Int J Clin Exp Med 20169(2)2166-2174wwwijcemcom ISSN1940-5901IJCEM0016829

Original Article Gemcitabine induces apoptosis via JAK2STAT3 signaling pathways in lung cancer

Ze-Yu Zhang Yan Zhang Hong Fu Jing Wang Zhi-Qin Zheng Qiu-An Dong

Department of Oncology Tongji Hospital 389 Xincun Road Putuo District Shanghai 200065 China

Received September 24 2015 Accepted December 17 2015 Epub February 15 2016 Published February 29 2016

Abstract Background This study was aimed to elucidate the antitumor effect of gemcitabine in lung cancer SPC-A-1 cells and the possible molecular mechanism involved Materials and methods Cell proliferation was measured by the Cell Count Kit-8 (CCK-8) Cell cycle apoptosis reactive oxygen species (ROS) production and mitochondrial membrane potential (MMP) level were assessed by flow cytometry analysis Real-time PCR and Western blot analy-sis were used to detect the ratio of BaxBcl-2 and the activation of JAK2 and STAT3 after cells were treated with different concentrations of gemcitabine Results The results revealed that gemcitabine could inhibit the growth of SPC-A-1 cells significantly in both a time and dose-dependent manner The cell cycle was blocked and ROS produc-tion was increased while MMP level was decreased after the cells were treated with different concentrations of gemcitabine Bcl-2 expression was down-regulated remarkably while Bax expression was increased after apoptosis occurred Additionally the activation of JAK2 and STAT3 was significantly inhibited in gemcitabine treated SPC-A-1 cells Conclusions Gemcitabine may serve as a potential therapeutic agent for lung cancer

Keywords Lung cancer SPC-A-1 gemcitabine apoptosis JAK2STAT3

Introduction

Lung cancer is one of the most common lead-ing causes of death worldwide [1] and non-small cell lung cancer (NSCLC) represents the most frequent type of lung cancer [2] Although a combination of surgery chemotherapy and radiotherapy is available the prognostics are generally poor [3] Upon this reason it is cur-rently extensive to screen new bioactive com-pounds from either natural and synthetic sourc-es for potential antitumor activity In recent years many studies have observed that gem-citabine a novel nucleoside analog that is incorporated into DNA as a fraudulent base inhibits the activity of DNA polymerases respon-sible for DNA repair and synthesis [4] These characteristics suggest that gemcitabine may be a good candidate for cancer treatment In clinical study gemcitabine has demonstrated important antitumor activity in many human tumor xenografts including lung cancer [5]

The proliferation inhibitory effect of gem-citabine is strongly related to cancer research Recently it was demonstrated that gemcitabine

possessed an anti-proliferative effect on human oral squamous cell carcinoma [6] and breast cancer in vitro [7] In cancer cell cycle homeostasis is generally disrupted by muta-tions leading to uncontrolled cell division and tumor growth For example gemcitabine induced arrest of pancreatic cancer cells in S phase both in vivo and in vivo [8] Model predic-tions and experimental data showed that gem-citabine induced cell cycle arrest in the S phase at low concentrations whereas higher concen-trations induced arrest in all cell cycle phases [9]

The abnormal production of reactive oxygen species (ROS) by exogenous sources or endog-enous mechanisms is among the main causes of cancer-related mutations [10] ROS also stimulate apoptosis through mitochondrial sig-nal mechanisms associated with cancer pro-gression It was previously reported that gem-citabine-induced apoptosis in breast cancer cells was associated with the mitochondrial apoptotic signaling pathway and induced mito-chondrial membrane potential (MMP) loss in pancreatic cancer cells [11 12]

Gemcitabine blocks JAK2STAT3 pathways

2167 Int J Clin Exp Med 20169(2)2166-2174

Signal transducer and activator of transcription 3 (STAT3) protein is a member of a family of latent cytoplasmic transcription factors trans-mitting signals from the cell surface to the nucleus activated by cytokines and growth fac-tors which results in dimerization of their cog-nate receptors and activation of tyrosine kinas-es such as Janus kinase (JAK) [13] Numerous experiments have demonstrated that normal physical functions of STATs are critical in regu-lating many aspects of cellular proliferation apoptosis and migration STAT3 is the most inti-mately linked to cell proliferation and tumori-genesis and indispensable for apoptosis in numbers of cancer cells including breast colon and lung cancer cells [14-16]

In this study we examined the effects of gem-citabine on proliferation apoptosis and JAK2STAT3 signaling in lung cancer cells We found that Gemcitabine inhibited the proliferation and induced apoptosis resulting in increase of BaxBcl-2 ratio and inhibition of JAK2 and STAT3 activation thus suggesting a potential utility for development of preclinical and clinical investigations

Materials and methods

Cell culture

Lung cancer cell line SPC-A-1 (Academia Sinica Cell Bank Shanghai China) were cultured in DMEM medium supplemented with 10 (vv) fetal bovine serum 100 unitsmL penicillin and 100 μgmL streptomycin (Life Technologies Carlsbad CA USA) and incubated at 37degC in a humidified atmosphere of 5 CO2

Cell proliferation assay

The cell proliferation was evaluated by Cell Counting Kit 8 (CCK-8 Dojindo Molecular Technologies Rockville USA) SPC-A-1 cells were plated in the 96-well plates (5times103 cellswell) and incubated for 24 h Gemcitabine (10 20 and 40 mM) was introduced separately to cells in culture medium Cells cultured in the medium without adding agents were taken as the control After 0 24 48 and 72 h incubation the cells were washed with D-Hanks buffer solution One hundred microlitres of CCK-8 solution was added to each well and incubated for an additional 3 h at 37degC The optical den-sity (OD) of each well at 450 nm was record-

ed on a microplate reader (Thermo Fisher Scientific Waltham MA USA)

Cell cycle detection

SPC-A-1 cells were seeded in 12-well plates at the density of 3times103 cellswell and then treat-ed with 10 20 and 40 mM of gemcitabine for 48 h After treatments the percentages of cells in the different phases of cell cycle were evalu-ated by determining the DNA content after propidium iodide staining Briefly cells were washed with PBS trypsinized and centrifuged at 400timesg at 4degC for 5 min Pellets were fixed overnight in 70 cold ethanol After fixation cells were washed twice with PBS and incubat-ed in PBS containing RNase (1 mgmL) for 10 min at room temperature Finally samples were stained with propidium iodide (1 mgmL) for 30 min at 4degC Data acquisition was done by flow cytometry (Epics XLMCL Beckman Coulter Fullerton CA USA) using Cell Quest software (BD Biosciences Franklin Lakes NJ USA)

Cell apoptosis detection

Apoptosis kit (FITC Annexin V Apoptosis Detection Kit I BD Biosciences) was employed to detect apoptotic cells following the manu-facturerrsquos instructions Briefly SPC-A-1 cells were plated in the 6-well plates (1times105 cellswell) and incubated for 24 h Gemcitabine (10 20 and 40 mM) were introduced to the cells and incubated for another 48 h A549 cells were collected washed twice with cold D-hanks buffer solution and re-suspended in binding buffer (1times106 cellsmL) After 100 microl of SPC-A-1 cells was transferred to a tube 5 microl of FITC-conjugated Annexin V and 5 microl of propidium iodide were added followed by incubation for 15 min at room temperature in the dark The stained SPC-A-1 cells were diluted by the bind-ing buffer and directly analyzed by flow cytom-etry using Cell Quest software

Mitochondria membrane potential (MMP)

Mitochondria Membrane Potential (MMP) was detected by using Tetramethylrhodamine methyl ester (TMRM) dye Cells (1times106 cellswell) were cultured in 6-well plate After a peri-od of treatment (48 h) with various concentra-tions of gemcitabine (10 20 and 40 mM) cells were washed with PBS incubated with TMRM (10 mM) and subsequently subjected to flow cytometry

Gemcitabine blocks JAK2STAT3 pathways

2168 Int J Clin Exp Med 20169(2)2166-2174

Detection of reactive oxygen species (ROS)

Detection of ROS was performed by flow cytom-etry analysis as described previously In brief (5times104 cellswell) were cultured in 6-well plate after a period of treatment (48 h) with various concentrations of gemcitabine (10 20 and 40 mM) cells were washed with PBS and resus-pended in complete medium followed by incu-bation with 10 μM DCFH-DA for 20 min at 37degC ROS fluorescence intensity was determined by cytometry with excitation at 480 nm and emis-sion at 525 nm

Real-time PCR

Total RNAs were extracted from SPC-A-1 cells using TRIZOL reagent (Invitrogen Life Tech- nologies) and stored at -80degC The DyNAmo Flash SYBR Green qPCR kit (Finnzymes Oy Espoo Finland) was used according to the manufacturerrsquos instructions The primers se- quences (senseantisense) used were as fol-lows Bax forward 5rsquo-GTCATCTCGCTCTGGTA- CGG-3rsquo and reverse 5rsquo-CACACACACAAAGCT- GCTCC-3rsquo Bcl-2 forward 5rsquo-CCACCTGTGGT- CCATCTGAC-3rsquo and reverse 5rsquo-CAATCCTCCC- CCAGTTCACC-3rsquo GAPDH forward 5rsquo-GGAAT- CCACTGGCGTCTTCA-3rsquo and reverse 5rsquo-GGTTC- ACGCCCATCACAAAC-3rsquo Relative quantification of the signals was performed by normalizing the signals of different genes with the GAPDH signal

Western blot

Following treatment with gemcitabine at the desired concentrations and time SPC-A-1 cells were harvested The expression of apoptosis-related proteins was detected by Western blot according to the manufacturerrsquos instructions and previous reports [17] Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody was used as an internal control for whole cell lysates The experiment was repeated three times independently

Statistical analysis

Experimental data were presented as mean plusmn SD of at least three independent replicates through analyzing with GraphPad Prism 5 (GraphPad Software La Jolla CA) The paired two-tailed Studentrsquos t-test was used to analyze the significance of difference between groups

Differences were considered significant at val-ues of Plt005

Results

Gemcitabine causes growth inhibitory and cell cycle arrest in lung cancer cells

Human lung cancer cell lines SPC-A-1 was employed as a model system to investigate the effect of gemcitabine on lung cancer cell prolif-eration We quantified the cytotoxic effects of gemcitabine by measuring percent proliferation using CCK-8 assay Our data demonstrated that gemcitabine induced a dose- and time-dependent decrease in proliferation of the lung cancer cell lines SPC-A-1 with 60 35 and 18 of control at the concentration of 40 mM after 24 48 and 72 h treatments respectively (Figure 1A) Suppression of cancer cell prolif-eration can be caused by arrest of cell cycle progression Our findings on cell cycle distribu-tion demonstrated that treatment with Gemcitabine resulted in enrichment of lung cancer cells in G1 and G2 phase with a con-comitant decrease in number of cells in S phase We observed a 13 19 and 64 folds decrease in number of cells in S phase at 10 20 and 40 mM concentrations of gemcitabine respectively (Figure 1B and 1C) Together these data indicate that gemcitabine has prolif-eration inhibitory effects on lung cancer cells through arresting cell cycle

Gemcitabine induces apoptosis in lung cancer cells

Induction of apoptosis is another cause of can-cer cell growth suppression In apoptosis assay our data demonstrated a considerable increase in apoptotic index in a dose-dependent manner after 48 h of gemcitabine treatment (Figure 2) At 10 20 and 40 mM concentrations of gem-citabine we observed 30 48 and 82-folds increase in apoptotic indices of SPC-A-1 cells respectively Altogether our findings demon-strate that gemcitabine has both cytostatic and cytotoxic properties against lung cancer cells

Gemcitabine induces apoptosis in the mito-chondrial pathway

To assess the effect of gemcitabine on the changes of MMP in SPC-A-1 cells flow cytome-try analysis was carried out to detect the fluo-

Gemcitabine blocks JAK2STAT3 pathways

2169 Int J Clin Exp Med 20169(2)2166-2174

Figure 1 Gemcitabine inhibited the proliferation and arrested cell cycle of SPC-A-1 cells The cells were treated with gemcitabine (10 20 and 40 mM) for 24 48 and 72 h (A) Cell proliferation was determined using CCK-8 assay and (B C) cell cycle was analyzed using a propidium iodide staining assay and flow cytometry Plt005 Plt001 Plt0001 compared with control cells

Gemcitabine blocks JAK2STAT3 pathways

2170 Int J Clin Exp Med 20169(2)2166-2174

Figure 2 Gemcitabine promoted the apoptosis of SPC-A-1 cells The cells were treated with gemcitabine (10 20 and 40 mM) for 48 h A B Cell apoptosis was determined using Annexin-VPI double stain assay and flow cytometry Plt0001 compared with control cells

rescence intensity of TMRM As shown in Figure 3A and 3B treatment of SPC-A-1 cells with gemcitabine at the concentrations of 10 20 and 40 μM for 48 h caused a moderate depo-larization of MMP in a dose-dependent manner We observed 12 19 and 24-folds decrease in MMP level of SPC-A-1 cells respectively On the other hand ROS generation is also linked to mitochondria Fluorescence probe DCFH-DA was used to determine the levels of ROS pro-duction in SPC-A-1 cells As shown in Figure 3C and 3D SPC-A-1 cells exposed to gemcitabine at 10 20 and 40 μM for 48 h caused a signifi-cant increase in the intracellular accumulation of ROS in a dose-dependent manner We observed 12 14 and 17-folds increase in ROS accumulation of SPC-A-1 cells respective-ly Taken together these data suggest that gemcitabine induces lung cancer cells apopto-sis through the mitochondrial pathway

Gemcitabine alters the expression of proteins in lung cancer cells

To investigate the mechanistic basis of prolif-eration inhibitory effects of gemcitabine we

next examined its effect on the expression of key proteins involved in cell apoptosis and sig-naling pathway Our data revealed a dose-dependent increase in the ratio of BaxBcl-2 both in mRNA and protein levels (Figure 4A-C) Among the signaling pathway proteins we observed a dose-dependent reduction in the levels the phosphorylation of JAK2 and STAT3 whereas the expression of total JAK2 and STAT3 was not changed (Figure 4D and 4E) These findings demonstrate that Gemcitabine alters the expression of proteins involved in the regulation of cell apoptosis and signaling path-way to confer its proliferation inhibitory effect

Discussion

Lung cancer remains a devastating malignancy due to lack of effective therapy for treatment The present study demonstrated that gem-citabine is effective in suppressing the prolifer-ation and arresting cell cycle of human lung cancer cells SPC-A-1 due to its cytostatic and cytotoxic properties Furthermore our studies provided evidence for a role of gemcitabine in

Gemcitabine blocks JAK2STAT3 pathways

2171 Int J Clin Exp Med 20169(2)2166-2174

Figure 3 Gemcitabine decreased the MMP level and increased the ROS production of SPC-A-1 cells The cells were treated with gemcitabine (10 20 and 40 mM) for 48 h A B Cell MMP level was determined using TMRM and flow cytometry C D Cell ROS production was determined using DCFH-DA and flow cytometry Plt0001 compared with control cells

Gemcitabine blocks JAK2STAT3 pathways

2172 Int J Clin Exp Med 20169(2)2166-2174

inducing the lung cancer cells apoptosis and in suppressing activation of JAK2 and STAT3

Deregulated growth in cancer cells is often attributed to loss of control in proliferation and apoptosis [18] In this study we observed that cells were incubated with different concentra-tions of gemcitabine significantly declining cell proliferation in a time- and dose-dependent manner Similar concentrations of gemcitabine were found to exert the same effects with dif-ferent cancer cell lines by other groups [19] In fact molecular studies have revealed that cell cycle associated with cell survival is frequently altered in multiple human cancers We observed that cells were incubated with differ-ent concentrations of gemcitabine significantly increased the number of cells in G1 and G2 phase while decreased the number of cells in S phase However low concentration of gem-citabine (lt05 mM) showed cell cycle arrest in S and G2 phases which similar to the report of Hamed and colleagues [9 20] These results

suggest that gemcitabine arrest cell cycle dependent on its concentrations and targeted cancer cells

Following cell cycle arrest cells may either undergo repair or enter the apoptotic pathway to maintain cellular integrity [21] Thus induc-tion of apoptosis is one of the protective mech-anisms against caner development and pro-gression In the present study we observed significant induction of apoptosis in gem-citabine-treated lung cancer cells Cell survival is maintained by a balance of the ratio of anti-apoptotic and pro-apoptotic protein The upreg-ulation of Bax and downregulation of Bcl-2 were observed in a dose-dependent manner in lung cancer cells after treated with different concen-trations of gemcitabine The permeabilization of the mitochondrial outer membrane is tightly controlled by Bcl-2 [22] and activation of Bax can trigger a sequence of events that leads to alterations in mitochondrial permeability transi-tion [23] Therefore we found that reduction of

Figure 4 Gemcitabine increased BaxBcl-2 ratio and blocked the JAK2STAT3 signaling The cells were treated with gemcitabine (10 20 and 40 mM) for 48 h The mRNA expression level of BaxBcl-2 was determined using Real-time PCR (A) and Western blot (B C) The cells were treated with gemcitabine (10 20 and 40 mM) for 6 h The protein ex-pression levels of p-JAK2JAK2 and p-STAT3STAT3 were determined using Western blot (D E) Plt005 Plt001 Plt0001 compared with control cells

Gemcitabine blocks JAK2STAT3 pathways

2173 Int J Clin Exp Med 20169(2)2166-2174

MMP level emerged in lung cancer cells after treated with gemcitabine Besides mitochon-dria are major sources of ROS and the reduc-tion in the level of Bcl-2 would promote the gen-eration of ROS [24] An increase of ROS production was observed in lung cancer cells treated with gemcitabine These results indi-cate that gemcitabine induce lung cancer cells apoptosis through the mitochondria pathway

Abnormal JAKSTAT signaling pathways are involved in the pathogenesis of several cancers including apoptosis [25] However the molecu-lar mechanism by which disordered JAK2STAT3 signaling pathways contributes to apop-tosis has not been clarified in lung cancer cells Therefore our work found that gemcitabine treatment could inhibit the activation of JAK2 and STAT3 in a dose-dependent manner evi-denced by decreased p-JAK2 and p-STAT3 lev-els Previous study found that the expression of Bcl-2 that is known to be downstream target of the STATs pathway is decreased and the MMP level is reduced after suppression of JAK2STAT3 signaling pathways [15] These findings suggest that abnormal expression of Bcl-2 may be the trigger involved in JAK2STAT3 signaling pathway induced MMP downregulation Indeed it has been reported earlier that anti-cancer effects of gemcitabine can be potentiated by inhibition of JAK2STAT3 signaling pathways in pancreatic cancer [26]

In conclusion several factors involving the modulation of cell cycle apoptosis and expres-sion of critical genes involved in drug activity may contribute to the effect of gemcitabine against lung cancer cells in vitro Here we have shown for the first time the growth inhibitory and apoptosis induction of gemcitabine in lung cancer cells Gemcitabine causes G1 and G2 phase cell cycle arrest and induction of apopto-sis by altering the expression of Bax and Bcl-2 and phosphorylation of JAK2 and STAT3 We therefore believe that gemcitabine could be a novel promising agent for the treatment of lung cancer

Disclosure of conflict of interest

None

Address correspondence to Dr Yan Zhang Depart- ment of Oncology Tongji Hospital 389 Xincun Road Putuo District Shanghai 200065 China Tel +86-

021-66111009 Fax +86-021-66111009 E-mail zhangyan_p163com

References

[1] Jemal A Siegel R Ward E Hao Y Xu J Murray T Thun MJ Cancer statistics 2008 CA Cancer J Clin 2008 58 71-96

[2] Siegel R Naishadham D Jemal A Cancer sta-tistics 2012 CACancer J Clin 2012 62 10-29

[3] Zhou J Zhao WY Ma X Ju RJ Li XY Li N Sun MG Shi JF Zhang CX Lu WL The anticancer efficacy of paclitaxel liposomes modified with mitochondrial targeting conjugate in resistant lung cancer Biomaterials 2013 34 3626-38

[4] Conroy T Desseigne F Ychou M Boucheacute O Guimbaud R Beacutecouarn Y Adenis A Raoul JL Gourgou-Bourgade S de la Fouchardiegravere C Bennouna JBachet JB Khemissa-Akouz F Peacutereacute-Vergeacute D Delbaldo C Assenat E Chauffert B Michel P Montoto-Grillot C Ducreux M Groupe Tumeurs Digestives of Unicancer PRODIGE Intergroup Folfirinox versus gem-citabine for metastatic pancreatic cancer N Engl J Med 2011 364 1817-25

[5] Wu YL Zhou C Hu CP Feng J Lu S Huang Y Li W Hou M Shi JH Lee KY Xu CR Massey D Kim M Shi Y Geater SL Afatinib versus cispla-tin plus gemcitabine for first-line treatment of asian patients with advanced non-small-cell lung cancer harbouring egfr mutations (lux-lung 6) An open-label randomised phase 3 trial Lancet Oncol 2014 15 213-22

[6] Wang J Guo J Wu S Feng H Sun S Pan J Zhang J Beebe SJ Synergistic effects of nano-second pulsed electric fields combined with low concentration of gemcitabine on human oral squamous cell carcinoma in vitro PLoS One 2012 7 e43213

[7] Yang XL Lin FJ Guo YJ Shao ZM Ou ZL Gem-citabine resistance in breast cancer cells regu-lated by pi3kakt-mediated cellular prolifera-tion exerts negative feedback via the mekmapk and mtor pathways Onco Targets Ther 2014 7 1033

[8] Eastman A Montano R Thompson R Khan N Hou H Lewis LD et al Dissecting the combina-tion of gemcitabine and the chk1 inhibitor mk-8776 in vitro and in vivo Cell cycle perturba-tion and the impact of administration schedule Cancer Research 2013 73 3413

[9] Hamed SS Straubinger RM Jusko WJ Phar-macodynamic modeling of cell cycle and apop-totic effects of gemcitabine on pancreatic ad-enocarcinoma cells Cancer Chemother Phar- macol 2013 72 553-63

[10] Wang X Liu JZ Hu JX Wu H Li YL Chen HL Bai H Hai CX Ros-activated p38 mapkerk-akt

Gemcitabine blocks JAK2STAT3 pathways

2168 Int J Clin Exp Med 20169(2)2166-2174

Detection of reactive oxygen species (ROS)

Detection of ROS was performed by flow cytom-etry analysis as described previously In brief (5times104 cellswell) were cultured in 6-well plate after a period of treatment (48 h) with various concentrations of gemcitabine (10 20 and 40 mM) cells were washed with PBS and resus-pended in complete medium followed by incu-bation with 10 μM DCFH-DA for 20 min at 37degC ROS fluorescence intensity was determined by cytometry with excitation at 480 nm and emis-sion at 525 nm

Real-time PCR

Total RNAs were extracted from SPC-A-1 cells using TRIZOL reagent (Invitrogen Life Tech- nologies) and stored at -80degC The DyNAmo Flash SYBR Green qPCR kit (Finnzymes Oy Espoo Finland) was used according to the manufacturerrsquos instructions The primers se- quences (senseantisense) used were as fol-lows Bax forward 5rsquo-GTCATCTCGCTCTGGTA- CGG-3rsquo and reverse 5rsquo-CACACACACAAAGCT- GCTCC-3rsquo Bcl-2 forward 5rsquo-CCACCTGTGGT- CCATCTGAC-3rsquo and reverse 5rsquo-CAATCCTCCC- CCAGTTCACC-3rsquo GAPDH forward 5rsquo-GGAAT- CCACTGGCGTCTTCA-3rsquo and reverse 5rsquo-GGTTC- ACGCCCATCACAAAC-3rsquo Relative quantification of the signals was performed by normalizing the signals of different genes with the GAPDH signal

Western blot

Following treatment with gemcitabine at the desired concentrations and time SPC-A-1 cells were harvested The expression of apoptosis-related proteins was detected by Western blot according to the manufacturerrsquos instructions and previous reports [17] Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody was used as an internal control for whole cell lysates The experiment was repeated three times independently

Statistical analysis

Experimental data were presented as mean plusmn SD of at least three independent replicates through analyzing with GraphPad Prism 5 (GraphPad Software La Jolla CA) The paired two-tailed Studentrsquos t-test was used to analyze the significance of difference between groups

Differences were considered significant at val-ues of Plt005

Results

Gemcitabine causes growth inhibitory and cell cycle arrest in lung cancer cells

Human lung cancer cell lines SPC-A-1 was employed as a model system to investigate the effect of gemcitabine on lung cancer cell prolif-eration We quantified the cytotoxic effects of gemcitabine by measuring percent proliferation using CCK-8 assay Our data demonstrated that gemcitabine induced a dose- and time-dependent decrease in proliferation of the lung cancer cell lines SPC-A-1 with 60 35 and 18 of control at the concentration of 40 mM after 24 48 and 72 h treatments respectively (Figure 1A) Suppression of cancer cell prolif-eration can be caused by arrest of cell cycle progression Our findings on cell cycle distribu-tion demonstrated that treatment with Gemcitabine resulted in enrichment of lung cancer cells in G1 and G2 phase with a con-comitant decrease in number of cells in S phase We observed a 13 19 and 64 folds decrease in number of cells in S phase at 10 20 and 40 mM concentrations of gemcitabine respectively (Figure 1B and 1C) Together these data indicate that gemcitabine has prolif-eration inhibitory effects on lung cancer cells through arresting cell cycle

Gemcitabine induces apoptosis in lung cancer cells

Induction of apoptosis is another cause of can-cer cell growth suppression In apoptosis assay our data demonstrated a considerable increase in apoptotic index in a dose-dependent manner after 48 h of gemcitabine treatment (Figure 2) At 10 20 and 40 mM concentrations of gem-citabine we observed 30 48 and 82-folds increase in apoptotic indices of SPC-A-1 cells respectively Altogether our findings demon-strate that gemcitabine has both cytostatic and cytotoxic properties against lung cancer cells

Gemcitabine induces apoptosis in the mito-chondrial pathway

To assess the effect of gemcitabine on the changes of MMP in SPC-A-1 cells flow cytome-try analysis was carried out to detect the fluo-

Gemcitabine blocks JAK2STAT3 pathways

2169 Int J Clin Exp Med 20169(2)2166-2174

Figure 1 Gemcitabine inhibited the proliferation and arrested cell cycle of SPC-A-1 cells The cells were treated with gemcitabine (10 20 and 40 mM) for 24 48 and 72 h (A) Cell proliferation was determined using CCK-8 assay and (B C) cell cycle was analyzed using a propidium iodide staining assay and flow cytometry Plt005 Plt001 Plt0001 compared with control cells

Gemcitabine blocks JAK2STAT3 pathways

2170 Int J Clin Exp Med 20169(2)2166-2174

Figure 2 Gemcitabine promoted the apoptosis of SPC-A-1 cells The cells were treated with gemcitabine (10 20 and 40 mM) for 48 h A B Cell apoptosis was determined using Annexin-VPI double stain assay and flow cytometry Plt0001 compared with control cells

rescence intensity of TMRM As shown in Figure 3A and 3B treatment of SPC-A-1 cells with gemcitabine at the concentrations of 10 20 and 40 μM for 48 h caused a moderate depo-larization of MMP in a dose-dependent manner We observed 12 19 and 24-folds decrease in MMP level of SPC-A-1 cells respectively On the other hand ROS generation is also linked to mitochondria Fluorescence probe DCFH-DA was used to determine the levels of ROS pro-duction in SPC-A-1 cells As shown in Figure 3C and 3D SPC-A-1 cells exposed to gemcitabine at 10 20 and 40 μM for 48 h caused a signifi-cant increase in the intracellular accumulation of ROS in a dose-dependent manner We observed 12 14 and 17-folds increase in ROS accumulation of SPC-A-1 cells respective-ly Taken together these data suggest that gemcitabine induces lung cancer cells apopto-sis through the mitochondrial pathway

Gemcitabine alters the expression of proteins in lung cancer cells

To investigate the mechanistic basis of prolif-eration inhibitory effects of gemcitabine we

next examined its effect on the expression of key proteins involved in cell apoptosis and sig-naling pathway Our data revealed a dose-dependent increase in the ratio of BaxBcl-2 both in mRNA and protein levels (Figure 4A-C) Among the signaling pathway proteins we observed a dose-dependent reduction in the levels the phosphorylation of JAK2 and STAT3 whereas the expression of total JAK2 and STAT3 was not changed (Figure 4D and 4E) These findings demonstrate that Gemcitabine alters the expression of proteins involved in the regulation of cell apoptosis and signaling path-way to confer its proliferation inhibitory effect

Discussion

Lung cancer remains a devastating malignancy due to lack of effective therapy for treatment The present study demonstrated that gem-citabine is effective in suppressing the prolifer-ation and arresting cell cycle of human lung cancer cells SPC-A-1 due to its cytostatic and cytotoxic properties Furthermore our studies provided evidence for a role of gemcitabine in

Gemcitabine blocks JAK2STAT3 pathways

2171 Int J Clin Exp Med 20169(2)2166-2174

Figure 3 Gemcitabine decreased the MMP level and increased the ROS production of SPC-A-1 cells The cells were treated with gemcitabine (10 20 and 40 mM) for 48 h A B Cell MMP level was determined using TMRM and flow cytometry C D Cell ROS production was determined using DCFH-DA and flow cytometry Plt0001 compared with control cells

Gemcitabine blocks JAK2STAT3 pathways

2172 Int J Clin Exp Med 20169(2)2166-2174

inducing the lung cancer cells apoptosis and in suppressing activation of JAK2 and STAT3

Deregulated growth in cancer cells is often attributed to loss of control in proliferation and apoptosis [18] In this study we observed that cells were incubated with different concentra-tions of gemcitabine significantly declining cell proliferation in a time- and dose-dependent manner Similar concentrations of gemcitabine were found to exert the same effects with dif-ferent cancer cell lines by other groups [19] In fact molecular studies have revealed that cell cycle associated with cell survival is frequently altered in multiple human cancers We observed that cells were incubated with differ-ent concentrations of gemcitabine significantly increased the number of cells in G1 and G2 phase while decreased the number of cells in S phase However low concentration of gem-citabine (lt05 mM) showed cell cycle arrest in S and G2 phases which similar to the report of Hamed and colleagues [9 20] These results

suggest that gemcitabine arrest cell cycle dependent on its concentrations and targeted cancer cells

Following cell cycle arrest cells may either undergo repair or enter the apoptotic pathway to maintain cellular integrity [21] Thus induc-tion of apoptosis is one of the protective mech-anisms against caner development and pro-gression In the present study we observed significant induction of apoptosis in gem-citabine-treated lung cancer cells Cell survival is maintained by a balance of the ratio of anti-apoptotic and pro-apoptotic protein The upreg-ulation of Bax and downregulation of Bcl-2 were observed in a dose-dependent manner in lung cancer cells after treated with different concen-trations of gemcitabine The permeabilization of the mitochondrial outer membrane is tightly controlled by Bcl-2 [22] and activation of Bax can trigger a sequence of events that leads to alterations in mitochondrial permeability transi-tion [23] Therefore we found that reduction of

Figure 4 Gemcitabine increased BaxBcl-2 ratio and blocked the JAK2STAT3 signaling The cells were treated with gemcitabine (10 20 and 40 mM) for 48 h The mRNA expression level of BaxBcl-2 was determined using Real-time PCR (A) and Western blot (B C) The cells were treated with gemcitabine (10 20 and 40 mM) for 6 h The protein ex-pression levels of p-JAK2JAK2 and p-STAT3STAT3 were determined using Western blot (D E) Plt005 Plt001 Plt0001 compared with control cells

Gemcitabine blocks JAK2STAT3 pathways

2173 Int J Clin Exp Med 20169(2)2166-2174

MMP level emerged in lung cancer cells after treated with gemcitabine Besides mitochon-dria are major sources of ROS and the reduc-tion in the level of Bcl-2 would promote the gen-eration of ROS [24] An increase of ROS production was observed in lung cancer cells treated with gemcitabine These results indi-cate that gemcitabine induce lung cancer cells apoptosis through the mitochondria pathway

Abnormal JAKSTAT signaling pathways are involved in the pathogenesis of several cancers including apoptosis [25] However the molecu-lar mechanism by which disordered JAK2STAT3 signaling pathways contributes to apop-tosis has not been clarified in lung cancer cells Therefore our work found that gemcitabine treatment could inhibit the activation of JAK2 and STAT3 in a dose-dependent manner evi-denced by decreased p-JAK2 and p-STAT3 lev-els Previous study found that the expression of Bcl-2 that is known to be downstream target of the STATs pathway is decreased and the MMP level is reduced after suppression of JAK2STAT3 signaling pathways [15] These findings suggest that abnormal expression of Bcl-2 may be the trigger involved in JAK2STAT3 signaling pathway induced MMP downregulation Indeed it has been reported earlier that anti-cancer effects of gemcitabine can be potentiated by inhibition of JAK2STAT3 signaling pathways in pancreatic cancer [26]

In conclusion several factors involving the modulation of cell cycle apoptosis and expres-sion of critical genes involved in drug activity may contribute to the effect of gemcitabine against lung cancer cells in vitro Here we have shown for the first time the growth inhibitory and apoptosis induction of gemcitabine in lung cancer cells Gemcitabine causes G1 and G2 phase cell cycle arrest and induction of apopto-sis by altering the expression of Bax and Bcl-2 and phosphorylation of JAK2 and STAT3 We therefore believe that gemcitabine could be a novel promising agent for the treatment of lung cancer

Disclosure of conflict of interest

None

Address correspondence to Dr Yan Zhang Depart- ment of Oncology Tongji Hospital 389 Xincun Road Putuo District Shanghai 200065 China Tel +86-

021-66111009 Fax +86-021-66111009 E-mail zhangyan_p163com

References

[1] Jemal A Siegel R Ward E Hao Y Xu J Murray T Thun MJ Cancer statistics 2008 CA Cancer J Clin 2008 58 71-96

[2] Siegel R Naishadham D Jemal A Cancer sta-tistics 2012 CACancer J Clin 2012 62 10-29

[3] Zhou J Zhao WY Ma X Ju RJ Li XY Li N Sun MG Shi JF Zhang CX Lu WL The anticancer efficacy of paclitaxel liposomes modified with mitochondrial targeting conjugate in resistant lung cancer Biomaterials 2013 34 3626-38

[4] Conroy T Desseigne F Ychou M Boucheacute O Guimbaud R Beacutecouarn Y Adenis A Raoul JL Gourgou-Bourgade S de la Fouchardiegravere C Bennouna JBachet JB Khemissa-Akouz F Peacutereacute-Vergeacute D Delbaldo C Assenat E Chauffert B Michel P Montoto-Grillot C Ducreux M Groupe Tumeurs Digestives of Unicancer PRODIGE Intergroup Folfirinox versus gem-citabine for metastatic pancreatic cancer N Engl J Med 2011 364 1817-25

[5] Wu YL Zhou C Hu CP Feng J Lu S Huang Y Li W Hou M Shi JH Lee KY Xu CR Massey D Kim M Shi Y Geater SL Afatinib versus cispla-tin plus gemcitabine for first-line treatment of asian patients with advanced non-small-cell lung cancer harbouring egfr mutations (lux-lung 6) An open-label randomised phase 3 trial Lancet Oncol 2014 15 213-22

[6] Wang J Guo J Wu S Feng H Sun S Pan J Zhang J Beebe SJ Synergistic effects of nano-second pulsed electric fields combined with low concentration of gemcitabine on human oral squamous cell carcinoma in vitro PLoS One 2012 7 e43213

[7] Yang XL Lin FJ Guo YJ Shao ZM Ou ZL Gem-citabine resistance in breast cancer cells regu-lated by pi3kakt-mediated cellular prolifera-tion exerts negative feedback via the mekmapk and mtor pathways Onco Targets Ther 2014 7 1033

[8] Eastman A Montano R Thompson R Khan N Hou H Lewis LD et al Dissecting the combina-tion of gemcitabine and the chk1 inhibitor mk-8776 in vitro and in vivo Cell cycle perturba-tion and the impact of administration schedule Cancer Research 2013 73 3413

[9] Hamed SS Straubinger RM Jusko WJ Phar-macodynamic modeling of cell cycle and apop-totic effects of gemcitabine on pancreatic ad-enocarcinoma cells Cancer Chemother Phar- macol 2013 72 553-63

[10] Wang X Liu JZ Hu JX Wu H Li YL Chen HL Bai H Hai CX Ros-activated p38 mapkerk-akt

Gemcitabine blocks JAK2STAT3 pathways

2169 Int J Clin Exp Med 20169(2)2166-2174

Figure 1 Gemcitabine inhibited the proliferation and arrested cell cycle of SPC-A-1 cells The cells were treated with gemcitabine (10 20 and 40 mM) for 24 48 and 72 h (A) Cell proliferation was determined using CCK-8 assay and (B C) cell cycle was analyzed using a propidium iodide staining assay and flow cytometry Plt005 Plt001 Plt0001 compared with control cells

Gemcitabine blocks JAK2STAT3 pathways

2170 Int J Clin Exp Med 20169(2)2166-2174

Figure 2 Gemcitabine promoted the apoptosis of SPC-A-1 cells The cells were treated with gemcitabine (10 20 and 40 mM) for 48 h A B Cell apoptosis was determined using Annexin-VPI double stain assay and flow cytometry Plt0001 compared with control cells

rescence intensity of TMRM As shown in Figure 3A and 3B treatment of SPC-A-1 cells with gemcitabine at the concentrations of 10 20 and 40 μM for 48 h caused a moderate depo-larization of MMP in a dose-dependent manner We observed 12 19 and 24-folds decrease in MMP level of SPC-A-1 cells respectively On the other hand ROS generation is also linked to mitochondria Fluorescence probe DCFH-DA was used to determine the levels of ROS pro-duction in SPC-A-1 cells As shown in Figure 3C and 3D SPC-A-1 cells exposed to gemcitabine at 10 20 and 40 μM for 48 h caused a signifi-cant increase in the intracellular accumulation of ROS in a dose-dependent manner We observed 12 14 and 17-folds increase in ROS accumulation of SPC-A-1 cells respective-ly Taken together these data suggest that gemcitabine induces lung cancer cells apopto-sis through the mitochondrial pathway

Gemcitabine alters the expression of proteins in lung cancer cells

To investigate the mechanistic basis of prolif-eration inhibitory effects of gemcitabine we

next examined its effect on the expression of key proteins involved in cell apoptosis and sig-naling pathway Our data revealed a dose-dependent increase in the ratio of BaxBcl-2 both in mRNA and protein levels (Figure 4A-C) Among the signaling pathway proteins we observed a dose-dependent reduction in the levels the phosphorylation of JAK2 and STAT3 whereas the expression of total JAK2 and STAT3 was not changed (Figure 4D and 4E) These findings demonstrate that Gemcitabine alters the expression of proteins involved in the regulation of cell apoptosis and signaling path-way to confer its proliferation inhibitory effect

Discussion

Lung cancer remains a devastating malignancy due to lack of effective therapy for treatment The present study demonstrated that gem-citabine is effective in suppressing the prolifer-ation and arresting cell cycle of human lung cancer cells SPC-A-1 due to its cytostatic and cytotoxic properties Furthermore our studies provided evidence for a role of gemcitabine in

Gemcitabine blocks JAK2STAT3 pathways

2171 Int J Clin Exp Med 20169(2)2166-2174

Figure 3 Gemcitabine decreased the MMP level and increased the ROS production of SPC-A-1 cells The cells were treated with gemcitabine (10 20 and 40 mM) for 48 h A B Cell MMP level was determined using TMRM and flow cytometry C D Cell ROS production was determined using DCFH-DA and flow cytometry Plt0001 compared with control cells

Gemcitabine blocks JAK2STAT3 pathways

2172 Int J Clin Exp Med 20169(2)2166-2174

inducing the lung cancer cells apoptosis and in suppressing activation of JAK2 and STAT3

Deregulated growth in cancer cells is often attributed to loss of control in proliferation and apoptosis [18] In this study we observed that cells were incubated with different concentra-tions of gemcitabine significantly declining cell proliferation in a time- and dose-dependent manner Similar concentrations of gemcitabine were found to exert the same effects with dif-ferent cancer cell lines by other groups [19] In fact molecular studies have revealed that cell cycle associated with cell survival is frequently altered in multiple human cancers We observed that cells were incubated with differ-ent concentrations of gemcitabine significantly increased the number of cells in G1 and G2 phase while decreased the number of cells in S phase However low concentration of gem-citabine (lt05 mM) showed cell cycle arrest in S and G2 phases which similar to the report of Hamed and colleagues [9 20] These results

suggest that gemcitabine arrest cell cycle dependent on its concentrations and targeted cancer cells

Following cell cycle arrest cells may either undergo repair or enter the apoptotic pathway to maintain cellular integrity [21] Thus induc-tion of apoptosis is one of the protective mech-anisms against caner development and pro-gression In the present study we observed significant induction of apoptosis in gem-citabine-treated lung cancer cells Cell survival is maintained by a balance of the ratio of anti-apoptotic and pro-apoptotic protein The upreg-ulation of Bax and downregulation of Bcl-2 were observed in a dose-dependent manner in lung cancer cells after treated with different concen-trations of gemcitabine The permeabilization of the mitochondrial outer membrane is tightly controlled by Bcl-2 [22] and activation of Bax can trigger a sequence of events that leads to alterations in mitochondrial permeability transi-tion [23] Therefore we found that reduction of

Figure 4 Gemcitabine increased BaxBcl-2 ratio and blocked the JAK2STAT3 signaling The cells were treated with gemcitabine (10 20 and 40 mM) for 48 h The mRNA expression level of BaxBcl-2 was determined using Real-time PCR (A) and Western blot (B C) The cells were treated with gemcitabine (10 20 and 40 mM) for 6 h The protein ex-pression levels of p-JAK2JAK2 and p-STAT3STAT3 were determined using Western blot (D E) Plt005 Plt001 Plt0001 compared with control cells

Gemcitabine blocks JAK2STAT3 pathways

2173 Int J Clin Exp Med 20169(2)2166-2174

MMP level emerged in lung cancer cells after treated with gemcitabine Besides mitochon-dria are major sources of ROS and the reduc-tion in the level of Bcl-2 would promote the gen-eration of ROS [24] An increase of ROS production was observed in lung cancer cells treated with gemcitabine These results indi-cate that gemcitabine induce lung cancer cells apoptosis through the mitochondria pathway

Abnormal JAKSTAT signaling pathways are involved in the pathogenesis of several cancers including apoptosis [25] However the molecu-lar mechanism by which disordered JAK2STAT3 signaling pathways contributes to apop-tosis has not been clarified in lung cancer cells Therefore our work found that gemcitabine treatment could inhibit the activation of JAK2 and STAT3 in a dose-dependent manner evi-denced by decreased p-JAK2 and p-STAT3 lev-els Previous study found that the expression of Bcl-2 that is known to be downstream target of the STATs pathway is decreased and the MMP level is reduced after suppression of JAK2STAT3 signaling pathways [15] These findings suggest that abnormal expression of Bcl-2 may be the trigger involved in JAK2STAT3 signaling pathway induced MMP downregulation Indeed it has been reported earlier that anti-cancer effects of gemcitabine can be potentiated by inhibition of JAK2STAT3 signaling pathways in pancreatic cancer [26]

In conclusion several factors involving the modulation of cell cycle apoptosis and expres-sion of critical genes involved in drug activity may contribute to the effect of gemcitabine against lung cancer cells in vitro Here we have shown for the first time the growth inhibitory and apoptosis induction of gemcitabine in lung cancer cells Gemcitabine causes G1 and G2 phase cell cycle arrest and induction of apopto-sis by altering the expression of Bax and Bcl-2 and phosphorylation of JAK2 and STAT3 We therefore believe that gemcitabine could be a novel promising agent for the treatment of lung cancer

Disclosure of conflict of interest

None

Address correspondence to Dr Yan Zhang Depart- ment of Oncology Tongji Hospital 389 Xincun Road Putuo District Shanghai 200065 China Tel +86-

021-66111009 Fax +86-021-66111009 E-mail zhangyan_p163com

References

[1] Jemal A Siegel R Ward E Hao Y Xu J Murray T Thun MJ Cancer statistics 2008 CA Cancer J Clin 2008 58 71-96

[2] Siegel R Naishadham D Jemal A Cancer sta-tistics 2012 CACancer J Clin 2012 62 10-29

[3] Zhou J Zhao WY Ma X Ju RJ Li XY Li N Sun MG Shi JF Zhang CX Lu WL The anticancer efficacy of paclitaxel liposomes modified with mitochondrial targeting conjugate in resistant lung cancer Biomaterials 2013 34 3626-38

[4] Conroy T Desseigne F Ychou M Boucheacute O Guimbaud R Beacutecouarn Y Adenis A Raoul JL Gourgou-Bourgade S de la Fouchardiegravere C Bennouna JBachet JB Khemissa-Akouz F Peacutereacute-Vergeacute D Delbaldo C Assenat E Chauffert B Michel P Montoto-Grillot C Ducreux M Groupe Tumeurs Digestives of Unicancer PRODIGE Intergroup Folfirinox versus gem-citabine for metastatic pancreatic cancer N Engl J Med 2011 364 1817-25

[5] Wu YL Zhou C Hu CP Feng J Lu S Huang Y Li W Hou M Shi JH Lee KY Xu CR Massey D Kim M Shi Y Geater SL Afatinib versus cispla-tin plus gemcitabine for first-line treatment of asian patients with advanced non-small-cell lung cancer harbouring egfr mutations (lux-lung 6) An open-label randomised phase 3 trial Lancet Oncol 2014 15 213-22

[6] Wang J Guo J Wu S Feng H Sun S Pan J Zhang J Beebe SJ Synergistic effects of nano-second pulsed electric fields combined with low concentration of gemcitabine on human oral squamous cell carcinoma in vitro PLoS One 2012 7 e43213

[7] Yang XL Lin FJ Guo YJ Shao ZM Ou ZL Gem-citabine resistance in breast cancer cells regu-lated by pi3kakt-mediated cellular prolifera-tion exerts negative feedback via the mekmapk and mtor pathways Onco Targets Ther 2014 7 1033

[8] Eastman A Montano R Thompson R Khan N Hou H Lewis LD et al Dissecting the combina-tion of gemcitabine and the chk1 inhibitor mk-8776 in vitro and in vivo Cell cycle perturba-tion and the impact of administration schedule Cancer Research 2013 73 3413

[9] Hamed SS Straubinger RM Jusko WJ Phar-macodynamic modeling of cell cycle and apop-totic effects of gemcitabine on pancreatic ad-enocarcinoma cells Cancer Chemother Phar- macol 2013 72 553-63

[10] Wang X Liu JZ Hu JX Wu H Li YL Chen HL Bai H Hai CX Ros-activated p38 mapkerk-akt

Gemcitabine blocks JAK2STAT3 pathways

2170 Int J Clin Exp Med 20169(2)2166-2174

Figure 2 Gemcitabine promoted the apoptosis of SPC-A-1 cells The cells were treated with gemcitabine (10 20 and 40 mM) for 48 h A B Cell apoptosis was determined using Annexin-VPI double stain assay and flow cytometry Plt0001 compared with control cells

rescence intensity of TMRM As shown in Figure 3A and 3B treatment of SPC-A-1 cells with gemcitabine at the concentrations of 10 20 and 40 μM for 48 h caused a moderate depo-larization of MMP in a dose-dependent manner We observed 12 19 and 24-folds decrease in MMP level of SPC-A-1 cells respectively On the other hand ROS generation is also linked to mitochondria Fluorescence probe DCFH-DA was used to determine the levels of ROS pro-duction in SPC-A-1 cells As shown in Figure 3C and 3D SPC-A-1 cells exposed to gemcitabine at 10 20 and 40 μM for 48 h caused a signifi-cant increase in the intracellular accumulation of ROS in a dose-dependent manner We observed 12 14 and 17-folds increase in ROS accumulation of SPC-A-1 cells respective-ly Taken together these data suggest that gemcitabine induces lung cancer cells apopto-sis through the mitochondrial pathway

Gemcitabine alters the expression of proteins in lung cancer cells

To investigate the mechanistic basis of prolif-eration inhibitory effects of gemcitabine we

next examined its effect on the expression of key proteins involved in cell apoptosis and sig-naling pathway Our data revealed a dose-dependent increase in the ratio of BaxBcl-2 both in mRNA and protein levels (Figure 4A-C) Among the signaling pathway proteins we observed a dose-dependent reduction in the levels the phosphorylation of JAK2 and STAT3 whereas the expression of total JAK2 and STAT3 was not changed (Figure 4D and 4E) These findings demonstrate that Gemcitabine alters the expression of proteins involved in the regulation of cell apoptosis and signaling path-way to confer its proliferation inhibitory effect

Discussion

Lung cancer remains a devastating malignancy due to lack of effective therapy for treatment The present study demonstrated that gem-citabine is effective in suppressing the prolifer-ation and arresting cell cycle of human lung cancer cells SPC-A-1 due to its cytostatic and cytotoxic properties Furthermore our studies provided evidence for a role of gemcitabine in

Gemcitabine blocks JAK2STAT3 pathways

2171 Int J Clin Exp Med 20169(2)2166-2174

Figure 3 Gemcitabine decreased the MMP level and increased the ROS production of SPC-A-1 cells The cells were treated with gemcitabine (10 20 and 40 mM) for 48 h A B Cell MMP level was determined using TMRM and flow cytometry C D Cell ROS production was determined using DCFH-DA and flow cytometry Plt0001 compared with control cells

Gemcitabine blocks JAK2STAT3 pathways

2172 Int J Clin Exp Med 20169(2)2166-2174

inducing the lung cancer cells apoptosis and in suppressing activation of JAK2 and STAT3

Deregulated growth in cancer cells is often attributed to loss of control in proliferation and apoptosis [18] In this study we observed that cells were incubated with different concentra-tions of gemcitabine significantly declining cell proliferation in a time- and dose-dependent manner Similar concentrations of gemcitabine were found to exert the same effects with dif-ferent cancer cell lines by other groups [19] In fact molecular studies have revealed that cell cycle associated with cell survival is frequently altered in multiple human cancers We observed that cells were incubated with differ-ent concentrations of gemcitabine significantly increased the number of cells in G1 and G2 phase while decreased the number of cells in S phase However low concentration of gem-citabine (lt05 mM) showed cell cycle arrest in S and G2 phases which similar to the report of Hamed and colleagues [9 20] These results

suggest that gemcitabine arrest cell cycle dependent on its concentrations and targeted cancer cells

Following cell cycle arrest cells may either undergo repair or enter the apoptotic pathway to maintain cellular integrity [21] Thus induc-tion of apoptosis is one of the protective mech-anisms against caner development and pro-gression In the present study we observed significant induction of apoptosis in gem-citabine-treated lung cancer cells Cell survival is maintained by a balance of the ratio of anti-apoptotic and pro-apoptotic protein The upreg-ulation of Bax and downregulation of Bcl-2 were observed in a dose-dependent manner in lung cancer cells after treated with different concen-trations of gemcitabine The permeabilization of the mitochondrial outer membrane is tightly controlled by Bcl-2 [22] and activation of Bax can trigger a sequence of events that leads to alterations in mitochondrial permeability transi-tion [23] Therefore we found that reduction of

Figure 4 Gemcitabine increased BaxBcl-2 ratio and blocked the JAK2STAT3 signaling The cells were treated with gemcitabine (10 20 and 40 mM) for 48 h The mRNA expression level of BaxBcl-2 was determined using Real-time PCR (A) and Western blot (B C) The cells were treated with gemcitabine (10 20 and 40 mM) for 6 h The protein ex-pression levels of p-JAK2JAK2 and p-STAT3STAT3 were determined using Western blot (D E) Plt005 Plt001 Plt0001 compared with control cells

Gemcitabine blocks JAK2STAT3 pathways

2173 Int J Clin Exp Med 20169(2)2166-2174

MMP level emerged in lung cancer cells after treated with gemcitabine Besides mitochon-dria are major sources of ROS and the reduc-tion in the level of Bcl-2 would promote the gen-eration of ROS [24] An increase of ROS production was observed in lung cancer cells treated with gemcitabine These results indi-cate that gemcitabine induce lung cancer cells apoptosis through the mitochondria pathway

Abnormal JAKSTAT signaling pathways are involved in the pathogenesis of several cancers including apoptosis [25] However the molecu-lar mechanism by which disordered JAK2STAT3 signaling pathways contributes to apop-tosis has not been clarified in lung cancer cells Therefore our work found that gemcitabine treatment could inhibit the activation of JAK2 and STAT3 in a dose-dependent manner evi-denced by decreased p-JAK2 and p-STAT3 lev-els Previous study found that the expression of Bcl-2 that is known to be downstream target of the STATs pathway is decreased and the MMP level is reduced after suppression of JAK2STAT3 signaling pathways [15] These findings suggest that abnormal expression of Bcl-2 may be the trigger involved in JAK2STAT3 signaling pathway induced MMP downregulation Indeed it has been reported earlier that anti-cancer effects of gemcitabine can be potentiated by inhibition of JAK2STAT3 signaling pathways in pancreatic cancer [26]

In conclusion several factors involving the modulation of cell cycle apoptosis and expres-sion of critical genes involved in drug activity may contribute to the effect of gemcitabine against lung cancer cells in vitro Here we have shown for the first time the growth inhibitory and apoptosis induction of gemcitabine in lung cancer cells Gemcitabine causes G1 and G2 phase cell cycle arrest and induction of apopto-sis by altering the expression of Bax and Bcl-2 and phosphorylation of JAK2 and STAT3 We therefore believe that gemcitabine could be a novel promising agent for the treatment of lung cancer

Disclosure of conflict of interest

None

Address correspondence to Dr Yan Zhang Depart- ment of Oncology Tongji Hospital 389 Xincun Road Putuo District Shanghai 200065 China Tel +86-

021-66111009 Fax +86-021-66111009 E-mail zhangyan_p163com

References

[1] Jemal A Siegel R Ward E Hao Y Xu J Murray T Thun MJ Cancer statistics 2008 CA Cancer J Clin 2008 58 71-96

[2] Siegel R Naishadham D Jemal A Cancer sta-tistics 2012 CACancer J Clin 2012 62 10-29

[3] Zhou J Zhao WY Ma X Ju RJ Li XY Li N Sun MG Shi JF Zhang CX Lu WL The anticancer efficacy of paclitaxel liposomes modified with mitochondrial targeting conjugate in resistant lung cancer Biomaterials 2013 34 3626-38

[4] Conroy T Desseigne F Ychou M Boucheacute O Guimbaud R Beacutecouarn Y Adenis A Raoul JL Gourgou-Bourgade S de la Fouchardiegravere C Bennouna JBachet JB Khemissa-Akouz F Peacutereacute-Vergeacute D Delbaldo C Assenat E Chauffert B Michel P Montoto-Grillot C Ducreux M Groupe Tumeurs Digestives of Unicancer PRODIGE Intergroup Folfirinox versus gem-citabine for metastatic pancreatic cancer N Engl J Med 2011 364 1817-25

[5] Wu YL Zhou C Hu CP Feng J Lu S Huang Y Li W Hou M Shi JH Lee KY Xu CR Massey D Kim M Shi Y Geater SL Afatinib versus cispla-tin plus gemcitabine for first-line treatment of asian patients with advanced non-small-cell lung cancer harbouring egfr mutations (lux-lung 6) An open-label randomised phase 3 trial Lancet Oncol 2014 15 213-22

[6] Wang J Guo J Wu S Feng H Sun S Pan J Zhang J Beebe SJ Synergistic effects of nano-second pulsed electric fields combined with low concentration of gemcitabine on human oral squamous cell carcinoma in vitro PLoS One 2012 7 e43213

[7] Yang XL Lin FJ Guo YJ Shao ZM Ou ZL Gem-citabine resistance in breast cancer cells regu-lated by pi3kakt-mediated cellular prolifera-tion exerts negative feedback via the mekmapk and mtor pathways Onco Targets Ther 2014 7 1033

[8] Eastman A Montano R Thompson R Khan N Hou H Lewis LD et al Dissecting the combina-tion of gemcitabine and the chk1 inhibitor mk-8776 in vitro and in vivo Cell cycle perturba-tion and the impact of administration schedule Cancer Research 2013 73 3413

[9] Hamed SS Straubinger RM Jusko WJ Phar-macodynamic modeling of cell cycle and apop-totic effects of gemcitabine on pancreatic ad-enocarcinoma cells Cancer Chemother Phar- macol 2013 72 553-63

[10] Wang X Liu JZ Hu JX Wu H Li YL Chen HL Bai H Hai CX Ros-activated p38 mapkerk-akt

Gemcitabine blocks JAK2STAT3 pathways

2171 Int J Clin Exp Med 20169(2)2166-2174

Figure 3 Gemcitabine decreased the MMP level and increased the ROS production of SPC-A-1 cells The cells were treated with gemcitabine (10 20 and 40 mM) for 48 h A B Cell MMP level was determined using TMRM and flow cytometry C D Cell ROS production was determined using DCFH-DA and flow cytometry Plt0001 compared with control cells

Gemcitabine blocks JAK2STAT3 pathways

2172 Int J Clin Exp Med 20169(2)2166-2174

inducing the lung cancer cells apoptosis and in suppressing activation of JAK2 and STAT3

Deregulated growth in cancer cells is often attributed to loss of control in proliferation and apoptosis [18] In this study we observed that cells were incubated with different concentra-tions of gemcitabine significantly declining cell proliferation in a time- and dose-dependent manner Similar concentrations of gemcitabine were found to exert the same effects with dif-ferent cancer cell lines by other groups [19] In fact molecular studies have revealed that cell cycle associated with cell survival is frequently altered in multiple human cancers We observed that cells were incubated with differ-ent concentrations of gemcitabine significantly increased the number of cells in G1 and G2 phase while decreased the number of cells in S phase However low concentration of gem-citabine (lt05 mM) showed cell cycle arrest in S and G2 phases which similar to the report of Hamed and colleagues [9 20] These results

suggest that gemcitabine arrest cell cycle dependent on its concentrations and targeted cancer cells

Following cell cycle arrest cells may either undergo repair or enter the apoptotic pathway to maintain cellular integrity [21] Thus induc-tion of apoptosis is one of the protective mech-anisms against caner development and pro-gression In the present study we observed significant induction of apoptosis in gem-citabine-treated lung cancer cells Cell survival is maintained by a balance of the ratio of anti-apoptotic and pro-apoptotic protein The upreg-ulation of Bax and downregulation of Bcl-2 were observed in a dose-dependent manner in lung cancer cells after treated with different concen-trations of gemcitabine The permeabilization of the mitochondrial outer membrane is tightly controlled by Bcl-2 [22] and activation of Bax can trigger a sequence of events that leads to alterations in mitochondrial permeability transi-tion [23] Therefore we found that reduction of

Figure 4 Gemcitabine increased BaxBcl-2 ratio and blocked the JAK2STAT3 signaling The cells were treated with gemcitabine (10 20 and 40 mM) for 48 h The mRNA expression level of BaxBcl-2 was determined using Real-time PCR (A) and Western blot (B C) The cells were treated with gemcitabine (10 20 and 40 mM) for 6 h The protein ex-pression levels of p-JAK2JAK2 and p-STAT3STAT3 were determined using Western blot (D E) Plt005 Plt001 Plt0001 compared with control cells

Gemcitabine blocks JAK2STAT3 pathways

2173 Int J Clin Exp Med 20169(2)2166-2174

MMP level emerged in lung cancer cells after treated with gemcitabine Besides mitochon-dria are major sources of ROS and the reduc-tion in the level of Bcl-2 would promote the gen-eration of ROS [24] An increase of ROS production was observed in lung cancer cells treated with gemcitabine These results indi-cate that gemcitabine induce lung cancer cells apoptosis through the mitochondria pathway

Abnormal JAKSTAT signaling pathways are involved in the pathogenesis of several cancers including apoptosis [25] However the molecu-lar mechanism by which disordered JAK2STAT3 signaling pathways contributes to apop-tosis has not been clarified in lung cancer cells Therefore our work found that gemcitabine treatment could inhibit the activation of JAK2 and STAT3 in a dose-dependent manner evi-denced by decreased p-JAK2 and p-STAT3 lev-els Previous study found that the expression of Bcl-2 that is known to be downstream target of the STATs pathway is decreased and the MMP level is reduced after suppression of JAK2STAT3 signaling pathways [15] These findings suggest that abnormal expression of Bcl-2 may be the trigger involved in JAK2STAT3 signaling pathway induced MMP downregulation Indeed it has been reported earlier that anti-cancer effects of gemcitabine can be potentiated by inhibition of JAK2STAT3 signaling pathways in pancreatic cancer [26]

In conclusion several factors involving the modulation of cell cycle apoptosis and expres-sion of critical genes involved in drug activity may contribute to the effect of gemcitabine against lung cancer cells in vitro Here we have shown for the first time the growth inhibitory and apoptosis induction of gemcitabine in lung cancer cells Gemcitabine causes G1 and G2 phase cell cycle arrest and induction of apopto-sis by altering the expression of Bax and Bcl-2 and phosphorylation of JAK2 and STAT3 We therefore believe that gemcitabine could be a novel promising agent for the treatment of lung cancer

Disclosure of conflict of interest

None

Address correspondence to Dr Yan Zhang Depart- ment of Oncology Tongji Hospital 389 Xincun Road Putuo District Shanghai 200065 China Tel +86-

021-66111009 Fax +86-021-66111009 E-mail zhangyan_p163com

References

[1] Jemal A Siegel R Ward E Hao Y Xu J Murray T Thun MJ Cancer statistics 2008 CA Cancer J Clin 2008 58 71-96

[2] Siegel R Naishadham D Jemal A Cancer sta-tistics 2012 CACancer J Clin 2012 62 10-29

[3] Zhou J Zhao WY Ma X Ju RJ Li XY Li N Sun MG Shi JF Zhang CX Lu WL The anticancer efficacy of paclitaxel liposomes modified with mitochondrial targeting conjugate in resistant lung cancer Biomaterials 2013 34 3626-38

[4] Conroy T Desseigne F Ychou M Boucheacute O Guimbaud R Beacutecouarn Y Adenis A Raoul JL Gourgou-Bourgade S de la Fouchardiegravere C Bennouna JBachet JB Khemissa-Akouz F Peacutereacute-Vergeacute D Delbaldo C Assenat E Chauffert B Michel P Montoto-Grillot C Ducreux M Groupe Tumeurs Digestives of Unicancer PRODIGE Intergroup Folfirinox versus gem-citabine for metastatic pancreatic cancer N Engl J Med 2011 364 1817-25

[5] Wu YL Zhou C Hu CP Feng J Lu S Huang Y Li W Hou M Shi JH Lee KY Xu CR Massey D Kim M Shi Y Geater SL Afatinib versus cispla-tin plus gemcitabine for first-line treatment of asian patients with advanced non-small-cell lung cancer harbouring egfr mutations (lux-lung 6) An open-label randomised phase 3 trial Lancet Oncol 2014 15 213-22

[6] Wang J Guo J Wu S Feng H Sun S Pan J Zhang J Beebe SJ Synergistic effects of nano-second pulsed electric fields combined with low concentration of gemcitabine on human oral squamous cell carcinoma in vitro PLoS One 2012 7 e43213

[7] Yang XL Lin FJ Guo YJ Shao ZM Ou ZL Gem-citabine resistance in breast cancer cells regu-lated by pi3kakt-mediated cellular prolifera-tion exerts negative feedback via the mekmapk and mtor pathways Onco Targets Ther 2014 7 1033

[8] Eastman A Montano R Thompson R Khan N Hou H Lewis LD et al Dissecting the combina-tion of gemcitabine and the chk1 inhibitor mk-8776 in vitro and in vivo Cell cycle perturba-tion and the impact of administration schedule Cancer Research 2013 73 3413

[9] Hamed SS Straubinger RM Jusko WJ Phar-macodynamic modeling of cell cycle and apop-totic effects of gemcitabine on pancreatic ad-enocarcinoma cells Cancer Chemother Phar- macol 2013 72 553-63

[10] Wang X Liu JZ Hu JX Wu H Li YL Chen HL Bai H Hai CX Ros-activated p38 mapkerk-akt

Gemcitabine blocks JAK2STAT3 pathways

2172 Int J Clin Exp Med 20169(2)2166-2174

inducing the lung cancer cells apoptosis and in suppressing activation of JAK2 and STAT3

Deregulated growth in cancer cells is often attributed to loss of control in proliferation and apoptosis [18] In this study we observed that cells were incubated with different concentra-tions of gemcitabine significantly declining cell proliferation in a time- and dose-dependent manner Similar concentrations of gemcitabine were found to exert the same effects with dif-ferent cancer cell lines by other groups [19] In fact molecular studies have revealed that cell cycle associated with cell survival is frequently altered in multiple human cancers We observed that cells were incubated with differ-ent concentrations of gemcitabine significantly increased the number of cells in G1 and G2 phase while decreased the number of cells in S phase However low concentration of gem-citabine (lt05 mM) showed cell cycle arrest in S and G2 phases which similar to the report of Hamed and colleagues [9 20] These results

suggest that gemcitabine arrest cell cycle dependent on its concentrations and targeted cancer cells

Following cell cycle arrest cells may either undergo repair or enter the apoptotic pathway to maintain cellular integrity [21] Thus induc-tion of apoptosis is one of the protective mech-anisms against caner development and pro-gression In the present study we observed significant induction of apoptosis in gem-citabine-treated lung cancer cells Cell survival is maintained by a balance of the ratio of anti-apoptotic and pro-apoptotic protein The upreg-ulation of Bax and downregulation of Bcl-2 were observed in a dose-dependent manner in lung cancer cells after treated with different concen-trations of gemcitabine The permeabilization of the mitochondrial outer membrane is tightly controlled by Bcl-2 [22] and activation of Bax can trigger a sequence of events that leads to alterations in mitochondrial permeability transi-tion [23] Therefore we found that reduction of

Figure 4 Gemcitabine increased BaxBcl-2 ratio and blocked the JAK2STAT3 signaling The cells were treated with gemcitabine (10 20 and 40 mM) for 48 h The mRNA expression level of BaxBcl-2 was determined using Real-time PCR (A) and Western blot (B C) The cells were treated with gemcitabine (10 20 and 40 mM) for 6 h The protein ex-pression levels of p-JAK2JAK2 and p-STAT3STAT3 were determined using Western blot (D E) Plt005 Plt001 Plt0001 compared with control cells

Gemcitabine blocks JAK2STAT3 pathways

2173 Int J Clin Exp Med 20169(2)2166-2174

MMP level emerged in lung cancer cells after treated with gemcitabine Besides mitochon-dria are major sources of ROS and the reduc-tion in the level of Bcl-2 would promote the gen-eration of ROS [24] An increase of ROS production was observed in lung cancer cells treated with gemcitabine These results indi-cate that gemcitabine induce lung cancer cells apoptosis through the mitochondria pathway

Abnormal JAKSTAT signaling pathways are involved in the pathogenesis of several cancers including apoptosis [25] However the molecu-lar mechanism by which disordered JAK2STAT3 signaling pathways contributes to apop-tosis has not been clarified in lung cancer cells Therefore our work found that gemcitabine treatment could inhibit the activation of JAK2 and STAT3 in a dose-dependent manner evi-denced by decreased p-JAK2 and p-STAT3 lev-els Previous study found that the expression of Bcl-2 that is known to be downstream target of the STATs pathway is decreased and the MMP level is reduced after suppression of JAK2STAT3 signaling pathways [15] These findings suggest that abnormal expression of Bcl-2 may be the trigger involved in JAK2STAT3 signaling pathway induced MMP downregulation Indeed it has been reported earlier that anti-cancer effects of gemcitabine can be potentiated by inhibition of JAK2STAT3 signaling pathways in pancreatic cancer [26]

In conclusion several factors involving the modulation of cell cycle apoptosis and expres-sion of critical genes involved in drug activity may contribute to the effect of gemcitabine against lung cancer cells in vitro Here we have shown for the first time the growth inhibitory and apoptosis induction of gemcitabine in lung cancer cells Gemcitabine causes G1 and G2 phase cell cycle arrest and induction of apopto-sis by altering the expression of Bax and Bcl-2 and phosphorylation of JAK2 and STAT3 We therefore believe that gemcitabine could be a novel promising agent for the treatment of lung cancer

Disclosure of conflict of interest

None

Address correspondence to Dr Yan Zhang Depart- ment of Oncology Tongji Hospital 389 Xincun Road Putuo District Shanghai 200065 China Tel +86-

021-66111009 Fax +86-021-66111009 E-mail zhangyan_p163com

References

[1] Jemal A Siegel R Ward E Hao Y Xu J Murray T Thun MJ Cancer statistics 2008 CA Cancer J Clin 2008 58 71-96

[2] Siegel R Naishadham D Jemal A Cancer sta-tistics 2012 CACancer J Clin 2012 62 10-29

[3] Zhou J Zhao WY Ma X Ju RJ Li XY Li N Sun MG Shi JF Zhang CX Lu WL The anticancer efficacy of paclitaxel liposomes modified with mitochondrial targeting conjugate in resistant lung cancer Biomaterials 2013 34 3626-38

[4] Conroy T Desseigne F Ychou M Boucheacute O Guimbaud R Beacutecouarn Y Adenis A Raoul JL Gourgou-Bourgade S de la Fouchardiegravere C Bennouna JBachet JB Khemissa-Akouz F Peacutereacute-Vergeacute D Delbaldo C Assenat E Chauffert B Michel P Montoto-Grillot C Ducreux M Groupe Tumeurs Digestives of Unicancer PRODIGE Intergroup Folfirinox versus gem-citabine for metastatic pancreatic cancer N Engl J Med 2011 364 1817-25

[5] Wu YL Zhou C Hu CP Feng J Lu S Huang Y Li W Hou M Shi JH Lee KY Xu CR Massey D Kim M Shi Y Geater SL Afatinib versus cispla-tin plus gemcitabine for first-line treatment of asian patients with advanced non-small-cell lung cancer harbouring egfr mutations (lux-lung 6) An open-label randomised phase 3 trial Lancet Oncol 2014 15 213-22

[6] Wang J Guo J Wu S Feng H Sun S Pan J Zhang J Beebe SJ Synergistic effects of nano-second pulsed electric fields combined with low concentration of gemcitabine on human oral squamous cell carcinoma in vitro PLoS One 2012 7 e43213

[7] Yang XL Lin FJ Guo YJ Shao ZM Ou ZL Gem-citabine resistance in breast cancer cells regu-lated by pi3kakt-mediated cellular prolifera-tion exerts negative feedback via the mekmapk and mtor pathways Onco Targets Ther 2014 7 1033

[8] Eastman A Montano R Thompson R Khan N Hou H Lewis LD et al Dissecting the combina-tion of gemcitabine and the chk1 inhibitor mk-8776 in vitro and in vivo Cell cycle perturba-tion and the impact of administration schedule Cancer Research 2013 73 3413

[9] Hamed SS Straubinger RM Jusko WJ Phar-macodynamic modeling of cell cycle and apop-totic effects of gemcitabine on pancreatic ad-enocarcinoma cells Cancer Chemother Phar- macol 2013 72 553-63

[10] Wang X Liu JZ Hu JX Wu H Li YL Chen HL Bai H Hai CX Ros-activated p38 mapkerk-akt

Gemcitabine blocks JAK2STAT3 pathways

2173 Int J Clin Exp Med 20169(2)2166-2174

MMP level emerged in lung cancer cells after treated with gemcitabine Besides mitochon-dria are major sources of ROS and the reduc-tion in the level of Bcl-2 would promote the gen-eration of ROS [24] An increase of ROS production was observed in lung cancer cells treated with gemcitabine These results indi-cate that gemcitabine induce lung cancer cells apoptosis through the mitochondria pathway

Abnormal JAKSTAT signaling pathways are involved in the pathogenesis of several cancers including apoptosis [25] However the molecu-lar mechanism by which disordered JAK2STAT3 signaling pathways contributes to apop-tosis has not been clarified in lung cancer cells Therefore our work found that gemcitabine treatment could inhibit the activation of JAK2 and STAT3 in a dose-dependent manner evi-denced by decreased p-JAK2 and p-STAT3 lev-els Previous study found that the expression of Bcl-2 that is known to be downstream target of the STATs pathway is decreased and the MMP level is reduced after suppression of JAK2STAT3 signaling pathways [15] These findings suggest that abnormal expression of Bcl-2 may be the trigger involved in JAK2STAT3 signaling pathway induced MMP downregulation Indeed it has been reported earlier that anti-cancer effects of gemcitabine can be potentiated by inhibition of JAK2STAT3 signaling pathways in pancreatic cancer [26]

In conclusion several factors involving the modulation of cell cycle apoptosis and expres-sion of critical genes involved in drug activity may contribute to the effect of gemcitabine against lung cancer cells in vitro Here we have shown for the first time the growth inhibitory and apoptosis induction of gemcitabine in lung cancer cells Gemcitabine causes G1 and G2 phase cell cycle arrest and induction of apopto-sis by altering the expression of Bax and Bcl-2 and phosphorylation of JAK2 and STAT3 We therefore believe that gemcitabine could be a novel promising agent for the treatment of lung cancer

Disclosure of conflict of interest

None

Address correspondence to Dr Yan Zhang Depart- ment of Oncology Tongji Hospital 389 Xincun Road Putuo District Shanghai 200065 China Tel +86-

021-66111009 Fax +86-021-66111009 E-mail zhangyan_p163com

References

[1] Jemal A Siegel R Ward E Hao Y Xu J Murray T Thun MJ Cancer statistics 2008 CA Cancer J Clin 2008 58 71-96

[2] Siegel R Naishadham D Jemal A Cancer sta-tistics 2012 CACancer J Clin 2012 62 10-29

[3] Zhou J Zhao WY Ma X Ju RJ Li XY Li N Sun MG Shi JF Zhang CX Lu WL The anticancer efficacy of paclitaxel liposomes modified with mitochondrial targeting conjugate in resistant lung cancer Biomaterials 2013 34 3626-38

[4] Conroy T Desseigne F Ychou M Boucheacute O Guimbaud R Beacutecouarn Y Adenis A Raoul JL Gourgou-Bourgade S de la Fouchardiegravere C Bennouna JBachet JB Khemissa-Akouz F Peacutereacute-Vergeacute D Delbaldo C Assenat E Chauffert B Michel P Montoto-Grillot C Ducreux M Groupe Tumeurs Digestives of Unicancer PRODIGE Intergroup Folfirinox versus gem-citabine for metastatic pancreatic cancer N Engl J Med 2011 364 1817-25

[5] Wu YL Zhou C Hu CP Feng J Lu S Huang Y Li W Hou M Shi JH Lee KY Xu CR Massey D Kim M Shi Y Geater SL Afatinib versus cispla-tin plus gemcitabine for first-line treatment of asian patients with advanced non-small-cell lung cancer harbouring egfr mutations (lux-lung 6) An open-label randomised phase 3 trial Lancet Oncol 2014 15 213-22

[6] Wang J Guo J Wu S Feng H Sun S Pan J Zhang J Beebe SJ Synergistic effects of nano-second pulsed electric fields combined with low concentration of gemcitabine on human oral squamous cell carcinoma in vitro PLoS One 2012 7 e43213

[7] Yang XL Lin FJ Guo YJ Shao ZM Ou ZL Gem-citabine resistance in breast cancer cells regu-lated by pi3kakt-mediated cellular prolifera-tion exerts negative feedback via the mekmapk and mtor pathways Onco Targets Ther 2014 7 1033

[8] Eastman A Montano R Thompson R Khan N Hou H Lewis LD et al Dissecting the combina-tion of gemcitabine and the chk1 inhibitor mk-8776 in vitro and in vivo Cell cycle perturba-tion and the impact of administration schedule Cancer Research 2013 73 3413

[9] Hamed SS Straubinger RM Jusko WJ Phar-macodynamic modeling of cell cycle and apop-totic effects of gemcitabine on pancreatic ad-enocarcinoma cells Cancer Chemother Phar- macol 2013 72 553-63

[10] Wang X Liu JZ Hu JX Wu H Li YL Chen HL Bai H Hai CX Ros-activated p38 mapkerk-akt

Gemcitabine blocks JAK2STAT3 pathways

2174 Int J Clin Exp Med 20169(2)2166-2174

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[11] Qiao Z Ren S Li W Wang X He M Guo Y Sun L He Y Ge Y Yu Q Chidamide a novel histone deacetylase inhibitor synergistically enhances gemcitabine cytotoxicity in pancreatic cancer cells Biochem Biophys Res Commun 2013 434 95-101

[12] Zheng RR Hu W Sui CG Ma N Jiang YH Ef-fects of doxorubicin and gemcitabine on the induction of apoptosis in breast cancer cells Oncol Rep 2014 32 2719-25

[13] Hedvat M Huszar D Herrmann A Gozgit JM Schroeder A Sheehy A Buettner R Proia D Kowolik CM Xin H Armstrong B Bebernitz G Weng S Wang L Ye M McEachern K Chen H Morosini D Bell K Alimzhanov M Ioannidis S McCoon P Cao ZA Yu H Jove R Zinda M The jak2 inhibitor azd1480 potently blocks stat3 signaling and oncogenesis in solid tumors Cancer Cell 2009 16 487-97

[14] Deng X-S Wang S Deng A Liu B Edgerton SM Lind SE Wahdan-Alaswad R Thor AD Metfor-min targets stat3 to inhibit cell growth and in-duce apoptosis in triple-negative breast can-cers Cell Cycle 2012 11 367-76

[15] Du W Hong J Wang YC Zhang YJ Wang P Su WY Lin YW Lu R Zou WP Xiong H Fang JY In-hibition of jak2stat3 signalling induces colorectal cancer cell apoptosis via mitochon-drial pathway J Cell Mol Med 2012 16 1878-88

[16] Takezawa K Okamoto I Nishio K Jaumlnne PA Nakagawa K Role of erk-bim and stat3-sur-vivin signaling pathways in alk inhibitor-in-duced apoptosis in eml4-alk-positive lung can-cer Clin Cancer Res 2011 17 2140-8

[17] Zhao Y Yang LF Ye M Gu HH Cao Y Induction of apoptosis by epigallocatechin-3-gallate via mitochondrial signal transduction pathway Prev Med 2004 39 1172-9

[18] Hanahan D Weinberg RA Hallmarks of can-cer The next generation Cell 2011 144 646-74

[19] Arora S Bhardwaj A Srivastava SK Singh S McClellan S Wang B Singh AP Honokiol ar-rests cell cycle induces apoptosis and poten-tiates the cytotoxic effect of gemcitabine in hu-man pancreatic cancer cells PLoS One 2011 6 e21573

[20] Giovannetti E Mey V Nannizzi S Pasqualetti G Marini L Del Tacca M Danesi R Cellular and pharmacogenetics foundation of synergis-tic interaction of pemetrexed and gemcitabine in human non-small-cell lung cancer cells Mol Pharmacol 2005 68 110-8

[21] Chen CY Hsu YL Chen YY Hung JY Huang MS Kuo PL Isokotomolide a a new butanolide ex-tracted from the leaves of cinnamomum kotoense arrests cell cycle progression and induces apoptosis through the induction of p53p21 and the initiation of mitochondrial system in human non-small cell lung cancer a549 cells Eur J Pharmacol 2007 574 94-102

[22] Adams J Cory S The bcl-2 apoptotic switch in cancer development and therapy Oncogene 2007 26 1324-37

[23] Wallace DC Mitochondria and cancer Nat Rev Cancer 2012 12 685-98

[24] Alexandre J Hu Y Lu W Pelicano H Huang P Novel action of paclitaxel against cancer cells Bystander effect mediated by reactive oxygen species Cancer Res 2007 67 3512-7

[25] Alvarez JV Greulich H Sellers WR Meyerson M Frank DA Signal transducer and activator of transcription 3 is required for the oncogenic effects of non-small-cell lung cancer-associat-ed mutations of the epidermal growth factor receptor Cancer Res 2006 66 3162-8

[26] Bao GQ Shen BY Pan CP Zhang YJ Shi MM Peng CH Andrographolide causes apoptosis via inactivation of stat3 and akt and potenti-ates antitumor activity of gemcitabine in pan-creatic cancer Toxicol Lett 2013 222 23-35