Journal of Surgical Oncology2012;106:680–688 AMPK Inhibitor Compound C Suppresses Cell Proliferation by Induction of Apoptosis and Autophagy in Human Colorectal Cancer Cells
WENG-LANG YANG,PhD,1,2*WILLIAM PERILLO,BS,1DEANNA LIOU,HS,1PHILIPPE MARAMBAUD,PhD,3
AND PING WANG,MD1,2
1Department of Surgery,North Shore University Hospital and Long Island Jewish Medical Center,Manhasset,New York 2Laboratory of Surgical Research,The Feinstein Institute for Medical Research,Manhasset,New York 3Litwin-Zuker Research Center for the Study of Alzheimer’s Disease,The Feinstein Institute for Medical Research,Manhasset,New York
Background and Objectives:AMP-activated protein kinase(AMPK)is a main regulator of energy metabolism through the inhibition of biosynthetic pathways and enhancement of ATP-generating pathways.However,targeting AMPK as anti-tumor therapy remains controversial. In this study,we examined the effect of compound C,a small molecule inhibitor of AMPK,on the proliferation of several human colorectal cancer cell lines with diverse characteristics.
Methods:Four human colorectal cancer cell lines(HCT116,DLD-1,SW480,and KM12C)were treated with compound C.Cell viability was determined by MTS assay.Cell cycle prolife was analyzed by?ow cytometry.Acidic vesicular organelles were detected by acridine orange staining.Protein levels were measured by western blotting.
Results:Compound C inhibited the growth of four cell lines in a dose-dependent manner and caused G2/M https://www.doczj.com/doc/029684843.html,pound C increased sub-G1cell population and induced chromatin condensation and cleavage of PARP in HCT116and KM12C cells,while it induced acidic vesicular formation and conversion of LC3-I to autophagosome-associated LC3-II in DLD-1and SW480cells.Survivin,an anti-apoptotic protein,was down-regulated in all cell lines treated with compound C.
Conclusions:Compound C induces apoptotic or autophagic death in colorectal cancer cells and the preferred death mode is cell type-dependent.
J.Surg.Oncol.2012;106:680–688.?2012Wiley Periodicals,Inc.
K EY W ORDS:AMPK;compound C;apoptosis;autophagy;colorectal cancer
INTRODUCTION
Colorectal cancer is the second leading cause of cancer death in the United States,with140,000new cases diagnosed each year[1]. Nearly half of the patients will develop liver metastases during the course of their disease and subsequently die[1].For a long period of time,5-?uorouracil(5-FU)was the only chemotherapeutic drug to treat colorectal cancer.In the1990s,two other chemotherapeutic drugs,irinotecan and oxaliplatin,along with5-FU were used for treating patients with advanced colorectal cancer.However,the response rates of these two drugs are still lower than50%and the improvement in median survival does not exceed3.5months in com-parison to5-FU treatment alone[2–4].A new class of drug emerged during the2000s,which consisted of monoclonal antibodies, bevacizumab,cetuximab,and panitumumab,were approved by US Food and Drug Administration.These drugs interfere with the growth of cancer cells by targeting vascular endothelial growth factor or epidermal growth factor receptor[5–7].Although these drugs showed a signi?cant extent in survival time during the trials, the improvement of5-year survival rate has not been achieved.
Recent studies show that diabetic patients treated with metformin have a lower incidence of cancer than the ones who receive other treatment regimens[8,9].These observations point out another potential strategy to treat colorectal cancer.Although the exact molecular mechanism on how metformin inhibits tumorigenesis is not very clear,it is known that it can activate AMP-activated protein kinase(AMPK)[10].AMPK is a key regulator for balancing intracellular energy when cells are under metabolic stresses,such as hypoxia,glucose deprivation,heat shock,or mitochondrial dysfunction,which result in depleting cellular ATP and elevating AMP levels[11].Under these stress conditions,AMPK will be activated to inhibit biosynthetic pathways that consume ATP and enhance ATP-generating pathways involving glucose transport, glycolysis,and fatty acid oxidation[12].Therefore,it is still unclear the correlation between AMPK activation and development and progression of tumor.
Targeting AMPK to control tumor growth is still controversial.In line with the observation in diabetic patients,metformin has shown an anti-tumor effect on the mouse xenograft model and cell culture of human colorectal,prostate,and breast cancer cells[13–15]. Another AMPK activator,5-aminoimidazole-4-carboxyamide ribonu-cleoside(AICAR)[16,17],has also been shown to inhibit the growth of various types of cancer cells,including colon,renal,leukemia, and glioblastoma[13,18–20].On the other hand,several studies have also reported that administration of a pharmacological AMPK inhibi-tor,compound C(cell-permeable pyrrazolopyrimidine derivative) [10],results in inhibiting the proliferation of prostate,breast,myelo-ma,and giloma cancer cells[21–24]or sensitizing cancer cells to other anti-cancer drugs in combined treatments[25–27].Such a dis-Grant sponsor:Faculty Practice Plan Research Fund of North Shore-Long Island Jewish Health System;Grant sponsor:National Institutes of Health grant;Grant number:GM057468.
Con?icts of interest:None.
*Correspondence to:Weng-Lang Yang,PhD,Department of Surgery, The Feinstein Institute for Medical Research,350Community Drive, Manhasset,NY11030;Fax:t151********.
E-mail:wlyang@https://www.doczj.com/doc/029684843.html,
Received30November2011;Accepted14May2012
DOI10.1002/jso.23184
Published online4June2012in Wiley Online Library (https://www.doczj.com/doc/029684843.html,).
pute indicates that further studies are required to determine whether AMPK pharmacological manipulation can interfere with cancer progression.
In this study,we examined the cellular responses to compound C in a panel of four human colorectal cancer cell lines(HCT116, DLD-1,SW480,and KM12C)with different p53statuses and geno-typic/phenotypic characteristics.We?rst determined the effect of compound C on cell proliferation.We then analyzed the cell cycle pro?le and the mode of cell death in these four cell lines treated with compound C.We also explored the molecular mechanisms of regulating cell cycle progression and death in these cell lines exposed to compound C.
MATERIALS AND METHODS
Cell Lines and Cell Culture
Human colorectal cancer cell lines HCT116,SW480,and DLD-1 were obtained from the American Type Culture Collection (Manassas,V A)and KM12C was a gift from Dr.I.J.Fidler(M.D. Anderson Cancer Center,Houston,TX).The characteristics of these four cell lines are listed in Table I.HCT116cells were cultured in McCoy’s5A medium(Invitrogen,Carlsbad,CA)and SW480,DLD-1,and KM12C cells were cultured in DMEM medium(Invitrogen) containing10%fetal bovine serum and supplemented with penicillin and streptomycin.The cells were maintained in a humidi?ed incubator at378C with5%CO2.
Cell Viability Assay and Cell Cycle Analysis Cells were plated in a96-well plate and treated with various con-centrations of compound C(Tocris Bioscience,Ellisville,MO)for an indicated time.Cell viability was assessed in quadruplicate by using the CellTiter96cell proliferation assay kit(Promega,Madison, MI)in accordance with the manufacturer’s instructions.This assay is referred to here as the MTS assay.The plates were read at490and 690nm(background).For cell cycle analysis,cells either untreated or treated with various concentrations of compound C for24or 48hr were harvested and?xed in75%cold ethanol.The?xed cells were incubated with PBS containing RNase(100m g/ml)and propi-dium iodide(10m g/ml)and then subjected to a?ow cytometer(BD Biosciences,San Jose,CA).Two thousand events were collected per sample.Data acquisition and cell cycle analysis were performed using FlowJo software.
Western Blot Analysis
Cells were harvested and lysed on ice for30min in RIPA buffer (10mM Tris-HCl pH7.5,120mM NaCl,1%NP-40,1%sodium deoxycholate,and0.1%SDS)containing a protease inhibitor cocktail(Roche Diagonstics,Indianapolis,IN)and then centrifuged at10,000g for10min.Protein concentration was determined by the BCA protein assay reagent(Pierce,Rockford,IL).Cell lysates were electrophoresed on SDS–polyacrylamide gels and transferred onto nitrocellulose membranes.Membranes were blocked with5%nonfat dry milk in TTBS buffer(0.1%Tween-20,20mM Tris-HCl pH7.5, and140mM NaCl)and incubated with a primary antibody against PARP,caspase-3,p53,p21,actin(Santa Cruz Biotechnology,Santa Cruz,CA),LC3,survivin,or p-ACC(Ser79;Cell signaling,Danvers, MA),followed by a secondary antibody-horseradish peroxidase con-jugate(Pierce)and detected using chemiluminescence(Pierce)and autoradiography.
Immunofluorescence Staining
To identify apoptosis,cells were?xed with ice-cold methanol for 10min,stained with Hoechst33258(10m g/ml)in PBS for15min, and evaluated the morphological changes of chromatin under a ?uorescence microscope.To detect formation of acidic vesicular organelles,live cells in a culture plate were stained with1m g/ml acridine orange(Sigma-Aldrich,St.Louis,MO)for15min and examined under a?uorescence microscope at green and red channels.In order to compare the?uorescent intensity between cell lines with or without compound C treatment,the exposure time of each image was kept constant when it was recorded.
RESULTS
Effects of Compound C on Cell Viability and
Clonogenicity in Colorectal Cancer Cells We?rst compared the sensitivity of a panel of colorectal cancer cell lines,HCT116,DLD-1,SW480,and KM12C,to compound C. Cells were treated with2,4,10,20,and30m M of compound C for 48hr.The double time of these cell lines is ranging from16to 24hr.The effect of compound C on these cells was evaluated after two cell https://www.doczj.com/doc/029684843.html,pound C induced a dose-dependent reduction in cell proliferation in all cell lines,as measured by the MTS assay (Fig.1).KM12C cells were the most sensitive to compound C-induced growth inhibition among the tested cell lines.At2m M of compound C,the survival rate of KM12C cells was only 56.6?3.7%,while there was no signi?cant change of survival rate in the rest of cell lines(Fig.1).
We examined whether the growth inhibition induced by compound C was reversible.Cells were?rst incubated with various
TABLE I.Characteristics of Human Colorectal Cancer Cell Lines Used in This Study
Cell line
p53status
Differentiation Dukes’Codon change a.a.change
HCT116Wild-type Wild-type Poorly D DLD-1C!T S241F Poorly C SW480G!A R273H Poorly B C!T P309S
KM12C A!G H179R Poorly
B https://www.doczj.com/doc/029684843.html,pound
C inhibited the proliferation of human colorectal cancer cells.HCT116,DLD-1,SW480,and KM12C cells were incu-bated with compound C at the indicated concentration for48hr.Cell viability was determined by MTS assay.The cell survival of each individual untreated cell lines was considered to be100%.Data are shown as means?SD.
Anti-Growth on Colon Cancer by Compound C681
concentrations of compound C for 24hr.After treatment,the drug was removed and cells were allowed to recover without the drug for a further 48hr before subjecting to MTS assay.Concentrations in excess of 10m M markedly reduced the recovery of all cell lines,indicating substantial loss of viability (Fig.2).KM12C cells showed the lowest tolerance and could only be recovered from 2and 4m M of compound C (Fig.2).These results indicated that higher concen-trations of compound C induced cytotoxicity in these cancer cells.
Effects of Compound C on Cell Cycle Progression
Since compound C caused growth inhibition,we examined the cell-cycle prolife of each cell line after exposure to various concen-trations for 48hr (Fig.3).At 4m M of compound C,an increased cell population at G 2/M was observed in all four cell lines.The ratios of G 2to G 1phase of SW480and KM12C cells were increased from 0.21to 3.12and 0.31to 1.56,respectively,compared with the un-treated cells (Fig.3).HCT116cells were the most resistant to G 2/M arrest induced by compound C at lower concentrations;however,their G 2/G 1ratio was markedly increased from 0.28to 1.89at 20m M,compared with the untreated cells (Fig.3).Interestingly,KM12C cells,the most sensitive cell line,started showing sub-G 1population at 4m M and reached 65%of this population at 20m M (Fig.3).A high population in sub-G 1phase (45%)was also observed in HCT116cells treated with 20m M of compound C (Fig.3).We also detected 28.2and 32.2%of sub-G 1population in HCT116and KM12C cells,respectively,after 24hr-treatment with 20m M of compound C by ?ow cytometry (data not shown).
Induction of Apoptosis by Compound C
After detecting the sub-G 1cell population at 48hr in compound C-treated HCT116and KM12C cells,we further validated the occur-rence of apoptosis in these cells.We ?rst stained all cancer cell lines treated with 20m M of compound C for 48hr by Hoechst 33258and examined their nuclear patterns under a ?uorescence microscope.
Only the nuclei of HCT116and KM12C cells exhibited chromosome condensation and fragmentation,while that of DLD1and SW480cells did not (Fig.4A).We then analyzed the cleavage of poly(ADP-ribose)polymerase (PARP)from 116to 85kDa and caspase-3deg-radation by western blotting.As shown in Figure 4B,an 85kDa PARP fragment was detected in KM12C cells at 4m M and its inten-sity was increased in a dose-dependent manner.In HCT116cells,an 85kDa fragment was only observed at 20m M.No degradation of PARP was observed in DLD-1and SW480cells treated with com-pound C.Multiple degraded caspase-3products could be detected in KM12C cells,while no caspase-3degradation was observed in the rest of three cell lines (Fig.4B).In this study,we did not detect cleaved caspase-3in compound C-treated HCT116cells.The occur-rence of caspase-independent apoptosis has been reported in ovarian carcinoma cells treated with paclitaxel [28].Therefore,we conclude that apoptosis occurred in HCT116and KM12C cells,but not in DLD1and SW480cells,in response to compound C.
Induction of Autophagy by Compound C
To investigate other mechanisms involved in the anti-proliferation effect of compound C,we examined the morphology of four cell lines after treating with 20m M of compound C for 24hr under a light microscope.HCT116,DLD1,and SW480cells showed various degree of cytoplasmic vacuolization after treatment,while absent in their untreated cells (Fig.5A).KM12C cells were clumped together after treatment and the individual cell morphology was dif?cult to identify (Fig.5A).To further validate that the compound C-treated cells had formed acidic vesicular organelles,one of the main charac-teristics described for autophagy,we stained cells with acridine or-ange [29].With this staining,acidic compartments of cells will exhibit bright red ?uorescence,while the cytoplasm and nucleus will show bright green and dim red ?uorescence under a ?uorescence microscope.We observed all the untreated cancer cells exhibited green ?uorescence with hardly detectable red ?uorescence (Fig.5B).All cancer cells treated with compound C showed red
?uorescence
Fig.2.Cell viability of human colorectal cancer cells recovered from compound C treatment.HCT116,DLD-1,SW480,and KM12C cells were treated at the indicated concentration of compound C for 24hr (&)or recovered in a fresh medium without compound C for a further 48hr (&).Cell viability was determined by MTS assay.The cell survival of each individual untreated cell lines at 24hr was considered to be 100%.Data are shown as means ?SD.
682Yang et al.
in the vesicles.However,the intensity in DLD-1and SW480cells was brighter than that in HCT116and KM12C cells (Fig.5B).
Another characteristic hallmark of autophagy is the conversion of LC3from the 18-kDa cytosolic form (LC3-I)to the proteolytic and lipidated form (LC3-II)[30].We then examined the expression pat-tern of LC3in cell lysates treated with different concentrations of compound C for 24hr by western blotting (Fig.5C).Increased LC3-II expression was detected with as low as 4m M of compound C in DLD1and SW480cells.In HCT116cells,the LC3-II expression could only be clearly detected at 10m M.A minor LC3-II expression was observed at 10and 20m M in KM12C cells.Taken together,compound C induces autophagy in DLD1and SW480cells rather than in HCT116and KM12C cells.
Changes of Molecular Expression in Response to
Compound C
To explore molecular mechanisms on the differential responses of these four cell lines to compound C,we analyzed the expression levels of several proteins at different concentrations of compound C for 24hr by western blotting.p53and its downstream target,p21,
were key regulators of cell cycle progression and cell death [31].Survinin,a member of the inhibitors-of-apoptosis protein (IAPs)family,also plays a signi?cant role in regulating cell survival and death [32].Compound C induced expression of wild-type p53in HCT116cells,while it had no effect on expression of mutant p53in the rest of cell lines (Fig.6).The expression level of p21was elevat-ed in a dose-dependent manner in HCT116cells,whereas it could not be detected in DLD-1and KM12C cells.Intriguingly,the p21expression was decreased in a dose-dependent manner in SW480cells (Fig.6).Although p53is mutated,constitutive expression of p21in SW480cells has been reported in the absence of stress [33].A recent study demonstrates that p21plays an essential role in deter-mining the type of cell death,positively for apoptosis and negatively for autophagy in ?broblast cells treated with C 2-ceramide [34].The effect of down-regulation of p21expression by compound C on cell death mode of SW480cells needs further investigation.Survivin was down-regulated in all four cancer cell lines and it was decreased more dramatically in SW480cells (Fig.6).Finally,we validated the pharmacological activity of compound C by examining the expres-sion of phosphorylated acetyl-CoA carboxylase (ACC),a down-stream substrate of AMPK [35].As shown in Figure 6,compound
C
Fig.3.Effect of compound C on cell-cycle distribution.HCT116,DLD-1,SW480,and KM12C cells were treated at the indicated concen-tration of compound C for 48hr and subjected to ?ow cytometry staining with propidium iodide.The percentage of each cycle phase was indicated at the right and the ratio of G 2to G 1was indicated at the up-left corner.The data represent three independent experiments.
Anti-Growth on Colon Cancer by Compound C 683
effectively reduced the phosphorylation levels of ACC in all four cancer cell lines.
DISCUSSION
The difference in metabolism between cancer and normal cells has been noticed for a century [36].The utilization of different meta-bolic pathways makes cancer cells able to survive and proliferate at low oxygen and low nutrient environments [37].In consideration of its biological activity in regulating energy metabolism,activation of AMPK should be a survival factor for cancer cells.Correspondingly,AMPK is highly activated in solid-tumor microenvironments as demonstrated by a mouse model [38].In contrast,AMPK has been considered as an anti-tumorigenesis molecule due to its association with other tumor suppressor proteins,such as LKB1,tu-berous sclerosis complex,and p53,in the AMPK signaling network [39,40].
In this study,we demonstrated that compound C possessed an anti-proliferation activity to all four colorectal cancer cell lines,HCT116,DLD-1,SW480,and KM12C.The inhibition of cell growth by compound C has also been reported in other various types
of cancer cells [21–24].As expected,compound C effectively inhibited AMPK activity in our studied colorectal cancer cells,as demonstrated by reduction of phosphorylated ACC.In addition to targeting AMPK,other mechanisms triggered by compound C for inhibiting cancer cell growth have been proposed.For example,compound C induces apoptotic cell death in human breast cancer MCF7cells through enhancing production of ceramide [22].Compound C-stimulated TRAIL-induced apoptosis is dependent on the generation of reactive oxygen species in human renal cancer Caki cells [26].
We further identi?ed that compound C at higher concentrations arrested the cell cycle progression at G 2/M phase in all colorectal cancer cell lines,which was consistent with a previous report shown in glioma cells [24].Noticeably,at 10m M of compound C,HCT116cells carrying wild-type p53were more dominant at G 1than G 2/M phase,whereas other cells lines carrying mutant p53were more dominant at G 2/M than G 1phase.Correspondingly,we observed that compound C could activate the expression of p53and p21in HCT116cells,which was also reported in human gastric cancer AGS cells [25].These results indicate that p53may play a signi?cant role in governing the cell cycle progression of cancer cells
exposed
Fig.4.Induction of apoptosis in human colorectal cancer cells by compound C.HCT116,DLD-1,SW480,and KM12C cells were treated with 20m M compound C for 48h.A :Cells were ?xed and viewed with ?uorescence microscopy after staining with DNA-binding dye Hoechst 33258.Apoptotic characteristics with condensed and fragmented chromatin are indicated by arrows.a,HCT116;b,DLD-1;c,SW480;d,KM12C.Magni?cation,400?.B :Total lysates from cells treated with various concentrations of compound C were subjected to western blotting against PARP and caspase-3antibodies.Actin was served as a loading control.The data represent three independent experiments.
684Yang et al.
Fig.5.Induction of autophagy in human colorectal cancer cells by compound C.HCT116,DLD-1,SW480,and KM12C cells were untreat-ed (Unt)or treated with 20m M compound C (CC)for 24hr.A :Images were taken using a phase-contrast microscope.Vesicular organelles are formed in compound C-treated cells.Magni?cation,400?.B :Images were viewed with ?uorescence microscopy after staining with acridine orange.Cytoplasm and nucleus show bright green and dim red ?uorescence.Formation of acidic vesicular organelles exhibits bright red ?uorescence.Magni?cation,400?.C :Total lysates from cells treated with various concentrations of compound C for 24hr were subjected to western blotting against LC3antibody.Actin was served as a loading control.The data represent three independent experiments.
Anti-Growth on Colon Cancer by Compound C 685
to lower concentrations of compound C.Thus,the status of p53in cancer cells will be an important factor to be considered if com-pound C is to be combined with other chemotherapeutic drugs for the treatment,since most of the anti-cancer drugs are targeting on a speci?c cell cycle phase [41].
In determining the mode of cell death,we found that both apopto-sis and autophagy were induced by compound C in colorectal cancer cells.Apoptosis mostly occurred in HCT116and KM12C cells,while autophagy was observed mainly in DLD-1and https://www.doczj.com/doc/029684843.html,pound C-induced apoptosis has also been described in other types of cancer cells [22–24]as well as autophagy [42].The occur-rence of apoptosis leading to cell death has been well recognized;however,the role of autophagy in cell death is still not conclusive.Autophagy is a process that sequesters cytoplasmic proteins or organelles into a lytic compartment that facilitates degradation and re-cycling [43].This can promote cell survival during cell stress [44];however,excessive autophagy can lead to cell death also called non-apoptotic type II programmed cell death [45].To further con?rm the cytotoxic effect of compound C,we also counted the cell number by hemocytometer and observed a reduction of cell number in all cancer cell lines after the treatment,corresponding to the result of MTS assay (data not shown).
Tumor suppressor gene p53is considered to be a critical regulator in determining the cell fate under various types of stimuli.By study-ing four colorectal cancer cell lines with different p53statuses,we did not observe a clear link of functional wild-type p53to a speci?c cell death mode induced by compound C.Apoptosis was detected in cells either with wild-type or mutated p53(HCT116vs.KM12C)at 20m M of compound C.Intriguingly,expression of LC3-II,a hall-mark of autophagy,was detected in HCT116cells treated with 10m M of compound C.Recently,it has been proposed that p53exerts an ambiguous function in the regulation of autophagy.Within the nucleus,p53acts as an autophagy-inducing transcription factor,while p53in the cytoplasm exerts a tonic autophagy-inhibitory function [46].The control of autophagy by p53location is also true for p53mutants [47].Thus,the different death modes induced by compound C might be due to the redistribution of wild-type p53localization in HCT116cells and also due to the different locations of mutated p53expressing in the other three cancer cell lines.Whether these phenomena can be applied to cancer cells responding to compound C needs further investigation.
To explore other potential molecule in governing the death mode,we examined the expression of survivin and shown its down-regulation in all four cancer cell lines treated with compound C.Survivin is a well-known anti-apoptotic protein [48],which corre-sponds to our observation of its reduction in compound C-induced apoptotic cells (HCT116and KM12C).Recently,studies have shown another aspect of survivin on protecting cells from autophagic death in prostate and gastric cancer cells [49,50],where the results are consistent with its decrease in autophagic cells (DLD-1and SW480).In addition to regulating cell death,survivin also participates in con-trolling cell division during mitosis [51].Knocking-down expression of survivin results in G 2/M arrest in SW480and human chondrosar-coma cells [52,53].Thus,in addition to activation of wild-type p53,down-regulation of survivin may be another mechanism in contribut-ing to compound C-induced G 2/M arrest.
In summary,AMPK inhibitor compound C causes G 2/M arrest and inhibits cell growth in colorectal cancer cells regardless of their p53status and histological https://www.doczj.com/doc/029684843.html,pound C induces either apoptotic or autophagic cell death in colorectal cancer cells and the preference of cell death mode is cell line-dependent.Down-regulation of survivin is a common factor in all four colorectal cancer cells after treated with compound C.
CONCLUSIONS
The capability of compound C in executing its cytotoxic effect on cancer cells can be mediated through different death pathways.Thus,compound C has a broad spectrum of anti-tumor activity where some tumors possess resistance to a certain death pathway.
ACKNOWLEDGMENTS
The authors thank Dr.Asha Jacob and Lana M.Corbo for critical-ly reviewing the manuscript.
REFERENCES
1.Fong Y ,Salo J:Surgical therapy of hepatic colorectal metasta-sis.Semin Oncol 1999;26:514–523.
2.Saltz LB,Cox JV ,Blanke C,et al.:Irinotecan plus ?uorouracil and leucovorin for metastatic colorectal cancer Irinotecan Study Group.N Engl J Med 2000;343:905–914.
3.Giacchetti S,Perpoint B,Zidani R,et al.:Phase III multicenter randomized trial of oxaliplatin added to chronomodulated ?uorouracil-leucovorin as ?rst-line treatment of metastatic colorectal cancer.J Clin Oncol
2000;18:136–147.
Fig.6.Effect of compound C on the protein expression in human colorectal cancer cells.HCT116,SW480,DLD-1,and KM12C cells were treated at the indicated concentration of compound C for 24hr.Total cell lystates were isolated and subjected to western blotting against p53,p21,survivin,or phosphorylated ACC (p-ACC).Actin was served as a loading control.The data represent three independent experiments.
686Yang et al.
4.Grothey A,Sargent D,Goldberg RM,et al.:Survival of patients
with advanced colorectal cancer improves with the availability of?uorouracil-leucovorin,irinotecan,and oxaliplatin in the course of treatment.J Clin Oncol2004;22:1209–1214.
5.Grothey A,Ellis LM:Targeting angiogenesis driven by vascular
endothelial growth factors using antibody-based therapies.
Cancer J2006;14:170–177.
6.Baselga J:The,EGFR as a target for anticancer therapy–focus
on cetuximab.Eur J Cancer2001;37:S16–S22.
7.Yang XD,Jia XC,Corvalan JR,et al.:Eradication of
established tumors by a fully human monoclonal antibody to the epidermal growth factor receptor without concomitant che-motherapy.Cancer Res1999;59:1236–1243.
8.Evans JM,Donnelly LA,Emslie-Smith AM,et al.:Metformin
and reduced risk of cancer in diabetic patients.BMJ2005;
330:1304–1305.
9.Bowker SL,Majumdar SR,Veugelers P,et al.:Increased
cancer-related mortality for patients with type2diabetes who use sulfonylureas or insulin.Diabetes Care2006;29:254–258. 10.Zhou G,Myers R,Li Y,et al.:Role of AMP-activated protein
kinase in mechanism of metformin action.J Clin Invest2001;
108:1167–1174.
11.Kemp BE,Mitchelhill KI,Stapleton D,et al.:Dealing with en-
ergy demand:The AMP-activated protein kinase.Trends Bio-chem Sci1999;24:22–25.
12.Hardie DG:The AMP-activated protein kinase pathway–new
players upstream and downstream.J Cell Sci2004;117:5479–5487.
13.Buzzai M,Jones RG,Amaravadi RK,et al.:Systemic treatment
with the antidiabetic drug metformin selectively impairs p53-de?cient tumor cell growth.Cancer Res2007;67:6745–6752. 14.Ben Sahra I,Laurent K,Loubat A,et al.:The antidiabetic drug
metformin exerts an antitumoral effect in vitro and in vivo through a decrease of cyclin D1level.Oncogene2008;27:3576–3586.
15.Zakikhani M,Dowling R,Fantus IG,et al.:Metformin is an
AMP kinase-dependent growth inhibitor for breast cancer cells.
Cancer Res2006;66:10269–10273.
16.Sullivan JE,Brocklehurst KJ,Marley AE,et al.:Inhibition of
lipolysis and lipogenesis in isolated rat adipocytes with AICAR,
a cell-permeable activator of AMP-activated protein kinase.
FEBS Lett1994;353:33–36.
17.Corton JM,Gillespie JG,Hawley SA,et al.:5-aminoimidazole-
4-carboxamide ribonucleoside.A speci?c method for activating AMP-activated protein kinase in intact cells?Eur J Biochem 1995;229:558–565.
18.Woodard J,Joshi S,Viollet B,et al.:AMPK as a therapeutic
target in renal cell carcinoma.Cancer Biol Ther2010;10:1168–1177.
19.Robert G,Ben Sahra I,Puissant A,et al.:Acadesine kills
chronic myelogenous leukemia(CML)cells through PKC-dependent induction of autophagic cell death.PLoS One2009;
18:e7889.
20.Guo D,Hildebrandt IJ,Prins RM,et al.:The AMPK agonist
AICAR inhibits the growth of EGFRvIII-expressing glioblasto-mas by inhibiting lipogenesis.Proc Natl Acad Sci USA2009;
106:12932–12937.
21.Park HU,Suy S,Danner M,et al.:AMP-activated protein ki-
nase promotes human prostate cancer cell growth and survival.
Mol Cancer Ther2009;8:733–741.
22.Jin J,Mullen TD,Hou Q,et al.:AMPK inhibitor Compound C
stimulates ceramide production and promotes Bax redistribution and apoptosis in MCF7breast carcinoma cells.J Lipid Res 2009;50:2389–2397.
23.Baumann P,Mandl-Weber S,Emmerich B,et al.:Inhibition of
adenosine monophosphate-activated protein kinase induces apo-ptosis in multiple myeloma cells.Anticancer Drugs2007;18: 405–410.
24.Vucicevic L,Misirkic M,Janjetovic K,et al.:AMP-activated
protein kinase-dependent and-independent mechanisms underlying in vitro antiglioma action of compound C.Biochem Pharmacol2009;77:1684–1693.25.Kim HS,Hwang JT,Yun H,et al.:Inhibition of AMP-activated
protein kinase sensitizes cancer cells to cisplatin-induced apoptosis via hyper-induction of p53.J Biol Chem2008;283: 3731–3742.
26.Jang JH,Lee TJ,Yang ES,et al.:Compound C sensitizes Caki
renal cancer cells to TRAIL-induced apoptosis through reactive oxygen species-mediated down-regulation of c-FLIPL and Mcl-1.Exp Cell Res2010;316:2194–2203.
27.Jung SN,Park IJ,Kim MJ,et al.:Down-regulation of AMP-
activated protein kinase sensitizes DU145carcinoma to Fas-in-duced apoptosis via c-FLIP degradation.Exp Cell Res2009;
315:2433–2441.
28.Ahn HJ,Kim YS,Kim JU,et al.:Mechanism of taxol-induced
apoptosis in human SKOV3ovarian carcinoma cells.J Cell Biochem2004;91:1043–1052.
29.Paglin S,Hollister T,Delohery T,et al.:A novel response of
cancer cells to radiation involves autophagy and formation of acidic vesicles.Cancer Res2001;61:439–444.
30.Mizushima N,Yoshimori T:How to interpret LC3immunoblot-
ting.Autophagy2007;3:542–545.
31.V ousden KH,Lane DP:p53in health and disease.Nat Rev Mol
Cell Biol2007;8:275–283.
32.Cheung CH,Cheng L,Chang KY,et al.:Investigations of
survivin:The past,present and future.Front Biosci2011;16: 952–961.
33.Rochette PJ,Bastien N,Lavoie J,et al.:SW480,a p53double-
mutant cell line retains pro?ciency for some p53functions.
J Mol Biol2005;352:44–57.
34.Fujiwara K,Daido S,Yamamoto A,et al.:Pivotal role of the
cyclin-dependent kinase inhibitor p21WAF1/CIP1in apoptosis and autophagy.J Biol Chem2008;283:388–397.
35.Ha J,Daniel S,Broyles SS,et al.:Critical phosphorylation sites
for acetyl-CoA carboxylase activity.J Biol Chem1994;269: 22162–22168.
36.Warburg O:Metabolism of tumours.Biochem Zeitschr1923;
142:317–333.
37.Kroemer G,Pouyssegur J:Tumor cell metabolism:Cancer’s
Achilles’heel.Cancer Cell2008;13:472–482.
https://www.doczj.com/doc/029684843.html,deroute KR,Amin K,Calaoagan JM,et al.:50-AMP-activated
protein kinase(AMPK)is induced by low-oxygen and glucose deprivation conditions found in solid-tumor microenvironments.
Mol Cell Biol2006;26:5336–5347.
39.Shackelford DB,Shaw RJ:The LKB1-AMPK pathway:Metab-
olism and growth control in tumour suppression.Nat Rev Can-cer2009;9:563–575.
40.Wang W,Guan KL:AMP-activated protein kinase and cancer.
Acta Physiol2009;196:55–63.
41.Johnson DG,Walker CL:Cyclins and cell cycle check-points.
Annu Rev Pharmacol Toxicol1999;39:295–312.
42.Vucicevic L,Misirkic M,Janjetovic K,et al.:Compound C
induces protective autophagy in cancer cells through AMPK in-hibition-independent blockade of Akt/mTOR pathway.Autoph-agy2011;7:40–50.
43.Mizushima N,Levine B,Cuervo AM,et al.:Autophagy?ghts
disease through cellular self-digestion.Nature2008;451:1069–1075.
44.White E:Autophagic cell death unraveled:Pharmacological
inhibition of apoptosis and autophagy enables necrosis.
Autophagy2008;4:399–401.
45.Galluzzi L,Vicencio JM,Kepp O,et al.:To die or not to die:
That is the autophagic question.Curr Mol Med2008;8: 78–91.
46.Maiuri MC,Galluzzi L,Morselli E,et al.:Autophagy regulation
by p53.Curr Opin Cell Biol2010;22:181–185.
47.Morselli E,Tasdemir E,Maiuri MC,et al.:Mutant p53protein
localized in the cytoplasm inhibits autophagy.Cell Cycle2008;
7:3056–3061.
48.Ambrosini G,Adida C,Altieri DC:A novel anti-apoptosis
gene,survivin,expressed in cancer and lymphoma.Nat Med 1997;3:917–921.
49.Roca H,Varsos Z,Pienta KJ:CCL2protects prostate cancer
PC3cells from autophagic death via phosphatidylinositol Anti-Growth on Colon Cancer by Compound C687
3-kinase/AKT-dependent survivin up-regulation.J Biol Chem 2008;283:25057–25073.
50.Chiou SK,Hoa N,Hodges A:Sulindac sul?de induces
autophagic death in gastric epithelial cells via Survivin down-regulation:A mechanism of NSAIDs-induced gastric injury.
Biochem Pharmacol2011;81:1317–1323.
51.Lens SM,Vader G,Medema RH:The casefor survivinas mitotic
regulator.Curr Opin Cell Biol2006;18:616–622.52.Zhonghong L,Lianjie L,Changqing Z,et al.:The in?uence of
survivin shRNA on the cell cycle and the invasion of SW480 cells of colorectal carcinoma.J Exp Clin Cancer Res2008;
27:20.
53.Lechler P,Renkawitz T,Campean V,et al.:The antiapoptotic
gene survivin is highly expressed in human chondrosarcoma and promotes drug resistance in chondrosarcoma cells in vitro.
BMC Cancer2011;11:120.
688Yang et al.