apoptosis related molecular differences for response to tyrosin kinase inhibitors in drug sensitive and drug resistant human bladder cancer cells

edu.cn Access this article online Website: Website: www.cancerjournal.net DOI: DOI: 10.4103/0973-1482.126478 PMID: PMID: 24518715 Quick Response Code: Original Article Apoptosis-relate

Jixia Li,

Bo Lv 1 , Xiangyong Li, Zhiwei He 2 , Keyuan Zhou 2

Institute of Biochemistry and Molecular Biology,

2 Key Laboratory for Medical Molecular Diagnostics of Guangdong Province, Guangdong Medical College, Dongguan,

1 Department of Emergency and Critical Care Medicine, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong, China

For correspondence:

Dr Keyuan Zhou, Key Laboratory for Medical Molecular Diagnostics

of Guangdong Province, Guangdong Medical College, Dongguan-523808, Guangdong, China E-mail: kyz@ gdmc edu.cn

Access this article online Website:

Website: www.cancerjournal.net DOI:

DOI: 10.4103/0973-1482.126478 PMID:

PMID: 24518715 Quick Response Code:

Original Article

Apoptosis-related molecular differences

for response to tyrosin kinase inhibitors in

drug-sensitive and drug-resistant human

bladder cancer cells

ABSTRACT

Context: The epidermal growth factor receptor (EGFR) family is reportedly overexpressed in bladder cancer, and tyrosine kinaseinhibitors

(TKIs) have been suggested as treatment Gefitinib is a selective inhibitor of the EGFR and lapatinib is a dual inhibitor of both the EGFR and

HER2 (human EGFR type 2 receptor) Both compounds compete with the binding of adenosine triphosphate (ATP) to the tyrosine kinase

domain of the respective receptors to inhibit receptor autophosphorylation causing suppression of signal transduction Unfortunately,

resistance to these inhibitors is a major clinical problem.

Aims: To compare the apoptosis signaling pathway(s) induced by gefitinib and lapatinib, in UM-UC-5 (drug-sensitive) and UM-UC-14

(drug-resistant) bladder cancer cells and to identify molecular differences that might be useful predictors of their efficacy.

Materials and Methods: Cell proliferation, cell cycle and apoptosis assay were used to detect the effect of TKIs on UM-UC-5 and

UM-UC-14 cells Molecular differences for response to TKIs were examined by protein array.

Results: TKIs strongly inhibited cell proliferation and induced cell cycle G1 arrest and apoptosis in UM-UC-5 cells Most notable apoptosis

molecular differences included decreased claspin, trail, and survivin by TKIs in the sensitive cells In contrast, TKIs had no effect on resistant cells.

Conclusions: Claspin, trail, and survivin might be used to determine the sensitivity of bladder cancers to TKIs.

KEY WORDS: Bladder cancer, gefitinib, lapatinib, tyrosine kinase inhibitor

INTRODUCTION

Bladder cancer is a common malignant disease in

oncogenes, such as the epidermal growth factor

receptor (EGFR) and tumor suppressor genes, are one

of the risk factors for the development of bladder

cancer Bladder cancer highly expresses EGFR and/

the EGFR within the bladder enhances tumor

progression in mice, providing direct support for

EGFR is a cell-surface receptor, belonging to the

EGFR family of receptor tyrosine kinases The

EGFR family comprises four members, EGFR

(HER1, ErbB1), ErbB2 (HER2), ErbB3 (HER3), and

ErbB4 (HER4) The EGFR family members have four

ectodomains, a single transmembrane domain and

a cytoplasmic tail containing the active tyrosine

kinase domain Following the kinase domain,

a C-terminal tail contains autophosphorylation

EGFR-associated signaling pathway plays an important

role in the development and progression of cancers

It has become one of the most important targets for anticancer drug discovery, and a large number

of different small molecule and antibody-based EGFR antagonists have been tested in clinical trials Three small molecule EGFR tyrosine kinase inhibitors (TKIs) are in clinical use and include gefitinib (ZD1839, Iressa), erlotinib (Tarceva) and lapatinib (GW 572016, Tykerb) All are based on

a 4-anilinoquinazoline scaffold and target the ATP site to inhibit receptor autophosphorylation causing suppression of signal transduction

Gefitinib and erlotinib, selective EGFR inhibitors, target the active form of the kinase and have been approved for nonsmall-cell lung cancer Lapatinib,

a dual inhibitor of EGFR and HER2, preferentially targets the inactive conformation and has been

Although TKIs display a survival advantage in clinical trials, only a minority of patients seem

to respond to this approach Resistance to TKIs has become a major clinical problem Research is needed to identify and validate predictive factors that can be used to select patients with disease

Li, et al.: Molecular differences for response to tyrosine kinase inhibitors in drug-sensitive and resistant human bladder cancer cells

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Journal of Cancer Research and Therapeutics - October-December 2013 - Volume 9 - Issue 4

likely to respond to TKIs Recent data showed that tumor

response is not associated with a higher proportion of

EGFR-positive tumor cells or more intensive EGFR staining in lung

markers is extremely important

In this study, we compared the apoptosis signaling pathway(s)

induced by TKIs (gefitinib and lapatinib), in UM-UC-5

(drug-sensitive) and UM-UC-14 (drug-resistant) bladder cancer cell

lines and identified molecular differences as predictors of

their efficacy Here, we report that claspin, trail, and survivin

substantially suppressed by TKIs in UM-UC-5 cells

MATERIALS AND METHODS

Eagle’s minimum essential medium (MEM) was purchased

from Invitrogen (Carlsbad, CA) Fetal bovine serum (FBS)

was purchased from Gemini Bio-products (Calabasa, CA)

and the antibiotics (penicillin and streptomycin) were from

Invitrogen (Carlsbad, CA) The human apoptosis array kit

was purchased from R&D (Minneapolis, MN) The protein

assay kit was from Bio-Rad (Hercules, CA) The CellTiter96

Aqueous One Solution Cell Proliferation Assay Kit was from

Promega (Madison, WI)

The bladder cancer cell lines (UM-UC-5 and UM-UC-14,

Department of Urology, University of Texas M D Anderson

Cancer Center) were cultured in monolayers at 37°C in a 5%

incubator overnight Cells were then fed with fresh medium

and treated with gefitinib (2.5 μM) or lapatinib (2.5 μM) After

culturing for various times, 20 μl of Cell Titer 96 Aqueous One

Solution were added to each well, and the cells were then

was measured at 490 and 690 nm

incubator Cells were then starved in serum-free medium for

24 h followed by treatment for 24 h with gefitinib (2.5 μM) or

lapatinib (2.5 μM) in 10% FBS/MEM The cells were trypsinized

and then washed twice with cold PBS, and fixed with ice-cold

70% ethanol at -20°C overnight Cells were then washed twice

with PBS, incubated with 20 mg/mL RNase A and 200 mg/mL

propidium iodide in PBS at room temperature for 30 min in the

dark and subjected to flow cytometry using the FACSCalibur

flow cytometer Data were analyzed using ModFit LT (Verity

Software House, Inc., Topsham, ME)

Annexin V and propidium iodide staining was used to

visualize apoptotic cells in a similar procedure as described

above but the cells were not prefixed with 70% ethanol

Cells were stained using the Annexin V-FITC Apoptosis

Detection Kit (MBL International Corporation, Watertown,

MA) and propidium iodide according to the manufacturer's instructions Cells were analyzed by two-color flow cytometer The emission fluorescence of the Annexin V conjugate was detected and recorded through a 530/30 bandpass filter in the FL1 detector Propidium iodide was detected in the FL2 detector through a 585/42 bandpass filter Apoptotic cells were only those that stained positive for Annexin V and negative for propidium iodide, located in the bottom right quadrant

Each cell line was cultured to 90% confluence and then starved

24 h in serum-free media They were treated, respectively, with 2.5 μM gefitinib or lapatinib in culture medium containing 10% FBS for 24 h and then harvested Cell samples were disrupted and then proteins were extracted The protein concentration was determined using a dye-binding protein assay kit (Bio-Rad) as described in the manufacturer’s manual Following the instructions provided with the protein arrays, cell lysates were subjected to Proteome Profiler™ Array analysis

As necessary, data are expressed as means ± standard error

(S.E.) and significant differences were determined using

one-way ANOVA A probability value of p < 0.05 was used as the

criterion for statistical significance

RESULTS

We first examined the effects of TKIs on UM-UC-5 and UM-UC-14 cell proliferation Cells were treated with 2.5 μM of the agents (gefitinib and lapatinib) dissolved in DMSO (vehicle) for 72 h

Gefitinib and lapatinib caused a decrease to 44% and 33% (P <

0.05) of control, respectively [Figure 1] in UM-UC-5 cells; while in UM-UC-14 cells, both drugs had little effect on cell proliferation

We next assessed the effects of TKIs on cell cycle progression and apoptosis in both cells After treatment with TKIs, cells were stained with propidium iodide and analyzed by flow cytometry Cell cycle distribution analysis showed that the TKIs treatments for 24 h result in an increased accumulation

of cells in G1-phase [Figure 2] in UM-UC-5 cells but do

Figure 1: TKIs inhibit cell proliferation in UM-UC-5 cells Cells were

treated with gefi tinib (2.5 μM) or lapatinib (2.5 μM) in 10% FBS/MEM for various times At the end of each treatment time, cell growth was measured by MTS assay Data are shown as means ±S.E The

asterisks (*) indicate a signifi cant difference (P < 0.05) between groups

treated with drugs and the group treated with DMSO

Li, et al.: Molecular differences for response to tyrosine kinase inhibitors in drug-sensitive and resistant human bladder cancer cells

not affect on UM-UC-14 cells After gefitinib or lapatinib

treatment for 24 h, no differences in numbers of apoptotic

cells were observed in UM-UC-14 cells However, treatment

with TKIs resulted in significant apoptosis in UM-UC-5 cells

[Figure 3] These results suggested that TKIs inhibited cell

proliferation by inducing G1 arrest and apoptosis in

UM-UC-5 cells

To assess the direct effects of TKIs on apoptosis signaling

pathway, we used protein array to measure the level of

apoptosis-related proteins Results showed in sensitive cells

both drugs strongly downregulated antiapoptotic proteins

such as claspin, survivin, and death receptors (TRAIL R1,

TRAIL R2), but in resistant cells, TKIs had no effect [Figure

4] These suggest claspin, TRAIL, and Survivin might be

used to determine the sensitivity of bladder cancers to TKIs

DISCUSSION

Apoptosis can be activated through two pathways: The extrinsic pathway (mediated by death receptors) or the intrinsic pathway (mediated by mitochondria) The death receptor pathway is activated in response to the engagement

of ligands such as TNF-α with their receptors (TRAIL-R1 and TRAIL-R2) This in turn induces the recruitment of adapter proteins (FADD) to form the death-inducing signal complex (DISC), which activates caspase-8 In turn, caspase 8 activates

proteins (IAPs) family consists of an evolutionarily conserved

family include c-IAP-1, c-IAP-2, XIAP, surivivin, livin, and NAIP

In general, the IAP proteins function through direct interactions

to inhibit the activity of several caspases, including caspase-3,

study, our data showed EGFR inhibitors reverses TRAIL

Claspin is a mediator of Chk1 signal transduction at the replication checkpoint and in response to DNA damage Its expression is negatively regulated by both proteosome- and

tumor suppressor since downregulation promotes apoptosis

behave as an oncogene in other instances since overexpression

been suggested to be a sensitive marker of abnormally

induced apoptosis by suppressing Claspin protein

In sensitive cell, both drugs significantly downregulated TRAIL R1, TRAIL R2, survivin, and claspin TKIs were the apoptosis inducer mainly by death receptor pathways In resistant cell, both drugs had little effect on these proteins The reason is that these proteins are downstream molecular of Akt and MAP kinases signaling pathway Upstream signaling unchanged after inhibitors treated resistant cell; thus, downstream proteins had no change

Figure 3: TKIs induce apoptosis in UM-UC-5 cells (a) Cells were

starved in serum-free medium for 24 h and then treated with gefi tinib (2.5 μM) or lapatinib (2.5 μM) for 24 h Apoptosis was analyzed by fl ow cytometry Data are shown as means ± S.E The asterisk (*) indicates

a signifi cant difference (P < 0.05) between groups treated with TKIs

and the group treated with DMSO

Figure 4: UM-UC-5 cells are sensitive to TKIs and show decreased

expression of TRAIL, claspin, and survivin (a) Results of apoptosis

protein array analysis using UM-UC-5 and UM-UC-14 cells treated

or not treated with gefi tinib (b) Results of apoptosis protein array

analysis using UM-UC-5 and UM-UC-14 cells treated or not treated

with lapatinib For A and B, both cell lines were treated with 2.5 mM

gefi tinib or lapatinib for 24 h, and the cell lysates were hybridized to

the apoptosis array In the array, each signal is spotted in duplicate

Hybridization signals at the corners serve as controls

Figure 2: TKIs induce signifi cant G1 arrest in UM-UC-5 cells Cells

were starved in serum-free medium for 24 h and then treated with

gefi tinib (2.5 μM) or lapatinib (2.5 μM) for 24 h Cell cycle analysis was

performed using fl ow cytometry Data are shown as means ± S.E The

asterisks (*) indicate a signifi cant difference (P < 0.05) between groups

treated with drugs and the group treated with DMSO

Li, et al.: Molecular differences for response to tyrosine kinase inhibitors in drug-sensitive and resistant human bladder cancer cells

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Journal of Cancer Research and Therapeutics - October-December 2013 - Volume 9 - Issue 4

CONCLUSION

In summary, we first compared the signaling pathway(s)

induced by gefitinib and lapatinib in human bladder cancer

cell and confirmed that TKIs were effective in suppressing

EGFR/ErbB2 and inducing apoptosis by mainly inhibiting death

receptor (TRAIL), claspin and survivin proteins expression in

sensitive cells, whereas TKIs had no effect in resistant cells

These molecular differences could be predictors of their efficacy

in human bladder cancer cells

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Cite this article as: Li J, Lv B, Li X, He Z, Zhou K Apoptosis-related molecular

differences for response to tyrosin kinase inhibitors in drug-sensitive and drug-resistant human bladder cancer cells J Can Res Ther 2013;9:668-71.

Source of Support: Grants from National Natural Science Foundation of China

(81201710), Guangdong Natural Science Foundation (S2012010008259) and Guangdong Medical College for the research grant (no XG-1101 and GX0306),

Confl ict of Interest: None declared.

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