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Experimental Therapeutics, Molecular Targets, and Chemical Biology

Enhanced Expression of Asparagine Synthetase under Glucose-Deprived Conditions Protects Pancreatic Cancer Cells from Apoptosis Induced by Glucose Deprivation and Cisplatin

Hongyan Cui, Stephanie Darmanin, Mitsuteru Natsuisaka, Takeshi Kondo, Masahiro Asaka, Masanobu Shindoh, Fumihiro Higashino, Junji Hamuro, Futoshi Okada, Masataka Kobayashi, Koji Nakagawa, Hideyuki Koide and Masanobu Kobayashi
Hongyan Cui
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Stephanie Darmanin
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Mitsuteru Natsuisaka
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Takeshi Kondo
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Masahiro Asaka
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Masanobu Shindoh
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Fumihiro Higashino
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Junji Hamuro
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Futoshi Okada
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Masataka Kobayashi
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Koji Nakagawa
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Hideyuki Koide
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Masanobu Kobayashi
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DOI: 10.1158/0008-5472.CAN-06-2519 Published April 2007
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    Figure 1.

    Expression of ASNS under different conditions. A, BxPC3, MiaPaCa2, and PCI43 cells were incubated under normal glucose conditions (N) and glucose-deprived conditions (L). The glucose concentration of the normal glucose group was 100 mg/dL and that of the glucose-deprived group was 10 mg/dL. After incubation for 16 h, the mRNA level of ASNS was analyzed by real-time PCR. β-Actin mRNA was used to standardize the total amount of cDNA. ASNS mRNA expressions were enhanced under low-glucose conditions. Representative results of three independent experiments. Columns, mean of triplicate wells; bars, SD. *, P < 0.05. B, MiaPaCa2 cells were incubated under the two different conditions for the indicated times. After incubation, the mRNA level of ASNS was analyzed by real-time PCR. The increase in ASNS mRNA expression was sustained under glucose-deprived conditions in time course experiments (from 16 h up to 48 h). Representative results of three independent experiments. Points, mean of triplicate wells; bars, SD. C, MiaPaCa2 cells were cultured in medium containing varying concentrations of glucose for 16 h and the mRNA level of ASNS was then analyzed by real-time PCR. Varying glucose concentrations (from 50 to 10 mg/dL) enhanced ASNS expression. Representative results of three independent experiments. Columns, mean of triplicate wells; bars, SD. *, P < 0.05. D, MiaPaCa2 cells were incubated under the two different conditions for 48 h and the protein levels of ASNS in whole-cell lysates were determined by Western blot analysis. ASNS protein expression was also enhanced under low-glucose conditions. Representative results of three independent experiments. E, after preincubation with an AMPK inhibitor, compound C, for 60 min, MiaPaCa2 cells were incubated under normal glucose conditions and glucose-deprived conditions for 16 h. ASNS mRNA was analyzed by real-time PCR. Compound C showed no effect on the expression of ASNS mRNA. Representative results of two independent experiments. Columns, mean of triplicate wells; bars, SD. F, MiaPaca2 cells were transfected with 5 nmol/L siRNA for ATF-4, under normal glucose and glucose-deprived conditions, and total RNA was extracted 48 h after transfection. ASNS mRNA was analyzed by real-time PCR. A mixture of siRNAs for ATF-4 completely suppressed the expression of ASNS mRNA. Representative results of three independent experiments. Columns, mean of triplicate wells; bars, SD. *, P < 0.05.

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    Figure 2.

    Effects of siRNAs for ASNS on the sensitivity to apoptosis. A, MiaPaca2 cells were transfected with 5 nmol/L siRNA for ASNS, under normal glucose and glucose-deprived conditions, and total RNA was extracted 48 h after transfection. ASNS mRNAs were analyzed by real-time PCR. The relative copy number to β-actin in the cells treated with control siRNA, under normal or low-glucose conditions, was artificially defined as one. All siRNAs for ASNS efficiently suppressed the expression of ASNS mRNA. Representative results of three independent experiments. Columns, mean of triplicate wells; bars, SD. *, P < 0.05. B, MiaPaca2 cells were transfected with 5 nmol/L siRNA for ASNS, under normal glucose and glucose-deprived conditions, and whole-cell lysates were prepared 48 h after transfection. Protein levels of ASNS were analyzed by Western blot analysis. Two siRNAs for ASNS efficiently suppressed the expression of ASNS protein. Representative results of three independent experiments. C, MiaPaca2 cells were transfected with 5 nmol/L siRNA for ASNS under normal glucose and glucose-deprived conditions for 48 h. Viable cells were analyzed by FACS two-color analysis using propidium iodide– and FITC-conjugated Annexin V. siRNAs for ASNS decreased the percentages of viable cells under low-glucose conditions. Representative results of four independent experiments. D, MiaPaCa2 cells, transfected with 5 nmol/L siRNA for ASNS, were cultured under normal glucose and glucose-deprived conditions with indicated concentrations of CDDP for 48 h. Viable cells were analyzed by a colorimetric MTS assay. siRNAs for ASNS decreased the percentages of viable cells following CDDP treatment only under glucose-deprived conditions. Representative results of three independent experiments. Columns, mean of triplicate wells; bars, 95% CI. *, P < 0.05.

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    Figure 3.

    Establishment of ASNS transfectants. A, the uncloned ASNS-transfected and mock-transfected cells were cultured under normal glucose and varying glucose concentrations for 48 h. Viable cells were analyzed by means of a colorimetric MTS assay. The uncloned ASNS transfectant is more resistant to apoptosis induced by glucose deprivation than the mock transfectant. Representative results of three independent experiments. Columns, mean of triplicate wells; bars, 95% CI. *, P < 0.05. B, digital images acquired by fluorescence microscopy. Staining with Hoechst-33342 reveals that more than half of the mock-transfected cells cultured under low-glucose conditions show bright fluorescence (representing apoptosis), whereas nuclei of the ASNS-transfected cells display only dim staining, except for a small fraction. C, MiaPaCa2 cells were transfected with the p3XFLAG-CMVTM-14 expression vector with the use of Lipofectamine 2000. Expression levels of ASNS protein in the transfectants are shown. Clone 7 and clone 26 were chosen for further experiments. D, the transfectants (ASNS-transfected clones 7 and 26 and a mock transfectant) were cultured under normal glucose and glucose-deprived conditions for 48 h. Viable cells were analyzed by FACS two-color analysis using propidium iodide– and FITC-conjugated Annexin V. The ASNS transfectants are resistant to apoptosis induced by glucose deprivation. E, JNK/SAPK activation by low glucose in mock and ASNS transfectants is shown by Western blotting. ASNS clearly inhibits JNK/SAPK activation under glucose-deprived conditions. F, MiaPaCa2 parent cells were incubated under glucose-deprived conditions for 24 h in the presence (+) or absence (−) of SP600125, an inhibitor of JNK. After incubation, viable cells were analyzed by a colorimetric MTS assay. SP600125 increased the number of viable cells. Columns, mean of three different experiments; bars, 95% CI. Viability is compared with the cells cultured without SP600125 (−), under normal glucose conditions (−). *, P < 0.05.

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    Figure 4.

    Sensitivity of ASNS transfectants to several anticancer drugs. ASNS transfectants were incubated under normal glucose conditions with the indicated concentrations of CDDP (A), carboplatin (B), 5-FU (C), VP-16 (D), paclitaxel (E), and gemcitabine (F) for 48 h. After incubation, viable cells were analyzed by a colorimetric MTS assay. The two transfectant clones are resistant to both CDDP and carboplatin but not to 5-FU, VP-16, paclitaxel, or gemcitabine. Representative results of three independent experiments. Columns, mean of triplicate wells; bars, 95% CI. *, P < 0.05.

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    Figure 5.

    Roles of asparagine and JNK/SAPK in CDDP resistance. A, asparagine added in the culture medium up to 20 mmol/L had no effect on the MiaPaCa2 cell sensitivity to CDDP following 48 h of incubation under normal glucose conditions. Viable cells were analyzed by a colorimetric MTS assay. Representative results of three independent experiments. Columns, mean of triplicate wells; bars, 95% CI. B, JNK/SAPK activation induced by CDDP and 5-FU. CDDP but not 5-FU clearly activated JNK/SAPK after 12-h incubation under normal glucose conditions. The activation of JNK/SAPK was suppressed in the ASNS transfectant. Representative results of three independent experiments. C, MiaPaCa2 parent cells were cultured under normal glucose conditions for 48 h in the presence (+) or absence (−) of CDDP, 5-FU, and SP600125 (an inhibitor of JNK), as illustrated. After incubation, viable cells were analyzed by a colorimetric MTS assay. Representative results of three independent experiments. Columns, mean of triplicate wells; bars, 95% CI. *, P < 0.05.

Tables

  • Figures
  • Table 1.

    Overexpressed genes under glucose-deprived conditions

    GeneSwiss ProtProtein Bank
    Cell signaling
        EIF2AK3Eukaryotic initiation factor 2α kinase 3Q9NZJ5
        PPEF2Protein phosphatase EF hand 2O14830
        CPR8Cell cycle progression 8 proteinAAB69314.1
        DNAJC3DnaJ (Hsp40) homologue subfamily C member 3 (protein kinase inhibitor p58)BAA74859.1
        PRKCZProtein kinase Cζ isoformQ05513
        CDC2Cell division cycle 2, cyclin-dependent protein kinaseP06493
        SGKLSerum/glucocorticoid regulated kinase-likeAAF27051.2
        ZAP70ζ-chain-associated 70 kDa proteinP43403
        TOPKPDZ-binding kinaseBAA99576.1
        CDT1DNA replication factor (double parked)AAG45181.1
        CNKCytokine inducible kinaseQ9H4B4
    Transcriptional regulation
        TRPS1Trichorhinophalangeal syndromeQ9UHF7
        TGIFTG-interacting factorQ15583
        ARNTLAryl hydrocarbon receptor nuclear translocator-likeAAC24353.1
        RELBReticuloendotheliosis viral (v-rel) oncogene related BQ01201
        PREBProlactin regulatory element binding proteinQ9HCU5
        ELF3E74 like factor 3AAB58075.1
    Enzymes
        ART3ADP-ribosyltransferase 3Q13508
        FUT1Fucosyltransferase 1 [α(1,2) fucosyltransferase, galactoside 2-α-l-fucosyltransferase]P19526
        NEU1Neuraminidase 1 (lysosomal neuraminidase, sialidase 1)Q99519
        GOT1Soluble glutamate-oxaloacetate transaminase (cytosolic aspartate aminotransferase)P17174
        ASNSAsparagine synthetaseP08243
        GFPT1Glutamine-fructose-6-phosphate transaminase 1Q06210
        PCK2Phosphoenolpyruvate carboxykinase 2Q16822
        CBR3Carbonyl reductase 3O75828
        PYCR1Pyrroline-5-carboxylate reductase 1P32322
        SARSSeryl-tRNA synthetaseP49591
        ERO1-L(β)Endoplasmic reticulum oxidoreductin 1-LβAAF97547.1
        NDUFB3NADH dehydrogenase (ubiquinone) 1β subcomplex 3 (12 kDa B12)O43676
        RRM2Ribonucleotide reductase subunit M2P31350
        B3GNT6β-1,3-N-AcetylglucosaminyltransferaseAAC39538.1
        PIGAGPI GlcNAc transferase aP37287
        TXNRD1Thioredoxin reductase 1Q16881
    Others
        PPIBCyclophilin BAAH20800.1
        PRAMEPreferentially expressed antigen of melanomaP78395
        FKBP2FK506-binding protein 2 (FKBP13)AAA36563.1
        DFNA5Deafness autosomal dominant 5O60443
        RTP801HIF-1 responsive RTP801AAL38424.1
        SEMG1Semenogelin IP04279
        H4FEH4 histone family member EP02304
        TFRCTransferrin receptorP02786
        GRO2Macrophage inflammatory protein 2P19875
        GADD45DNA damage inducible transcript 1P24522
        TGIFTG-interacting factorQ15583
    Transporter proteins
        ACATNAcetyl-CoA transporterAAH14416.1
        CLGNCalmeginO14967
        DNAJB11DnaJ (Hsp40) homologue subfamily B member 11 (human endoplasmic reticulum–associated DNAJ)Q9UBS4
        CALRCalreticulin,P27797
        CANXCalnexinP27824
        SLC3A2Heavy chain of 4F2 cell-surface antigenP08195
        UGTREL1UDP-galactose transporter–related 1BAA13525.1
        KDELR2KDEL (Lys-Asp-Glu-Leu) endoplasmic reticulum protein retention receptor 2P33947
        KCNF1Potassium voltage channel subfamily F1AAH26110.1
    Redox-related proteins
        PDIA4Protein disulfide isomerase–related protein (calcium-binding protein, intestinal related)P13667
        TXNIPVitamin D3 up-regulated protein-BAB18859.1
        SPS2Selenophosphate synthetase 2Q99611
        PDIA6Protein disulfide isomerase–related protein P5Q15084
        GRP58Glucose regulated 58 kDa proteinP30101
    Cytoskeletal proteins
        GFAPGlial fibrillary acidic proteinP14136BAB31108.1
        GABARAPL3GABA(A) receptor–associated protein like 3AAK16237.1
        OCLNOccludinQ16625
        KIF5CKinesin heavy chain 5CO60282
        ANLNAnilineAAF75796.1
    • (Continued on the following page)

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Cancer Research: 67 (7)
April 2007
Volume 67, Issue 7
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Enhanced Expression of Asparagine Synthetase under Glucose-Deprived Conditions Protects Pancreatic Cancer Cells from Apoptosis Induced by Glucose Deprivation and Cisplatin
Hongyan Cui, Stephanie Darmanin, Mitsuteru Natsuisaka, Takeshi Kondo, Masahiro Asaka, Masanobu Shindoh, Fumihiro Higashino, Junji Hamuro, Futoshi Okada, Masataka Kobayashi, Koji Nakagawa, Hideyuki Koide and Masanobu Kobayashi
Cancer Res April 1 2007 (67) (7) 3345-3355; DOI: 10.1158/0008-5472.CAN-06-2519

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Enhanced Expression of Asparagine Synthetase under Glucose-Deprived Conditions Protects Pancreatic Cancer Cells from Apoptosis Induced by Glucose Deprivation and Cisplatin
Hongyan Cui, Stephanie Darmanin, Mitsuteru Natsuisaka, Takeshi Kondo, Masahiro Asaka, Masanobu Shindoh, Fumihiro Higashino, Junji Hamuro, Futoshi Okada, Masataka Kobayashi, Koji Nakagawa, Hideyuki Koide and Masanobu Kobayashi
Cancer Res April 1 2007 (67) (7) 3345-3355; DOI: 10.1158/0008-5472.CAN-06-2519
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