smoking aflatoxin E exposure ormonal intake occupational exposures and cronic medical conditions suc as metabolic syndrome diabetes mellitus obesity or even tyroid diseases [6]. ost cases of epatocellular carcinoma are diagnosed in an advanced stage because of te relatively scarce symptoms. Te patients survival depends not only on tumor caraceristics but also on te extent of te undelying disease. Furermore te terapeutic agents available for HCC and te indications for aggresive terapy expand and an increase of an interdisciplinary approac between surgery ra-

8 dioterapy and cemoterapy is expected to callenge our understanding of prognostic factors for HCC []. Encouraging results ave recently emerged from data publised on breast cancer and point towards metformin an oral-antidiabetic used for decades in te treatment of type diabetes mellitus as an efcient anticancer drug [8]. etformin as already been investigated by our team for te treatment of WHO grade III and IV malignant gliomas in te laboratory settings [9] results being conrmed by retrospective epidemiological studies tat reported a decrease in cancer risk and also a better response to cemoterapy of diabetic patients wit breast cancer treated wit metformin []. In te current researc paper we studied te effect of metformin plus PIAF Cisplatin/Interferon -b/ Doxorubicin/5-Fluorouracil wit Capecitabine replacing 5-Fluorouracil wit combination cemoterapy on malignant liver stem-like cells in order to furter investigate te basic mecanisms by wic tis association of drugs exerts its action on te cancer cell. Te aim of te current paper is to investigate te correlation between an old drug and te best standardof-care for unresectable epatocellular carcinoma wit te final goal of improving te terapeutic index of suc patients.

Experimental Oncology arc approximately to 6 . Tese cells were previusly reported to express te stem cell specific markers

albumin -antitrypsin -fetoprotein cytokeratin-8 telomerase CD9 and CD [ ].

MATERIALS AND METHODSReactives. etformin -dimetylbiguanide ydrocloride was purcesed from Wurwag Parma Bucarest Romania and used in a final concentration of m diluted in pospate buffer solution PBS. It was added prior to a sligtly modiffied PIAF regimen wit capecitabine replacing 5-fluorouracil as according to te results of von Delius et al []. Doxorubicin was used at .5 g/mL cisplatin at .5 g/mL capecitabine at g/mL and interferon -b at g/mL corresponding to te concentrations used in te preclinical models tat were later on followed by clinical trials []. N-Acetyl-L-Cysteine NAC was obtained from Organika Bucarest Romania and used a final concentration of 5 g/ml in order to fully assess te role of increased oxidative stress in te arm wit metformin plus PIAF combination cemoterapy. Cell culture. Cancer stem-like cells isolated as previusly described [] CSC cell line were cultured along wit normal liver stem cells LIV cell line and te non-stem tumor cell line HepG bot kindly provided by te Assistant Professor Sergiu Susman D PD at Department of Histology Iuliu Hatieganu University of edicine and Parmacy from Cluj Napoca Romania and maintained in Hams F- and Dulbeccos odified Essential edium at : ratio supplemented wit % fetal calf serum FCS U/mL penicillin and g/mL streptomycin all from Sigma Aldric St Louis O USA in a C umidified incubator wit a mixture of 95% air and 5% carbon dioxide. All experiments were performed on exponentially growing cells wit a doubling time of

Proliferation assay. Cell survival was assessed using te TT assay. For -5-dimetyltiazol-yl-5-dipenyltetrazolium bromide TT assays cancer cells in monolayer culture were cultivated at subconfluence in DE:F- media supplemented wit 5% FCS U/mL penicillin and g/mL streptomycin complete media before being wased twice wit pospate buffer solution PBS. Cells were ten incubated wit trypsin-EDTA resuspended in culture medium wit FCS counted and plated in L media at 5 x cells/well in 96-well microtiter plates. After cells were wased and treated wit eiter metformin or N-Acetyl-L-Cysteine before drugs being added. Te control arms were compared wit te corresponding conventional cytostatics at identical concentrations. Absorbance of te TT was measured at 9 nm using a BioTek Synergy fluorescence microplate reader Winooski VT USA. Lipid peroxidation assay. For bot te determination of lipid peroxidation and reduced glutation cells were seeded in 5 ml flasks at a density of milions cells/flask. At and 8 after treatments cells were detaced wit trypsin and centrifugated 5 min 5 rpm at 9C. Te supernatant was removed and te pellet was suspended in ml lysis solution. Ten eac probe was sonicated to break te cellular membranes. Peroxidase activity was determined by using te indirect metod tat measures te rate f disappearance of NADPH adapted to laboratory conditions. Te reaction mixture consists of m GSH . U/ml glutatione 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. Te assay was initially performed wit m t-butyl ydroperoxide solution before a decrease in absorbance at nm for anoter 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 according to Korde et al. [5]. Reduced glutathione assay. Reduced GSH is an in-

tracellular antioxidant and is known to maintain cellular redox balance. We terefore measured intracellular GSH levels. Cell lysates were prepared as described above and analyzed for GSH levels troug a fluori-

metric metod due to its property to make a fluorescent reaction wit o-ptalaldeyde. Cells are mixed wit acid tricloracetic TCA % and after min centrifuged. Te supernatant was separated and . ml pospate buffer wit pH 8 and ml of o-parafolmaldeyde were added. After 5 min te intensity of emission at nm on an excitation of 5 nm was measured. Glutatione concentration was measured using a calibration curve realized wit known concentrations of glutatione obtained in te same way. Concentration values are expressed in nmol/ml.

Experimental Oncology arc Dicloro-Fluorescein diacetate viability assay. 96 wells plates were prepared in te same way as described in proliferation TT assay seeded wit cells and incubated for seven days. For te viability test wit fluorescein diacetate FDA-an indicator of cell viability cell monolayers were wased twice wit PBS supplemented wit g+ and Ca + and stained for 5 min wit FDA in te dark at C at a final concentration of . Te wells were wased 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 te experiments were performed in triplicate. Apoptosis quantification by chip flow cytometry. Te cells were treated as described above and cultured for anoter . After incubation te cells were trypsinized collected and stained wit Anexinn V-Cy5 Apoptosis Detection Kit BioVision for min in te dark. Calcein A was used as a viability marker te cells were incubated wit Calcein A for min at C wased and quantified by on-cip flow cytometry according to te manufacturer instructions Agilent Tecnologies. Te number of apoptotic cells was assessed wit Agilent Lab-on-a-cip Bioanalyzer Agilent Tecnologies as percent of apoptotic cells in live cells. Autophagy assay. Te autopagy assay was carried out using fluorescence microscopy and igcontent imaging and analysis using cells previously fixed in .% paraformaldeyde in PBS and permeabilized wit .% Triton X- diluted in PBS. Ten te cells were treated wit cloroquine for 6 before te LCB rabbit polyclonal antibody was diluted in blocking buffer in order to prepare .5 g/ml working solution. .% formaldeyde in PBS was added and incubated for 5 min at room temperature before te fixative was removed and cells wased tree times. Te next step was to add .% Triton X- in PBS and cells were incubated for 5 min at room temperature. Te permeabilization buffer was removed te primary antibody added to te cells and incubated for before being once again removed and cells wased tree times. Cells were ten incubated wit an anti-rabbit secondary antibody for 5 min wased tree times once more and additional DNA counterstaining was carried out. Images were taken using a Zeiss Axiovert fluorescence microscope and autopagosomes looked like vesicular structures located in te perinuclear region. Data analysis. Statistical significance values were obtained by a one-way analysis of variance ANOVA wit 95% confidence level using GrapPad Prism 5 statistics program La Jolla CA USA. Data were analyzed using one-way ANOVA wit te Bonferroni multiple comparison test Kruskal-Wallis as nonparametric. Statistical significance was set at p < .5 and all experiments were performed in triplicate.

9 a and b. Neverteless te results are better wen metformin plus PIAF combination cemoterapy was used and statistically signifficant data were obained as seen in Table at and Table at 8 . Tis is especially important because epatocarcinoma stemlike cells ave proven to be resistant to conventional treatment wit cisplatin/doxorubicin/capecitabine.2.0 MTT 24h a

1.5

1.0

0.5

0.0Co ntr ol tf Me or mi n PIA Me F r n+ mi PIA F NA Me C r n+ mi NA C P M + IAF NA C N + AC PIA F

tfo

tfo

or etf

mi

n+

1.5

MTT 48h

b

1.0

0.5

0.0Co ntr ol M o etf rm in PIA Me F r n+ mi PIA F NA Me C r n+ mi NA C P + IAF r tfo NA C N + AC PIA F

tfo

tfo

Me

mi

n+

Fig. 1. Te TT cell proliferation assay sows tat te combination of metformin plus PIAF is te best combination cemoterapy 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 Control vs Metformin Control vs Metformin + PIAF Control vs NAC + Metformin + PIAF Control vs NAC + Metformin + PIAF Metformin vs PIAF Metformin vs NAC Metformin vs NAC + PIAF PIAF vs Metformin + PIAF PIAF vs NAC + Metformin + PIAF PIAF vs NAC + Metformin + PIAF Metformin + PIAF vs NAC Metformin + PIAF vs NAC + PIAF NAC vs NAC + Metformin + PIAF NAC vs NAC + Metformin + PIAF NAC + Metformin + PIAF vs NAC + PIAF NAC + PIAF vs NAC + Metformin + PIAF 95% CI of difference 0.4726 to 1.058 0.5284 to 1.114 0.3860 to 0.9714 0.4878 to 1.073 -1.293 to -0.6168 -1.267 to -0.5906 -1.124 to -0.4476 0.6726 to 1.349 0.5302 to 1.206 0.6320 to 1.308 -1.322 to -0.6464 -1.179 to -0.5034 0.5040 to 1.180 0.6058 to 1.282 -1.037 to -0.3610 0.4628 to 1.139

RESULTSCell proliferation assay. etformin doesnt seem to inibit epatocellular carcinoma-derived cancer stemlike cell proliferation bot at and after 8 Fig.

Determination of lipid peroxidation. etformin increases te values of lipid peroxidation bot wen used alone or wen combined wit conventional ce-

moterapy drugs at and 8 as seen in Fig. a and b. Te most increased values of oxidative stress correspond to te lowest proliferation rates obtained wit te treatment metformin plus PIAF cemoterapy.Table 2. MTT assay at 48 h Statistically significant groups Control vs Metformin Control vs Metformin + PIAF Control vs NAC + Metformin + PIAF Control vs NAC + PIAF Control vs NAC + Metformin + PIAF Metformin vs PIAF Metformin vs NAC Metformin vs NAC + PIAF PIAF vs Metformin + PIAF PIAF vs NAC + Metformin + PIAF PIAF vs NAC + Metformin + PIAF Metformin + PIAF vs NAC Metformin + PIAF vs NAC + PIAF NAC vs NAC + Metformin + PIAF NAC vs NAC + Metformin + PIAF NAC + Metformin + PIAF vs NAC + PIAF NAC + PIAF vs NAC + Metformin + PIAF 1.0 0.8 0.6 0.4 0 0.2 0.0 95% CI of difference 0.3236 to 0.5920 0.3742 to 0.6426 0.3206 to 0.5890 -0.2736 to -0.005188 0.3686 to 0.6370 -0.6104 to -0.3060 -0.6648 to -0.3604 -0.7494 to -0.4450 0.3566 to 0.6610 0.3030 to 0.6074 0.3510 to 0.6554 -0.7154 to -0.4110 -0.8000 to -0.4956 0.3574 to 0.6618 0.4054 to 0.7098 -0.7464 to -0.4420 0.4900 to 0.7944 a 2 6

Experimental Oncology arcReduced glutathione 24h a

4

2

0

Co

ntr

ol M

o etf

rm

in

PIA

F or m

tf Me

in+

PIA

F

NA NA

C M

C+

o etf

rm

in N

+ AC C+

PIA

F or m

NA

tf Me

in+

PIA

F

4

Reduced glutathione 48h

b

3 Lipid peroxidation 24h

1

n Co tro l M mi n PIA Me F rm IAF NA Me C rm AC PIA Me t AC in+ NA IAF

tro

l M

o etf

rm

in

PIA Me

F rm

tfo

P in+

IAF

NA NA

C M

C+

o etf

rm

in NA NA

P C+

IAF tfo r

n Co

or etf

tfo

P in+

tfo

N in+

N F+

P C+

C

e +M

n+ mi

PIA

F

Fig. 3. Determination of reduced glutatione at a and 8 b.

m for

1.0 0.8 0.6 0.4

Lipid peroxidation 48h

b

Dicloro-FDA assay. Te FDA assay confirms our previous results confirming increased oxidative stress in cells tat were treated wit metformin plus cytostatics in comparison wit cells treated eiter wit metformin or PIAF as illustrated in Fig. . Data are sown in Table .4000 FDA Assay

3000 OD

0.2 0.0

2000

Co

ntr

ol M

o etf

rm

in

PIA Me

F m

r tfo

in+

PIA

F

NA Me

C

r tfo

n+ mi

NA

C P

+ IAF

NA

C +N

M Fig. 2. Lipid peroxidation values at a and 8 b

o etf

rm

in

+ AC

PIA

F

1000

0r tfo Me Fig. 4. Determination of increased oxidative stress using te FDA assay n Co tro l tf Me or mi n PIA F n+ mi PIA F

Determination of reduced glutathione. Te initial ypotesis tat oxidative stress plays a key role in te action of cytostatic drugs plus metformin in liver cancer cell inibition is confirmed bot by te data obtained from te determination of lipid peroxidation and reduced glutatione. Suc oxidative stress is most increased wen using tis combination cemoterapy tat is also corresponding to te lowest proliferation rates at Fig. a and 8 Fig. b.

Apoptosis assay. After te incubation and staining wit Anexinn V-Cy5 Apoptosis Detection Kit ad using calcein A as a viability marker te Agilent Lab-on-a-cip Bioanalyzer tecnology allowed us to assess te percentage of cells in apoptosis

Experimental Oncology arc as furter seen in Fig. 5. All te determinations were carried out in triplicate according to data provided in Table .Table 3. FDA assay Statistically significant groups Control vs PIAF Control vs Metformin + PIAF Metformin vs PIAF Metformin vs Metformin + PIAF PIAF vs Metformin + PIAF 50 40 OD 30 20 10 0Co ntr

Table 4. Apoptosis assay Statistically significant groups Control vs Metformin + PIAF Metformin vs Metformin + PIAF PIAF vs Metformin + PIAF 95% CI of difference -41.52 to -33.35 -41.62 to -33.45 -40.02 to -31.85

95% CI of difference -632.2 to -267.8 -871.2 to -506.8 -545.6 to -181.2 -784.6 to -420.2 -421.2 to -56.77

Apoptosis assay

Autophagy assay. Autopagy is confirmed by Fig. 6 a b in comparison wit te control as seen in Fig. 6 c d. In te case of metformin plus PIAF combination cemoterapy vesicular-like structures are seen in te perinuclear region of te cells after incubation wit te secondary anti-rabbit monoclonal antibody and counterstaining of te cell nucleus wit DAPI diamindino--penylindole dye. Tus our initial ypotesis ans previous data obtained are confirmed.

DISCUSSIONost cases of HCC are diagnosed in an advanced stage because of te relatively scarce symptoms. Tus te oncologist must rely liver imaging in order to proper assess te exact number and size of liver lesions te location relative to te surrounding vessels te nature or origin of te tumor and most important te presence of extraepatic spread. To te present day tere is no internationally-accepted consensus regarding te best strategy for te imaging of liver tumors and it depends mostly on te request of te clinician te condition of te patient and te equipment available in te ospital. ost centers use ultrasound US computed tomograpy CT and magentic resonanceb

ol M

o etf

rm

in

PIA

F Me tfo r

n+ mi

PIA

F

Fig. 5. Apoptosis assay compares in triplicate all te arms of our experiment proving an increased apoptosis via oxidative stress in cells treated wit metformin plus temozolomide. Te data presented by using Agilent Lab-on-a-cip Bioanalyzer is seen in te image presented bellow a

c

d

Fig. 6. Autopagy assay confirms te presence of perinuclear autopagososmes after staining wit te secondary antibody a and counterstaining wit DAPI of te nucleus b. Controls are seen in figures c and d

imaging RI but some oters tecniques are available. Tese include positron emission tomograpgy PET a CT during an arterial portograpy CTAP or during arteriograpy CTHA and even laparoscopy wit intraoperative ultrasound [68]. CT-based scanning is a more sensitive examination for te detection of focal liver lesions. If its still not clear enog RI as an inerent ig soft tussue contrast because tissue-specific electromagnetic paramaters suc as te T and T relaxation time effect dominate te signal. So RI uses te many types of sequences tat investigate a different tissue for eac differential diagnosis between ealty and patological liver parencyma. By using different contrast mecanisms te RI can provide bot anatomical and functional information te cemical sift imaging being an example were information is provided wit regard to te intracellular fat content of te liver parencyma or a focal liver lesion [9 ]. Staging of te malignant liver mass is aimed to stratify patients into groups wit similar prognoses in order to elp coose te best treatment aid in patient counseling allow comparisons of te outcome of different terapy protocols and last but certainly not least facilitate a good selection or randomization for reserac protocols. In te cse of HCC te most widely used in Europe and te US is te Barcelona Clinic Liver Cancer BCLC system proposed in 999 due to its efficacy in bot predicting prognosis and as a guide to select te most appropiate terapy []. Even if te BCLC staging was proven to be an efficient tool in prognostics te treatment algoritm is mostly based on te Spanis experience and is overly conservative wit respect to te 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 ablation and etanol injection recommended for tose wit multifocal disease tat respect te ilan criteria []. Te AJCC/UICC 6t edition TN staging system is based on a study of te International Cooperative Study Group on Hepatocellular Carcinoma a analysis of 59 cases from te US Japan and France wo all underwent surgical resection [ ]. Te major strengt is te use of centralized patological review focusing on tumor multifocality size and presence of microvascular or major vascular caracteristics of prognostic significance. It also brings togeter te data of liver fibrosis and cirrosis based on te Isak istological grading but te most important limitation is tat it was developed based only on resected tissue and its applicability to patients undergoing oter nonsugical locoregional treatments is questionable [5]. But still even toug we take into consideration te most permissive staging possible most of te patients diagnosed wit a HCC are not eligible for surgical resection. Tese patients are considered to be incurable and in order to acieve a good quality of life troug increased toxicity for te cancer cells and decresed toxicity for te ealty tissue modern

Experimental Oncology arc epatology must develop a differet approac based on recently publised data. Tis very concept in used in te current study. By experimenting on cancer cells previously proven to ave stem-like caracteristics our preliminary in vitro results aim to target te small but very aggressive subpopulation tougt to be responsible for clinical relapse and patient deat. Encouraging results ave emerged from recent data publised on breast cancer and point towards metformin an oral-antidiabetic used for decades in te treatment of type diabetes mellitus as an efcient anticancer drug. etformin as been investigated by our team for te treatment of WHO grade III and IV malignant gliomas in te laboratory setting and te results were furter conrmed by retrospective epidemiological studies tat reported a decrease in cancer risk and also a better response to cemoterapy of diabetic patients wit breast cancer treated wit metformin [6 ]. Still despite compelling evidence of a role of metformin as an anticancer drug te mecanisms by wic metformin exerts its oncostatic actions are yet not fully understood. In te current researc paper we studied te effect of metformin plus PIAF combination cemoterapy on HCC-derived stem-like cells in order to furter investigate te basic mecanisms by wic tis association of drugs exerts its action on te cancer cell. Tis study demonstrates for te first time tat te association between metformin and conventional is linked to te generation of oxidative stress enancing its anti-neoplastic action. Recent evidence sows tat metformin decreases te reactive oxigen species ROS in ealty cells because of its antioxidant and free radical scavenging abilities. Suc is te case of normal rat pancreatic islets were

metformin as been proven to reduce te oxidative stress induced by cronic exposure to ig free fatty acids and restore te antioxidant status

in type diabetic patients. Our results confirm tis experiment and furtermore even if metformin as demonstrated powerful anti-oxidant properties in normal cells in cancer stem-like ones isolated from a liver tumor it stimulates te production of oxidative stress bot alone or in combination wit cemoterapy. Increased values of oxidative stress are correlated wit decreased proliferation rates tus contradicting te observation tat oxidative stress contributes to drug resistance. Cellular redox omeostasis is tus maintained by a fine balance between antioxidants and prooxidants. Glutatione GSH is a critical intracellular antioxidant responsible for maintaining redox balance tat can be oxidized to form a more complex structure GSSG wit te ratio GSH/GSSG being a excellent indicator of oxidative stress in te cells []. Decreased GSH levels will indicate te sift of redox equilibrium towards a pro-oxidant state. Our results sow tat te association between metformin and temozolomide as low values of reduced glutatione wic correlates wit te increase production of lipid peroxids.

Experimental Oncology arc It is well known tat most of te liver patology may it be non-alcoolic fatty liver disease NAFLD nonalcoolic steatoepatitis NASH syndrome cirrosis and even epatocellular carcinoma develop partially due to an underlying constellation of closely related risk factors known as metabolic syndrome or syndrome X [8]. Wit a rapidly growing prevalence in te Western world tis syndrome is caracterized by obesity insulin resistance yperinsulinemia yperglycaemia dyslipidemia and ypertension. As te first step in disease appearance and development is te accumulation of triglycerides in te epatocyte as a result of periperic insulin resistance. In time tis will result in increased oxidative stress witin te liver cell due to excessive production of ROS by te mitocondria and te cytocrome P-5 system and afterwards an increse in lipid peroxidation pro-inflammatory cytokine and Fas ligand induction promotes te progression from steatosis to NASH fibrosis and finally cirrosis [5]. Wen cirrosis appears te natural istory of te liver parencyma most often evolves to eiter epatocellular carcinoma or colnagiocarcinoma. Our data is supported by tis circuit of patological canges of te epatic tissue and it is only natural tat a drugs known for its action against various features and risk factors of metabolic syndrome via oxidative stress to enance te effect of various cytostsic drugs and increase toxicity in te malignant cell wile decreasing te same toxic effects in te ealty tissue.

USA and Professor Victor Cristea D PD Head of Department and Attending pysician in Clinical Gastroenterology and Hepatology at te Octavian Fodor Regional Institute of Gastroenterology and Hepatology Cluj Napoca Romania.

CONFLICTS OF INTERESTNone declared.1. Papavramidou N, Fee E, Christopoulou-Aletra H. Jaundice in the Hippocratic corpus. J Gastrointest Surg 2007; 11: 172831. 2. Lykouras E, Poulakou-Rebelakou E, Ploumpidis DN. Searching the seat of the soul in Ancient Greek and Byzantine medical literature. Acta Cardiol 2010; 65: 61926. 3. Reuben A. The way to a mans heart is through his liver. Hepatology 2003; 37: 15002. 4. Hammerschmidt DE. Prometheus. J Lab Clin Med 2000; 135: 498. 5. Altekruse SF, McGlynn KA, Reichman ME. Hepatocellular carcinoma incidence, mortality, and survival trends in the United States from 1975 to 2005. J Clin Oncol 2009; 27: 148591. 6. El-Serag HB. Hepatocellular carcinoma. N Engl J Med 2011; 365: 111827. 7. Ryder SD. British Society of Gastroenterology. Guidelines for the diagnosis and treatment of hepatocellular carcinoma (HCC) in adults. Gut 2003; 52: iii18. 8. Gonzalez-Angulo AM, Meric-Bernstam F. Metformin: a therapeutic opportunity in breast cancer. Clin Cancer Res 2010; 16: 1695700. 9. Soritau O, Tomuleasa C, Aldea M, et al. Metformin plus temozolomide-based chemotherapy as adjuvant treatment for WHO grade III and IV malignant gliomas. J BUON 2011; 16: 2829. 10. Jiralerspong S, Palla SL, Giordano SH, et al. Metformin and pathologic complete responses to neoadjuvant chemotherapy in diabetic patients with breast cancer. J Clin Oncol 2009; 27: 3297302. 11. von Delius S, Lersch C, Mayr M, et al. Capecitabine for treatment of advanced hepatocellular carcinomma. Hepatogastroenterology 2007; 54: 23104. 12. Yeo W, Mok TS, Zee B, et al. A randomized phase III study of doxorubicin versus cisplatin/interferon alpha-2b/ doxorubicin/fluorouracil (PIAF) combination chemotherapy for unresectable hepatocellular carcinoma. J Natl Cancer Inst 2005; 97: 15328. 13. Tomuleasa C, Soritau O, Rus-Ciuca D, et al. Isolation and characterization of hepatic cancer cells with stem-like properties from hepatocellular carcinoma. J Gastrointestin Liver Dis 2010; 19: 617. 14. Tomuleasa C, Soritau O, Fischer-Fodor E, et al. Arsenic trioxide plus cisplatin/interferon -2b/doxorubicin/ capecitabine combination chemotherapy for unresectable hepatocellular carcinoma. Hematol Oncol Stem Cell Ther 2011; 4: 606. 15. Korde SD, Basak A, Chaudhary M, et al. Enhanced nitrosative and oxidative stress with decreased total antioxidant capacity in patients with oral precancer and oral squamous cell carcinoma. Oncology 2011; 80: 3829. 16. El-Serag HB, Marrero JA, Rudolph L, Reddy KR. Diagnosis and treatment of hepatocellular carcinoma. Gastroenterology 2008; 134: 175263.

REFERENCES

CONCLUSIONSHepatocellular carcinomas are eterogeneous tumors wit an inpredicable and most often letal clinical course. As te molecular approac previusly tougt to cange te management of a patient diagnosed wit tis dreadfull disease as proven most often less effective tan te classic approac our researc team turned to a drug used in te clinic for decades and combines tis oral antidiabetic wit best supportive care for advanced stage HCC. Our resuls confirm previusly publised papers tat report te sensitizing effect of metformin to tamoxifen terapy for women wit HER/neu + breast cancer. Still furter data on animal models and retrospective analysis are needed before we make a step forward in te field to pase I clinical trials and start prescribing metformin along wit PIAF combination cemoterapy for epatocellular carcinoma tteatment.

ACKNOWLEDGEMENTS AND FUNDINGTis researc as never been publised before and was financially supported by a researc grant of te Iuliu Hatieganu University of edicine and Parmacy granted to Bobe Petrusev D. All te experiments were carried out bot at te Department of Cancer Immunology Ion Ciricuta Compreensive Cancer Center in Cluj Napoca and at te G.I. Core Center at Jons Hopkins University under te supervision of Ciprian Tomuleasa D Postdoctoral Researcer at Te Jons Hopkins University in Baltimore D

17. Forner A, Reig ME, de Lope CR, Bruix J. Current strategy for staging and treatment: the BCLC update and future prospects. Semin Liver Dis 2010; 30: 6174. 18. Silva MA, Hegab B, Hyde C, et al. Needle track seeding following biopsy of liver lesions in the diagnosis of hepatocellular cancer: a systematic review and meta-analysis. Gut 2008; 57: 15926. 19. Suh YJ, Kim MJ, Choi JY, et al. Differentiation of hepatic hyperintense lesions seen on gadoxetic acidenhanced hepatobiliary phase MRI. Am J Roentgenol 2011; 197: W4452. 20. El-Serag HB. Surveillance for hepatocellular carcinoma: in whom and how? Therap Adv Gastroenterol 2011; 4: 510. 21. Llovet JM, Br C, Bruix J. Prognosis of hepatocellular carcinoma: the BCLC staging classification. Semin Liver Dis 1999; 19: 32938. 22. Forner A, Reig ME, de Lope CR, Bruix J. Current strategy for staging and treatment: the BCLC update and future prospects. Semin Liver Dis 2010; 30: 6174. 23. Varotti G, Ramacciato G, Ercolani G, et al. Comparison between the fifth and sixth editions of the AJCC/UICC TNM staging systems for hepatocellular carcinoma: multicentric study on 393 cirrhotic resected patients. Eur J Surg Oncol 2005; 31: 7607. 24. Zhou L, Rui JA, Wang SB, et al. LCSGJ-T classification, 6th or 5th edition TNM staging did not independently predict the long-term prognosis of HBV-related hepatocellular carcinoma after radical hepatectomy. J Surg Res 2010; 159: 53844. 25. Zhou L, Rui JA, Ye DX, et al. Edmondson-Steiner grading increases the predictive efficiency of TNM staging for long-term survival of patients with hepatocellular carcinoma after curative resection. World J Surg 2008; 32: 174856.

Experimental Oncology arc26. Dowling RJ, Goodwin PJ, Stambolic V. Understanding the benefit of metformin use in cancer treatment. BMC Med 2011; 9: 33. 27. Hadad S, Iwamoto T, Jordan L, et al. Evidence for biological effects of metformin in operable breast cancer: a preoperative, window-of-opportunity, randomized trial. Breast Cancer Res Treat 2011; 128: 78394. 28. Burhans WC, Heintz NH. The cell cycle is a redox cycle: linking phase-specific targets to cell fate. Free Radic Biol Med 2009; 47: 128293. 29. Donohoe CL, Pidgeon GP, Lysaght J, Reynolds JV. Obesity and gastrointestinal cancer. Br J Surg 2010; 97: 62842. 30. Lackner C. Hepatocellular ballooning in nonalcoholic steatohepatitis: the pathologists perspective. Expert Rev Gastroenterol Hepatol 2011; 5: 22331. 31. Lebovics E, Rubin J. Non-alcoholic fatty liver disease (NAFLD): why you should care, when you should worry, what you should do. Diabetes Metab Res Rev 2011; 27: 41924. 32. Liu Q, Bengmark S, Qu S. The role of hepatic fat accumulation in pathogenesis of non-alcoholic fatty liver disease (NAFLD). Lipids Health Dis 2010; 9: 42. 33. Mirza MS. Obesity, visceral fat, and NAFLD: querying the role of adipokines in the progression of nonalcoholic fatty liver disease. ISRN Gastroenterol 2011; 2011: 592404. 34. Kashyap SR, Diab DL, Baker AR, et al. Triglyceride levels and not adipokine concentrations are closely related to severity of nonalcoholic fatty liver disease in an obesity surgery cohort. Obesity (Silver Spring) 2009; 17: 1696701. 35. Welzel TM, Graubard BI, Zeuzem S, et al. Metabolic syndrome increases the risk of primary liver cancer in the United States: a study in the SEER-Medicare database. Hepatology 2011; 54: 46371.

Copyright Experimental Oncology, 2012