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Inhibition of Grade Dependent Autophagy in Urothelial CarcinomaIncreases Cell Death under Nutritional Limiting Condition andPotentiates the Cytotoxicity of Chemotherapeutic Agent
Rani Ojha, Shrawan K. Singh,* Shalmoli Bhattacharyya, Rakesh Singh Dhanda,
Aruna Rakha, Arup K. Mandal and Vivekanand Jha
From the Departments of Urology (RO, SKS, AKM), Biophysics (SB) and Translational and Regenerative Medicine (RSD, AR, VJ),
Post Graduate Institute of Medical Education and Research, Chandigarh, India
Abbreviations
and Acronyms
3-MA ¼ 3-methyladenine
AMPK ¼ adenosinemonophosphate activated proteinkinase
AO ¼ acridine orange
AV ¼ autophagic vesicle
CQ ¼ chloroquine
FACS ¼ flow cytometry
FBS ¼ fetal bovine serum
HG ¼ high grade
LG ¼ low grade
mTOR ¼ mammalian target ofrapamycin
p ¼ phosphorylated
PARP ¼ poly adenosinediphosphate-ribose polymerase
PI3K ¼ phosphoinositide 3-kinase
UC ¼ urothelial carcinoma
Wm ¼ wortmannin
z-VAD-fmk ¼ carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone
Purpose: We evaluated the status of autophagy in different grades of urothelialcarcinoma and explored autophagy modulators as a potential adjunctive thera-peutic agent for urothelial carcinoma.
Materials and Methods: The study was performed in tumor tissue from patientswith low and high grade urothelial carcinoma, in normal urothelial tissueand in the T24 cell line. Autophagic vesicles and the expression of variousautophagic proteins were studied in tissue samples by transmission electronmicroscopy and Western blot, respectively. The effect of autophagy induction andinhibition was evaluated by measuring AMPK and mTOR expression, cellviability and mitochondrial membrane potential. The therapeutic implication ofautophagy was studied using cisplatin alone or combined with an autophagyinhibitor.
Results: High grade urothelial carcinoma showed a higher number of autophagicvesicles and significantly higher expression of autophagic proteins. Upon star-vation cells cultured from high and low grade urothelial carcinoma demonstratedsignificant autophagy induction associated with AMPK activation and mTORinhibition. AMPK inhibition decreased the autophagic response and increasedcell death. Autophagy inhibition by wortmannin, 3-methyladenine and chloro-quine increased mitochondrial hypopolarization as well as caspase-9 and3 dependent cell death. Combined treatment with cisplatin and an autophagyinhibitor resulted in greater cell death than cisplatin treatment alone.
Conclusions: Autophagy is related to urothelial carcinoma grade and regulatedvia the AMPK pathway for tumor cell survival. Autophagy inhibition leads tocancer cell death through an intrinsic apoptotic pathway. The potential appli-cation of autophagy inhibitors as an adjunct to chemotherapy for urothelialcarcinoma must be explored.
Key Words: urinary tract, urothelium, carcinoma, apoptosis, autophagy
Accepted for publication January 6, 2014.Study received institutional ethics committee approval.Supported by the Research and Education Cell of Post Graduate Institute of Medical Education and Research, Chandigarh and Indian Council
of Medical Research, New Delhi, India.* Correspondence: Unit-II, Department of Urology, Post Graduate Institute of Medical Education and Research, Chandigarh 160012, India
(telephone: 91 172 2756321; FAX: 91 172 2744401; e-mail: [email protected]).
See Editorial on page 1644.
0022-5347/14/1916-1889/0
THE JOURNAL OF UROLOGY®
© 2014 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION AND RESEARCH, INC.
http://dx.doi.org/10.1016/j.juro.2014.01.006
Vol. 191, 1889-1898, June 2014
Printed in U.S.A.www.jurology.com j 1889
1890 INHIBITION OF GRADE DEPENDENT AUTOPHAGY IN UROTHELIAL CANCER
UROTHELIAL carcinoma, one of the most commonlydiagnosed urinary tract malignancies, is character-ized by low sensitivity to chemotherapy and ahigh recurrence rate.1,2 The molecular basis of UCinitiation, progression and recurrence is notclearly understood. UC grows as a solid mass andmost solid tumors are under constant stress, suchas hypoxia, nutrient deficiency, etc. Tumor cellsadopt various strategies against these adverse con-ditions, including higher glycolytic flux, glutamineaddiction and induction of autophagy, to meet theirmetabolic requirements.3
Autophagy is an evolutionary conserved catabolicprocess in which cytoplasmic components, includinglong lived or aggregated proteins and damaged or-ganelles, are engulfed in the double membranestructures called autophagosomes and targeted tolysosomes for degradation.4 Autophagy is a complexprocess functionally and there is currently intensedebate on the role of autophagy in cancer. Autophagyacts as a tumor suppressor and tumor enhancermechanism based on the cancer cell type and phys-iological context.5 Autophagy functions as a tumorsuppressor by reducing inflammation, preventinggenomic instability and removing damaged organ-elles. Moreover, cancer cells resistant to apoptosisundergo autophagic cell death.6 On the other hand,autophagy promotes tumor progression by providingnutrients, thus maintaining bioenergetic homeosta-sis during the nutritional limiting conditions underwhich cancer cells usually grow.7
Various studies describe strong crosstalk betweenautophagy and apoptosis. Some anticancer drugs,such as cisplatin, gemcitabine, etc, induce auto-phagy, which was proposed as a protective mecha-nism against chemotherapy in cancer cells. Theeffect of autophagy modulation by chemotherapeuticagents has been studied in different tumor models,including glioma, myeloma, and breast, colon andprostate cancer.8 We evaluated the status of auto-phagy in different grades of UC and the effect ofmodulating autophagy by starvation and cisplatin.
MATERIALS AND METHODSThe supplementary data (http://jurology.com/) provide afull description of materials.
Tumor SamplesUrothelial tumor tissue samples were collected frompatients with UC undergoing transurethral resectionof bladder tumor. Tumor grade was established histo-pathologically according to WHO criteria.9 Three normalbladder urothelial tissue samples were collected frompatients undergoing transurethral prostate resection forbenign prostatic hyperplasia. The study was approved bythe institutional ethics committee and written informedconsent was obtained from patients. Supplementary table 1
(http://jurology.com/) shows study inclusion/exclusioncriteria and the patient demographic profile.
A total of 30 tumor tissue samples were obtained frompatients with HG (15) and LG (15) UC. Supplementarytable 2 (http://jurology.com/) shows the patient sampledistribution for the experimental setup.
Cell CulturePrimary cultures were established from cells obtainedfrom patients with LG or HG UC as well as normal uro-thelial tissue according to previously described protocols.10
Normal urothelial cells were cultured in KeratinocyteSerum-Free Medium (catalogue No. 10785, Gibco�) andUC cells were grown in Ham’s F12 medium (catalogue No.N6760, Sigma-Aldrich�) containing 10% FBS. To charac-terize UC cells cytokeratin-7 staining was performed usingFACS (supplementary fig. 1, http://jurology.com/). In allexperiments cells from passage 3 and thereafter wereused. In all experiments we used 3 normal urothelial cell,and LG and HG UC cell isolates of primary tissue culturefrom 3 patients each. The T24 human bladder cancer cellline (National Centre of Cell Sciences, Pune, India) wasmaintained in RPMI-1640 medium containing 10% FBS inhumidified atmosphere at 37C.
Autophagy MeasurementTransmission electron microscopy was used to study dou-ble membrane AVs in tissue samples. In situ expression ofautophagic proteins was performed by Western blot.Densitometry of protein expression was performed usingImageJ (http://imagej.nih.gov/ij/). Autophagy was alsostudied in ex vivo and in vitro conditions under nutrientstarvation induced by Hanks’ Balanced Salt Solution(catalogue No. H1387, Sigma-Aldrich) without addingFBS or by cisplatin treatment. Monodansylcadaverine andAO staining was used to study autophagosomes.
Cell Death AssayCell death was measured by AO/EtBr staining and subG1analysis using PI/RNAse Staining Buffer (catalogueNo. 550825) and staining with Annexin V/7-AAD (cata-logue No. 556547, BD PharmingenTM) according to themanufacturer protocol. Mitochondrial membrane poten-tial was quantified by DiOC6 staining (catalogue No.D-273, Molecular Probes�) using FACS according to themanufacturer protocol. Caspase activation was measuredby colorimetric assay and immunoblot. FACS data werequantified by FlowJo (TreeStar, Ashland, Oregon) andCellQuest� Pro.
Statistical AnalysisData are shown as the mean � SD. Data were comparedusing 1-way and 2-way ANOVA with p <0.05 consideredstatistically significant.
RESULTS
Grade Dependent Increase in UC
More AVs were found in HG and LG UC specimensthan in normal urothelial cells. Significantly moreAVswere found inHGUC(fig. 1,a).To further confirmautophagy status we examined the expression of
Figure 1. Increased grade dependent autophagy in UC. a, representative transmission electron micrographs reveal AVs. Arrowheads
indicate autophagosomes. Scale bar indicates 500 nm. Bar graph demonstrates number of AVs per cell in tissue samples of normal
urothelial cells (NUC ), and LG and HG UC from 3 patients each. b, immunoblots show LC3, Beclin1 and Atg7 expression in tissue
samples with expression level of proteins in normal urothelial cells from patient (N1) considered onefold. Values below each lane
indicate intensity ratios of autophagy (Atg) proteins. L1, L2 and L3, 3 individual LG UC samples from different patients. HI, H2 and
H3, 3 individual HG UC samples from different patients. c, bar graph indicates autophagy protein ratio in 3 normal urothelial cell
tissue, 15 LG UC and 15 HG UC samples. Values are shown as mean � SD. Single asterisk indicates p <0.05 vs normal urothelial
cells. Double asterisks indicate p <0.01 vs normal urothelial cells. Triple asterisks indicate p <0.001 vs normal urothelial cells.
Pound sign indicates p <0.05 LG vs HG UC.
INHIBITION OF GRADE DEPENDENT AUTOPHAGY IN UROTHELIAL CANCER 1891
1892 INHIBITION OF GRADE DEPENDENT AUTOPHAGY IN UROTHELIAL CANCER
various autophagic proteins, suchasLC3,Beclin1 andAtg7, by immunoblot in tissue samples. Conversion ofLC3-I to LC3-II served as a marker of autophagy in-duction.With the increase inUCgrade fromLG toHGthere was an increase in LC3-I conversion to LC3-II.We also observed an increase in the expression ofBeclin1 and Atg7, 2 key autophagic proteins involvedin the initial stepsofautophagosomebiogenesis, inHGand LG UC (fig. 1, b and c).
Induced by Starvation in UC
Starvation is a known autophagy inducing stimulusand most solid tumors grow under nutritionallimiting conditions.11 We determined how cells fromdifferent grades of UC would respond to a commonstress such as starvation. Thus, primary tumor LGandHGUCcellswere grownunder starvation for 0 to48 hours (fig. 2, a). Autophagy induction started at 6hours in HG and LG UC cells, and continuously
Figure 2. Starvation induced autophagy in UC. a, immunoblots show L
cells. b, representative FACS cytograms reveal AO staining in primary
Wm. FITC, fluorescein isothiocyanate. LGUCC, LU UC cells. HGUCC
autophagic proteins at 24 hours of starvation. d, bar graph indic
ImageJ. Untreated cells in complete medium served as control. Valu
vs control. Double asterisks indicate p <0.01 vs control. Triple asteri
starvation vs starvation plus Wm.
increased up to 24 hours. However, the fold changewas more obvious in HG UC cells. In normal urothe-lial cells autophagy was induced only after 24 hours(fig. 2, a).More autophagosomeswere observed inUCcells than in normal urothelial cells at 24 hours. Thenumber of autophagosomes were significantly higherin HG than in LG UC cells (p <0.001 and <0.05,respectively, fig. 2, b). Levels of LC3-II, Beclin1 andAtg7 were also significantly higher in HG UC cells(fig. 2, c). Wm, a class III PI3K inhibitor that inhibitsautophagy, blocked starvation induced autophagy inUC cells and normal urothelial cells (fig. 2, b to d ).
As Cell Survival Mechanism
To determine the role of autophagy in UC andnormal urothelial cells during starvation we incu-bated primary cultured cells with various autophagyinhibitors, including Wm, 3-MA and CQ. Cell deathwas monitored after UC cells and normal urothelial
C3 expression in primary cultured cells. NUC, normal urothelial
cultured cells during starvation (Starv) with or without 200 ng/ml
, HG UC cells. APC, allophycocyanin. c, immunoblots reveal
ates percent intensity of monodansylcadaverine stain using
es are shown as mean � SD. Single asterisk indicates p <0.05
sks indicate p <0.001 vs control. Pound sign indicates p <0.05
INHIBITION OF GRADE DEPENDENT AUTOPHAGY IN UROTHELIAL CANCER 1893
cells were incubated for 24 hours in starvation me-dium. Starvation caused increased death of normalurothelial cells but had no effect on HG or LG UCcells (fig. 3, a). However, upon 3-MA/Wm/CQ treat-ment there was increased HG and LG UC cell deathbut no further increase in normal urothelial cell
Figure 3. Autophagy as cell survival mechanism. a, representative
autophagy inhibitors, including 200 ng/ml Wm, 5 mM 3-MA and 50
areas represent apoptotic cells. Starv, starvation. AO/EtBr stain, re
cytograms of 1 primary cultured cell isolate each of normal urothe
FITC, fluorescein isothiocyanate. Annexin V/7-AAD stain.
death. Annexin V/7-AAD staining confirmed thatautophagy inhibition increased the apoptotic popu-lation (annexinþ and annexinþ-7AADþ) in UC cells(fig. 3, b). The proportion of the population that wasapoptotic was greater in HG than in LG UC cells(55% vs 37%).
fluorescent images show staining in presence or absence of
mM CQ. Green areas represent viable, healthy cells. Orange/red
duced from �20. b, representative photographs show FACS
lial cell (NUC ), and LG (LGUCC ) and HG (HGUCC ) UC cells.
1894 INHIBITION OF GRADE DEPENDENT AUTOPHAGY IN UROTHELIAL CANCER
Inhibition Led to Cell Death Via Intrinsic Apoptotic
Pathway
Having established that treatment with autophagyinhibitors caused an increase in UC apoptotic pop-ulations, we investigated the mechanism of thisapoptotic cell death. Caspases are the central proteinsinvolved in apoptosis. Therefore, we measured theactivation of caspase-8 and 9. Caspase-8 activationwas not observed inUC cells or normal urothelial cellsduring starvation alone orwhen autophagy inhibitorswere present in starvation medium. We also found no
Figure 4. Autophagy inhibition led to cell death via intrinsic apop
of caspase-9 inhibitor along with 200 ng/ml Wm/5 mM 3-MA. O.D.,
cells. HGUCC, HG UC cells. L-fmk, z-LEHD-fmk. b, representative FA
as positive control. C, control. S, starvation. PE/PI, phycoerythrin
c, immunoblots show cytochrome c (Cyt C ) release in cytosolic frac
and PARP expression. e, bar graph shows mean � SD total cell dea
z-VAD, z-VAD-fmk. Single asterisk indicates p <0.05 vs control. Dou
p <0.01 starvation vs starvation plus Wm/3-MA plus caspase inhibito
change in the expressionofBid, a substrateof caspase-8 (data not shown). However, caspase-9 was activatedin normal urothelial cells during starvation and Wm/3-MA had no further effect on caspase-9 activation(fig. 4, a). Caspase-9 was not activated by starvationalone but adding Wm/3-MA significantly activatedcaspase-9 in LG and HG UC cells.
Caspase-9 is the initiator caspase involved in theintrinsic apoptotic pathway. The intrinsic apoptoticpathway is characterized by permeabilization ofthe mitochondria and release of cytochrome c into
totic pathway. a, caspase-9 activity in presence and absence
optical density. NUC, normal urothelial cells. LGUCC, LG UC
CS cytograms show DiOC6 staining with 50 mM CCCP serving
/propidium iodide. MMP, mitochondrial membrane potential.
tions (CE ). z-fmk, z-VAD-fmk. d, immunoblots reveal caspase-3
th measured by Annexin V/7-AAD staining. D-fmk, z-DEVD-fmk.
ble asterisks indicate p <0.01 vs control. Pound signs indicate
r. f, immunoblots demonstrate LC3 expression.
INHIBITION OF GRADE DEPENDENT AUTOPHAGY IN UROTHELIAL CANCER 1895
the cytoplasm.12 Therefore, we measured the chan-ges in mitochondrial membrane potential (fig. 4, b).Starvation caused mitochondrial hypopolarizationin normal urothelial cells and 3-MA had no furthereffect at 24 hours. In contrast, starvation alone didnot alter mitochondrial membrane permeabilizationbut 3-MA treatment caused mitochondrial hypo-polarization in UC cells. HG UC cells were moreprone to mitochondrial hypopolarization than LGUC cells in the presence of autophagy inhibitor(from 90.8% to 20.59% vs from 92.7% to 41.9%).
We then assessed cytochrome c release in thecytosolic fraction. Cytochrome c was released onlywhen UC cells were treated with Wm/3-MA at24 hours (fig. 4, c). Caspase-3, the executionercaspase, has many substrates that are crucial forapoptosis.12 Caspase-3 activation and cleavage of itsclassic substrate PARP were measured. Starvationalone had no effect on caspase-3 or PARP in UCcells (fig. 4, d ). Adding Wm/3-MA to starvation me-dium caused caspase-3 activation and cleavageof PARP in UC cells. Caspase-3 activation and PARP
Figure 5. Autophagy activation via AMPK helped cell survival durin
primary cultured normal urothelial cells (NUC ), and LG (LGUCC ) an
expression during starvation in primary cultured cells. c and d, repr
at 24 hours in T24 cells. Experiments were repeated 3 times. C, contro
cleavage were blocked by the broad range caspaseinhibitor z-VAD-fmk (fig. 4, d ). Cell death induced byautophagy inhibitors was effectively blocked bycaspase-9 and 3, and z-VAD-fmk (fig. 4, e). WhenUC and normal urothelial cells were pretreated withz-VAD-fmk and Wm together, autophagy wasrestored at 24 hours of starvation (fig. 4, f ).
Activation Via AMPK Helped Cell Survival During
Starvation
To evaluate AMPK and mTOR status we incubatedprimary cultured cells in starvation medium fordifferent intervals. The expression of pAMPK andpmTOR was then analyzed (fig. 5, a and b). Obviousactivation of AMPK (pAMPK) and mTOR inhibition(pmTOR) started at 6 hours in HG UC cells, at 12hours in LG UC cells and at 24 hours in normalurothelial cells.
We further investigated the role of AMPKdependent autophagy regulation on cell survivalduring starvation. To do this we used dorsomorphin,an inhibitor of AMPK, and studied its effect on T24
g starvation. a, pAMPK/AMPK expression during starvation in
d HG (HGUCC ) UC cells. b, immunoblots show pmTOR/mTOR
esentative FACS cytograms of subG1 analysis using PI/RNAse
l. Dor, 30 mM dorsomorphin. Wm, 200 ng/ml Wm. S, starvation.
Figure 6. Autophagy inhibitor potentiated cisplatin cytotoxicity.
a, immunoblots show LC3 expression in primary normal
urothelial cells (NUC), and LG (LGUCC ) and HG (HGUCC ) UC
cells at different intervals of treatment with 25 mM cisplatin.
b, representative FACS cytograms of Annexin V/7-AAD
staining at 24 hours of treatment with 25 mM cisplatin (Cisp)
alone and combined with 50 mM CQ in 1 each of normal
urothelial cells, and LG and HG UC cell isolates from primary
culture. FITC, fluorescein isothiocyanate. c, representative
FACS cytograms of subG1 population at 24 hours of treatment
with 25 mM cisplatin alone and combined with 50 mM CQ in
1 each of normal urothelial cells, and LG and HG UC cell
isolates from primary culture.
1896 INHIBITION OF GRADE DEPENDENT AUTOPHAGY IN UROTHELIAL CANCER
cell survival. AMPK inhibition by dorsomorphindecreased LC3-I to LC3-II conversion and causedincreased apoptotic population to starvation alone(supplementary fig. 2, a and fig. 5, c). Adding Wmto dorsomorphin further increased this cell death(supplementary fig. 2, b and c, http://jurology.com/and fig. 5, c).
Inhibitor Potentiated Cisplatin Cytotoxic Effect
To determine whether the autophagy inhibitor CQwould augment the effect of cisplatin, a knownchemotherapeutic agent for UC, we measuredcisplatin cytotoxicity in T24 cells by MTT assay(supplementary fig. 3, a, http://jurology.com/).Cisplatin induced cell death in a dose and timedependent manner in T24 cells. We then studied theautophagic responseofnormalurothelial andUCcellsto cisplatin in a time dependent manner. Autophagywas induced by cisplatin at 6 and 12 hours in HG andLG UC cells, respectively. It attained the maximumlevel at 24 hours in LG andHGUC cells. However, innormal urothelial cells autophagy increased only at24 hours (supplementary fig. 3, b, http://jurology.com/and fig. 6, a).
To determine the importance of cisplatin inducedautophagy we treated cell isolates from primarycultures with cisplatin alone or combined with CQ.We then measured its effect on cell viability. AddingCQ to cisplatin increased LG and HG UC cell deathfrom 15% to 45% and 23% to 60%, respectively.Combined cisplatin and CQ treatment had no effecton the viability of normal urothelial cells comparedto cisplatin treatment alone (fig. 6, b and c).
Observations made during T24 cell experimentswere similar to the results described (data notshown).
DISCUSSIONThe role of autophagy in cancer development andprogression continues to be better defined andcurrently appears to be cell type dependent.13 Theinvolvement of autophagy contributes to tumori-genesis14 but whether it acts as a promoter or sup-pressor during tumorigenesis seems to be contextspecific.15 Since cancer cells are a heterogeneousmass of cells and each cancer has a different etiologyand mutational status, they may modulate theautophagic response in various ways depending ontumor microenvironment, genetic makeup andaggressiveness.16
We observed higher autophagic flux (AVs andautophagic proteins) in UC tissue than in normalurothelial tissue. More autophagic flux in HG thanin LG UC suggests that in UC autophagy is relatedto tumor grade. This might be due to a higher stresslevel in high grade UC. Autophagy is a stress
responsive mechanism17 and, therefore, it mayreflect UC aggressiveness. However, to our knowl-edge it remains to be seen whether autophagy has arole in UC progression. In parallel with our findingsMa et al noted increased autophagy in aggressivecompared with indolent melanoma.18
HG solid tumors usually grow under constantnutritional limiting conditions. Therefore, HG tu-mors may have a strong built-in adaptive mecha-nism for these stressing conditions.16 Starvationacts as a potent inducer of autophagy in variouscancer cells.11 In the current study when cancer cellsderived from primary HG and LG UC, and normalurothelial tissue were cultured under similarstress condition (starvation), UC cells respondedearly to starvation by inducing autophagy, incontrast to normal urothelial cells. HG UC cellsshowed significantly higher autophagic responsethan LG UC cells, suggesting that the autophagicresponse may increase with tumor aggressivenessdue to greater energy and metabolic demands. The
INHIBITION OF GRADE DEPENDENT AUTOPHAGY IN UROTHELIAL CANCER 1897
lower autophagic responsiveness to starvation ofnormal urothelial cells may be attributable to thelower energy requirement of normal urothelial cellscompared to UC cells, which resort to autophagy forcell survival during an energy crisis.
Intracellular adenosine triphosphate was consis-tently maintained in UC cells during starvation.However, it became depleted when autophagy wasinhibited by PI3K inhibitors such as Wm and 3-MA,leading to LG and HGUC cell death (supplementaryfig. 4, http://jurology.com/). These findings suggestthat autophagy acts as a catabolic, energy yieldingstress response during energy collapse by providingalternative pathways that circumvent energylimiting conditions in UC cells. Lum et al also re-ported that growth factor deprivation caused auto-phagy induction to degrade biosynthetic moleculesto maintain adenosine triphosphate levels.19
Autophagy may influence apoptosis in variouscancer cells.20 Salazar et al reported that autophagyinduced apoptotic cell death in human glioma cells.21
In contrast, Chen and Debnath observed that inhib-iting autophagy accelerated apoptotic cell death inlung, lymphoma and squamous carcinoma cells.22
Chen23 and Zhang24 et al suggested that autophagyis a UC cell survival mechanism and, therefore, in-hibition would cause cancer cell death. Boya et alnoted that inhibiting autophagy in a cervical cancercell line led to cancer cell death from caspase activa-tion.25 We observed mitochondrial hypopolarization,release of cytochrome c, and activation of caspase-9and 3 mediated cell death in UC cells upon treat-ment with autophagy inhibitors. This suggests thatapoptotic UC cell death occurs through an intrinsicpathway after autophagy inhibition.
Autophagy is also known as type II programmedcell death, and autophagy and apoptosis are inter-connected stress responses. While the role ofapoptosis is strictly cell death, autophagy may alsoact as a cell survival response.20 The molecularinterplay between these 2 pathways seems to becomplex and it is not well understood. In our studycaspase inhibition by z-VAD-fmk along with Wm notonly inhibited cell death but also restored the pro-cess of autophagy. This finding highlights the factthat caspases are the potential molecular devicesthat switch the cell to the apoptotic pathway byinhibiting the autophagic cell survival response.22
Our study illustrates the mechanism behindautophagy induction in UC cells during starvation.AMPK mediated phosphorylation of mTOR had aninhibitory effect on autophagy during stress condi-tions in a breast cancer cell line.26 We observed thatstarvation caused AMPK activation and mTOR in-hibition along with increased autophagy in UC cellsas well as normal urothelial cells. AMPK inhibitionwith dorsomorphin led to a decreased autophagic
response and, thus, induced cell death in T24 cells.Adding the autophagy inhibitor Wm to the AMPKinhibitor caused further increased T24 cell death.These findings suggest involvement of the PI3K/AMPK pathway in starvation induced autophagy inUC cells. Therefore, autophagy inhibitors mightexert antitumor effects by modulating this pathway.Our finding is consistent with those in a recentreport showing that autophagy protected bladdercancer cells from the cytotoxic effect of a PI3K/Akt/mTOR pathway inhibitor.27
In light of the mentioned findings it appears thatan autophagy inhibitor may be useful as an effectivetargeted therapeutic agent. Therefore, there is aneed to evaluate the role of autophagy inhibitors inpreclinical models of UC cells. Autophagy inhibitionenhanced the efficacy of anticancer agents in pre-clinical models of various other cancers, such asglioblastoma and lymphoma.28,29 Moreover, theautophagy inhibitors hydrochloroquine, a lysomo-tropic agent that inhibits autophagy, is currently inclinical trials for other malignancies.9
Cisplatin, the most commonly used chemothera-peutic agent for UC, induced autophagy in esopha-geal squamous cell carcinoma.30 We also noted thatcisplatin treatment led to the induction of auto-phagy in UC cells, delaying the apoptotic responseand, thus, compromising cisplatin cytotoxicity. Itwas suggested that autophagy induction in cancercells may be one of the mechanism of resistance tochemotherapeutic agents.9 In the current studyautophagy inhibitors sensitized the cytotoxic effectof cisplatin, resulting in more cell death thancisplatin alone. Our observation raises the hope thatautophagy inhibitors have the potential to enhancethe antineoplastic effect of certain chemothera-peutic agents.
CONCLUSIONSTo our knowledge this is the first report to showthat autophagy is related to UC grade, and con-tributes to the maintenance of cellular bioenergeticsand cancer cell survival. Because cisplatin inducedautophagy acts as a protective mechanism in UCcells, autophagy inhibitors can be useful adjuncts tocurrent chemotherapeutic agents for UC. Our studydemonstrates the potential therapeutic implicationsof autophagy inhibitors in ex vivo conditions. How-ever, validation of our observations is required inin vivo studies since the ex vivo model may not wellrepresent human disease.
ACKNOWLEDGMENTSProf. Kusum Joshi assisted with transmissionelectron microscopy.
1898 INHIBITION OF GRADE DEPENDENT AUTOPHAGY IN UROTHELIAL CANCER
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