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Introduction

Esophageal cancer is a common human gastrointestinal cancer, accounting for 400,000 mortalities and 480,000 new cases in 2008 worldwide (1). Despite advances in early detection and standardized treatment regimens, relatively low 5-year survival rates for esophageal cancer have been observed (2,3). In Eastern Asia and Africa, the incidence rates of esophageal cancer are extremely high (4). Countries in this region are developing countries and their patients may not be able to afford advanced treatments. Therefore, low-cost treatments, such as hyperthermia have been considered.

In the past decade, anticancer trials with multimodal therapies, including hyperthermia have been reported (5,6). These clinical trials demonstrated that hyperthermia increased overall survival by making normally inoperable tumors candidates for surgery and by improving the efficacy of chemo- or radiotherapy with reduced toxicity (7–9). Hyperthermia is considered the fifth pillar of cancer treatment following surgery, chemotherapy, radiotherapy and biotherapy. However, the mechanisms by which hyperthermia improves clinical outcomes remain unclear. Recent findings suggest that hyperthermia impairs protein synthesis, leading to apoptosis (10).

Apoptosis is a conserved and regulated cell suicide process, which is activated and executed by cysteine aspartate-specific proteases (caspases) (11). Survivin, a protein that is often overexpressed in cancer, suppresses apoptosis by inhibiting caspase activation (12). Due to its upregulation in almost all human tumors and its key role in apoptosis, proliferation and angiogenesis, Survivin is considered a crucial target for anticancer therapies (13). Downregulation of Survivin in human cancer cell lines and mouse models inhibits tumor growth and sensitizes tumor cells to chemo- or radiotherapy (14,15).

In the present study, we investigated the function of Survivin in hyperthermia-induced apoptosis in EC109 esophageal cancer cells and examined Survivin expression in patients with esophageal cancer, correlating its expression with clinico pathological characteristics. The results showed that, hyperthermia decreased the expression of Survivin, prevented its binding to XIAP, activated caspase-3 and induced apoptosis. In addition, Survivin expression was higher in esophageal cancer than in normal tissues, and this higher level of expression was associated with poor prognosis. Therefore, Survivin may be a crucial target for hyperthermia in the esophageal cancer treatment.

Materials and methods

Construction of plasmids and transfection

Full-length Survivin and XIAP were generated by PCR from EC109 cell cDNA using the primers: Survivin sense, 5′-AGATCTGGATCCGGTGCCCCGACGTTGCCCCC-3′ and antisense, 5′-TCTAGAGCGGCCGCTCAATCCATGGCAGCCAGCTGCTCG-3′; XIAP sense, 5′-AGATCTGGATCCACTTTTAACAGTTTTGAAGG-3′ and antisense, 5′-TCTAGAGCGGCCGCTTAAGACATAAAAATTTTTTGCTTG-3′. The resulting fragments were digested by BamHI and NotI (Thermo Scientific, Rockford, IL, USA) and cloned into pcDNA3.0-Flag. The resulting or control plasmids were transfected into the EC109 cells with Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA). Western blotting was used to identify protein overexpression in EC109 cells. The siRNAs for XIAP and the control siRNA were designed and produced by Shanghai GenePharma Co., Ltd. (Shanghai, China).

Cell culture and heat treatment

The human EC109 cells were obtained from the American Type Culture Collection; Manassas, VA, USA). The cells were maintained at 37°C and 5% CO2 in RPMI-1640 medium, supplemented with 10% fetal bovine serum (FBS) (both from GE Healthcare Life Sciences, Logan, UT, USA), 100 U/ml penicillin and 100 g/ml streptomycin (both from Sigma, St. Louis, MO, USA). For heat treatment, EC109 cells were collected into 1.5 ml Eppendorf tubes and then incubated in a hot water bath at 37°, 39°, 41°, 42°, 43° or 45°C for 40 min. Following heat treatment, the cells were recovered under standard culture conditions.

MTT assay

After heat treatment, 5×103 EC109 cells were plated in each well of 96-well plates. The following day, 20 µl of MTT (Sigma) was added to each well, and the cells were incubated for an additional 4 h at 37°C, prior to the addition of 150 µl dimethylsulfoxide (DMSO). After 20 min, absorbance at 490 nm (A490) was measured with a microplate reader (Bio-Rad Laboratories, Richmond, CA, USA). Cell viability (%) was calculated as: the average A490 in an experimental chemotherapy group/the average A490 in the blank control group × 100%.

Flow cytometry

The cells were incubated in a water bath at different temperatures for 40 min, and then plated into 24-well plates at a concentration of 1×105 cells/well. After 24 h, the cells were trypsinized and incubated with 5 µl propidium iodide (PI) and 5 µl Annexin V-FITC (both from Invitrogen Life Technologies) for 15 min. The samples were then analyzed for apoptosis using a FACScan flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

Western blot analysis

Following heat treatment, EC109 cells were collected into a lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA (pH 8.0), 1% Triton X-100 and protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN, USA)], and incubated for 30 min on ice. Protein (30 µg) for each sample were separated on 12% SDS-PAGE gels (Invitrogen Life Technologies) and transferred to PVDF membranes (EMD Millipore, Billerica, MA, USA). After blocking with 5% BSA (Shanghai Bioleaf Biotech Co., Ltd., Shanghai, China) at room temperature for 1 h, the membranes were incubated with primary antibody overnight at 4°C, incubated with horseradish peroxidase-labeled goat anti-rabbit IgG antibody (1:5,000; sc-45101; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 1 h at room temperature and developed using ECL detection (Thermo Scientific, Waltham, MA, USA). β-actin was used as a loading control. For immunodetection, the primary antibodies used were: anti-Survivin rabbit polyclonal IgG antibody (1:1,000; sc-10811; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) anti-Smac rabbit monoclonal antibody (1:1,000; 1012-1), anti-XIAP rabbit polyclonal antibody (1:2,000; S1022) (both from Epitomics, Burlingame, CA, USA), anti-bcl-2, anti-bax, anti-bcl-xl (rabbit polyclonal antibody, 1:1,000, sc-783, sc-493, sc-7195; Santa Cruz Biotechnology, Inc.), and anti-pro-caspase-3 rabbit monoclonal, anti-active caspase-3 rabbit polyclonal antibodies (1:1,000; ab32499; ab2302), and anti-β-actin rabbit polyclonal antibody (1:5,000; ab75186) (all from Abcam, Cambridge, UK).

Co-immunoprecipitation

The EC109 cells were transfected with Flag-Survivin, Flag-XIAP or Flag control vector for 48 h, collected in lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA (pH 8.0), 1% Triton X-100 and protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN, USA)], and then incubated for 30 min on ice. Protein (500 µg) from the cell lysates was incubated with 20 µl Anti-FLAG M2 Affinity Gel (Sigma) for 2 h at 4°C. The collected beads were washed three times with lysis buffer and then eluted with SDS-PAGE sample buffer. Immunoblotting was detected using western blot analysis.

Clinical samples

Primary tumor specimens were obtained from 86 patients diagnosed with esophageal squamous cell carcinomas, who underwent complete resection at the First Affiliated Hospital of Xi'an Jiaotong University from February, 2009 to February, 2011. Normal esophageal tissues were collected at >5 cm from the edge of the tumor. The tissues were fixed in 10% formalin immediately and embedded in paraffin within 24 h after surgical resection. Follow-up information was obtained from a review of the patient medical records. There were 64 males and 22 females and their mean age was 59.8 years (42–77 years). The patients had a single tumor, without distant metastasis and none of them had been previously treated with chemo- or radiotherapy. After surgical resection, the patients underwent standard therapeutic procedures according to the Clinical Oncology Information Network guidelines. Carcinomas were classified in accordance with the tumor-node-metastasis (TNM) classification system of the International Union Against Cancer (UICC) (16). The study design and procedure were approved by the Ethics Committee of the hospital and each participant signed an informed consent document prior to enrollment.

Immunohistochemistry

Resected specimens were fixed with 10% formaldehyde and embedded in paraffin blocks. Sections (5 µm) were deparaffinized with xylene and rehydrated in a series of ethanol concentrations. Endogenous peroxidase activity was blocked by immersion in 0.3% methanolic peroxide for 15 min. Immunoreactivity of the target antigens was enhanced by microwaving the sections for 10 min in 0.1 M citrate buffer, pH 6.0. Test sections were incubated with anti-Survivin rabbit polyclonal IgG antibody (sc-10811; Santa Cruz Biotechnology, Inc.) at a dilution of 1:100 in the blocking solution at 4°C overnight and goat anti-rabbit IgG/HRP secondary antibody (bs-0295G-HRP; Beijing Biosynthesis Inc., Beijing, China) was used at a dilution of 1:250 for 1 h at room temperature. After washing, the signal was detected using a DAB kit (Beijing Biosynthesis, Inc.). The section was counterstained with hematoxylin and photographed under a light microscope (Nikon, Tokyo, Japan). For the negative controls, the anti-Survivin antibody was replaced with 1% bovine serum albumin (Shanghai Bioleaf Biotech Co., Ltd.) in phosphate-buffered saline (PBS). No staining was detected in any control section.

Immunohistochemical scoring

The stained slides were assessed by two independent pathologists, without knowledge of the patient clinical data. Ten randomly-selected fields for each slide were scored for the area and intensity of positively stained (brown) cytoplasm and/or cell membrane under light microscopy. The intensity of Survivin staining was scored as: 0, no signal; 1, weak; 2, moderate; and 3, marked. Percentage scores were assigned as: 1, 1–25%; 2, 26–50%; 3, 51–75%; and 4, 76–100%. The scores of each tumor sample were multiplied to give a final score of 0–12, and the tumors were designated as negative expression (−), score 0–4; weak expression (+), score 5–8; high expression (++), score 9–12. Tumor samples scored (+) to (++) were considered positive.

Statistical analysis

The χ2 and Fisher's exact tests were used to compare frequencies. The Kaplan-Meier method was used to assess prognosis after surgery, and the log-rank test was used to compare survival curves. Univariate analysis was performed with the Cox regression model. Most pathological variables were used as dichotomized variables: Age (<60 vs. ≥60 years), location (upper+middle vs. lower thoracic), tumor differentiation (high+middle vs. low), tumor size (<5 vs. ≥5 cm), lymph node metastasis (N0 vs. N1+N2+N3) and stage (I+II vs. III). Statistical analyses were performed using SPSS version 16.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant result.

Results

Hyperthermia induces apoptosis and necrosis in EC109 cells

To analyze the effect of hyperthermia in esophageal cancer cells, we first treated EC109 cells at different temperatures (37°C, 39°C, 41°C, 42°C, 43°C and 45°C) for 40 min. After the cells were incubated at 37°C in 5% CO2 for another 24 h, cell viability was assessed using an MTT assay. With higher temperatures, EC109 cells showed significantly decreased viability (Fig. 1). To determine whether EC109 cells treated with hyperthermia underwent apoptosis, we treated cells as before, and after 24 h the cells were stained with Annexin V-FITC and PI to assess necrosis and apoptosis induction by flow cytometry. As shown in Fig. 2A, necrosis was calculated as Annexin V (−) and PI (+) while apoptosis was indicated by Annexin V (+) and PI (±). Necrotic EC109 cells increased with higher temperatures, whereas apoptotic cells reached their peak at 43°C. The levels of apoptosis were similar between the last two groups, but at 45°C the number of necrotic EC109 cells was significantly higher than at 43°C (Fig. 2B).

Figure 1 Hyperthermia reduces the viability of EC109 cells. Cells were treated with hyperthermia for 40 min and then incubated at 37°C for 24 h. Cell viability was determined using MTT assays, and data are presented as the mean ± SD. With higher temperatures, EC109 cells showed significant decreases in viability.

Figure 2 Hyperthermia induces apoptosis and necrosis of EC109 cells. (A) Cells were treated with hyperthermia for 40 min and were then incubated at 37°C for 24 h. Annexin V/PI staining was used to detect apoptosis. (B) Statistical analysis of FACS in (A). The necrotic EC109 cells increased with higher temperatures, whereas the apoptotic cells reached their peak at 43°C.

Hyperthermia inhibits Survivin and activates caspase-3 in EC109 cells

To investigate the increased induction of apoptosis in EC109 cells following heat treatment, western blotting was performed for components of the apoptotic pathway. Following treatment with increased temperature, the expression of XIAP and Smac did not change, whereas active caspase-3 and Survivin were significantly altered. Hyperthermia reduced Bcl-2, while Bax and Bcl-xL were unaffected. Notably, the activation of caspase-3 coincided with the decrease of Survivin expression (Fig. 3). Survivin is a member of the IAP familiy, which inhibits caspase activation. It is possible that a reduced Survivin expression leads to caspase-3 activation during hyperthermia.

Figure 3 Hyperthermia promotes apoptosis by inhibiting Survivin and activating caspase-3. EC109 cells were treated with hyperthermia for 40 min and then incubated at 37°C for 24 h. Western blotting was used to analyze the changes in protein expression of members of the apoptotic pathway. The expression of XIAP and Smac did not change, whereas activated caspase-3 and Survivin changed significantly. Hyperthermia reduced Bcl-2, while Bax and Bcl-xL were not affected.

Overexpression of Survivin inhibits hyperthermia-induced apoptosis through caspase-3 inactivation

To investigate the function of Survivin in EC109 cells with heat treatment, we transfected pcDNA3.0-Flag-Survivin (FL-Sur) or empty vector (EV) into EC109 cells for 48 h and then incubated the cells at 43°C for 40 min. The following day, western blotting confirmed that the expression of Survivin in the Survivin-transfected cells was higher than that in the control cells, and heat-induced active caspase-3 was inhibited by the overexpression of Survivin (Fig. 4A and B). Furthermore, Survivin-overexpressing and control cells were stained with PI and Annexin V-FITC to assess apoptosis induction by flow cytometry. At 43°C cultures, the number of apoptotic EC109 cells with a higher Survivin expression was significantly lower than that in the control cells (P<0.05, Fig. 4C). These results indicated that the overexpression of Survivin inhibited the activation of caspase-3, resulting in decreased hyperthermia-induced apoptosis.

Figure 4 Overexpression of Survivin inhibits hyperthermia-induced apoptosis. (A) EC109 cells were transfected with pcDNA3.0-Flag-Survivin (FL-Sur) or empty vector (EV) for 48 h and then incubated at 43°C for 40 min. The protein expression was evaluated by western blotting. (B) Values are presented as the mean ± SD, *P<0.05. (C) Annexin V/PI staining was used to detect apoptosis and the apoptotic rates were provided as mean ± SD, *P<0.05.

Survivin inactivates caspase-3 by binding to XIAP

We determined whether Survivin and caspase-3 physically interacted in EC109 cells. EC109 cells were transfected with Flag-Survivin, although immunoprecipitates of Flag from transfected cells rarely contained caspase-3 by western blotting. By contrast, XIAP was detected in Flag-Survivin immunoprecipitates from cells (Fig. 5A). When we overexpressed Flag-XIAP in EC109 cells and performed Flag immunoprecipitations, there was an interaction between XIAP and Survivin or caspase-3 (Fig. 5B). To demonstrate the interaction of XIAP with Survivin and caspase-3, we co-transfected Flag-Survivin and SiXIAP into EC109 cells, and then treated cells with hyperthermia (43°C for 40 min). The inhibitions of active caspase-3 and apoptosis by Survivin were reversed in the absence of XIAP (Fig. 5C–E). These results supported the model that Survivin inactivated caspase-3 by binding XIAP.

Figure 5 Survivin affects caspase-3 by binding XIAP. (A) EC109 cells were transfected with pcDNA3.0-Flag-Survivin (FL-Sur) or empty vector (EV) for 48 h. The cell supernatant was collected and immunoprecipitated with Anti-FLAG M2 Affinity Gel. Western blotting was performed for XIAP and caspase-3. (B) EC109 cells transfected with pcDNA3.0-Flag-XIAP (FL-XIAP) or EV were immunoprecipitated with Anti-FLAG M2 Affinity Gel and western blotting with Survivin and caspase-3. (C) EC109 cells were transfected with Flag-Survivin and SiXIAP or Si control. The cells were incubated at 43°C for 40 min. The following day, protein expression was evaluated by western blotting. (D) Values are presented as the mean ± SD, *P<0.05. (E) Annexin V/PI staining was used to detect apoptosis and the apoptotic rates were provided as mean ± SD, *P<0.05.

Expression of Survivin in esophageal cancer and para-tumor tissues

Using immunohistochemistry and patient samples, we found that the expression rate of Survivin in the esophageal cancer tissues was 57.0% (49 of 86 patients), and positive staining was identified in the cytoplasm. Of the 62 para-tumor tissues, 16 samples exhibited positive staining, and the remaining stained negative. The positive staining rate was 25.8% (16 of 62 patients) (Fig. 6). The expression of Survivin in esophageal cancer was significantly higher than that in the para-tumor tissues (P<0.01) (Table I).

Figure 6 Survivin expression in esophageal cancer and para-tumor tissues by immunohistochemistry. (A) Negative expression of Survivin in normal esophageal tissue. (B) Negative expression (score 0–4) of Survivin in esophageal cancer with stage I. (C) Weak positive expression (score 5–8) of Survivin in esophageal cancer with stage II. (D) Strong positive expression (score 9–12) of Survivin in esophageal cancer with stage III. Original magnification, ×100.

Table I Comparison of Survivin expression in esophgeal cancer and para-tumor tissues.

Table I

Comparison of Survivin expression in esophgeal cancer and para-tumor tissues.

Tissues	Total	Survivin		Positive rate (%)
		+	−
Esophageal cancer	86	49	37	57.0
Para-tumor	62	16	46	25.8

[i] χ²=14.212, P<0.001.

Correlation between Survivin expression and clinicopatho-logical characteristics

To examine the association between Survivin expression and the clinicopathological characteristics of patients, we compared Survivin expression with common parameters, such as age, gender, tumor size, tumor location, pathological type, tumor differentiation, tumor size, depth of tumor invasion, lymph node metastasis and clinical stage (Table II). In the 86 esophageal squamous carcinoma samples, the expression of Survivin was directly correlated with clinical stage and lymph node metastasis (P<0.05), but not age, gender, tumor location, pathological type, tumor differentiation, tumor size or depth of tumor invasion (P>0.05).

Table II Relationship between the expression of Survivin and the characteristics of patients.

Table II

Relationship between the expression of Survivin and the characteristics of patients.

Characteristics	Total	The expression of Survivin			χ2	P-value
		Positive	Negative	Positive rate (%)
Age (years)
<60	45	27	18	60.0	0.352	0.553
≥60	41	22	19	53.7
Gender
Male	64	36	28	56.3	0.054	0.861
Female	22	13	9	59.1
Location
Upper+middle thoracic	43	25	18	58.1	0.047	0.828
Lower thoracic	43	24	19	55.8
Pathological type
Polypoid	9	5	4	55.6	0.051a	0.829a
Medullary	41	24	17	58.5
Ulceration	29	18	11	62.1
Erosive	7	2	5	28.6
Tumor differentiation
High+middle	60	33	27	55.0	0.316	0.574
Low	26	16	10	61.5
Tumor size (cm)
<5	59	35	24	59.3	0.422	0.516
≥5	27	14	13	51.9
Depth of invasion
T1 (submucosa)	12	5	7	41.7	1.251	0.263
T2 (muscle)	15	8	7	53.3
T3 (serosa)	41	26	15	63.4
T4 (adjacent organs)	18	10	8	55.6
Lymph node metastasis
Yes	37	28	9	75.7	9.263	0.002
No	49	21	28	42.9
Stage
I+II	46	20	26	43.5	7.351	0.007
III	40	29	11	72.5
Total	86	49	37	57.0

a Fisher's exact test.

Prognostic value of Survivin and other clinical characteristics

We used the Kaplan-Meier survival curves and the log-rank test to examine the prognostic value of Survivin and other clinical characteristics (Fig. 7). We found that the patients with a negative expression of Survivin had a significantly longer overall survival than those with a positive expression of Survivin (P=0.007). Lymph node metastasis and clinical stage also showed a significant relationship with overall survival (P<0.001). Other clinical characteristics, such as age, gender, tumor location, had no correlation with overall survival of patients (P>0.05). Univariate Cox regression analysis results revealed that, Survivin expression (P=0.009), tumor differentiation (P=0.028), lymph node metastasis (P<0.001) and clinical stage (P<0.001) were the best predictors of survival of esophageal cancer patients.

Figure 7 Association between the expression of Survivin and survival of patients with esophageal cancer. Patients with a negative expression of Survivin had a significantly longer overall survival than those with a positive expression of Survivin (P=0.007). Analyses were constructed using the Kaplan-Meier method. The log-rank test was used to compare survival curves between Survivin positive and negative. P<0.05, significant difference.

Discussion

Survivin is an essential regulator of cell death, apoptosis, cell division and proliferation, which is rarely expressed in normal tissues. However, it is upregulated in the majority of cancers (17,18). As a tumor-specific molecule, Survivin promoted angiogenesis, antagonized apoptosis and facilitated resistance to chemotherapy/radiotherapy (19). Survivin is a good diagnostic biomarker for estimating the prognosis of cancer patients (20). Therefore, it is a potentially valuable target for drug identification. In our experiments, we found that Survivin is expressed at higher levels in esophageal cancer tissues than in normal tissues, and its high expression correlated with poor prognosis, suggesting that Survivin is also a target for esophageal cancer treatment.

Hyperthermia, the heating of tumors to 42–43°C, is considered the fifth pillar of cancer treatment and is strongly supported by evidence-based medicine (21,22). However, the mechanisms by which hyperthermia improves clinical outcomes remain unclear. There are possible mechanisms that are to be considered. First, hyperthermia induces apoptosis via the cell death pathway. Second, hyperthermia alters the cell cycle by disrupting S phase and blocking of mitosis. Third, hyperthermia causes chronic ischemia inside the tumor and reduces vessel regulation (23,24). In the present study, hyperthermia was found to induce esophageal cancer apoptosis by inhibiting Survivin. Due to the high expression of Survivin in esophageal cancer tissues, hyperthermia is a valuable therapy for targeting Survivin in esophageal cancer.

Survivin plays a crucial role in inhibiting caspase-dependent apoptosis in different tumors, such as lung and thyroid tumors. As an IAP family member, Survivin contains a BIR domain that may bind to caspases and interfere with their functions (12). There is a debate concerning the Survivin ability to directly bind to caspases (such as other IAP members) with its single BIR domain. Previous findings have demonstrated that Survivin binds to caspase-3 and -7 under specific conditions (25). By contrast, other studies have shown that Survivin inhibited the activation of caspases by binding and stabilizing XIAP rather than directly binding to caspases (26,27). In the present study, we found that hyperthermia inhibits Survivin, activates caspase-3 and induces apoptosis. Overexpression of Survivin reduced hyperthermia-induced apoptosis by inhibiting the activation of caspase-3. However, Survivin did not interact with caspase-3 directly. Caspase-3 and Survivin bind to XIAP and the inhibition of activated caspase-3 by Survivin was reversed in the absence of XIAP. Therefore, Survivin assisted XIAP in inactivating caspase-3 and inhibiting apoptosis.

It has been shown that inhibiting Survivin increases the sensitivity of cancer cells to chemo- or radiotherapy, in vitro and in vivo. For example, YM155, a small molecule inhibitor of Survivin, is potent in targeting various renal cancer and lymphoma cell lines. It inhibits the transcription of Survivin and participates in numerous antitumor activities (28–30). LY2181308, a Survivin antisense oligonucleotide, has been proven to be effective by itself or in combination with chemotherapy in pre-clinical and clinical studies. However, these small molecules have many adverse effects in patients, including fatigue, nausea, pyrexia or even renal failure (31,32). In the present study, we reported that hyperthermia is a new method to treat cancer by inhibiting Survivin. Due to its low adverse effects, hyperthermia behaved as a better Survivin inhibitor and improved the function of chemotherapy or radiotherapy for esophageal cancer patients. However, more studies are required to precisely identify the effects of hyperthermia in esophageal cancer models and to determine whether there is synergy in combination with chemotherapy or radiotherapy.

In conclusion, hyperthermia decreases the expression of Survivin, prevents its binding to XIAP, activates caspase-3 and induces apoptosis (Fig. 8). Additionally, the expression of Survivin is higher in esophageal cancer than in the normal tissues, and its higher level in cancer is associated with poor prognosis. Therefore, Survivin may be an essential target for hyperthermia in the treatment of esophageal cancer.

Figure 8 A model for hyperthermia induction of apoptosis by targeting Survivin. Hyperthermia decreases the expression of Survivin, prevents its binding to XIAP, activates caspase-3 and induces apoptosis.

Acknowledgments

We would like to thank J.R. Skaar for critically reading the manuscript. The present study was supported by the National Natural Science Foundation of China, approved ID nos.: 81402506 and 81272418.

References