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Abstract

BACKGROUND:

Expressions of Caspase-8 and Caspase-3 have been identified as important markers in many malignant tumors, but their roles in colorectal cancer (CRC) have not been confirmed. The purpose of this study was to investigate the role of Caspase-8 and Caspase-3 in CRC.

METHODS:

We enrolled 470 CRC patients in this study. Archival paraffin-embedded CRC tissue samples were used to construct tissue microarray (TMA), expressions of Caspase-8 and Caspase-3 that were stained by immunohistochemistry. Prognostic and predictive role of Caspase-8 and Caspase-3 expressions, alone or united, were evaluated by univariate and multivariate analysis respectively.

RESULTS:

In comparison with adjacent normal tissues, Caspase-8 and Caspase-3 protein levels were upregulated in CRC tissues significantly, furthermore, high expressions of Caspase-8 and Caspase-3 were correlated with decreased overall survival (OS) ( $p<$ 0.05), and also with unfavorable clinicopathologic characteristics. Cox regression analysis showed that high Caspase-8 and Caspase-3 expressions were independent negative markers of OS.

CONCLUSION:

Caspase-8 and Caspase-3 expressions in tumor tissues are novel candidate prognostic markers for CRC patients. It was the first time to be identified that Caspase-8 and Caspase-3 expressions had synergistic role as efficient prognostic indicators for CRC patients.

Keywords

Caspase-8 Caspase-3 colorectal cancer (CRC)diagnosis prognosis

1. Introduction

Colorectal cancer (CRC) is the fourth most commonly cancer in Asia [1]. The CRC incidence trend in some regions, for example Beijing in China, is increasing especially in older groups of patients, while in other regions such as New York, it is decreasing in older age groups [2]. However, among younger adults in the whole world, the incidence trend of CRC is Increasing [3, 4]. Most people are diagnosed with CRC at the advanced stage usually with local lymph node or distant metastasis in the whole world. Despite the application of multidisciplinary treatment strategies including surgical operation, perioperative chemotherapy, radiotherapy and targeted therapy, the actual 5-year survival rate for advanced CRC is 60% or less, because of its invasion and metastasis [5, 6, 7]. In the past recent years, some genes have been identified as significant biomarkers such as septin9, which is a good diagnostic biomarker complementary to fecal occult blood test (FOBT) as a screening tool for CRC [8], the sensitivity for CRC detection was 73.3% (95% CI 63.9–80.9%) and 68.0% (95% CI 58.2–76.5%) for Septin9 and fecal immunochemical test (FIT), respectively. Specificity of the Epi proColon test was 81.5% (95% CI 75.5–86.3%) compared with 97.4% (95% CI 94.1–98.9%) for FIT [9]. Nevertheless, there is still no definite prognostic factors for CRC to guide therapy. Therefore, we need to find some molecular markers associated with CRC to guide clinical therapy urgently.

Cell apoptosis or named programmed cell death (PCD), is a process of cell physiological self-destruc-tion, which plays a very important role in embryonic development, biological homeostasis and defense against of the external and internal injury of multicellular organisms [10, 11]. Proteases of the caspase family, including Caspase-2, 3, 6, 7, 8, 9, 10 et al. are the initiators (2, 8, 9, 10) and executioners (3, 6, 7) of proteolysis [12, 13, 14]. Caspase-8 is one of initiators of proteolysis that not only can cut PARP (poly ADP-ribose polymerase) but also is involved in the activation of other proteases such as Caspase-3 (32 KD) [15, 16]. Executioner Caspase-3 is the key implementation protease of PCD, in which it functions in many ways in apoptotic signal transduction [17, 18, 19, 20]. Caspase-3 exists in the form of zymogen in cytoplasm, which is activated in the early stage of apoptosis, eventually lead to cell apoptosis. But in the late stage of apoptosis and death cells, the activity of Caspase-3 significantly decreased. It was reported that brewers’ rice can induce apoptosis and inhibit the proliferation of HT-29 cells via activation of Caspase-8 and Caspase-3 and down regulation the Wnt/ $\beta$ -catenin downstream signaling in vivo [21]. Caspase-8 activity assay was proposed as a functional predictive biomarker that could allow better prediction of the response to IAPi $+$ TRA-based therapies than the analysis of expression levels of protein biomarkers in breast cancer cell lines [22]. The fact that the activity of Caspase-3 was significantly increased by Coptis japonica treatment, suggests that Coptis japonica could induce apoptotic anticancer effect through Caspase-3 activation on SNU-668 human gastric cancer cells [18]. Imaging Caspase-3 activation is a marker of apoptosis-targeted treatment response in cancer [23]. However, so far, no one has studied the correlation between Caspase-8 and Caspase-3 in CRC.

Our previous studies have shown that cullin1, MMP-2 and c-Myc expressions are novel diagnostic and prognostic markers for CRC. CRC patients with low expression of both cullin1 and c-Myc, or both cullin1 and MMP-2 had a favorable survival outcome [24, 25]. In this study, we aimed to elucidate the expression patterns of Caspase-8 and Caspase-3 in a CRC patients cohort, and to examine the possibility of Caspase-8 and Caspase-3 alone or together as a prognostic and predictive biomarkers of CRC.

2. Materials and methods

2.1 Patients and samples

The study was approved by Institutional Review Boards of Yixing Hospital Affiliated to Medical College of Yangzhou University. All 470 CRC patients with histopathologically confirmed CRC were retrospectively recruited to this study in our hospital from January 2000 to December 2006 in our hospital .

Finally, Caspase-8 and Caspase-3 expressions were assessed in 464 and 462 patient tissues, respectively, because of some missing samples. All the patients were followed up for at least 5 years. Overall survival (OS) was the primary endpoint of this study, and the OS was calculated from the date of surgery to the date of death or to the last follow-up. The clinicopathological features of the CRC patients including age, gender, pathological classification, tumor-node-metastasis (TNM) stage et al. were summarized in Table S1. The database of patients’ death was obtained from follow-up by telephone calls, and was verified by local civil affairs department.

2.2 Tissue microarray (TMA) construction and immunohistochemistry

The CRC TMAs were constructed by contract service at the National Engineering Center for Biochip, Shanghai, China. Briefly, duplicate 1.0-mm diameter cores of tissue from each sample were punched from paraffin tumor block and corresponding non-tumor tissues. As a tissue control, the biopsies of normal colorectal epithelium tissues were inserted in the 4 angles and the center of each slide. A standard protocol used for immunostaining was provided in our previous study [26, 27]. The omission of the primary antibody served as the negative control.

2.3 Assessment of immunohistochemistry

Staining of Caspase-8 and Caspase-3 in the tissues were scored by two independent pathologists who were blinded to the clinical data. Semi-quantitative immunoreactivity score (IRS) was applied as was previously reported [26, 27]. The intensity of immunostaining was shown in Figs S1 and S2. Category A documented the intensity of immunostaining as 0–3 (0, negative; 1, weak; 2, moderate; 3, strong). Category B documented the percentage of immunoreactive cells as 1 (0%–25%), 2 (26%–50%), 3 (51%–75%), and 4 (76%–100%). The concordance for IRS staining score of Caspase-8 and Caspase-3 between the 2 pathologists was 450 (95.7%) and 446(94.9%) in 470 samples respectively and the discrepancies were resolved by consensus using a multihead microscope.

The optimum cutoff value of IRS is obtained by receiver operator characteristic (ROC) analysis, the area under the curve (AUC) at different cutoff values of Caspase-8 and Caspase-3 IRS for 1, 3, and 5 years of OS time was calculated. The optimal value of the cutoff point for IRS of the Caspase-8 was 4 and for Caspase-3 was 6 and these cutoff points were the best for evaluation of relation of IRS with death date (Supplementary Figs S3 and S4). Under these conditions, samples with IRS 0–3 and IRS 4–12 as well as IRS 0–5 and IRS 6–12 were classified as low and high expression of Caspase-8 or Caspase-3 in CRC tumors, respectively.

2.4 Western blotting

Western blot analyses were performed as previously described [24, 27]. The monoclonal rabbit anti-Caspase-8 antibody (1:1,000 dilution, Epitomics, California, USA), the rabbit polyclonal anti-Caspase-3 antibody (1:1,000, dilution; Abcam), and monoclonal mouse anti- $\beta$ -actin antibody (1:2,000 dilution, Cell Signaling Technology, MA, USA) were used as the primary antibodies. Immunoreactive bands were detected with a Phototo pe-horseradish peroxide Western blot detection kit (Cell Signaling Technology Inc.). The intensity of Caspase-8 and Caspase-3 protein bands were analyzed by densitometry, after normalization to the corresponding $\beta$ -actin level.

2.5 Statistical analysis

The associations between Caspase-8/Caspase-3 expressions and clinicopathologic data were evaluated by Fisher’s exact test. The statistical processing was executed using SPSS 20.0 software (SPSS, Inc, Chicago, IL,USA). The significance of correlations of Caspase-8 and Caspase-3 staining in primary CRC tumors and their corresponding non-tumors counterparts were assessed by paired Wilcoxon test (grouped) and Spearman rank-order correlation (raw scores). The correlation of Caspase-8 and Caspase-3 expressions was established by Spearman rank-order correlation (raw scores) and Fisher’s exact test (grouped). Probability of differences in OS as a function of time was ascertained by use of the Kaplan–Meier method, with a log-rank test probe for significance. Univariate or multivariate Cox regression analysis was used to estimate the crude hazard ratios (HRs), adjusted HRs and their 95% confidence intervals (CIs), with adjustment for potential confounders. Then we analyzed the predictive value of the parameters using time-dependent ROC curve analysis for censored data and calculated AUC of the ROC curves. We evaluated the performances of different scores by plotting (t, AUC[t]) for different values of follow-up time (t). All the statistical analyses were conducted by STATA statistical software (version 10.1; StataCorp College Station, TX, USA). P-value of $<$ 0.05 was considered statistically significant, and all tests were two-sided.

3. Results

3.1 Association of Caspase-8 and Caspase-3 expressions with clinicopathologic characteristics in CRC patients

The expression of Caspase-8 protein was significantly associated with clinicopathologic characteristics including depth of invasion (T-category) and lymph node metastasis (N-category) in the CRC tissues (Table 1), the significant association was also seen in protein expression of Caspase-3 with depth of invasion (T-category) and lymph node metastasis (N-category) in the CRC tissues (Table 2). Nevertheless, the expressions of Caspase-8 and Caspase-3 had no correlation with other clinicopathologic characteristics of CRC patients such as age, gender, pathological classification of CRC, tumor diameter and distant metastases.

Table 1
Relationship between expression levels of Caspase-8 and clinicopathological features in CRC patients

Variables	$n=$ 462 cases
	Low (%)	Hsigh (%)	$P^{\rm a}$
All patients	127 (27.4)	337 (72.6)
Age (years)			0.519
$\leqslant$ 65	72 (27.3)	192 (72.7)
$>$ 65	55 (27.5)	145 (72.5)
Gender			0.311
Males	73 (26.4)	204 (73.6)
Females	54 (28.9)	133 (71.1)
Pathological classification ${}^{\rm b}$			0.272
I	3 (60.0)	2 (40.0)
II	113 (27.1)	305 (72.9)
III	9 (25.0)	27 (75.0)
Depth of invasion ${}^{\rm b}$			$<$ 0.001
T1/T2	52 (50.5)	51 (49.5)
T3/T4	74 (20.7)	283 (79.3)
Lymph node metastasis ${}^{\rm b}$			0.010
N0	86 (31.5)	187 (68.5)
N1/N2	40 (21.3)	148 (78.7)
TNM stage ${}^{\rm b}$			$<$ 0.001
I	45 (51.1)	43 (48.9)
II	40 (22.7)	136 (77.3)
III	40 (22.5)	138 (77.5)
IV	1 (5.9)	16 (94.1)
Tumor diameter ${}^{\rm b}$			0.216
$\leqslant$ 5 cm	105 (28.1)	268 (71.9)
$>$ 5 cm	21 (23.3)	69 (76.7)
Distant metastasis			0.070
M0	125 (28.1)	320 (71.9)
M1	2 (10.5)	17 (89.5)

${}^{\rm a}$ Two-sided Fisher’s exact tests; ${}^{\rm b}$ Some patients missing these clinical pathological parameters.

Table 2

Relationship between expression levels of Caspase-3 and clinicopathological features in CRC patients

Variables	$n=$ 462 cases
	Low (%)	High (%)	$P^{\rm a}$
All patients	270 (58.4)	192 (41.6)
Age (years)			0.498
$\leqslant$ 65	152 (58.2)	109 (41.8)
$>$ 65	118 (58.7)	83 (41.3)
Gender			0.395
Males	160 (57.8)	117 (42.2)
Females	110 (59.5)	75 (40.5)
Pathological classification ${}^{\rm b}$			0.505
I	4 (80.0)	1 (20.0)
II	243 (58.4)	173 (41.6)
III	19 (52.8)	17 (47.2)
Depth of invasion ${}^{\rm b}$			$<$ 0.001
T1/T2	85 (83.3)	17 (16.7)
T3/T4	182 (51.1)	174 (48.9)
Lymph node metastasis ${}^{\rm b}$			$<$ 0.001
N0	189 (69.5)	83 (30.5)
N1/N2	78 (41.7)	109 (58.3)
TNM stage ${}^{\rm b}$			$<$ 0.001
I	74 (85.1)	13 (14.9)
II	108 (61.4)	68 (38.6)
III	77 (43.5)	100 (56.5)
IV	7 (41.2)	10 (58.8)
Tumor diameter ${}^{\rm b}$			0.543
$\leqslant$ 5 cm	217 (58.3)	155 (41.7)
$>$ 5 cm	52 (58.4)	37 (41.6)
Distant metastasis			0.222
M0	261 (58.9)	182 (41.1)
M1	9 (47.4)	10 (52.6)

${}^{\rm a}$ Two-sided Fisher’s exact tests; ${}^{\rm b}$ Some patients missing these clinical pathological parameters.

3.2 Caspase-8 and Caspase-3 expressions in CRC versus noncancer tissues

Eight pairs of human CRC samples, including tumor tissues and adjacent normal tissues were used to test Caspase-8 and Caspase-3 proteins expressions by Western blot. Tested by Western blot, Caspase-8 and Caspase-3 proteins expressions, compared with the paired adjacent normal tissues, significantly increased in all of CRC tissues (Fig. 1A). Caspase-8 and Caspase-3 proteins expressions were also detected by immunohistochemical staining of the colorectal TMA in 450 and 446 CRC patients respectively as well as matched normal mucosa (Fig. 1B and C). Tested by immunohistochemical staining, Caspase-8 and Caspase-3 expressions were both upregulated in tumor tissues compared with that noted in the paired adjacent noncancer tissues ( $P<$ 0.001; Fig. 1D and E).

3.3 Correlation of Caspase-8, Caspase-3 expressions with OS in CRC patients

Our data revealed that significantly higher Caspase-8 and Caspase-3 expression in colorectal cancer tissues was correlated with shorter OS in CRC patients using Kaplan-Meier survival assay ( $P<$ 0.001 and $P<$ 0.001, respectively, log-rank test; Fig. 2). Caspase-8 and Caspase-3 expressions in colorectal cancer tissues were independent markers for the prognosis of CRC patients by univariate and multivariate Cox regression analysis. The univariate Cox regression analysis also showed that age, pathological classification, depth of invasion, lymph node metastasis, TNM stage , distant metastasis and Caspase-8 as well as Caspase-3 expression were positively negatively associated with OS of the CRC patients (Table 3) .The multivariate Cox regression analysis also revealed that high Caspase-8 expressions was an independent positive prognostic factors for CRC patients (HR, 0.579, 95% CI, 0.400-0.839, $P<$ 0.05; Table 4).Similarly, Caspase-3 expression was also an independent and unfavorable prognostic factor for CRC patients (HR, 0.539,95% CI 0.401–0.723, $P<$ 0.001; Table 4).

Table 3
Univariate Cox regression analysis of Caspase-8 or Caspase-3 expression and clinicopathological variables predicting survival in patients with CRC patients

Variables	$n=$ 470 cases
	HR (95 % CI)	$P$
Age ( $\leqslant$ 65 vs. $>$ 65)	1.607 (1.215–2.126)	0.001
Gender (male vs. female)	1.013 (0.762–1.347)	0.927
Pathological classification (I/II vs. III)	2.475 (1.587–3.860)	$<$ 0.001
Depth of invasion (T1/T2 vs. T3/T4)	3.687 (2.270–5.990)	$<$ 0.001
Lymph node metastasis (N0 vs. N1/N2)	2.807 (2.112–3.731)	$<$ 0.001
TNM stage (I/II vs. III/IV)	3.214 (2.407–4.291)	$<$ 0.001
Distant metastasis (M0 vs. M1)	8.150 (4.849–13.699)	$<$ 0.001
Tumor diameter ( $\leqslant$ 5 cm vs. $>$ 5 cm)	1.196 (0.848–1.688)	0.307
Caspase-8 expression (low vs. high)	0.534 (0.371–0.767)	0.001
Caspase-3 expression (low vs. high)	0.437 (0.329–0.582)	$<$ 0.001

Figure 1.

A: Caspase-8 or Caspase-3 was elevated in CRC and associated with poor prognosis of CRC patients. Expression of Caspase-8 or Caspase-3 was increased in cancer tissues (C) compared with paired normal colonic tissues (N) by Western blot. B, D: Representative images of Caspase-8 or Caspase-3 immunohistochemical staining in TMA are showed, respectively. Note: Top panel: original magnification, 40 $\times$ ; bottom panel: 200 $\times$ . C, E: The distribution of the difference of Caspase-8 Caspase-3 staining in CRC compared with paired normal tissues, respectively.

Table 4

Multivariate Cox regression analysis of Caspase-8, Caspase-3, Caspase-8/Caspase-3 expression and clinicopathological variables predicting survival in patients with CRC

Variables	HR (95% CI)	$P^{\rm a}$
Caspase-8
Age ( $\leqslant$ 65 vs. $>$ 65)	1.886 (1.413–2.517)	$<$ 0.001
Gender (male vs. female)	0.916 (0.685–1.226)	0.557
Pathological classification (I/II vs. III)	1.849 (1.128–3.032)	0.015
Distant metastasis (M0 vs. M1)	4.312 (2.467–7.537)	$<$ 0.001
TNM stage (I/II vs. III/IV)	3.054 (2.249–4.147)	$<$ 0.001
Tumor diameter ( $\leqslant$ 5 cm vs. $>$ 5 cm)	1.073 (0.738–1.561)	0.712
Caspase-8 expression (low vs. high)	0.579 (0.400–0.839)	0.004
Caspase-3
Age ( $\leqslant$ 65 vs. $>$ 65)	1.885 (1.415–2.510)	$<$ 0.001
Gender (male vs. female)	0.872 (0.650–1.168)	0.358
Pathological classification (I/II vs. III)	1.779 (1.092–2.899)	0.021
Distant metastasis (M0 vs. M1)	4.578 (2.624–7.987)	$<$ 0.001
TNM stage (I/II vs. III/IV)	2.812 (2.059–3.841)	$<$ 0.001
Tumor diameter ( $\leqslant$ 5 cm vs. $>$ 5 cm)	1.108 (0.762–1.609)	0.591
Caspase-3 expression (low vs. high)	0.539 (0.401–0.723)	$<$ 0.001
Caspase-8/Caspase-3
Age ( $\leqslant$ 65 vs. $>$ 65)	1.916 (1.442–2.545)	$<$ 0.001
Gender (male vs. female)	0.875 (0.655–1.168)	0.364
Pathological classification (I/II vs. III)	1.725 (1.067–2.789)	0.026
Distant metastasis (M0 vs. M1)	4.542 (2.615–7.889)	$<$ 0.001
TNM stage (I/II vs. III/IV)	2.838 (2.088–3.858)	$<$ 0.001
Tumor diameter ( $\leqslant$ 5 cm vs. $>$ 5 cm)	1.146 (0.796–1.649)	0.463
Caspase-8/Caspase-3 expression
Both low vs. one low	0.769 (0.649–0.910)	0.002
Both low vs. both high	0.430 (0.283–0.655)	$<$ 0.001

${}^{\rm a}$ Multivariate Cox regression analysis including age, gender, pathological classification, distant metastasis TNMstage, tumor diameter, Caspase-8 or Caspase-3 or combined 2 proteins expression status.

Figure 2.

Kaplan-Meier curves depicting OS according to expression patterns of Caspase-8, Caspase-3, and combined with Caspase-8/Caspase-3 expression in training cohort (A–C). P values were calculated with the log-rank test.

3.4 Synergistic effect of Caspase-8 and Caspase-3 expression on OS

We conducted a time-dependent ROC analysis for the censored data in order to further evaluate whether Caspase-8 and Caspase-3 expressions have a synergetic effect on the prognosis of CRC patients. The results of above ROR analysis indicated that combination of clinical risk score (TNM stage, histological type and tumor diameter) and Caspase-8 or Caspase-3 as well as Caspase-8 plus Caspase-3 expressions contributed much more to prognosis CRC patients than clinical parameters alone (Fig. 3). For instance, the AUC at year 5 was 0.633 (95% CI: 1.249–1.841) for clinical risk score, whereas it was significantly increased to 0.717 (95% CI: 1.434–2.778) when combination of the clinical risk score with Caspase-8 plus Caspase-3 risk score.

Figure 3.

Time-dependent ROC analyses for clinical risk score (TNM stage, histologic type, and tumor diameter), or the combination of Caspase-8, Caspase-3, or Caspase-8 plus Caspase-3. AUC $=$ area under the curve.

The stratified analysis showed that CRC patients with both high expressions of Caspase-8 and Caspase-3 had a more unfavorable outcome of survival ( $P<$ 0.001; log-rank test; Fig. 2) compared with one caspase low or both caspases low expression groups. The prognosis was best when CRC patients had low expressions of Caspase-8 and Caspase-3. As well, the multivariate Cox regression analysis indicated that low Caspase-8 or Caspase-3 expression alone was a favorable independent prognostic factor for CRC patients ( $P<$ 0.05 for both; Table 4).

4. Discussion

CRC was the third most commonly diagnosed cancers (1.36 million) worldwide in 2012 reported by GLOBOCAN [28, 29, 30]. Nowadays, TNM staging system for CRC is the main prognostic system [31]. However, it is not sufficiently reliable because of the cell heterogeneity of CRC tumors. Therefore, novel prognostic and predictive biomarkers are demanded to predict outcomes and determine personalized treatment plan.

Caspases family, especially Caspase-8 and Caspase-3, plays an important role in tumors apoptosis [21, 32, 33]. The activation of Caspase-8 could form a metastasis and invasion signaling complex, resulting in activation of ERK and JNK/AP-1 signaling pathway that mediate the elevation and activation of matrix metalloproteinase-1 (MMP1) and eventual promotion of cancer invasion and metastasis [32]. Caspase-8 activation is the proapoptotic pathway in human colon cancer cell lines in contact with proanthocyanidins extracted from various berries [34]. In the same time, Caspase-3 activation is confirmed to be related to the NF- $\kappa$ B-mediated Bcl-2 downregulation which may be the underlying mechanism of IFN- $\gamma$ - and TNF- $\alpha$ -mediated MIN6 cells apoptosis [35]. MiR-143 could inhibit Bcl-2 expression, causing Caspase-3 activation, thus inducing apoptosis in osteosarcoma cells [36]. Although there are more studies on the roles of Caspase-8 and Caspase-3 of tumor cells apoptosis, the relationship between Caspase-8, Caspase-3 and CRC prognosis has not been studied.

In present study, our results showed that Caspase-8 expression is higher in CRC tumor tissues versus matched adjacent normal tissues. High expression of Caspase-8 correlated significantly with depth of invasion, lymph node metastasis and TNM stage in CRC tumor tissues. Furthermore, the patients with high Caspase-8 expression in tumor tissues had poor OS in CRC patients using Kaplan-Meier survival analysis. Univariate and multivariate Cox proportional hazards regression analyses showed that Caspase-8 expression is an independent negative prognostic factor of CRC.

Caspase-3 expression was also upregulated in tumor tissues compared with that noted in the paired adjacent normal tissues in both CRC fresh tissues and a TMA cohort. Our results demonstrated that high Caspase-3 expression in CRC cancer tissues was correlated significantly with depth of invasion, lymph node metastasis and TNM stage. High Caspase-3 expression was also correlated with worse OS and was found to be an independent negative prognostic factor in CRC patients.

In our cohort of CRC patients, significantly increased expressions of Caspase-8 and Caspase3 correlated significantly with unfavorable clinicopathologic parameters and worse OS. It was the first time to be found that significantly increased Caspase-8 expression combined with significantly increased Caspase-3 expression had synergistic potential role in predicting the unfavorable prognosis of CRC patients and may be more effective than Caspase-8 or Caspase-3 expressions alone

In conclusion, our findings clearly demonstrated that high Caspase-8 and Caspase-3 expressions in CRC tumor tissues are associated with poor prognosis in CRC patients. We identified a combined value of Caspase-8 with Caspase-3 expressions as potential prognostic and predictive biomarkers for CRC for the first time.

Footnotes

Acknowledgments

The present study was supported in part by Young Medicine Focus Talent Foundation of Jiangsu Province (NO. QNRC2016206).

Conflict of interest

None.

Supplementary data

Table S1

The patients’ clinicopathological features in CRC

Variables	n
All patients	470	(%)
Age (years)
⩽ 65	267	56.8
> 65	203	43.2
Gender
Males	281	59.8
Females	189	40.2
Pathological classification b
I	5	1.1
II	423	91.2
III	36	7.7
Depth of invasion b
T1	9	2.0
T2	94	20.2
T3	347	74.6
T4	15	3.2
Lymph node metastasis b
N0	276	59.2
N1	126	27.0
N2	64	13.8
TNM stage b
I	88	18.9
II	179	38.6
III	180	38.8
IV	17	3.7
Tumor diameter b
⩽ 5 cm	378	80.6
> 5 cm	91	19.4
Distant metastasis
M0	451	95.9
M1	19	4.1

b Some patients missing these clinical pathological parameters.

Figure S1.

Representative images of Caspase-8 immunohistochemical staining in normal colorectal tissue and colorectal cancer tissue. A, B, C, D is adjacent normal tissue; E, F, G, H is cancer tissue. A, E, Negative staining. B, F, Weak staining. C, G, Moderate staining. D, H, Strong staining. All panels: original magnification, 40 × .

Figure S2.

Representative images of Caspase-3 immunohistochemical staining in normal colorectal tissue and colorectal cancer tissue. A,B,C,D is adjacent normal tissue; E,F,G,H is cancer tissue. A, E, Negative staining. B, F, Weak staining. C, G, Moderate staining. D, H, Strong staining. All panels: original magnification, 40 × .

Figure S3.

Receiver operating characteristic (ROC) curves were obtained to show the relation between area under the curve (AUC) at different cutoff values of Caspase-8 immunoreactivity score (IRS) for 1, 3 and 5 years of overall survival time.

Figure S4.

Receiver operating characteristic (ROC) curves were obtained to show the relation between area under the curve (AUC) at different cutoff values of Caspase-3 immunoreactivity score (IRS) for 1, 3 and 5 years of overall survival time.

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Synergistic role of Caspase-8 and Caspase-3 expressions: Prognostic and predictive biomarkers in colorectal cancer

Abstract

BACKGROUND:

METHODS:

RESULTS:

CONCLUSION:

Keywords

1. Introduction

2. Materials and methods

2.1 Patients and samples

2.2 Tissue microarray (TMA) construction and immunohistochemistry

2.3 Assessment of immunohistochemistry

2.4 Western blotting

2.5 Statistical analysis

3. Results

3.1 Association of Caspase-8 and Caspase-3 expressions with clinicopathologic characteristics in CRC patients

Table 1 Relationship between expression levels of Caspase-8 and clinicopathological features in CRC patients

3.3 Correlation of Caspase-8, Caspase-3 expressions with OS in CRC patients

Table 3 Univariate Cox regression analysis of Caspase-8 or Caspase-3 expression and clinicopathological variables predicting survival in patients with CRC patients

Footnotes

Acknowledgments

Conflict of interest

Supplementary data

References

Table 1
Relationship between expression levels of Caspase-8 and clinicopathological features in CRC patients

Table 3
Univariate Cox regression analysis of Caspase-8 or Caspase-3 expression and clinicopathological variables predicting survival in patients with CRC patients