Periostin induces chemoresistance in colon cancer cells through activation of the PI3K/Akt/survivin pathway
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Zhi-ming Xiao1, Xiao-yan Wang1, Ai-min Wang2,*
1
Department of Gastroenterology, the third hospital of Xiangya, Central South University,
Changsha 410013, China 2
Department of Emergency, Xiangya Hospital, Central South University, Changsha 410008,
China
*
Corresponding author.
Department of Emergency, Xiangya Hospital, Central South University, Changsha 410008, China Tel: +8673188618211 Fax: +8673188618211 E-mail: [email protected]
Running title: Periostin in colon cancer
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as DOI:10.1002/bab.1193.
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Abstract
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In the present study, we aimed to explore the effects of periostin, a cell adhesion protein, on chemoresistance in colon cancer cells. Reverse transcription-polymerase chain reaction and western blot analyses were employed to detect periostin expression in SW480 and HT-29 colon cancer cells treated with oxaliplatin or fluorouracil (5-FU). Small interfering RNA was used to downregulate endogenous periostin. Annexin-V/propidium iodide (PI) staining was performed to analyze the effects of periostin on drug-induced apoptosis. The results showed that treatment with oxaliplatin or 5-FU elevated both the mRNA and protein levels of periostin in SW480 and HT-29 cells. Silencing of periostin significantly (P < 0.01) augmented drug-induced apoptosis in colon cancer cells, coupled with enhanced cleavage of caspase-3 and poly(ADP-ribose) polymerase. Mechanistic studies revealed that periostin silencing significantly (P < 0.01) suppressed the expression of survivin, an antiapoptotic protein in colon cancer cells. Enforced expression of survivin repressed drug-induced apoptosis
in periostin-depleted SW480 and
HT-29 cells.
Additionally,
periostin
overexpression increased the expression of survivin and the phosphorylation of Akt, which was reversed by pretreatment with the phosphatidylinositol 3-kinase (PI3K) specific inhibitor LY294002. Taken together, our data demonstrate that periostin induces chemoresistance in colon cancer cells through activation of the PI3K/Akt/survivin pathway. Key words: apoptosis; chemotherapy; drug resistance; periostin; signaling pathway.
Abbreviations
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2
EMT
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Epithelial-mesenchymal transition FITC Fluorescein isothiocyanate 5-FU Fluorouracil GAPDH Glyceraldehyde 3-phosphate dehydrogenase HNSCC Head and neck squamous cell carcinoma PARP Poly(ADP-ribose) polymerase PI
Propidium iodide PI3K Phosphatidylinositol 3-kinase RT-PCR Reverse transcription-polymerase chain reaction
1. Introduction
Colon cancer is one of the leading causes of cancer-related deaths worldwide (1). Surgery is
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the primary treatment approach for early-stage colon cancer; but the risk of recurrence is still high due to undetected micrometastases (2). Chemotherapy, usually oxaliplatin and
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fluorouracil (5-FU), has been proposed to reduce the risk of postoperative recurrence in colon cancer. Intrinsic or acquired chemoresistance remains a major obstacle to successful cancer treatment. The survival benefits of adjuvant chemotherapy are only achieved in a minor population of patients with colon cancer (3). Therefore, identification of the molecular mechanisms involved in chemoresistance is of great significance in developing novel therapeutic modalities for this malignancy.
Periostin, a 93.3-kDa secreted protein, is also named osteoblast-specific factor 2 (4). It is upregulated in various types of human cancers, including head and neck, pancreatic, breast, ovarian, prostate, papillary thyroid, lung, liver and colon carcinoma (5). Hypoxia induces the upregulation of periostin in non-small-cell lung cancer cells, which promotes cancer cell survival through the activation of Akt/PKB pathway (6). Similarly, Baril et al (7) reported that periostin enhances the survival of pancreatic cancer cells under hypoxic conditions. Nicotine-mediated induction of periostin promotes the proliferation, invasion and epithelial-mesenchymal transition (EMT) of gastric cancer cells (8). Conversely, monoclonal antibody neutralization of periostin inhibits anchorage-independent growth and survival of ovarian cancer cells (9). These findings suggest that overexpression of periostin is extensively associated with tumor growth, progression, and chemoresistance.
In colon cancer, periostin has been shown to promote metastatic growth through augmenting
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cell survival via the Akt/PKB pathway (10). In the present study, we sought to explore the effects of periostin on the chemosensitivity of colon cancer cells and associated molecular
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mechanisms.
2. Materials and Methods
2.1. Cell culture SW480 and HT-29 cells were treated with oxaliplatin or 5-FU alone for 24 and 48 h. If not stated otherwise, the half-maximal inhibitory concentration (IC50) of the chemotherapeutic
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agents were used (11): i.e., 7 μM oxaliplatin and 27 μM 5-FU for SW480 cells and 9 μM oxaliplatin and 20 μM 5-FU for HT-29 cells. To investigate the involvement of PI3K/Akt
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pathway in periostin-mediated chemoresistance, 20 μM PI3K inhibitor (LY294002) was used to pretreat SW480 and HT-29 cells for 1 h.
2.2. Plasmids and siRNAs TRIzol reagent (Invitrogen) was used to isolate total RNA from human colon cancer cell line SW480 cells according to the manufacture’s protocol. Human periostin (GenBank NM_006475) full-length cDNA (2511 bp) and human survivin (GenBank NM_001168) full-length cDNA (429 bp) were synthesized by reverse transcription of the extracted total RNA and amplified by polymerase chain reaction (PCR). The PCR primers for periostin were as
follows:
forward,
5′-GCCGAAGCTTACCATGATTCCC-3′;
reverse,
5′-GCGCCTCGAGTCACTGAGAACG-3′. The PCR primers for survivin were as below: forward,
5′-GCCGAAGCTTACCATGGGTGCC-3′;
reverse,
5′-GCGCCTCGAGTCAATCCATGGC-3′. After digestion with Hind III and Xho I, the resulting restriction fragments were cloned into the Hind III/Xho I sites of the pcDNA3.1 (+) expression vector (invitrogen). The constructed plasmids (pcDNA3.1-periostin and pcDNA3.1-survivin) were verified by DNA sequencing.
A pool of three siRNA duplexes targeted to different regions of human periostin mRNA (sc-61324) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Non-targeting siRNA duplexes (Santa Cruz Biotechnology) were used as control.
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2.3. Cell transfection
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For overexpression of periostin, SW480 and HT-29 cells pretreated with LY294002 at a 70-80% confluence were transfected with vector or pcDNA3.1-periostin by Lipofectamine 2000 and incubated for 24 h. The cells were then collected and subjected to Western blot analysis.
For silencing of periostin with siRNAs, SW480 and HT-29 cells at a 70-80% confluence were transfected with control versus periostin siRNA by lipofectamine 2000 and incubated for 24 h. The cells were then exposed to oxaliplatin or 5-FU for additional 48 h and subjected to apoptosis detection and gene expression analysis.
For restoration of survivin, SW480 and HT-29 cells at a 70-80% confluence were transfected with control versus periostin siRNA with or without survivin by Lipofectamine 2000 and incubated for 24 h. The cells were then exposed to oxaliplatin or 5-FU for additional 48 h and subjected to apoptosis detection and gene expression analysis.
2.4. Assessment of apoptosis by annexin-V staining Cell apoptosis was assessed using an Annexin-V-FITC Apoptosis Detection Kit (BioVision, Mountain View, CA, USA) according to the manufacturer’s recommendations. Briefly, cells were detached, resuspended in binding buffer, and added with fluorescein isothiocyanate (FITC)-conjugated annexin-V and propidium iodide (PI). After incubation for 10 min at room
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temperature in the dark, the cells were analyzed for annexin-V-FITC binding by a flow cytometer (Becton Dickinson, San Jose, CA, USA). Early apoptosis was determined by
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measurement of annexin-V-positive and PI-negative cells. Each experiment was repeated three times.
2.5. Reverse transcription-PCR Total RNA was isolated from cells with indicated treatments using TRIzol reagent (Invitrogen, Carlsband, CA) according to the manufacturer’s protocol. RT-PCR was performed to analyze periostin mRNA expression. 5 μg of total RNA was used to synthesize cDNA. The periostin forward primer was 5'-CTTCAATGTATAACAATTTGG-3' and the periostin reverse primer was 5’-CGATGTGATGGCAGCTGCTC-3'. The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward primer was 5'-CTGCACCACCAACTGCTTAG-3' and the GAPDH reverse primer was 5'-TGAAGTCAGAGGAGACCACC-3'. The amplicons for periostin and GAPDH were 215 and 407 bp, respectively. PCR products were electrophorized on 1.5% agarose gel and visualized by ethidium-bromide staining.
2.6. Western blot analysis Cells with indicated treatments were lysed with ice-cold lysis buffer (pH7.4) containing 25 mM HEPES, 1% Triton X-100, 50 mM NaCl, 1mM EDTA, 1mM EGTA, 1mM PMSF and 1 μg/ml leupeptin. Protein quantification was performed by BCA assay. Protein extracts (50 μg/lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto PVDF membrane. Membranes were blocked in 5% (w/v) non-fat dried milk in
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Tris-buffered saline for 1 h at room temperature. Then membranes were incubated with appropriate primary antibodies followed by incubation with HRP-conjugated secondary
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antibodies. Immunoreactive proteins were visualized using an enhanced chemiluminescent detection
system
immunoreactive
(Amersham bands
were
Biosciences, measured
Piscataway, by
NJ).
The
computerized
intensities
image
of
analysis
(QuantityOne-software; Bio-Rad, Hercules, CA) and normalized to β-actin levels. Antibodies used in the present studies were as follows: anti-periostin, survivin, pAkt, total Akt and β-actin primary antibodies and HRP-conjugated goat-antirabbit IgG, and goat-anti-mouse IgG and donkey-anti-goat IgG were purchased from Santa Cruz Biotechnology. Anti-cleaved caspase-3 and cleaved PARP antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). 2.7. Statistical analysis Data are expressed as mean ± standard deviation (SD). Difference between group means were evaluated by student’s t-test or one-way analysis of variance (ANOVA) followed by the Tukey post-hoc test. P-value less than 0.05 were considered as significant.
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3. Results
3.1. Chemotherapeutic drugs upregulates periostin in colon cancer cells Western blot analysis revealed that oxaliplatin treatment for 24 or 48 h significantly raised periostin expression at protein levels in both SW480 and HT-29 cells (Fig. 1A). Likewise, there was a marked elevation in periostin expression at protein levels in SW480 and HT-29 cells exposed to 5-FU for 24 or 48 h (Fig. 1B). RT-PCR analysis confirmed the upregulation of periostin by chemotherapeutic drugs at the mRNA levels (Fig. 1C and D).
3.2. Inhibition of periostin sensitizes colon cancer cells to drug-induced apoptosis Having identified the elevation of periostin upon the exposure to chemotherapeutic agents, we next explored the effects of periostin inhibition on colon cancer cell chemosensitivity. As
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shown in Fig. 2A and B, the pretreatment of SW480 and HT-29 cells with periostin specific siRNAs resulted in a marked reduction in the periostin mRNA abundance in comparison with
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control siRNA-transfected counterparts, in the presence of oxaliplatin or 5-FU. Upon the treatment with the chemotherapeutic agents, periostin siRNA-transfected SW480 cells showed a significant increase in the percentage of apoptotic cells when compared to control siRNA-transfected SW480 cells (Fig. 2C). Similar findings were observed in HT-29 cells (Fig. 2D). Additionally, the delivery of periostin specific siRNAs resulted in a significant enhancement in the cleavage of caspase-3 and PARP compared to the transfection with control siRNAs (Fig. 3). Taken together, these results demonstrate that depletion of periostin increases the susceptibility to chemotherapeutic agents in colon cancer cells.
3.3.
Downregulation
of
survivin
is
critical
for periostin
inhibition-induced
chemosensitization Previous studies have shown that upregulation of survivin confers to cancer cells resistance against drug-induced cell death (12). Next, we examined whether such chemosensitization induced by periostin inhibition was mediated by modulation of survivin expression. As shown in Fig. 4A and B, siRNA-mediated depletion of periostin profoundly diminished the expression of survivin at protein levels in SW480 and HT-29 cells treated with oxaliplatin or 5-FU, compared to control siRNA-transfected counterparts. Most importantly, transfection with wild-type survivin gene significantly repressed chemotherapeutic drug-induced apoptosis in periostin-depleted SW480 and HT-29 cells by about 50% (P < 0.05; Fig. 4C and D).
Western
blot
analysis
confirmed
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the
overexpression
of
survivin
in
11
pcDNA3.1-survivin-transfected SW480 and HT-29 cells (Fig. 4C and D).
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3.4. Activation of the PI3K/Akt pathway contributes to the induction of survivin by periostin It has been documented that the PI3K/Akt pathway is implicated in the regulation of survivin expression (13). Finally, we checked the role of the PI3K/Akt pathway in the modulation of survivin by periostin. Western blot analysis showed that overexpression of periostin markedly increased the protein level of survivin and the phosphorylation of Akt at site 473 without altering the expression of total Akt in SW480 cells (Fig. 5). Notably, the phosphorylation of Akt and induction of survivin by periostin was significantly inhibited by the pretreatment with the PI3K specific inhibitor LY294002 (Fig. 5), suggesting the involvement of the PI3K/Akt pathway in periostin-induced survivin elevation.
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4. Discussion
To determine the role of periostin in the chemoresistance of colon cancer, in this study we have chosen two widely used conon cancer cell lines SW480 and HT-29 that have shown resistance to chemotherapeutic agents (14,15). Our data showed that exposure to chemotherapeutic drugs enhanced the expression of periostin at both mRNA and protein levels in human colon cancer cells. Targeting periostin rendered cancer cells more susceptible to drug-induced apoptosis, which was associated with enhanced cleavage of caspase-3 and PARP. Periostin silencing inhibited the expression of survivin in colon cancer cells. Restoration of survivin significantly reversed the induction of apoptosis by periostin depletion. Additionally, ectopic expression of periostin elevated the expression of survivin in colon cancer cells via activation of the PI3K/Akt pathway. Our results collectively indicate that acquired expression of periostin confers chemoresistance in colon cancer cells, which is largely associated with activation of the PI3K/Akt/survivin pathway.
Periostin is frequently upregulated in various types of human cancers and implicated in
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cancer cell proliferation, invasion, and metastasis (6-8). Kudo et al (16) showed that periostin is elevated in head and neck squamous cell carcinoma (HNSCC) and promotes invasion and
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anchorage-independent growth of HNSCC. Overexpression of periostin has been found to facilitate tumor progression and angiogenesis in breast cancer (17). Several lines of evidence suggest periostin upregulation as a result of stress (18,19). Tanabe et al (18) reported that periostin inhibits cell death through up-regulation of Bcl-xL expression by maintaining the Notch1 protein level under the stress condition. Hypoxic induction of periostin has been found to promote cell survival in non-small-cell lung cancer (6). Nicotine-induced upregulation of periostin in gastric cancer cells confers nicotine-mediated gastric cancer cell growth, invasion, drug resistance and EMT (8). These results suggest that upregulation of periostin represents a survival strategy for cells under stress conditions. In line with this view, our data revealed that there is an upregulation of periostin expression in colon cancer cells exposed to chemotherapeutic drugs and targeted reduction of periostin sensitizes colon cancer cells to chemotherapeutic agents. These results suggest a cytoprotective role for periostin against chemotherapy in colon cancer cells. Therefore, targeting periostin may be an attractive strategy to improve the efficacy of chemotherapy in colon cancer. Indeed, pre-clinical studies revealed that blocking periostin activity using a monoclonal antibody significantly suppresses tumor growth and metastasis in a mouse model of ovarian cancer (9). Lee et al (20) also reported that interfering with periostin action with periostin-binding DNA aptamer significantly reduces primary tumor growth and distant metastasis in an orthotopic mouse model of breast cancer.
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Survivin, an antiapoptotic protein, is initially identified as an inhibitor of caspase-9 and functions as a key modulator of programmed cell death (21). Growing evidence indicates that
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survivin is highly expressed in various types of human cancers and contributes to chemoresistance
(22-24).
Survivin
participates
in
fibronectin/PI3K/Akt2
signal
pathway-mediated docetaxel-resistance in breast and ovarian cancer (24). Inhibition of survivin expression is causally linked to the growth suppression and apoptosis induction by anticancer agents in colon cancer cells (25,26). Thus, we examined whether survivin is involved in periostin-mediated chemoresistance in colon cancer. Western blot analysis showed that the expression of survivin was significantly suppressed in periostin siRNA-transfected SW480 and HT-29 cells, compared to mock transfectants. Moreover, enforced expression of survivin significantly inhibited chemotherapeutic agent-induced apoptosis in periostin-depleted colon cancer cells. These results suggest that survivin is involved in periostin-mediated chemoresistance in colon cancer cells.
The PI3K/Akt pathway is implicated in cancer cell survival and outgrowth (27). Accumulating evidence suggests survivin as a downstream effector of PI3K/Akt signaling (28,29). Activation of the PI3K/Akt/survivin pathway usually results in resistance to apoptosis caused by various apoptotic stimuli (30), thus representing a promising target for anticancer therapy. Wang et al (31) reported that transforming growth factor beta causes apoptosis of colon cancer cells via suppression of the PI3K/Akt/survivin pathway. A number studies have indicated that activation of PI3K/Akt signaling mediates the tumor-promoting effects of periostin. For instance, Bao et al (10) showed that activation of the Akt/PKB
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pathway is involved in periostin-mediated colon cell survival. Similarly, Baril et al (7) reported that periostin enhances the survival of pancreatic cancer cells exposed to hypoxic
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conditions via activation of PI3K signaling. Most interestingly, we found that periostin overexpression led to enhanced Akt phosphorylation and survivin expression and such effects were blocked by pretreatment with the PI3K inhibitor LY294002. Taken together, these results suggest that periostin-induced apoptosis resistance is largely mediated through activation of the PI3K/Akt/survivin pathway in colon cancer cells.
In
conclusion,
our
data
indicate
that
upregulation
of
periostin
antagonizes
chemotherapy-induced apoptosis and confers survival advantages to colon cancer cells, which is, at least partially, ascribed to the activation of the PI3K/Akt/survivin signaling pathway. Periostin may thus represent a potential therapeutic target for improving the efficacy of adjuvant chemotherapy against colon cancer.
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Declaration of interest
The authors have no conflict of interest to declare.
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Figure 1. Effects of oxaliplatin and 5-FU on the expression of periostin at both mRNA and protein levels. SW480 and HT-29 cells were treated with either oxaliplatin or 5-FU for 24 and 48 h and subjected to periostin expression analysis. Western blot analysis showed the protein expression of periostin in SW480 and HT-29 cells incubated with oxaliplatin (A) and 5-FU (B). RT-PCR was examined to analyze the periostin mRNA expression in SW480 and HT-29 cells exposed to oxaliplatin (C) and 5-FU (D). Results from one representative experiment are shown.
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Figure 2. Silencing of periostin makes SW480 and HT-29 cells more susceptible to oxaliplatin and 5-FU. Periostin siRNA or non-targeting control siRNA duplexes were transfected into SW480 and HT-29 cells and incubated for 24 h. Then cells were treated with oxaliplatin or 5-FU for another 48 h and collected for the measurement of periostin expression and apoptosis detection. Western blot analysis of the expression of periostin in SW480 (A) and HT-29 (B) cells with indicated treatments. Annexin V and PI double staining was performed to detect apoptosis in SW480 (C) and HT-29 (D) cells with indicated treatments. Annexin V-positive and PI-negative cells are regarded as early apoptotic cells. Results from one representative experiment are shown.
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Figure 3. Knockdown of periostin enhanced the accumulation of cleaved caspase-3 and PARP in SW480 and HT-29 cells exposed to oxaliplatin or 5-FU. SW480 and HT-29 cells were transfected with periostin siRNA or control siRNA for 24 h, followed by incubation with oxaliplatin or 5-FU for another 48 h. Representative Western blots (top panel) showing that expression of cleaved caspase-3 and PARP in SW480 (A) and HT-29 cells (B) with indicated treatments. The expression of cleaved caspase-3 and PARP were normalized to β-actin (bottom panel). Data are shown as mean ± SD of three independent experiments. *P < 0.05.
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Figure 4. Effects of survivin overexpression on periostin-mediated chemoresistance. (A) SW480 and (B) HT-29 cells were treated with oxaliplatin or 5-FU for 48 h after transfection with periostin siRNA (psiRNA) or control siRNA (csiRNA) for 24 h. The expression of survivin was analyzed by Western blot analysis. Representative blots of three independent experiments are shown. SW480 and HT-29 cells were transfected with csiRNA or psiRNA alone or in combination with pcDNA3.1-survivin for 24 h. The cells were then exposed to oxaliplatin (C) or 5-FU (D) for another 48 h. Cells were tested for survivin expression or apoptosis. Representative Western blots of three independent experiments are shown in top panels. Bottom panels show the results of apoptosis analysis by annexin-v/PI staining. Data are shown as mean ± SD of three independent experiments. *P
Accepted Article
Zhi-ming Xiao1, Xiao-yan Wang1, Ai-min Wang2,*
1
Department of Gastroenterology, the third hospital of Xiangya, Central South University,
Changsha 410013, China 2
Department of Emergency, Xiangya Hospital, Central South University, Changsha 410008,
China
*
Corresponding author.
Department of Emergency, Xiangya Hospital, Central South University, Changsha 410008, China Tel: +8673188618211 Fax: +8673188618211 E-mail: [email protected]
Running title: Periostin in colon cancer
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as DOI:10.1002/bab.1193.
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1
Abstract
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In the present study, we aimed to explore the effects of periostin, a cell adhesion protein, on chemoresistance in colon cancer cells. Reverse transcription-polymerase chain reaction and western blot analyses were employed to detect periostin expression in SW480 and HT-29 colon cancer cells treated with oxaliplatin or fluorouracil (5-FU). Small interfering RNA was used to downregulate endogenous periostin. Annexin-V/propidium iodide (PI) staining was performed to analyze the effects of periostin on drug-induced apoptosis. The results showed that treatment with oxaliplatin or 5-FU elevated both the mRNA and protein levels of periostin in SW480 and HT-29 cells. Silencing of periostin significantly (P < 0.01) augmented drug-induced apoptosis in colon cancer cells, coupled with enhanced cleavage of caspase-3 and poly(ADP-ribose) polymerase. Mechanistic studies revealed that periostin silencing significantly (P < 0.01) suppressed the expression of survivin, an antiapoptotic protein in colon cancer cells. Enforced expression of survivin repressed drug-induced apoptosis
in periostin-depleted SW480 and
HT-29 cells.
Additionally,
periostin
overexpression increased the expression of survivin and the phosphorylation of Akt, which was reversed by pretreatment with the phosphatidylinositol 3-kinase (PI3K) specific inhibitor LY294002. Taken together, our data demonstrate that periostin induces chemoresistance in colon cancer cells through activation of the PI3K/Akt/survivin pathway. Key words: apoptosis; chemotherapy; drug resistance; periostin; signaling pathway.
Abbreviations
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2
EMT
Accepted Article
Epithelial-mesenchymal transition FITC Fluorescein isothiocyanate 5-FU Fluorouracil GAPDH Glyceraldehyde 3-phosphate dehydrogenase HNSCC Head and neck squamous cell carcinoma PARP Poly(ADP-ribose) polymerase PI
Propidium iodide PI3K Phosphatidylinositol 3-kinase RT-PCR Reverse transcription-polymerase chain reaction
1. Introduction
Colon cancer is one of the leading causes of cancer-related deaths worldwide (1). Surgery is
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3
the primary treatment approach for early-stage colon cancer; but the risk of recurrence is still high due to undetected micrometastases (2). Chemotherapy, usually oxaliplatin and
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fluorouracil (5-FU), has been proposed to reduce the risk of postoperative recurrence in colon cancer. Intrinsic or acquired chemoresistance remains a major obstacle to successful cancer treatment. The survival benefits of adjuvant chemotherapy are only achieved in a minor population of patients with colon cancer (3). Therefore, identification of the molecular mechanisms involved in chemoresistance is of great significance in developing novel therapeutic modalities for this malignancy.
Periostin, a 93.3-kDa secreted protein, is also named osteoblast-specific factor 2 (4). It is upregulated in various types of human cancers, including head and neck, pancreatic, breast, ovarian, prostate, papillary thyroid, lung, liver and colon carcinoma (5). Hypoxia induces the upregulation of periostin in non-small-cell lung cancer cells, which promotes cancer cell survival through the activation of Akt/PKB pathway (6). Similarly, Baril et al (7) reported that periostin enhances the survival of pancreatic cancer cells under hypoxic conditions. Nicotine-mediated induction of periostin promotes the proliferation, invasion and epithelial-mesenchymal transition (EMT) of gastric cancer cells (8). Conversely, monoclonal antibody neutralization of periostin inhibits anchorage-independent growth and survival of ovarian cancer cells (9). These findings suggest that overexpression of periostin is extensively associated with tumor growth, progression, and chemoresistance.
In colon cancer, periostin has been shown to promote metastatic growth through augmenting
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4
cell survival via the Akt/PKB pathway (10). In the present study, we sought to explore the effects of periostin on the chemosensitivity of colon cancer cells and associated molecular
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mechanisms.
2. Materials and Methods
2.1. Cell culture SW480 and HT-29 cells were treated with oxaliplatin or 5-FU alone for 24 and 48 h. If not stated otherwise, the half-maximal inhibitory concentration (IC50) of the chemotherapeutic
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5
agents were used (11): i.e., 7 μM oxaliplatin and 27 μM 5-FU for SW480 cells and 9 μM oxaliplatin and 20 μM 5-FU for HT-29 cells. To investigate the involvement of PI3K/Akt
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pathway in periostin-mediated chemoresistance, 20 μM PI3K inhibitor (LY294002) was used to pretreat SW480 and HT-29 cells for 1 h.
2.2. Plasmids and siRNAs TRIzol reagent (Invitrogen) was used to isolate total RNA from human colon cancer cell line SW480 cells according to the manufacture’s protocol. Human periostin (GenBank NM_006475) full-length cDNA (2511 bp) and human survivin (GenBank NM_001168) full-length cDNA (429 bp) were synthesized by reverse transcription of the extracted total RNA and amplified by polymerase chain reaction (PCR). The PCR primers for periostin were as
follows:
forward,
5′-GCCGAAGCTTACCATGATTCCC-3′;
reverse,
5′-GCGCCTCGAGTCACTGAGAACG-3′. The PCR primers for survivin were as below: forward,
5′-GCCGAAGCTTACCATGGGTGCC-3′;
reverse,
5′-GCGCCTCGAGTCAATCCATGGC-3′. After digestion with Hind III and Xho I, the resulting restriction fragments were cloned into the Hind III/Xho I sites of the pcDNA3.1 (+) expression vector (invitrogen). The constructed plasmids (pcDNA3.1-periostin and pcDNA3.1-survivin) were verified by DNA sequencing.
A pool of three siRNA duplexes targeted to different regions of human periostin mRNA (sc-61324) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Non-targeting siRNA duplexes (Santa Cruz Biotechnology) were used as control.
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2.3. Cell transfection
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For overexpression of periostin, SW480 and HT-29 cells pretreated with LY294002 at a 70-80% confluence were transfected with vector or pcDNA3.1-periostin by Lipofectamine 2000 and incubated for 24 h. The cells were then collected and subjected to Western blot analysis.
For silencing of periostin with siRNAs, SW480 and HT-29 cells at a 70-80% confluence were transfected with control versus periostin siRNA by lipofectamine 2000 and incubated for 24 h. The cells were then exposed to oxaliplatin or 5-FU for additional 48 h and subjected to apoptosis detection and gene expression analysis.
For restoration of survivin, SW480 and HT-29 cells at a 70-80% confluence were transfected with control versus periostin siRNA with or without survivin by Lipofectamine 2000 and incubated for 24 h. The cells were then exposed to oxaliplatin or 5-FU for additional 48 h and subjected to apoptosis detection and gene expression analysis.
2.4. Assessment of apoptosis by annexin-V staining Cell apoptosis was assessed using an Annexin-V-FITC Apoptosis Detection Kit (BioVision, Mountain View, CA, USA) according to the manufacturer’s recommendations. Briefly, cells were detached, resuspended in binding buffer, and added with fluorescein isothiocyanate (FITC)-conjugated annexin-V and propidium iodide (PI). After incubation for 10 min at room
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7
temperature in the dark, the cells were analyzed for annexin-V-FITC binding by a flow cytometer (Becton Dickinson, San Jose, CA, USA). Early apoptosis was determined by
Accepted Article
measurement of annexin-V-positive and PI-negative cells. Each experiment was repeated three times.
2.5. Reverse transcription-PCR Total RNA was isolated from cells with indicated treatments using TRIzol reagent (Invitrogen, Carlsband, CA) according to the manufacturer’s protocol. RT-PCR was performed to analyze periostin mRNA expression. 5 μg of total RNA was used to synthesize cDNA. The periostin forward primer was 5'-CTTCAATGTATAACAATTTGG-3' and the periostin reverse primer was 5’-CGATGTGATGGCAGCTGCTC-3'. The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward primer was 5'-CTGCACCACCAACTGCTTAG-3' and the GAPDH reverse primer was 5'-TGAAGTCAGAGGAGACCACC-3'. The amplicons for periostin and GAPDH were 215 and 407 bp, respectively. PCR products were electrophorized on 1.5% agarose gel and visualized by ethidium-bromide staining.
2.6. Western blot analysis Cells with indicated treatments were lysed with ice-cold lysis buffer (pH7.4) containing 25 mM HEPES, 1% Triton X-100, 50 mM NaCl, 1mM EDTA, 1mM EGTA, 1mM PMSF and 1 μg/ml leupeptin. Protein quantification was performed by BCA assay. Protein extracts (50 μg/lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto PVDF membrane. Membranes were blocked in 5% (w/v) non-fat dried milk in
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8
Tris-buffered saline for 1 h at room temperature. Then membranes were incubated with appropriate primary antibodies followed by incubation with HRP-conjugated secondary
Accepted Article
antibodies. Immunoreactive proteins were visualized using an enhanced chemiluminescent detection
system
immunoreactive
(Amersham bands
were
Biosciences, measured
Piscataway, by
NJ).
The
computerized
intensities
image
of
analysis
(QuantityOne-software; Bio-Rad, Hercules, CA) and normalized to β-actin levels. Antibodies used in the present studies were as follows: anti-periostin, survivin, pAkt, total Akt and β-actin primary antibodies and HRP-conjugated goat-antirabbit IgG, and goat-anti-mouse IgG and donkey-anti-goat IgG were purchased from Santa Cruz Biotechnology. Anti-cleaved caspase-3 and cleaved PARP antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). 2.7. Statistical analysis Data are expressed as mean ± standard deviation (SD). Difference between group means were evaluated by student’s t-test or one-way analysis of variance (ANOVA) followed by the Tukey post-hoc test. P-value less than 0.05 were considered as significant.
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3. Results
3.1. Chemotherapeutic drugs upregulates periostin in colon cancer cells Western blot analysis revealed that oxaliplatin treatment for 24 or 48 h significantly raised periostin expression at protein levels in both SW480 and HT-29 cells (Fig. 1A). Likewise, there was a marked elevation in periostin expression at protein levels in SW480 and HT-29 cells exposed to 5-FU for 24 or 48 h (Fig. 1B). RT-PCR analysis confirmed the upregulation of periostin by chemotherapeutic drugs at the mRNA levels (Fig. 1C and D).
3.2. Inhibition of periostin sensitizes colon cancer cells to drug-induced apoptosis Having identified the elevation of periostin upon the exposure to chemotherapeutic agents, we next explored the effects of periostin inhibition on colon cancer cell chemosensitivity. As
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10
shown in Fig. 2A and B, the pretreatment of SW480 and HT-29 cells with periostin specific siRNAs resulted in a marked reduction in the periostin mRNA abundance in comparison with
Accepted Article
control siRNA-transfected counterparts, in the presence of oxaliplatin or 5-FU. Upon the treatment with the chemotherapeutic agents, periostin siRNA-transfected SW480 cells showed a significant increase in the percentage of apoptotic cells when compared to control siRNA-transfected SW480 cells (Fig. 2C). Similar findings were observed in HT-29 cells (Fig. 2D). Additionally, the delivery of periostin specific siRNAs resulted in a significant enhancement in the cleavage of caspase-3 and PARP compared to the transfection with control siRNAs (Fig. 3). Taken together, these results demonstrate that depletion of periostin increases the susceptibility to chemotherapeutic agents in colon cancer cells.
3.3.
Downregulation
of
survivin
is
critical
for periostin
inhibition-induced
chemosensitization Previous studies have shown that upregulation of survivin confers to cancer cells resistance against drug-induced cell death (12). Next, we examined whether such chemosensitization induced by periostin inhibition was mediated by modulation of survivin expression. As shown in Fig. 4A and B, siRNA-mediated depletion of periostin profoundly diminished the expression of survivin at protein levels in SW480 and HT-29 cells treated with oxaliplatin or 5-FU, compared to control siRNA-transfected counterparts. Most importantly, transfection with wild-type survivin gene significantly repressed chemotherapeutic drug-induced apoptosis in periostin-depleted SW480 and HT-29 cells by about 50% (P < 0.05; Fig. 4C and D).
Western
blot
analysis
confirmed
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the
overexpression
of
survivin
in
11
pcDNA3.1-survivin-transfected SW480 and HT-29 cells (Fig. 4C and D).
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3.4. Activation of the PI3K/Akt pathway contributes to the induction of survivin by periostin It has been documented that the PI3K/Akt pathway is implicated in the regulation of survivin expression (13). Finally, we checked the role of the PI3K/Akt pathway in the modulation of survivin by periostin. Western blot analysis showed that overexpression of periostin markedly increased the protein level of survivin and the phosphorylation of Akt at site 473 without altering the expression of total Akt in SW480 cells (Fig. 5). Notably, the phosphorylation of Akt and induction of survivin by periostin was significantly inhibited by the pretreatment with the PI3K specific inhibitor LY294002 (Fig. 5), suggesting the involvement of the PI3K/Akt pathway in periostin-induced survivin elevation.
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4. Discussion
To determine the role of periostin in the chemoresistance of colon cancer, in this study we have chosen two widely used conon cancer cell lines SW480 and HT-29 that have shown resistance to chemotherapeutic agents (14,15). Our data showed that exposure to chemotherapeutic drugs enhanced the expression of periostin at both mRNA and protein levels in human colon cancer cells. Targeting periostin rendered cancer cells more susceptible to drug-induced apoptosis, which was associated with enhanced cleavage of caspase-3 and PARP. Periostin silencing inhibited the expression of survivin in colon cancer cells. Restoration of survivin significantly reversed the induction of apoptosis by periostin depletion. Additionally, ectopic expression of periostin elevated the expression of survivin in colon cancer cells via activation of the PI3K/Akt pathway. Our results collectively indicate that acquired expression of periostin confers chemoresistance in colon cancer cells, which is largely associated with activation of the PI3K/Akt/survivin pathway.
Periostin is frequently upregulated in various types of human cancers and implicated in
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13
cancer cell proliferation, invasion, and metastasis (6-8). Kudo et al (16) showed that periostin is elevated in head and neck squamous cell carcinoma (HNSCC) and promotes invasion and
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anchorage-independent growth of HNSCC. Overexpression of periostin has been found to facilitate tumor progression and angiogenesis in breast cancer (17). Several lines of evidence suggest periostin upregulation as a result of stress (18,19). Tanabe et al (18) reported that periostin inhibits cell death through up-regulation of Bcl-xL expression by maintaining the Notch1 protein level under the stress condition. Hypoxic induction of periostin has been found to promote cell survival in non-small-cell lung cancer (6). Nicotine-induced upregulation of periostin in gastric cancer cells confers nicotine-mediated gastric cancer cell growth, invasion, drug resistance and EMT (8). These results suggest that upregulation of periostin represents a survival strategy for cells under stress conditions. In line with this view, our data revealed that there is an upregulation of periostin expression in colon cancer cells exposed to chemotherapeutic drugs and targeted reduction of periostin sensitizes colon cancer cells to chemotherapeutic agents. These results suggest a cytoprotective role for periostin against chemotherapy in colon cancer cells. Therefore, targeting periostin may be an attractive strategy to improve the efficacy of chemotherapy in colon cancer. Indeed, pre-clinical studies revealed that blocking periostin activity using a monoclonal antibody significantly suppresses tumor growth and metastasis in a mouse model of ovarian cancer (9). Lee et al (20) also reported that interfering with periostin action with periostin-binding DNA aptamer significantly reduces primary tumor growth and distant metastasis in an orthotopic mouse model of breast cancer.
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14
Survivin, an antiapoptotic protein, is initially identified as an inhibitor of caspase-9 and functions as a key modulator of programmed cell death (21). Growing evidence indicates that
Accepted Article
survivin is highly expressed in various types of human cancers and contributes to chemoresistance
(22-24).
Survivin
participates
in
fibronectin/PI3K/Akt2
signal
pathway-mediated docetaxel-resistance in breast and ovarian cancer (24). Inhibition of survivin expression is causally linked to the growth suppression and apoptosis induction by anticancer agents in colon cancer cells (25,26). Thus, we examined whether survivin is involved in periostin-mediated chemoresistance in colon cancer. Western blot analysis showed that the expression of survivin was significantly suppressed in periostin siRNA-transfected SW480 and HT-29 cells, compared to mock transfectants. Moreover, enforced expression of survivin significantly inhibited chemotherapeutic agent-induced apoptosis in periostin-depleted colon cancer cells. These results suggest that survivin is involved in periostin-mediated chemoresistance in colon cancer cells.
The PI3K/Akt pathway is implicated in cancer cell survival and outgrowth (27). Accumulating evidence suggests survivin as a downstream effector of PI3K/Akt signaling (28,29). Activation of the PI3K/Akt/survivin pathway usually results in resistance to apoptosis caused by various apoptotic stimuli (30), thus representing a promising target for anticancer therapy. Wang et al (31) reported that transforming growth factor beta causes apoptosis of colon cancer cells via suppression of the PI3K/Akt/survivin pathway. A number studies have indicated that activation of PI3K/Akt signaling mediates the tumor-promoting effects of periostin. For instance, Bao et al (10) showed that activation of the Akt/PKB
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15
pathway is involved in periostin-mediated colon cell survival. Similarly, Baril et al (7) reported that periostin enhances the survival of pancreatic cancer cells exposed to hypoxic
Accepted Article
conditions via activation of PI3K signaling. Most interestingly, we found that periostin overexpression led to enhanced Akt phosphorylation and survivin expression and such effects were blocked by pretreatment with the PI3K inhibitor LY294002. Taken together, these results suggest that periostin-induced apoptosis resistance is largely mediated through activation of the PI3K/Akt/survivin pathway in colon cancer cells.
In
conclusion,
our
data
indicate
that
upregulation
of
periostin
antagonizes
chemotherapy-induced apoptosis and confers survival advantages to colon cancer cells, which is, at least partially, ascribed to the activation of the PI3K/Akt/survivin signaling pathway. Periostin may thus represent a potential therapeutic target for improving the efficacy of adjuvant chemotherapy against colon cancer.
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Declaration of interest
The authors have no conflict of interest to declare.
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17
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References
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Q., Chen, J., Su, C. (2011) P16 reactivation induces anoikis and exhibits antitumour potency by downregulating Akt/survivin signalling in hepatocellular carcinoma cells. Gut 60,
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Figure 1. Effects of oxaliplatin and 5-FU on the expression of periostin at both mRNA and protein levels. SW480 and HT-29 cells were treated with either oxaliplatin or 5-FU for 24 and 48 h and subjected to periostin expression analysis. Western blot analysis showed the protein expression of periostin in SW480 and HT-29 cells incubated with oxaliplatin (A) and 5-FU (B). RT-PCR was examined to analyze the periostin mRNA expression in SW480 and HT-29 cells exposed to oxaliplatin (C) and 5-FU (D). Results from one representative experiment are shown.
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Figure 2. Silencing of periostin makes SW480 and HT-29 cells more susceptible to oxaliplatin and 5-FU. Periostin siRNA or non-targeting control siRNA duplexes were transfected into SW480 and HT-29 cells and incubated for 24 h. Then cells were treated with oxaliplatin or 5-FU for another 48 h and collected for the measurement of periostin expression and apoptosis detection. Western blot analysis of the expression of periostin in SW480 (A) and HT-29 (B) cells with indicated treatments. Annexin V and PI double staining was performed to detect apoptosis in SW480 (C) and HT-29 (D) cells with indicated treatments. Annexin V-positive and PI-negative cells are regarded as early apoptotic cells. Results from one representative experiment are shown.
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Accepted Article
Figure 3. Knockdown of periostin enhanced the accumulation of cleaved caspase-3 and PARP in SW480 and HT-29 cells exposed to oxaliplatin or 5-FU. SW480 and HT-29 cells were transfected with periostin siRNA or control siRNA for 24 h, followed by incubation with oxaliplatin or 5-FU for another 48 h. Representative Western blots (top panel) showing that expression of cleaved caspase-3 and PARP in SW480 (A) and HT-29 cells (B) with indicated treatments. The expression of cleaved caspase-3 and PARP were normalized to β-actin (bottom panel). Data are shown as mean ± SD of three independent experiments. *P < 0.05.
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Accepted Article
Figure 4. Effects of survivin overexpression on periostin-mediated chemoresistance. (A) SW480 and (B) HT-29 cells were treated with oxaliplatin or 5-FU for 48 h after transfection with periostin siRNA (psiRNA) or control siRNA (csiRNA) for 24 h. The expression of survivin was analyzed by Western blot analysis. Representative blots of three independent experiments are shown. SW480 and HT-29 cells were transfected with csiRNA or psiRNA alone or in combination with pcDNA3.1-survivin for 24 h. The cells were then exposed to oxaliplatin (C) or 5-FU (D) for another 48 h. Cells were tested for survivin expression or apoptosis. Representative Western blots of three independent experiments are shown in top panels. Bottom panels show the results of apoptosis analysis by annexin-v/PI staining. Data are shown as mean ± SD of three independent experiments. *P
