Tumor Biol. DOI 10.1007/s13277-016-5040-z

ORIGINAL ARTICLE

The roles of serum PDCD5 in circulating CD133 positive cells of the patients with gastric cancer Dan Wang 1 & Wei Wang 2 & Chang-Liang Song 3 & Pu Xia 4,5

Received: 5 February 2016 / Accepted: 28 March 2016 # International Society of Oncology and BioMarkers (ISOBM) 2016

Abstract Cancer stem cells are responsible for the development, metastasis, recurrence, and drug resistance of cancer. More and more studies exhibited that the circulating CD133+ cells is a marker for the prognosis of various malignancies. Programmed cell death protein 5 (PDCD5) can promote apoptosis in different tumor cell types in response to various stimuli. However, the impact of PDCD5 on circulating CD133+ cells of gastric cancer patients remains unclear. In this study, we detected serum PDCD5 level in blood samples of the patients with gastric cancer by using ELISA. MTT assay, sphere assay, and wound healing assay were used to test the anti-tumor effects of rhPDCD5 on CD133 + cells in vitro. Lower serum levels of PDCD5 protein were identified in the gastric cancer patients that with CD133+ fraction more than 1.6 %. No difference between healthy controls and the gastric cancer patients that with CD133 + fraction less than 1.6 %. Serum PDCD5 was correlated with the favorable prognosis of

* Pu Xia [email protected]

1

Department of Histology and Embryology, College of Basic Medical Science, Liaoning Medical University, Jinzhou, China

2

Department of Pathophysiology, College of Basic Medical Science, China Medical University, Shenyang, People’s Republic of China

3

Department of Radiotherapy, Center Hospital of Handan, Handan, China

4

Department of Cell Biology, College of Basic Medical Science, Liaoning Medical University, Jinzhou, China

5

Department of Cell Biology, Basic Medical College of Liaoning Medical University, Jinzhou 121000, Liaoning, People’s Republic of China

patients with gastric cancer. In the last, we confirmed that rhPDCD5 could induce apoptosis, and inhibit the proliferation, colony formation, and mobility of CD133+ cells in vitro by suppressing MEK/ERK pathway. Serum PDCD5 could be considered as a potential drug for the gastric cancer patients with circulating CD133+ cells. Keywords Gastric cancer . Circulating CD133+ cells . PDCD5 . Proliferation . Prognosis

Introduction Cancer stem cells (CSC) are a subgroup of tumor cells that have the capacity of self-renewal and multilineage differentiation [1]. CSCs are responsible for tumor development, metastasis, and recurrence, but also unsuccessful treatment of cancer [2]. It has been reported that CSCs possibly do express their own unique markers, such as aldehyde dehydrogenase 1 (ALDH1) [3], CD44 [4], and CD133 [5]. In our previous study, higher percentages of CD133 + cells were identified in the blood samples from the gastric cancer patients compared with healthy controls and the numbers of CD133+ cells correlated with poor prognosis of these patients [6]. Programmed cell death protein 5 (PDCD5) was first discovered in 1999 as a gene upregulated in TF-1 cells undergoing apoptosis [7]. Previous studies indicated that PDCD5 expression is downregulated in a variety of various human tumors, including hepatocellular carcinoma [8], breast cancer [9], and lung cancer [10], and overexpression of PDCD5 can promote apoptosis in different tumor cell types in response to various stimuli [11, 12]. Furthermore, increased serum PDCD5 was detected in

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patients with rheumatoid arthritis [13], multiple sclerosis [14], and systemic lupus erythematosus [15]. However, serum PDCD5 levels in the patients with gastrointestinal tract cancer were no statistical significance compared with that in healthy controls [16]. To our knowledge, the impact of PDCD5 on circulating CD133+ cells of gastric cancer patients remains unclear. In this study, we first explored the serum PDCD5 levels in patients with gastric cancer. Second, we compared the relationship between the serum PDCD5 levels and the number of circulating CD133+ cells from these patients. In the last, the mechanisms of PDCD5 in CD133+ cells were identified in vitro.

Materials and methods Gastric cancer patient blood samples The clinical investigation was conducted according to the principles expressed in the Helsinki Declaration of 1975. Blood samples were obtained from 36 patients with gastric cancer and 16 healthy individuals. The detailed information of these patients can be found in our previous paper [6].

Fig. 1 Serum PDCD5 in gastric cancer specimens. a PDCD5 serum levels from the patients with gastric cancer and healthy controls were detected by using enzyme-linked immunosorbent assay. b KaplanMeier curves of the cumulative survival rate of the patients with gastric cancer based on their PDCD5 serum levels. c ROC curves analysis for

Enzyme-linked immunosorbent assay Peripheral blood was obtained from the patients and healthy volunteers as described above. Samples were clotted for 30 min and then centrifuged for 10 min at 1000×g. Concentration of PDCD5 in serum was assayed using enzyme-linked immunosorbent assay (ELISA) kit for PDCD5 (USCN Life Science Inc., Houston, TX, USA). Cell proliferation As the methods of our previous study [6], CD133+ cells (1000 cells/well) were seeded on 96-well plate and allowed to adhere. After 24 h, cells were treated with various concentrations of rhPDCD5 protein (e.g., 0, 2.5, 5, 10, 20 μg/ml for each). After 24 h, 10 μl of CCK-8 solution (Dojindo Molecular Technologies, Rockville, MD) was added into each well of the plate, and the plates were incubated for 4 h in the incubator and absorbance rates were measured at 450 nm using a microplate reader (Bio-Rad, Hercules, CA). Sphere assay As the methods of Gibbs and his colleagues [15], CD133+ cells were plated at a density of 6 × 104 cells/well in six-well

PDCD5 diagnosis in these patients. d–f Correlation between serum PDCD5 and circulating CD133+ cells in healthy controls and gastric cancer patients. Control: healthy controls; Group 1: CD133+ cells/total cells <1.6 %; Group 2: CD133+ cells/total cells >1.6 %

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plates under serum-free, sphere-specific conditions. After 24 h, various concentrations of rhPDCD5 protein (e.g., 0, 2.5, 5, 10, 20 μg/ml) were added into each well. Fresh aliquots of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were added every day. After culture for 3 days, spheres were visible under a light microscope (Olympus CX31, Olympus, Tokyo, Japan). In vitro wound healing assay CD133 + cells treated with various concentrations of rhPDCD5 were grown in a six-well dish. A confluent monolayer of cells was scratched with a 200-μl pipette tip to simulate a wound. Cell migration into the wounded area was monitored microscopically. Images were captured at the interface of the unwounded and wounded areas. Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining To further determine the cytotoxic effect of exposure to PDCD5 on CD133+ cells, apoptotic rates were determined with AnnexinV-FITC/PI double staining assays (Apoptosis Detection Kit, KeyGEN, Nanjing, China). Cells were washed and resuspended in binding buffer prior to the addition of FITClabeled Annexin V and PI for 10 min. Suspensions were immediately analyzed by flow cytometry using a FACSCalibur machine (BD Biosciences, Baltimore, MD, USA). Western blot Proteins were extracted from cells by using the Total Protein Extraction Kit (KeyGEN, Nanjing, China) and measured using the BCA Protein Assay Kit (KeyGEN) according to the manufacture’s instruction. Proteins (45 μg) were denatured at 95 °C and then resolved on 8 % SDS-PAGE and transferred to the PVDF membranes (Millipore Corporation, Bedford, MA) using the wet transfer blotting system (Bio-Rad, Hercules, CA). Membranes were blocked with 2.5 % nonfat milk, and probed with antibodies. The primary antibodies were caspase 9, caspase 3, ERK, phospho-ERK, MEK, phospho-MEK, and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA) and the s e co n d a r y a n t i b o dy w a s a l k a l i n e p h o s ph a t a s e conjugated IgG (KeyGEN). Protein was detected using the ECL Western Blotting Analysis System (Amersham Biosciences, Piscataway, NJ). Statistical analysis Data were analyzed using GraphPad Prism 5 software (GraphPad Software, San Diego, CA). Statistical analysis was performed using a one-tailed Student’s t test (unilateral

and unpaired). Kaplan-Meier survival plots were generated and comparisons between survival curves were made using the log-rank statistical analysis. P values <0.05 were considered to indicate statistically significant differences.

Results The serum levels of PDCD5 in the patients with gastric cancer PDCD5 protein levels in blood samples were determined by using ELISA (Fig. 1a). The serum PDCD5 in 36 patients ranged from 0.8 to 11.4 ng/ml with a median of 2.6 ng/ml. In our previous study [6], we found that the CD133+ fraction comprised 0.4–4.1 % (mean = 1.6 %) total peripheral blood mononuclear cells in these gastric cancer patients. Based on Ta b l e 1 R e l a t i o n s h i p b e t w e e n s e r u m P D C D 5 a n d t h e clinicopathological parameters of the gastric cancer patients Clinicopathological features

Serum PDCD5 n

Low

High

13 23

9 11

4 12

11

3

8

25

17

8

Sex Female Male Age (years) <65 ≥65 Differentiation Differentiated Undifferentiated Lymphatic invasion − + Venous invasion − + Lymph node metastasis − + Tumor size <4 cm ≥4 cm pN category pN0 pN1 pN2 pN3

17 19

10 10

7 9

12

5

7

24

15

9

21 15

6 14

15 1

14 22

4 16

10 6

18 18

7 13

11 5

10 8

7 5

3 3

7 11

4 4

3 7

χ2

P

0.796

.372

3.615

.057

0.001

.970

0.689

.407

12.365

.0004

5.086

.024

2.813

.094

2.649

.449

χ2 value, chi-square distribution Italic values mean the values have statistical significance (<0.05)

Tumor Biol.

our previous results, we divided the patients into two groups (Group 1: CD133+ cells/total cells <1.6 %, Group 2: CD133+ cells/total cells >1.6 %). Lower serum levels of PDCD5 protein were identified in Group 2 (0.8 to 4.1 ng/ml, median = 2.2 ng/ml) compared with that in healthy controls (2.2 to 11.1 ng/ml, median = 5.3 ng/ml) (P < 0.05, Fig. 1a). No significant difference was found between Group 1 (1.1 to 11.4 ng/ml, median = 4.4 ng/ml) and healthy controls (P > 0.05, Fig. 1a). Furthermore, we found that serum PDCD5 were associated with lymphatic invasion (P = 0.024), and venous invasion (P = 0.0004) of gastric cancer. The results of the association of serum PDCD5 with the clinicopathological characteristics of these patients were summarized in Table 1. Serum PDCD5 was correlated with the favorable prognosis of the patients with gastric cancer by using Kaplan-Meier analysis (P < 0.05, Fig. 1b). The ROC curves of serum PDCD5 revealed a strong discrimination between gastric cancer patients with an AUC of 0.922 (P < 0.05, Fig. 1c). The serum levels of PDCD5 protein in these patients were significantly negatively correlated with circulating CD133+ cells in Group 2 (r = −0.7562) (P < 0.05, Fig. 1f), but not in control group (Fig. 1d) and Group 1 (Fig. 1e).

(>10 μg/ml) could inhibit the proliferation of CD133+ cells (P < 0.05, Fig. 2a). The results of sphere assay also showed that CD133+ cells treated with rhPDCD5 (>10 μg/ml) lost the ability of sphere formation (Fig. 2b). Then, we determined whether there were any mobility changes in rhPDCD5treated CD133+ cells by using the wound healing assay. The wound distance of CD133+ cells treated with rhPDCD5 (>10 μg/ml) was increased compared with untreated ones (Fig. 2c). Furthermore, quantitative analysis of apoptosis was conducted via AnnexinV/PI double staining and flow cytometry. The treatment of CD133+ cells with rhPDCD5 (>10 μg/ml) resulted in a significant increased apoptotic ratio compared with untreated ones (Fig. 2d). In the last, western blot was carried out to measure changes in the MEK/ERK signaling pathway. While total levels of MEK and ERK showed no changes, the levels of phosphoMEK and phospho-ERK were decreased in CD133+ cells treated with rhPDCD5 (>10 μg/ml) (Fig. 2e). Caspase 9 and caspase 3 were increased in rhPDCD5-treated CD133+ cells, followed with downregulated phospho-MEK and phosphoERK (Fig. 2e).

Anti-tumor roles and mechanisms of rhPDCD5 in CD133+ cells in vitro

Discussion

MTT assays were performed to detect the proliferation of CD133 + cells treated with various concentrations of rhPDCD5. The histogram indicated that rhPDCD5

Cancer metastasis originated from circulating tumor cells (CTCs) that detached into the vasculature from a primary tumor and can be found in the bloodstream of cancer patients [17]. CTCs harbor rare populations of CD133-positive

Fig. 2 The anti-tumor roles and mechanisms of rhPDCD5 in CD133+ cells in vitro. a The proliferation ratio of CD133+ cells treated with various concentrations of rhPDCD5 was determined by MTT assays. b Representative pictures of a sphere cluster formed by CD133+ cells treated with various concentrations of rhPDCD5. c The mobility of

CD133+ cells treated with various concentrations of rhPDCD5 was detected by using wound healing assay. d The proportion of apoptotic cells (early apoptosis) was determined by double-staining with AnnexinV/FITC and PI. e Western blot analysis of the ERK/MEK signaling pathway. β-actin was used as an internal loading control

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(CD133+) cells [18]. Circulating CD133+ cells have been identified in peripheral blood of patients with various malignancies, such as gastric cancer [6], breast cancer [19], and colorectal cancer [20]. Based on the results of our previous study, we divided the patients of this study into two groups according to the fraction of CD133 + cells/total cells. Interestingly, in this study, lower serum levels of PDCD5 protein were identified in the gastric cancer patients (CD133+ cells/total cells >1.6 %) compared with that in healthy controls. The similar serum PDCD5 levels were found in the gastric patients with CD133+ fraction less than 1.6 % and healthy controls. Increased serum PDCD5 was detected in patients with rheumatoid arthritis [21], multiple sclerosis [14], and hepatitis [22]. However, serum PDCD5 protein levels in cancers still remain unclear. Serum PDCD5 protein in the hepatocellular carcinoma (HCC) patients were significantly higher than that in the healthy controls [8]. In the study of Wang et al. [16], the serum PDCD5 levels in the patients with breast, gastrointestinal, or lung cancer showed no statistical difference compared with healthy controls. Our results that obtained from the gastric cancer patients with CD133+ fraction less than 1.6 % were consistent with the results of Wang et al. [16]. However, low serum level of PDCD5 protein was observed in the gastric cancer patients with CD133+ fraction more than 1.6 %. Furthermore, we found a positive relation between PDCD5 and CD133. Then we used recombinant human programmed cell death 5 (rhPDCD5) to simulate the roles of PDCD5 in CD133+ cells in vitro. In the study of Fu et al. [23], they found that rhPDCD5 inhibited cell proliferation, and induced apoptosis and S-phase arrest in HCC cells. Consistent with their results, we also confirmed that rhPDCD5 inhibited the proliferation and the colony formation, and induced apoptosis of CD133+ cells. In addition, both in vivo and in vitro results confirmed PDCD5 protein could inhibit the mobility of CD133+ cells. MAP kinase ERK kinase (MEK)/extracellular signal regulated kinase (ERK) pathway regulates diverse cellular functions, including cell proliferation, survival, differentiation, angiogenesis, and migration [24, 25]. Han et al. [26] found that PDCD5 mediates anti-tumor effects in the osteosarcoma cell line by suppressing the MEK/ERK signaling pathway. Our results identified rhPDCD5 could inhibit the activation of the MEK/ ERK signaling pathway for the first time. In conclusion, the principal findings of this study are that (1) we identified serum PDCD5 protein in the patients with gastric cancer by ELISA. We found that no difference between healthy controls and the gastric cancer patients that with CD133+ fraction less than 1.6 %. Lower serum levels of PDCD5 protein were identified in the gastric cancer patients that with CD133 + fraction more than 1.6 %. (2) We

demonstrated that serum PDCD5 protein was correlated with the survival rate of these patients. (3) In the last, we identified rhPDCD5 could inhibit the proliferation, colony formation, and mobility of CD133+ cells in vitro by suppressing MEK/ ERK pathway. Acknowledgments This study was supported by National Natural Scientific Foundation of China (No. 81502558) and Talents Introduction Projects of Liaoning Medical University. Compliance with ethical standards Conflicts of interest None

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