Open Access

Tetradecylthioacetic acid inhibits proliferation of human SW620 colon cancer cells - gene expression profiling implies endoplasmic reticulum stress

  • Anne G Lundemo1,
  • Caroline HH Pettersen1,
  • Kjetil Berge2,
  • Rolf K Berge2 and
  • Svanhild A Schønberg1Email author
Contributed equally
Lipids in Health and Disease201110:190

DOI: 10.1186/1476-511X-10-190

Received: 19 September 2011

Accepted: 25 October 2011

Published: 25 October 2011

Abstract

Background

Previous reports have shown an antiproliferative effect of the synthetic, 3-thia fatty acid tetradecylthioacetic acid (TTA) on different cancer cells in vitro and in vivo. The mechanisms behind the observed effects are poorly understood. We therefore wanted to explore the molecular mechanisms involved in TTA-induced growth inhibition of the human colon cancer cell line SW620 by gene expression profiling.

Methods

An antiproliferative effect of TTA on SW620 cells in vitro was displayed in real time using the xCELLigence System (Roche). Affymetrix gene expression profiling was performed to elucidate the molecular mechanisms behind the antiproliferative effect of TTA. Changes in gene expression were verified at protein level by western blotting.

Results

TTA reduced SW620 cell growth, measured as baseline cell index, by 35% and 55% after 48 h and 72 h, respectively. We show for the first time that TTA induces an endoplasmic reticulum (ER) stress response in cancer cells. Gene expression analysis revealed changes related to ER stress and unfolded protein response (UPR). This was verified at protein level by phosphorylation of eukaryote translation initiation factor 2 alpha (eIF2α) and downstream up-regulation of activating transcription factor 4 (ATF4). Transcripts for positive and negative cell cycle regulators were down- and up-regulated, respectively. This, together with a down-regulation of Cyclin D1 at protein level, indicates inhibition of cell cycle progression. TTA also affected transcripts involved in calcium homeostasis. Moreover, mRNA and protein level of the ER stress inducible C/EBP-homologous protein (CHOP), Tribbles homolog 3 (Drosophila) (TRIB3) and CCAAT/enhancer binding protein beta (C/EBPβ) were enhanced, and the C/EBPβ LIP/LAP ratio was significantly increased. These results indicate prolonged ER stress and a possible link to induction of cell death.

Conclusion

We find that TTA-induced growth inhibition of SW620 cells seems to be mediated through induction of ER stress and activation of the UPR pathway.

Keywords

Cell cycle Colon cancer Docosahexaenoic acid Endoplasmic reticulum stress Gene expression analysis Phosphorylated eIF2α Tetradecylthioacetic acid Thia fatty acids Unfolded protein response

Background

Colon cancer is one of the most common incident cancers across Europe [1]. There is an urgent need for finding novel, alternative or supplementary cancer treatments. Polyunsaturated fatty acids (PUFAs) are able to inhibit growth of both colon and several other types of cancer cells in vitro and in vivo ([25] and reviewed in [68]). During the last two decades, chemical modified fatty acid (FA) analogs have been produced in an attempt to achieve FAs having increased metabolic stability and more selective and targeted effects (reviewed in [9]). Among these is the bioactive, saturated 3-thia FA tetradecylthioacetic acid (TTA). TTA has been shown to have both a cardioprotective effect, as well as an antiproliferative effect on cancer cells (reviewed in [10]). TTA has been found to inhibit growth of glioma [11, 12], leukemia [1316] and colon cancer cell lines [11, 17]in vitro and in vivo, and hepatoma [18] and breast cancer cells [19]in vitro. TTA seems to be more potent in reducing cell growth compared with the omega-3 (n-3) PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) [20]. A diet containing TTA has also resulted in increased vascularisation of colon cancer xenografts in mice [17] and improved the survival of mice having leukemia xenografts [11, 14]. TTA food supplementation seems to be generally well tolerated by healthy persons [15, 21], and TTA therefore may have a potential in cancer treatment alone or in combination with other therapies.

The structure of TTA is equal to the saturated palmitic acid (PA), except that TTA has a sulphur atom inserted at the third position in the carbon chain [19]. The sulphur atom makes TTA resistant to mitochondrial β-oxidation and probably contributes substantial to its biological effects (reviewed in [10]). TTA is capable of reducing the growth of cancer cells that are not growth inhibited by PA [17, 20, 22]. TTA is degraded relatively slowly to various dicarboxylic acids via ω-oxidation and sulphur oxidation in the endoplasmic reticulum (ER) and subsequent β-oxidation in the peroxisomes. Except from blocked mitochondrial β-oxidation, the chemical properties and metabolism of TTA resembles those of normal FAs. TTA is activated by binding to coenzyme A and incorporated into various lipids, especially phospholipids (reviewed in [10, 23]).

Before any recommendations regarding use of TTA in cancer treatment can be given, it's important to elucidate the molecular mechanisms underlying the growth inhibitory effect of TTA. In some, but not all, cancer cells, TTA inhibits cancer cell growth via increased lipid peroxidation and oxidative stress [12], or partly via activation of peroxisome proliferator activated receptor gamma [22]. Also, TTA has been shown to induce apoptosis in several glioma [12, 13] and leukemia cell lines [13, 14]. Induction of apoptosis seems to be related to effects on mitochondria. TTA can induce a decrease in mitochondrial membrane potential [13, 24] and lead to release of cytochrome C (cyt C) and a reduction in mitochondrial glutathione, the latter indicating a selective modulation of the mitochondrial redox equilibrium [13]. Most of the biological effects of TTA assumed to be implicated in mediating the inhibitory effect of TTA on cancer cells do not seem to be specific for TTA, since they also are assumed to be involved in the growth inhibitory effect of other FAs like n-3 PUFAs [2527].

We have previously shown that TTA inhibits the growth of SW620 human colon cancer cells in vitro and in vivo[17]. SW620 cell growth is also inhibited by n-3 PUFAs [2, 4]. By using gene expression analysis, we found that DHA induces extensive changes in the expression of transcripts involved in biological pathways like ER stress and unfolded protein response (UPR), protein degradation, Ca2+ homeostasis, cell cycle progression and apoptosis [28]. Others have found that PA also can induce ER stress in human [29] and rat cancer cells [30].

The main functions of ER are protein synthesis and folding, lipid synthesis and maintenance of Ca2+ homeostasis. Disruption of these processes causes accumulation of misfolded proteins in ER lumen, leading to ER stress and activation of the cellular stress response UPR. The purpose of UPR is to restore cell homeostasis and promote cell survival, but during prolonged ER stress, apoptosis may be activated. During ER stress, one of the three ER stress sensors; eukaryotic translation initiation factor 2 alpha (eIF2α) kinase 3 (EIF2AK3/PERK) is known to phosphorylate eIF2α, thereby attenuating global protein synthesis to reduce the protein load of ER. Reduced synthesis of e.g. cyclin D1 promotes cell cycle arrest, creating time to cope with the stress. However, translation of certain mRNAs is allowed, like mRNAs for activating transcription factor 4 (ATF4) and its downstream target C/EBP-homologous protein (CHOP) [31, 32]. CHOP, which is also regulated by X-box binding protein 1 (XBP-1) (reviewed in [33]), promotes apoptosis by down-regulating anti-apoptotic factors like B-cell lymphoma 2 (Bcl-2) [34] and up-regulating pro-apoptotic factors like the Bcl-2-interacting protein Bim [35].

During ER stress XBP-1 also activates the transcription factor CCAAT/enhancer binding protein beta (C/EBPβ) [36], which participates in regulation of differentiation, cell growth, cell survival and apoptosis [37, 38]. Human C/EBPβ is mainly expressed as two 46 and 42 kDa liver enriched activator protein isoforms (LAP and LAP*) and a 20 kDa liver enriched inhibitor protein isoform (LIP) [39], which are synthesized from different AUG start codons within the C/EBPβ mRNA [40]. Since LIP lacks a transcription activation domain [41] and suppresses the transactivation by LAP, the LIP/LAP ratio influences on the extent of C/EBPβ-activated transcription [40].

LIP has been shown to decrease during early ER stress, and increase during prolonged ER stress, causing an increase in the LIP/LAP ratio [42]. Increased LIP seems to be essential for CHOP-induced apoptosis, since heterodimerization of LIP with CHOP promotes nuclear translocation of CHOP and pro-apoptotic gene regulation by CHOP [43]. In addition, CHOP-C/EBPβ [44] and CHOP-ATF4 [45] heterodimeres up-regulate the pro-apoptotic pseudo-kinase Tribbles homolog 3 (Drosophila) (TRIB3), which can promote apoptosis by inhibiting the anti-apoptotic kinase Akt [46] and the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) [47]. CHOP- and TRIB3-promoted cell death might be accompanied by the ER stress-induced caspase 4 (CASP4) and calpains [32, 48, 49].

In this report we have used Affymetrix gene expression analysis to search for possible molecular mechanisms underlying the growth inhibitory effect of TTA on SW620 cells in vitro. The results indicate that TTA reduces SW620 cell growth through activation of ER stress and UPR, by a similar mechanism as observed after treatment with DHA [28].

Results

TTA reduces growth of SW620 cells

In accordance with previous results [17], we verified a growth inhibitory effect of TTA (75 μM) on SW620 colon cancer cells by cell counting (data not shown) and by measuring the growth real time by using the xCELLigence System (Roche) (Figure 1A). The SW620 growth curve, which is presented as baseline cell index, showed that TTA (75 μM) significantly reduced the baseline cell index by 35% and 55% after 48 h and 72 h, respectively, compared to the control (Figure 1B).
Figure 1

Real time monitoring of TTA-induced growth inhibition of SW620 cells. SW620 colon cancer cells were seeded in 16 well E-plates. After ~24 h, TTA (75 μM) or NaOH (control) were added. Cell growth was monitored using the xCELLigence RTCA DP Instrument (Roche). (A) SW620 cell growth presented as baseline cell index from one representative experiment. (B) Average baseline cell index (± SD) of TTA-treated cells, presented as percent of control, at indicated time points. Mean was calculated from at least two duplicate measurements in four independent experiments. * Significantly different from control (one-tailed Student's t-test, P < 0.05).

TTA induces extensive changes at mRNA level indicating ER stress and UPR

In an attempt to unravel the mechanisms underlying the growth-inhibiting effect of TTA, SW620 cells were cultivated in the absence or presence of 75 μM TTA for 24 h, followed by gene expression profiling. Differentially expressed transcripts are presented and listed by full and short names in Table 1 and Additional file 1.
Table 1

Gene expression results

Gene Symbol

Affymetrix ID

Refseq NCBI ID

Transcript name

SW620

Fold Change

    

24 h

ER stress and unfolded protein response

  

ATF3

202672_s_at

NM_001030287

NM_001040619

NM_001674

NM_004024

Activating transcription factor 3

3.5

ATF4

200779_at

NM_001675

NM_182810

Activating transcription factor 4

1.7

ATF6

217550_at

231927_at

203952_at

NM_007348

Activating transcription factor 6

1.5

1.3

1.2

CEBPB

212501_at

NM_005194

CCAAT/enhancer binding protein (C/EBP), beta

3.2

CHOP/DDIT3

209383_at

NM_001130101

NM_001130102

NM_004083

NM_005693

DNA-damage-inducible transcript 3

2.7

GADD34

37028_at

202014_at

NM_014330

Growth arrest and DNA-damage-inducible 34

2.0

2.0

IRE1/ERN1

235745_at

NM_001433

Inositol-requiring enzyme 1/Endoplasmic reticulum to nucleus signaling 1

1.7

NRF2/NFE2L2

201146_at

NM_001145412

NM_001145413

NM_006164

Nuclear factor (erythroid-derived 2)-like 2

1.3

TRIB3

1555788_a_at

218145_at

NM_021158

Tribbles homolog 3 (Drosophila)

5.1

4.9

XBP1

242021_at

200670_at

NM_001079539

NM_005080

X-box binding protein 1

1.2

1.6

Chaperones/Protein folding/Unfolded protein response/Protein degradation/Amino acid synthesis

 

ASNS

205047_s_at

NM_001673

NM_133436

NM_183356

Asparagine synthetase

4.8

CREB3L2

212345_s_at

NM_194071

cAMP responsive element binding protein 3-like 2

1.4

CREB3L3

234361_at

NM_032607

cAMP responsive element binding protein 3-like 3

3.0

DNAJB2

202500_at

NM_001039550

NM_006736

DnaJ (Hsp40) homolog, subfamily B, member B2

1.3

DNAJB14

222850_s_at

NM_001031723

DnaJ (Hsp40) homolog, subfamily B, member B14

1.2

DNAJC24

242562_at

NM_181706

DnaJ (Hsp40) homolog, subfamily C, member 24

1.4

EDEM1

203279_at

NM_014674

ER degradation enhancer, mannosidase alpha-like 1

1.3

EDEM3

220342_x_at

NM_025191

ER degradation enhancer, mannosidase alpha-like 3

1.2

ERO1LB

231944_at

NM_019891

ERO1-like beta (S. cerevisiae)

1.3

HMOX1/HSP32

203665_at

NM_002133

Heme oxygenase (decycling) 1

1.6

HSPA13

202557_at

202558_s_at

NM_006948

Heat shock 70kDa protein 13

1.4

1.7

PDIA2

206691_s_at

NM_006849

Protein disulfide isomerase family A, member 2

1.2

PFDN2

218336_at

NM_012394

Prefoldin 2

1.4

PSMB1

214289_at

NM_002793

Proteasome subunit, beta type, 1

1.3

SQSTM1

201471_s_at

213112_s_at

239004_at

244804_at

NM_001142298

NM_001142299

NM_003900

Sequestosome 1

2.1

3.1

1.3

2.1

UBE2B

239163_at

NM_003337

Ubiquitin-conjugating enzyme E2B (RAD6 homolog)

1.5

SEC61A2

219499_at

228747_at

NM_001142627

NM_001142628

NM_018144

Sec61 alpha 2 subunit (S. cerevisiae)

1.3

1.2

SEC61B

244700_at

NM_006808

Sec61 beta subunit

1.6

SEC63

201914_s_at

201915_s_at

201916_s_at

NM_007214

SEC63 homolog (S. cerevisiae)

1.3

1.2

1.3

Ca2 + homeostasis, signaling and transport

 

ATP2B4/PMCA4

205410_s_at

212135_s_at

212136_at

NM_001001396

NM_001684

ATPase, Ca2+ transporting, plasma membrane 4

1.5

2.7

5.4

CAPN1

232012_at

NM_005186

Calpain 1, (mu/I) large subunit

1.4

CAPN2

208683_at

214888_at

NM_001146068

NM_001748

Calpain 2, large subunit

1.4

1.8

CAPN5

205166_at

NM_004055

Calpain 5

1.4

HERPUD1

217168_s_at

NM_001010989

NM_001010990

NM_014685

Homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1

1.9

ITPR1

244090_at

---

Inositol 1,4,5-triphosphate receptor, type 1

1.3

ITPR3

201187_s_at

201188_s_at

201189_s_at

NM_002224

Inositol 1,4,5-triphosphate receptor, type 3

1.3

1.5

1.5

PLCD3

234971_x_at

1552476_s_at

NM_133373

Phospholipase C, delta 3

1.3

1.3

S100A10

200872_at

238909_at

NM_002966

S100 calcium binding protein A10

1.2

2.4

S100P

204351_at

NM_005980

S100 calcium binding protein P

4.9

STC2

203438_at

203439_s_at

NM_003714

Stanniocalcin 2

2.1

2.7

Cell cycle/Apoptosis

    

ATF5

204998_s_at

204999_s_at

NM_012068

Activating transcription factor 5

1.5

1.7

AURKA

208080_at

NM_003600

NM_198433

NM_198434

NM_198435

NM_198436

NM_198437

Aurora kinase A

-1.3

BIRC5

202094_at

202095_s_at

210334_x_at

NM_001012270

NM_001012271

NM_001168

Baculoviral IAP repeat-containing 5/Survivin

-1.7

-1.3

-1.3

CASP4

213596_at

NM_001225

NM_033306

Caspase 4

1.4

CCNA2

203418_at

213226_at

NM_001237

Cyclin A2

-1.2

-1.2

CCND3

201700_at

NM_001136017

NM_001136125

NM_001136126

NM_001760

Cyclin D3

-2.0

CCNE1

213523_at

NM_001238

NM_057182

Cyclin E1

-1.3

CCNE2

205034_at

211814_s_at

NM_057749

Cyclin E2

-2.9

-3.1

CCNF

204827_s_at

NM_001761

Cyclin F

-1.2

CDC2/CDK1

203214_x_at

NM_001130829

NM_001786

NM_033379

Cell division cycle 2, G1 to S and G2 to M

-1.2

CDK2

204252_at

211804_s_at

NM_001798

NM_052827

Cyclin-dependent kinase 2

-1.3

-1.4

CDK4

202246_s_at

NM_000075

Cyclin-dependent kinase 4

-1.4

CDK5

204247_s_at

NM_001164410

NM_004935

Cyclin-dependent kinase 5

-1.5

CDK6

224847_at

224848_at

224851_at

243000_at

NM_001145306

NM_001259

Cyclin-dependent kinase 6

-1.4

-1.5

-1.3

-1.3

CDKN2A

211156_at

NM_000077

NM_058195

NM_058197

Cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4)

1.3

CDKN2B

207530_s_at

236313_at

NM_004936

NM_078487

Cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4)

1.5

2.0

CDKN2D

210240_s_at

NM_001800

NM_079421

Cyclin-dependent kinase inhibitor 2D (p19, inhibits CDK4)

1.2

CUL1

238509_at

NM_003592

Cullin 1

1.4

KLF4

220266_s_at

221841_s_at

NM_004235

Kruppel-like factor 4 (gut)

1.9

2.4

PDCD4

202731_at

212593_s_at

NM_014456

NM_145341

Programmed cell death 4 (neoplastic transformation inhibitor)

1.3

1.3

PDCD6

222152_at

222380_s_at

NM_013232

Programmed cell death 6

1.3

1.3

SFN

33322_i_at

33323_r_at

209260_at

NM_006142

Stratifin

1.6

1.7

1.6

Significantly differentially expressed transcripts in SW620 treated with TTA (75 μM) for 24 h as determined by Affymetrix microarray analysis (P < 0.05).

After incubation with TTA, the transcription level of several genes involved in ER stress and UPR were affected. The ER stress sensors inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6), and downstream targets of these, like XBP-1, were up-regulated (Table 1). However, most changes in the ER stress signaling pathway were found downstream of PERK. Phosphorylation of eIF2α by PERK is a hallmark of ER stress (reviewed in [31]). After TTA treatment, phosphorylated eIF2α (eIF2α-P) was found significantly increased after 6 h and 24 h (Figure 2A).
Figure 2

TTA induces proteins involved in ER stress and UPR. SW620 cells were treated with TTA (75 μM) or NaOH (control) for indicated time periods, and proteins were quantified by western blotting. (A) Analysis of eIF2α-P and Cyclin D1 from cytosolic protein extracts and ATF4 and CHOP from nuclear protein extracts. (B) Analysis of TRIB3 and the C/EBPβ protein isoforms LAP (45 kDa) and LIP (20 kDa) from nuclear protein extracts. Blots were quantified, and band intensities normalized relative to the respective loading control; β-actin (Cyclin D1), Lamin (nuclear extracts) or total level of eIF2α (eIF2α-P), to adjust for unequal protein loading within the membranes. Band intensities were related to the 24 h control band to adjust for differences in signal intensities between the membranes, except for TRIB3 where the band intensities are related to the 24 h TTA band (not expressed in control). Quantified results show mean fold change (± SD) of TTA-samples relative to control at indicated time periods for three independent experiments. One representative blot is shown. * Significantly different from control (one-tailed Student's t-test, P < 0.05).

Phosphorylation of eIF2α induces transcription of ATF4 and its downstream targets, like ATF3, CHOP, growth arrest and DNA-damage-inducible 34 (GADD34), TRIB3 [50, 51] and heme oxygenase 1 (HMOX1) [52]. All these transcripts were up-regulated upon TTA treatment (Table 1). Moreover, the protein levels of ATF4, CHOP (Figure 2A) and TRIB3 (Figure 2B) were also increased. ATF4 is known to induce genes involved in amino acid synthesis, and several transcripts involved in amino acid synthesis and transport were also up-regulated (Table 1 and Additional file 1).

When UPR is activated, cells try to up-regulate the folding capacity e.g. through up-regulation of chaperones and heat shock proteins (Hsps) [31]. Transcripts of the folding machinery were found to be up-regulated in the SW620 cells after incubation with TTA. These include prefoldin 2 (PFDN2), DnaJ (HSP40) homolog, subfamily B, member 2 (DNAJB2), DNAJB14, DnaJ (Hsp40) homolog, subfamily C, member 24 (DNAJC24), heat shock protein 13 (HSPA13), HSP32 (HMOX1) and hypoxia up-regulated 1 (HYOU1) (Table 1 and Additional file 1). Protein disulfide isomerase family A, member 2 (PDIA2) and ERO1-like beta (ERO1LB), which are known to catalyze protein folding [31, 53], were also up-regulated (Table 1). However, some DnaJ (Hsp40) homolog subfamily members and Hsps were also found to be down-regulated (Additional file 1).

During ER stress, improperly folded proteins are transferred to ER degradation enhancer, mannosidase alpha-like protein (EDEM) and translocated to cytosol for proteasomal degradation; a process called ER associated degradation (ERAD) [31]. EDEM1 and EDEM3, in addition to several proteasomal subunits and ubiquitin-related transcripts, were up-regulated upon TTA treatment. Also, proteasome subunit, beta type, 1 (PSMB1), ubiquitin-conjugating enzyme E2B (RAD6 homolog) (UBE2B) and cullin1 (CUL1) were up-regulated (Table 1 and Additional file 1). In addition, TTA also up-regulated sequestosome 1 (SQSTM1), which is able to sequester and shuttle polyubiquitinated, misfolded proteins to the proteasome (reviewed in [54]) (Table 1). Moreover, TTA up-regulated transcripts for parts of the ER translocation machinery which transports proteins to the cytosol, like Sec61 alpha 2 subunit (SEC61A2), Sec61 beta subunit (SEC61B) and SEC63 homolog (SEC63) (Table 1).

TTA affects Ca2+ homeostasis

Another indication of an ER stress condition in SW620 cells treated with TTA is the altered expression of transcripts coding for proteins involved in Ca2+ homeostasis. The more than 5-fold up-regulation of ATPase, Ca2+-transporting, plasma membrane 4 (ATP2B4/PMCA4) might indicate the presence of a high Ca2+ concentration in the cytosol (Table 1). A possible high Ca2+ level in the cytosol may be part of the ER stress response, with Ca2+ leaking from ER through the up-regulated IP3 receptors, type 1 and 3 (ITPR1 and 3, Table 1). We also found Phospholipase C D3 (PLCD3), which is known to participate in the generation of IP3 [55], to be up-regulated at mRNA level after TTA-treatment (Table 1).

Among other important Ca2+-related transcripts, we found several S100 Ca2+ binding proteins to be differentially expressed after TTA-treatment, e.g. S100P and S100A10 were up-regulated (Table 1 and Additional file 1). Also, TTA caused up-regulation of Stanniocalcin 2 (STC2), which is induced downstream of PERK and critical for survival after UPR [56]. TTA further up-regulated homocysteine-inducible endoplasmic reticulum stress-inducible ubiquitin-like domain member 1 (HERPUD1) (Table 1), which is known to counteract Ca2+ disturbances during ER stress [57].

Effect of TTA on cell cycle

TTA affected the mRNA level of several cell cycle transcripts, most of them having rather small fold change values. Positive cell cycle regulators were down-regulated and negative regulators were up-regulated, as outlined below.

The cell cycle is known to be positively regulated by cyclins (CCNs) and cyclin dependent kinases (CDKs). The mRNA level of cyclin D3 (CCND3), but not cyclin D1, was down-regulated upon TTA treatment (Table 1). However, the protein level of cyclin D1 was significantly decreased after incubation with TTA (Figure 2A). Moreover, transcripts for CCNA2, CCNE1, CCNE2, CCNF, CDK1/CDC2, CDK2, CDK4, CDK5 and CDK6 were down-regulated (Table 1), indicating inhibition of cell cycle progression. The CDK4 inhibitors CDK inhibitor 2A (CDKN2A), CDKN2B and CKDN2D, which are negative regulators of cell cycle, were increased (Table 1). In addition, the tumor suppressor programmed cell death 4 (PDCD4), which is able to reduce the protein level of CDK4 [58], and PDCD6 were up-regulated (Table 1).

Other important cell cycle related transcripts that were up-regulated by TTA were ATF5, stratifin (SFN/14-3-3 sigma) and kruppel like factor 4 (KLF4) (Table 1). TTA also induced down-regulation of aurora kinase A (AURKA) and survivin/baculoviral IAP repeat-containing 5 (BIRC5) (Table 1).

TTA changes expression of transcripts involved in ER stress-related apoptosis

Transcripts related to ER stress-induced apoptosis were also up-regulated upon TTA treatment. In addition to the up-regulation of CHOP and TRIB3, TTA also increased transcription of CASP4, calpain 1, large subunit (CAPN1), CAPN2 and CAPN5 (Table 1).

The C/EBPβ LIP isoform is known to augment ER stress-induced cell death by interfering with the expression of TRIB3 [59]. C/EBPβ (CEBPB) was up-regulated 3.2-fold at mRNA level (Table 1) after TTA treatment. Western blotting mainly displayed two C/EBPβ isoforms; a highly expressed 45 kDa LAP isoform and a lower expressed 20 kDa LIP isoform. The 42 kDa LAP* isoform was only found in very low amounts (Figure 2B). TTA did not affect the level of LAP, but induced a significant 6.9- and 11.2-fold induction of LIP after 24 and 48 h, respectively (Figure 2B). The accumulation of LIP at these time points caused a significant 6.6- and 13.3-fold increase in the LIP/LAP ratio after 24 h and 48 h, respectively, compared to control cells.

During ER stress, cAMP responsive element binding protein 3-like 2 and 3 (CREB3L2 and CREB3L3) are induced in order to attenuate ER stress-induced cell death [60]. These transcripts were also up-regulated (Additional file 1).

Discussion

Several studies have shown that TTA inhibits the growth of various cancer cells in vitro and in vivo[13, 20, 22]. We have previously shown that TTA inhibits DNA synthesis in SW620 human colon cancer cells in a time- and dose-dependent manner [17], and the growth inhibitory effect of TTA on this cell line was confirmed in the current study by xCELLigence proliferation assay. This assay monitors cell growth real time by measuring changes in electric impedance between two golden electrodes embedded in the bottom of the cell culture wells. The impedance, which is converted to a cell index value, is directly proportional to the number of cells and also reflects the cells' viability, morphology and adhesion strength [61]. The baseline cell index declined compared to the control from approximately 20 hours after TTA-supplementation. Previous studies have indicated that TTA affects multiple biochemical pathways that might play a role in its growth-limiting effect on different cancer cells. However, the exact mechanisms are not yet clear.

This is the first study demonstrating that TTA is able to induce ER stress in cancer cells. Transcripts involved in ER stress and downstream of all three ER stress sensors, like ATF6, IRE1, XBP-1 and ATF4, were found to be differentially expressed after treating SW620 cells with TTA. Also, the increased expression of Hsps and DNAJs indicates that the cells try to increase their protein folding capacity. The up-regulation of EDEM1 and EDEM3, SQSTM1, proteasomal subunits and ubiquitin-related transcripts indicates activation of ERAD.

The PERK branch of UPR was found to be activated, supported by the increased protein level of eIF2α-P and ATF4, as well as up-regulation of several downstream targets of ATF4, like ATF3, ASNS, CHOP and TRIB3, at mRNA level. CHOP and TRIB3, also induced at protein level, are known to link ER stress to ER stress-induced cell death (reviewed in [32]). The enhanced level of these proteins and transcripts for CASP4 and calpains indicates that TTA causes prolonged ER stress and direct the cell fate towards induction of death. We have previously shown that DHA induces ER stress with increased eIF2α-P, ATF4 and CHOP in the same cell line [28, 62]. We also found HL-60 leukemia cells to induce ER stress upon EPA treatment [63].

In SW620 cells, TTA up-regulated the C/EBPβ LIP isomer, while LAP was unchanged. LIP is capable of modulating the ER stress response by augmenting cell death, e.g. by enhancing the activity of CHOP [43]. On the contrary, LAP may attenuate ER stress and ER stress induced cell death by inhibiting CHOP [59, 64]. However, Li et al. found that LAP modulated the ER stress response by enhancing the expression of pro-apoptotic genes. They also proposed that the ER stress response may be modulated by regulation of the LIP/LAP ratio [42]. We found this ratio to be increased after 24 and 48 h of TTA treatment, supporting the assumption that TTA causes prolonged ER stress and a switch towards induction of cell death. Even if C/EBPβ mRNA was up-regulated, we only found an increase of LIP at protein level. Different regulation of LIP and LAP might be explained by different synthesis and degradation rates of these isomers [42].

Our results also indicate that TTA affects several transcripts involved in cell cycle progression. Transcripts involved in regulation of both the G1/S and G2/M phases were differentially expressed. In general, positive cell cycle regulators (like CDKs, cyclins, AURKA and survivin) were down-regulated, and negative cell cycle regulators (like ATF5, SFN and KLF4) were up-regulated, which indicates a stop in the cell cycle progression. We have previously demonstrated that DHA treatment of the SW620 cells affects several cell cycle transcripts and proteins like CDKs, cyclins and survivin etc. [62] and arrests the cells in the G2/M phase of the cell cycle [2]. Brewer et. al. stated that cell cycle arrest might be induced during ER stress to prevent cells from completing cell division when the conditions are compromising proper folding and assembly of proteins [65].

TTA also seems to induce changes in expression of transcripts involved in maintenance of Ca2+ homeostasis. The ER lumen contains a high Ca2+ concentration, and induction of ER stress may either come from and/or culminate with a release of Ca2+ from the ER (reviewed in [66, 67]). ER stress-accompanied changes in the Ca2+ metabolism may lead to a pro-apoptotic signal via the mitochondria [66]. We have previously shown that DHA and EPA affect Ca2+ homeostasis in cancer cells [28, 63]. Apoptosis has been observed in other cancer cells treated with TTA [13, 22], and it has been proposed that this effect is mediated via mitochondrial alterations, such as decrease in mitochondrial membrane potential and release of Cyt C [13]. CHOP and C/EBPβ may be involved in the regulation of mitochondrial stress genes like TRIB3 [44] in response to accumulation of unfolded proteins in mitochondria [68]. Their up-regulation by TTA may indicate that the SW620 cells are undergoing a mitochondrial stress after TTA treatment.

Madsen et al. found TTA to be incorporated mainly into phospholipids in liver after TTA supplementation to rats [69]. The inner mitochondrial membrane phospholipid cardiolipin (CL) is essential for the function of several mitochondrial protein complexes. It interacts with mitochondrial Cyt C, which might utilize reactive oxygen species and cause oxidation of CL. Oxidized CL is required for release of pro-apoptotic factors like Cyt C to cytosol [70]. Enrichment of EPA and DHA in CL isolated from colonic mucosa in rats fed fish oil or EPA/DHA ethyl esters might result in high susceptibility to lipid peroxidation due to the FAs' high degree of unsaturation. This may trigger release of pro-apoptotic factors from mitochondria (reviewed in [71]). Decreased synthesis of CL has also been related to Cyt C release from mitochondria during PA-induced apoptosis (reviewed in [9]). It remains to be investigated if this is the case with TTA.

Conclusions

TTA induces extensive changes in the expression of genes involved in several biological pathways. TTA causes accumulation of misfolded proteins in ER, causing ER stress and activation of UPR. The results show that TTA inhibits SW620 cell growth, at least partly, via the same mechanisms as DHA. Thus, cancer cells seem to cope with stress induced by various FAs through the same mechanisms.

Methods

Materials and cell culture

TTA was prepared at the Department of Chemistry, University of Bergen, Norway, as described previously [72] and in Supplementary experimental procedures (Additional file 2). All other solvents were of reagent grade from commercial sources. Antibody specifications are found in Additional file 2. The human colon carcinoma cell line SW620 was obtained from the American Type Culture Collection (ATCC, Rockville, MD) and cultivated in Leibovitz's L-15 medium (Cambrex, BioWhittaker, Walkersville, MD) supplemented with L-glutamine (2 mM), FBS (10%) and gentamicin (45 mg/l) (complete growth medium), in a humidified atmosphere of 5% CO2: 95% air at 37°C.

Cell proliferation assay by xCELLigence

SW620 cells were seeded in 16 well plates (E-plate 16, Roche, Mannheim, Germany) (12.000 cells in 150 μl medium/well), following the xCELLigence Real Time Cell Analyzer (RTCA) DP instrument manual as provided by the manufacturer (Roche). After ~24 h, TTA (75 μM, prepared as described in Additional file 2) or the vehicle NaOH (control medium) was added, and the experiment was allowed to run for 72 h. Average baseline cell index and percent reduction of baseline cell index for TTA-treated cells compared to control cells were calculated for at least two measurements from 4 replicate experiments ± SD. Significance was calculated using one-tailed Student's t-test (P < 0.05).

RNA isolation, gene expression profiling and statistical analysis

SW620 cells were seeded in 175 cm2 flasks (3.5 × 106 cells in 28 ml medium). After 24 h incubation, cells were treated with TTA (75 μM) or NaOH (control) for 24 h. Cell harvesting and total RNA isolation was performed as elaborated in [28], using the High Pure RNA Isolation Kit (Roche). Gene expression profiling was performed following the Eukaryote expression manual from Affymetrix (Santa Clara, CA), using the Human Genome U133 2.0 Plus Array (Affymetrix), as described in Additional file 2. Statistical analysis of gene expression results was performed using the RMA method. Differentially expressed genes were identified using a linear model with a modified T-statistic [73], and results were corrected for multiple testing using the method described in [74] with a false discovery rate of 0.05. Annotations of probe sets and biological information of genes were retrieved from the web-based NetAffx Analysis Center at affymetrix.com and eGOn at genetools.no. Signal values presented are mean values of 3 replicate experiments. All experiments have been submitted to Array-Express with accession number E-MEXP-2590.

Western blot analysis

Cells were treated with TTA (75 μM) or NaOH for 3, 6, 12, 24 and/or 48 h, as described in the gene expression profiling method. For detection of eIF2α-P and cyclin D1, cells were harvested as elaborated in [28], and cytosolic protein extracts were prepared immediately in lysis buffer as described in [63]. Nuclear protein extracts for detection of ATF4, CHOP, TRIB3 and C/EBPβ were obtained by using Nuclear Extract Kit (Active Motif, Rixensart, Belgium) according to the manufacturer's protocol. Protein concentrations were measured using Bio-Rad Protein Assay (Bio-Rad, Hercules, CA), and the protein extracts were snap-freezed in liquid nitrogen and stored at -80°C until further use. Cytosolic and nuclear proteins (80 or 50 μg per well, respectively) were separated on 10% precast denaturing NuPAGE® gels (Invitrogen, Carlsbad, US) and transferred onto Immobilon polyvinylidene difluoride membranes (Millipore, Billerica, MA). Membranes were blocked in phosphate buffered saline buffer with 0.1% (v/v) Tween® 20 (BioRad, Hercules, CA) and 5% blocker non-fat dry milk, before incubated with primary and secondary horse radish peroxidase-conjugated antibodies (DAKO, Carpinteria, CA) dissolved in this blocking buffer as described in Additional file 2. eIF2α-P incubations were performed using tris-buffered saline Tween 20 buffer as described in [63]. Blots were detected by chemiluminescense using SuperSignal® West Femto Maximum Sensitivity Substrate (Pierce, Rockford, IL) and visualized by Kodak Image Station 4000R (Eastman Kodak Co., Rochester, NY). Western blot band intensities were quantified using Kodak Molecular Imaging Software (version 4.0.1). Quantified western blot results, based on 3 independent replicates, were analysed as described in the proliferation assay method.

Notes

List of abbreviations

Transcript short names are to be found in Table 1 and Additional file 1.

Cyt C: 

Cytochrome C

DHA: 

Docosahexaenoic acid

eIF2α: 

Eukaryote translation initiation factor 2 alpha

EIF2AK3/PERK: 

eIF2α kinase 3

eIF2α-P: 

phoshorylated eIF2α

EPA: 

Eicosapentaenoic acid

ER: 

Endoplasmic reticulum

ERAD: 

ER-associated degradation

FAs: 

Fatty acids

Hsps: 

Heat shock proteins

LAP: 

Liver enriched activator protein

LIP: 

Liver enriched inhibitor protein

n-3: 

Omega-3

PA: 

Palmitic acid

PUFAs: 

Polyunsaturated fatty acids

TTA: 

Tetradecylthioacetic acid

UPR: 

Unfolded protein response

Declarations

Acknowledgements

The authors gratefully acknowledge Endre Anderssen, Norwegian Microarray Consortium (NMC), Norwegian University of Science and Technology (NTNU), Trondheim, Norway for microarray statistical analysis. The work was supported by The Faculty of Medicine, NTNU, The Cancer Research Fund, The Research Council of Norway through grants from the Functional Genomics Program (FUGE) and grants from Helse Vest, Norway. Microarray experiments were performed at the microarray core facility at the Norwegian Microarray Consortium (NMC), Trondheim, which is supported by the FUGE, The Norwegian Research Council.

Mailing address for reprints:

Svanhild A. Schonberg, Department of Laboratory Medicine, Children's and Women's Health, Norwegian University of Science and Technology, N-7006, Trondheim, Norway.

Authors’ Affiliations

(1)
Faculty of Medicine, Department of Laboratory Medicine, Children's and Women's Health, Norwegian University of Science and Technology
(2)
Institute of Medicine, Section of Medical Biochemistry, Haukeland University Hospital, University of Bergen

References

  1. Ferlay J, Autier P, Boniol M, Heanue M, Colombet M, Boyle P: Estimates of the cancer incidence and mortality in Europe in 2006. Ann Oncol. 2007, 18 (3): 581-592.View ArticlePubMedGoogle Scholar
  2. Schonberg SA, Lundemo AG, Fladvad T, Holmgren K, Bremseth H, Nilsen A, Gederaas O, Tvedt KE, Egeberg KW, Krokan HE: Closely related colon cancer cell lines display different sensitivity to polyunsaturated fatty acids, accumulate different lipid classes and downregulate sterol regulatory element-binding protein 1. FEBS J. 2006, 273 (12): 2749-2765. 10.1111/j.1742-4658.2006.05292.xView ArticlePubMedGoogle Scholar
  3. Finstad HS, Myhrstad MC, Heimli H, Lomo J, Blomhoff HK, Kolset SO, Drevon CA: Multiplication and death-type of leukemia cell lines exposed to very long-chain polyunsaturated fatty acids. Leukemia. 1998, 12 (6): 921-929. 10.1038/sj.leu.2401030View ArticlePubMedGoogle Scholar
  4. Bathen TF, Holmgren K, Lundemo AG, Hjelstuen MH, Krokan HE, Gribbestad IS, Schonberg SA: Omega-3 fatty acids suppress growth of SW620 human colon cancer xenografts in nude mice. Anticancer Res. 2008, 28 (6A): 3717-3723.PubMedGoogle Scholar
  5. Schonberg SA, Rudra PK, Noding R, Skorpen F, Bjerve KS, Krokan HE: Evidence that changes in Se-glutathione peroxidase levels affect the sensitivity of human tumour cell lines to n-3 fatty acids. Carcinogenesis. 1997, 18 (10): 1897-1904. 10.1093/carcin/18.10.1897View ArticlePubMedGoogle Scholar
  6. Roynette CE, Calder PC, Dupertuis YM, Pichard C: n-3 polyunsaturated fatty acids and colon cancer prevention. Clin Nutr. 2004, 23 (2): 139-151. 10.1016/j.clnu.2003.07.005View ArticlePubMedGoogle Scholar
  7. Cockbain AJ, Toogood GJ, Hull MA: Omega-3 polyunsaturated fatty acids for the treatment and prevention of colorectal cancer. Gut. 2011,Google Scholar
  8. Rose DP, Connolly JM: Omega-3 fatty acids as cancer chemopreventive agents. Pharmacol Ther. 1999, 83 (3): 217-244. 10.1016/S0163-7258(99)00026-1View ArticlePubMedGoogle Scholar
  9. Tronstad KJ, Berge K, Berge RK, Bruserud O: Modified fatty acids and their possible therapeutic targets in malignant diseases. Expert Opin Ther Targets. 2003, 7 (5): 663-677. 10.1517/14728222.7.5.663View ArticlePubMedGoogle Scholar
  10. Berge RK, Skorve J, Tronstad KJ, Berge K, Gudbrandsen OA, Grav H: Metabolic effects of thia fatty acids. Curr Opin Lipidol. 2002, 13 (3): 295-304. 10.1097/00041433-200206000-00010View ArticlePubMedGoogle Scholar
  11. Berge K, Tronstad KJ, Abdi-Dezfuli F, Ranheim T, Mahesparan R, Bjerkvig R, Berge RK: Poorly oxidizable fatty acid analogues inhibit the proliferation of cancer cells in culture. Adv Exp Med Biol. 1999, 466: 205-210.View ArticlePubMedGoogle Scholar
  12. Tronstad KJ, Berge K, Dyroy E, Madsen L, Berge RK: Growth reduction in glioma cells after treatment with tetradecylthioacetic acid: changes in fatty acid metabolism and oxidative status. Biochem Pharmacol. 2001, 61 (6): 639-649. 10.1016/S0006-2952(01)00525-1View ArticlePubMedGoogle Scholar
  13. Tronstad KJ, Gjertsen BT, Krakstad C, Berge K, Brustugun OT, Doskeland SO, Berge RK: Mitochondrial-targeted fatty acid analog induces apoptosis with selective loss of mitochondrial glutathione in promyelocytic leukemia cells. Chem Biol. 2003, 10 (7): 609-618. 10.1016/S1074-5521(03)00142-XView ArticlePubMedGoogle Scholar
  14. Iversen PO, Sorensen DR, Tronstad KJ, Gudbrandsen OA, Rustan AC, Berge RK, Drevon CA: A bioactively modified fatty acid improves survival and impairs metastasis in preclinical models of acute leukemia. Clin Cancer Res. 2006, 12 (11 Pt 1): 3525-3531.View ArticlePubMedGoogle Scholar
  15. Tronstad KJ, Bruserud O, Berge K, Berge RK: Antiproliferative effects of a non-beta-oxidizable fatty acid, tetradecylthioacetic acid, in native human acute myelogenous leukemia blast cultures. Leukemia. 2002, 16 (11): 2292-2301. 10.1038/sj.leu.2402698View ArticlePubMedGoogle Scholar
  16. Erikstein BS, McCormack E, Tronstad KJ, Schwede F, Berge R, Gjertsen BT: Protein kinase A activators and the pan-PPAR agonist tetradecylthioacetic acid elicit synergistic anti-leukaemic effects in AML through CREB. Leuk Res. 2010, 34 (1): 77-84. 10.1016/j.leukres.2009.09.005View ArticlePubMedGoogle Scholar
  17. Jensen LR, Berge K, Bathen TF, Wergedahl H, Schonberg SA, Bofin A, Berge RK, Gribbestad IS: Effect of dietary tetradecylthioacetic acid on colon cancer growth studied by dynamic contrast enhanced MRI. Cancer Biol Ther. 2007, 6 (11): 1810-1816. 10.4161/cbt.6.11.4887View ArticlePubMedGoogle Scholar
  18. Sorensen HN, Norrheim L, Spydevold O, Gautvik KM: Uptake and receptor binding of dexamethasone in cultured 7800 C1 hepatoma cells in relation to regulation of cell growth and peroxisomal beta-oxidation. Int J Biochem. 1990, 22 (10): 1171-1177. 10.1016/0020-711X(90)90117-LView ArticlePubMedGoogle Scholar
  19. Abdi-Dezfuli F, Berge RK, Rasmussen M, Thorsen T, Aakvaag A: Effects of saturated and polyunsaturated fatty acids and their 3-thia fatty acid analogues on MCF-7 breast cancer cell growth. Ann N Y Acad Sci. 1994, 744: 306-309. 10.1111/j.1749-6632.1994.tb52748.xView ArticlePubMedGoogle Scholar
  20. Abdi-Dezfuli F, Froyland L, Thorsen T, Aakvaag A, Berge RK: Eicosapentaenoic acid and sulphur substituted fatty acid analogues inhibit the proliferation of human breast cancer cells in culture. Breast Cancer Res Treat. 1997, 45 (3): 229-239. 10.1023/A:1005818917479View ArticlePubMedGoogle Scholar
  21. Pettersen RJ, Salem M, Skorve J, Ulvik RJ, Berge RK, Nordrehaug JE: Pharmacology and safety of tetradecylthioacetic acid (TTA): phase-1 study. J Cardiovasc Pharmacol. 2008, 51 (4): 410-417. 10.1097/FJC.0b013e3181673be0View ArticlePubMedGoogle Scholar
  22. Berge K, Tronstad KJ, Flindt EN, Rasmussen TH, Madsen L, Kristiansen K, Berge RK: Tetradecylthioacetic acid inhibits growth of rat glioma cells ex vivo and in vivo via PPAR-dependent and PPAR-independent pathways. Carcinogenesis. 2001, 22 (11): 1747-1755. 10.1093/carcin/22.11.1747View ArticlePubMedGoogle Scholar
  23. Skrede S, Sorensen HN, Larsen LN, Steineger HH, Hovik K, Spydevold OS, Horn R, Bremer J: Thia fatty acids, metabolism and metabolic effects. Biochim Biophys Acta. 1997, 1344 (2): 115-131.View ArticlePubMedGoogle Scholar
  24. Berge K, Tronstad KJ, Bohov P, Madsen L, Berge RK: Impact of mitochondrial beta-oxidation in fatty acid-mediated inhibition of glioma cell proliferation. J Lipid Res. 2003, 44 (1): 118-127. 10.1194/jlr.M200312-JLR200View ArticlePubMedGoogle Scholar
  25. Noding R, Schonberg SA, Krokan HE, Bjerve KS: Effects of polyunsaturated fatty acids and their n-6 hydroperoxides on growth of five malignant cell lines and the significance of culture media. Lipids. 1998, 33 (3): 285-293. 10.1007/s11745-998-0207-9View ArticlePubMedGoogle Scholar
  26. Calviello G, Serini S, Piccioni E, Pessina G: Antineoplastic effects of n-3 polyunsaturated fatty acids in combination with drugs and radiotherapy: preventive and therapeutic strategies. Nutr Cancer. 2009, 61 (3): 287-301. 10.1080/01635580802582777View ArticlePubMedGoogle Scholar
  27. Edwards IJ, O'Flaherty JT: Omega-3 Fatty Acids and PPARγ in Cancer. PPAR Research. 2008, 2008: 1-14.View ArticleGoogle Scholar
  28. Jakobsen CH, Storvold GL, Bremseth H, Follestad T, Sand K, Mack M, Olsen KS, Lundemo AG, Iversen JG, Krokan HE: DHA induces ER stress and growth arrest in human colon cancer cells: associations with cholesterol and calcium homeostasis. J Lipid Res. 2008, 49 (10): 2089-2100. 10.1194/jlr.M700389-JLR200PubMed CentralView ArticlePubMedGoogle Scholar
  29. Gu X, Li K, Laybutt DR, He ML, Zhao HL, Chan JC, Xu G: Bip overexpression, but not CHOP inhibition, attenuates fatty-acid-induced endoplasmic reticulum stress and apoptosis in HepG2 liver cells. Life Sci. 2010, 87 (23-26): 724-732. 10.1016/j.lfs.2010.10.012View ArticlePubMedGoogle Scholar
  30. Wei Y, Wang D, Pagliassotti MJ: Saturated fatty acid-mediated endoplasmic reticulum stress and apoptosis are augmented by trans-10, cis-12-conjugated linoleic acid in liver cells. Mol Cell Biochem. 2007, 303 (1-2): 105-113. 10.1007/s11010-007-9461-2View ArticlePubMedGoogle Scholar
  31. Schroder M, Kaufman RJ: ER stress and the unfolded protein response. Mutat Res. 2005, 569 (1-2): 29-63. 10.1016/j.mrfmmm.2004.06.056View ArticlePubMedGoogle Scholar
  32. Szegezdi E, Logue SE, Gorman AM, Samali A: Mediators of endoplasmic reticulum stress-induced apoptosis. EMBO Rep. 2006, 7 (9): 880-885. 10.1038/sj.embor.7400779PubMed CentralView ArticlePubMedGoogle Scholar
  33. Oyadomari S, Mori M: Roles of CHOP/GADD153 in endoplasmic reticulum stress. Cell Death Differ. 2004, 11 (4): 381-389. 10.1038/sj.cdd.4401373View ArticlePubMedGoogle Scholar
  34. McCullough KD, Martindale JL, Klotz LO, Aw TY, Holbrook NJ: Gadd153 sensitizes cells to endoplasmic reticulum stress by down-regulating Bcl2 and perturbing the cellular redox state. Mol Cell Biol. 2001, 21 (4): 1249-1259. 10.1128/MCB.21.4.1249-1259.2001PubMed CentralView ArticlePubMedGoogle Scholar
  35. Puthalakath H, O'Reilly LA, Gunn P, Lee L, Kelly PN, Huntington ND, Hughes PD, Michalak EM, McKimm-Breschkin J, Motoyama N: ER stress triggers apoptosis by activating BH3-only protein Bim. Cell. 2007, 129 (7): 1337-1349. 10.1016/j.cell.2007.04.027View ArticlePubMedGoogle Scholar
  36. Chen C, Dudenhausen EE, Pan YX, Zhong C, Kilberg MS: Human CCAAT/enhancer-binding protein beta gene expression is activated by endoplasmic reticulum stress through an unfolded protein response element downstream of the protein coding sequence. J Biol Chem. 2004, 279 (27): 27948-27956. 10.1074/jbc.M313920200View ArticlePubMedGoogle Scholar
  37. Piwien Pilipuk G, Galigniana MD, Schwartz J: Subnuclear localization of C/EBP beta is regulated by growth hormone and dependent on MAPK. J Biol Chem. 2003, 278 (37): 35668-35677. 10.1074/jbc.M305182200View ArticlePubMedGoogle Scholar
  38. Smink JJ, Leutz A: Rapamycin and the transcription factor C/EBPbeta as a switch in osteoclast differentiation: implications for lytic bone diseases. J Mol Med. 2010, 88 (3): 227-233. 10.1007/s00109-009-0567-8PubMed CentralView ArticlePubMedGoogle Scholar
  39. Baldwin BR, Timchenko NA, Zahnow CA: Epidermal growth factor receptor stimulation activates the RNA binding protein CUG-BP1 and increases expression of C/EBPbeta-LIP in mammary epithelial cells. Mol Cell Biol. 2004, 24 (9): 3682-3691. 10.1128/MCB.24.9.3682-3691.2004PubMed CentralView ArticlePubMedGoogle Scholar
  40. Descombes P, Schibler U: A liver-enriched transcriptional activator protein, LAP, and a transcriptional inhibitory protein, LIP, are translated from the same mRNA. Cell. 1991, 67 (3): 569-579. 10.1016/0092-8674(91)90531-3View ArticlePubMedGoogle Scholar
  41. Poli V, Mancini FP, Cortese R: IL-6DBP, a nuclear protein involved in interleukin-6 signal transduction, defines a new family of leucine zipper proteins related to C/EBP. Cell. 1990, 63 (3): 643-653. 10.1016/0092-8674(90)90459-RView ArticlePubMedGoogle Scholar
  42. Li Y, Bevilacqua E, Chiribau CB, Majumder M, Wang C, Croniger CM, Snider MD, Johnson PF, Hatzoglou M: Differential control of the CCAAT/enhancer-binding protein beta (C/EBPbeta) products liver-enriched transcriptional activating protein (LAP) and liver-enriched transcriptional inhibitory protein (LIP) and the regulation of gene expression during the response to endoplasmic reticulum stress. J Biol Chem. 2008, 283 (33): 22443-22456. 10.1074/jbc.M801046200PubMed CentralView ArticlePubMedGoogle Scholar
  43. Chiribau C-B, Gaccioli F, Huang CC, Yuan CL, Hatzoglou M: Molecular symbiosis of CHOP and C/EBP beta isoform LIP contributes to endoplasmic reticulum stress-induced apoptosis. Mol Cell Biol. 2010, 30 (14): 3722-3731. 10.1128/MCB.01507-09PubMed CentralView ArticlePubMedGoogle Scholar
  44. Ishikawa F, Akimoto T, Yamamoto H, Araki Y, Yoshie T, Mori K, Hayashi H, Nose K, Shibanuma M: Gene expression profiling identifies a role for CHOP during inhibition of the mitochondrial respiratory chain. J Biochem. 2009, 146 (1): 123-132. 10.1093/jb/mvp052View ArticlePubMedGoogle Scholar
  45. Ohoka N, Yoshii S, Hattori T, Onozaki K, Hayashi H: TRB3, a novel ER stress-inducible gene, is induced via ATF4-CHOP pathway and is involved in cell death. EMBO J. 2005, 24 (6): 1243-1255. 10.1038/sj.emboj.7600596PubMed CentralView ArticlePubMedGoogle Scholar
  46. Du K, Herzig S, Kulkarni RN, Montminy M: TRB3: a tribbles homolog that inhibits Akt/PKB activation by insulin in liver. Science. 2003, 300 (5625): 1574-1577. 10.1126/science.1079817View ArticlePubMedGoogle Scholar
  47. Wu M, Xu LG, Zhai Z, Shu HB: SINK is a p65-interacting negative regulator of NF-kappaB-dependent transcription. J Biol Chem. 2003, 278 (29): 27072-27079. 10.1074/jbc.M209814200View ArticlePubMedGoogle Scholar
  48. Hitomi J, Katayama T, Eguchi Y, Kudo T, Taniguchi M, Koyama Y, Manabe T, Yamagishi S, Bando Y, Imaizumi K: Involvement of caspase-4 in endoplasmic reticulum stress-induced apoptosis and Abeta-induced cell death. J Cell Biol. 2004, 165 (3): 347-356. 10.1083/jcb.200310015PubMed CentralView ArticlePubMedGoogle Scholar
  49. Matsuzaki S, Hiratsuka T, Kuwahara R, Katayama T, Tohyama M: Caspase-4 is partially cleaved by calpain via the impairment of Ca2+ homeostasis under the ER stress. Neurochem Int. 2010, 56 (2): 352-356. 10.1016/j.neuint.2009.11.007View ArticlePubMedGoogle Scholar
  50. Ron D, Walter P: Signal integration in the endoplasmic reticulum unfolded protein response. Nat Rev Mol Cell Biol. 2007, 8 (7): 519-529. 10.1038/nrm2199View ArticlePubMedGoogle Scholar
  51. Schroder M: Endoplasmic reticulum stress responses. Cell Mol Life Sci. 2008, 65 (6): 862-894. 10.1007/s00018-007-7383-5View ArticlePubMedGoogle Scholar
  52. He CH, Gong P, Hu B, Stewart D, Choi ME, Choi AM, Alam J: Identification of activating transcription factor 4 (ATF4) as an Nrf2-interacting protein. Implication for heme oxygenase-1 gene regulation. J Biol Chem. 2001, 276 (24): 20858-20865. 10.1074/jbc.M101198200View ArticlePubMedGoogle Scholar
  53. Pagani M, Fabbri M, Benedetti C, Fassio A, Pilati S, Bulleid NJ, Cabibbo A, Sitia R: Endoplasmic reticulum oxidoreductin 1-lbeta (ERO1-Lbeta), a human gene induced in the course of the unfolded protein response. J Biol Chem. 2000, 275 (31): 23685-23692. 10.1074/jbc.M003061200View ArticlePubMedGoogle Scholar
  54. Seibenhener ML, Geetha T, Wooten MW: Sequestosome 1/p62--more than just a scaffold. FEBS Lett. 2007, 581 (2): 175-179. 10.1016/j.febslet.2006.12.027PubMed CentralView ArticlePubMedGoogle Scholar
  55. Noh DY, Shin SH, Rhee SG: Phosphoinositide-specific phospholipase C and mitogenic signaling. Biochim Biophys Acta. 1995, 1242 (2): 99-113.PubMedGoogle Scholar
  56. Ito D, Walker JR, Thompson CS, Moroz I, Lin W, Veselits ML, Hakim AM, Fienberg AA, Thinakaran G: Characterization of stanniocalcin 2, a novel target of the mammalian unfolded protein response with cytoprotective properties. Mol Cell Biol. 2004, 24 (21): 9456-9469. 10.1128/MCB.24.21.9456-9469.2004PubMed CentralView ArticlePubMedGoogle Scholar
  57. Chigurupati S, Wei Z, Belal C, Vandermey M, Kyriazis GA, Arumugam TV, Chan SL: The homocysteine-inducible endoplasmic reticulum stress protein counteracts calcium store depletion and induction of CCAAT enhancer-binding protein homologous protein in a neurotoxin model of Parkinson disease. J Biol Chem. 2009, 284 (27): 18323-18333. 10.1074/jbc.M109.020891PubMed CentralView ArticlePubMedGoogle Scholar
  58. Jansen AP, Camalier CE, Colburn NH: Epidermal expression of the translation inhibitor programmed cell death 4 suppresses tumorigenesis. Cancer Res. 2005, 65 (14): 6034-6041. 10.1158/0008-5472.CAN-04-2119View ArticlePubMedGoogle Scholar
  59. Meir O, Dvash E, Werman A, Rubinstein M: C/EBP-beta regulates endoplasmic reticulum stress-triggered cell death in mouse and human models. PLoS One. 2010, 5 (3): e9516- 10.1371/journal.pone.0009516PubMed CentralView ArticlePubMedGoogle Scholar
  60. Kondo S, Saito A, Hino S, Murakami T, Ogata M, Kanemoto S, Nara S, Yamashita A, Yoshinaga K, Hara H: BBF2H7, a novel transmembrane bZIP transcription factor, is a new type of endoplasmic reticulum stress transducer. Mol Cell Biol. 2007, 27 (5): 1716-1729. 10.1128/MCB.01552-06PubMed CentralView ArticlePubMedGoogle Scholar
  61. Abassi YA, Xi B, Zhang W, Ye P, Kirstein SL, Gaylord MR, Feinstein SC, Wang X, Xu X: Kinetic cell-based morphological screening: prediction of mechanism of compound action and off-target effects. Chem Biol. 2009, 16 (7): 712-723. 10.1016/j.chembiol.2009.05.011PubMed CentralView ArticlePubMedGoogle Scholar
  62. Slagsvold JE, Pettersen CHH, Storvold GL, Follestad T, Krokan HE, Schonberg SA: DHA alters expression of target proteins of cancer therapy in chemoresistant SW620 colon cancer cells. Nutr Cancer. 2010, 62 (5): 611-621. 10.1080/01635580903532366View ArticlePubMedGoogle Scholar
  63. Slagsvold JE, Pettersen CH, Follestad T, Krokan HE, Schonberg SA: The antiproliferative effect of EPA in HL60 cells is mediated by alterations in calcium homeostasis. Lipids. 2009, 44 (2): 103-113. 10.1007/s11745-008-3263-5View ArticlePubMedGoogle Scholar
  64. Abreu MM, Sealy L: The C/EBPbeta isoform, liver-inhibitory protein (LIP), induces autophagy in breast cancer cell lines. Exp Cell Res. 2010, 316 (19): 3227-3238. 10.1016/j.yexcr.2010.07.021PubMed CentralView ArticlePubMedGoogle Scholar
  65. Brewer JW, Hendershot LM, Sherr CJ, Diehl JA: Mammalian unfolded protein response inhibits cyclin D1 translation and cell-cycle progression. Proc Natl Acad Sci USA. 1999, 96 (15): 8505-8510. 10.1073/pnas.96.15.8505PubMed CentralView ArticlePubMedGoogle Scholar
  66. Xu C, Bailly-Maitre B, Reed JC: Endoplasmic reticulum stress: cell life and death decisions. J Clin Invest. 2005, 115 (10): 2656-2664. 10.1172/JCI26373PubMed CentralView ArticlePubMedGoogle Scholar
  67. Verkhratsky A, Toescu EC: Endoplasmic reticulum Ca(2+) homeostasis and neuronal death. J Cell Mol Med. 2003, 7 (4): 351-361. 10.1111/j.1582-4934.2003.tb00238.xView ArticlePubMedGoogle Scholar
  68. Zhao Q, Wang J, Levichkin IV, Stasinopoulos S, Ryan MT, Hoogenraad NJ: A mitochondrial specific stress response in mammalian cells. EMBO J. 2002, 21 (17): 4411-4419. 10.1093/emboj/cdf445PubMed CentralView ArticlePubMedGoogle Scholar
  69. Madsen L, Froyland L, Grav HJ, Berge RK: Up-regulated delta 9-desaturase gene expression by hypolipidemic peroxisome-proliferating fatty acids results in increased oleic acid content in liver and VLDL: accumulation of a delta 9-desaturated metabolite of tetradecylthioacetic acid. J Lipid Res. 1997, 38 (3): 554-563.PubMedGoogle Scholar
  70. Bayir H, Fadeel B, Palladino MJ, Witasp E, Kurnikov IV, Tyurina YY, Tyurin VA, Amoscato AA, Jiang J, Kochanek PM: Apoptotic interactions of cytochrome c: redox flirting with anionic phospholipids within and outside of mitochondria. Biochim Biophys Acta. 2006, 1757 (5-6): 648-659. 10.1016/j.bbabio.2006.03.002View ArticlePubMedGoogle Scholar
  71. Chapkin RS, Seo J, McMurray DN, Lupton JR: Mechanisms by which docosahexaenoic acid and related fatty acids reduce colon cancer risk and inflammatory disorders of the intestine. Chem Phys Lipids. 2008, 153 (1): 14-23. 10.1016/j.chemphyslip.2008.02.011PubMed CentralView ArticlePubMedGoogle Scholar
  72. Spydevold O, Bremer J: Induction of peroxisomal beta-oxidation in 7800 C1 Morris hepatoma cells in steady state by fatty acids and fatty acid analogues. Biochim Biophys Acta. 1989, 1003 (1): 72-79.View ArticlePubMedGoogle Scholar
  73. Smyth GK: Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol. 2004, 3: Article 3-Google Scholar
  74. Benjamini Y, Hochberg Y: Controlling the false discovery rate: a practical and powerful approach to muliple testing. J R Stat Soc. 1995, 57 (1): 289-300. http://www.ncbi.nlm.nih.gov/pubmed/16740248Google Scholar

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© Lundemo et al; licensee BioMed Central Ltd. 2011

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