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ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 7  |  Page : 1476-1481

Combined effect of 125I and gemcitabine on PANC-1 cells: Cellular apoptosis and cell cycle arrest


1 Department of Interventional Medicine, The Second Hospital of Shandong University, Jinan, China
2 Department of Interventional Medicine, Linzi District People's Hospital, Jinan, China

Date of Web Publication19-Dec-2018

Correspondence Address:
Yu-Liang Li
Department of Interventional Medicine, The Second Hospital of Shandong University, 247 Beiyuan Road, Jinan 250033
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.JCRT_43_18

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 > Abstract 


Background: 125I seed implantation has recently become an effective, safe, and feasible treatment for advanced pancreatic cancer in China. Gemcitabine (GEM), superior to fluorouracil, has been widely proved as effective chemotherapy for many solid tumors and become the standard treatment for locally advanced and metastatic pancreatic cancer. The study aimed to evaluate the combined effect of 125I and GEM on pancreatic carcinoma cells (PANC-1) cells and explore the underlying molecular basis.
Subjects and Methods: PANC-1 cells were treated with 125I continuously at a low dose of radiation, combined with or without sensitizing concentration of GEM. The clonogenic capacity, cellular proliferation, cell cycle distribution, apoptosis, and molecular pathways of the cells following these treatments were analyzed in vitro.
Results: The cell growth could be significantly inhibited after the treatment with GEM or 125I alone, while the inhibition effects would be greater with combination therapy than either monotherapy (72 h, C vs. GEM, t = 16.59, P < 0.01; C vs. 125I, t = 9.808, P < 0.05; C vs. 125I + GEM, t = 17.87, P < 0.01; 125I vs. 125I+GEM, t = 8.191, P < 0.05). GEM increased radiation-induced apoptosis (4 Gy, 125I vs. 125I+GEM, t = 10.43, P < 0.01) and induced the arrest of G1. Caspase-3 expression and the Bax/Bcl2 ratio were lower in cells receiving combination treatment than that of in cells treated with 125I or GEM alone.
Conclusion: The combined treatment of 125I and GEM-induced stronger anti-proliferation effect than single-treatment, due to the cell cycle arrest and more cellular apoptosis in PANC-1 cells. The increased Bax/Bcl-2 ratio may lead to enhanced apoptosis.

Keywords: 125I, gemcitabine, pancreatic cancer


How to cite this article:
Li D, Jia YM, Cao PK, Wang W, Liu B, Li YL. Combined effect of 125I and gemcitabine on PANC-1 cells: Cellular apoptosis and cell cycle arrest. J Can Res Ther 2018;14:1476-81

How to cite this URL:
Li D, Jia YM, Cao PK, Wang W, Liu B, Li YL. Combined effect of 125I and gemcitabine on PANC-1 cells: Cellular apoptosis and cell cycle arrest. J Can Res Ther [serial online] 2018 [cited 2019 Jan 24];14:1476-81. Available from: http://www.cancerjournal.net/text.asp?2018/14/7/1476/247719




 > Introduction Top


Pancreatic cancer is a common digestive system tumor with poor prognosis, and the 5-year survival rate has been <5%.[1] In China, the 5-year survival rate was seven <4% due to the poor local control rate and high distant metastases rate.[2] About 80% of pancreatic cancer patients were not candidates of surgical resection, and most of them have to receive other treatments.[3] Although the pain of patients could be effectively relieved with external irradiation therapy, its sensitivity to pancreatic cancer has still been a problem. GEM, superior to fluorouracil, is the first-choice chemotherapy for pancreatic cancer. However, the drug resistance and complications of GEM has restricted its function, which is one of the reasons for poor prognosis.[4],[5],[6],[7] Due to better local control and less complications, 125I seed implantation has recently become an effective and safe treatment for advanced pancreatic cancer in China.[8] Although 125I or GEM was considered as feasible treatment for pancreatic cancer, their combined effect has not been investigated yet. The study aimed to evaluate the combined effect of 125I and GEM on PANC-1 cells and explore the underlying molecular mechanism of combined treatment.


 > Subjects and Methods Top


Cell culture

The human pancreas cancer cell line PANC-1 was purchased from the Shanghai Zhong Qiao Xin Zhou Biotechnology (Shanghai, China). The cells were incubated in 5% CO2 at 37°C and cultured in DMEM (Corning, NY, USA) supplemented with 10% fetal calf serum (Gibco, MA USA).

Radiation

125I seeds were provided by Ningbo Junan Pharmaceutical Technology Company (Ningbo, China). 125I seeds were seeded into an in vitro irradiation model, based on a previous paper.[9] The initial activity and dose rate were 2.0 mCi and 2.275 cGy/h.

Antibodies and other reagents

Primary antibodies were purchased from Abcam (Cambridge, Cambs, UK), including anti-Caspase-3 (ab13847), anti-Bax (ab32503), and anti-Bcl-2 (ab692). The following secondary antibodies were purchased from GenScript (Piscataway, NJ, USA), including goat anti-rabbit IgG (H and L) and goat anti-mouse IgG (H and L). GEM was purchased from Changan Hainan International Pharmaceutical Co., Ltd (Haikou, China).

Cell counting kit-8 assay and sensitizing concentration

Cells were seeded in 35 mm dishes. After treating with a concentration gradient of GEM from 0 to 100 ug/ml for 72 h, the optical density value was detected with cell counting kit-8 (CCK-8) (Dojindo, Japan). The IC50 was calculated with Prism 6.0 (GraphPad, CA, USA). According to a previously published paper, the sensitizing concentration of GEM was 10% of the IC50.[10]

Cell proliferation assay

After treating with 125I, GEM, or 125I+GEM, the cells were collected and prepared as single cell suspension. The cells were then seeded in 96-well plates, with 5 × 103 cells/well and each well containing 200 μL culture medium. After cells were attached for 24 h; at 24, 48, and 72 h, the medium was replaced with CCK-8 reagent and incubated at 37°C for 3 h. Finally, the plate was scanned with a microplate reader (Thermo, MA, USA).

Colony assay

After treating with 125I, GEM, or combined 125I and GEM, the cells were collected and prepared as single cell suspension. The cells were then seeded in 6-well plates, with 103 cells/well and each well containing 3 mL culture medium. After incubating for 10–14 days, the colonies were washed once with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for at least 1 h, and stained with crystal violet for 30 min. Colonies-containing >50 cells were counted in 3 replicate dishes.

Cell cycle and apoptosis analysis

Cells were seeded into 6-well plates at 2 × 105 cells per well and treated with 125I, GEM, or combined 125I and GEM. For the cell cycle test, after collection, the cells were washed with cold PBS, fixed with 70% cold ethanol and stored at −4°C overnight. Before analysis, the cells were stained with propidium iodide (PI) and RNase A (BD Bioscience, NJ, USA). For the apoptosis analysis, after harvest, cells were either stained with PI or Annexin V-FITC (BD Bioscience, NJ, USA). Both the cell cycle test and apoptosis, analysis were assessed with a flow cytometer (BD Bioscience, NJ, USA).

Transferase-dUTP nick-end labeling assay and 4,-6-diamidino-2-phenylindole staining

After treating with 125I, GEM, or combined 125I and GEM, the cells were washed once with PBS and incubated with PBS containing 0.3% Triton X-100 for 5 min. Then, the cells were fixed in 4% paraformaldehyde for at least 1 h, stained with Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick-End Labeling (TUNEL) reagent (Beyotime Biotechnology, Nanjing, China) for 1 h at 37°C and incubated with 4,-6-diamidino-2-phenylindole for another 15 min. The morphological changes were observed with a fluorescence microscope (Olympus, Tokyo, Japan). Three different fields were selected.

Western blot

After treating with 125I, GEM, or combined 125I and GEM, the cells were washed once with PBS, lysed with RIPA lysis buffer for 20 min on ice, and centrifuged at 12000 r/min for 10 min at 4°C. Protein was denatured at 95°C for 5 min. Samples (10 μg each) were loaded on a 10% SDS-PAGE and transferred onto nitrocellulose membranes (Millipore, MA, USA). Membranes were blocked with 5% nonfat milk in TBST-containing 1% Tween-20 for 1 h, and incubated with corresponding primary antibodies at 4°C overnight. Then, the membranes were washed with TBST 3 times (5 min each time), and incubated with special secondary antibodies for 1 h at room temperature. Protein bands were developed and detected with chemiluminescence (Millipore, MA, USA).

Statistical analysis

Data were expressed as the mean ± standard deviation every experiment was repeated at least twice. GraphPad Prism software (GraphPad, San Diego, CA, USA) version 6.0 was applied to analyze the statistics. A paired t-test was applied when appropriate. Differences were considered statistically significant if P < 0.05 and more statistically significant if P < 0.01.


 > Results Top


Effect of gemcitabine on PANC-1 cells viability

The CCK-8 assay suggested that GEM dose-dependently affected the viability of PANC-1 cells [Figure 1]. The estimated IC50 value of GEM at 72 h was 5.972 μg/ml.
Figure 1: Inhibitory rate of different concentrations of gemcitabine on PANC-1 cells

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The anti-proliferation effects of 125I and gemcitabine on PANC-1 cells

Proliferation assay was applied to analyze the antiproliferation effects of 125I and GEM on PANC-1 cells. As shown in [Figure 2]a, although the growth of PANC-1 cells was inhibited after treating with 125I or GEM alone, the combined treatment revealed a more significant inhibitory effect (72 h, C vs. GEM, t = 16.59, P < 0.01; C vs. 125I, t = 9.808, P < 0.05; C vs. 125I+GEM, t = 17.87, P < 0.01;125I vs. 125I+GEM, t = 8.191, P < 0.05). What's more, colony assay was performed to testify the anti-proliferation effects. In the [Figure 2]b, cells proliferation ability in the combined treatment group was less than that of in groups treated with 125I or GEM alone. To further investigate the anti-proliferation effects, a cell cycle analysis was performed. As shown in [Figure 2]c and [Figure 2]d, arrest of G1 was obviously induced by GEM. However, there was an arrest at G2/M induced by 125I (4 Gy, G2/M, C vs. GEM, t = 4.364, P < 0.05; C vs. 125I, t = 5.164, P < 0.05;125I vs. 125I+GEM, t = 7.070, P = 0.0194). In the combined treatment group, G1 cell cycle arrest was observed.
Figure 2: Gemcitabine increased the 125I-induced antiproliferation effect cells were treated with 125I, gemcitabine or concurrent 125I and gemcitabine. At a dose of 4 Gy, antiproliferation assay (a), colony formation assay (b) and cell cycle analysis (c and d) were used to analyze the antiproliferation effect. All experiments were performed in triplicate, and the data are presented as the mean ± standard deviation. The t-test was used for data analysis. *P < 0.05, **P < 0.01

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Gemcitabine increased 125I-induced cellular apoptosis

After treatment, the cells were tested by Annexin V-FITC/PI assay [Figure 3]a. When the cells were treated with 125I or GEM alone, cellular apoptosis was slightly increased compared to that of in the combined treatment group (4 Gy, C vs. GEM, t = 14.45, P < 0.05; C vs.125I, t = 19.36, P < 0.05; C vs.125I + GEM, t = 30.63, P < 0.01;125I vs.125I+GEM, t = 10.43, P < 0.01). Moreover, the results of TUNEL assay also showed that GEM increased 125I-induced cellular apoptosis. As shown in [Figure 3]b, apoptosis was higher in the combination treatment group than that of in either single-agent treatment group.
Figure 3: Gemcitabine increased the 125I-induced apoptosis cells were treated with 125I, gemcitabine or concurrent 125I and gemcitabine. At a dose of 4 Gy, apoptosis analysis (a) and transferase-dUTP nick-end labeling assay (b) were used to analyze the cellular apoptosis. All experiments were performed in triplicate, and the data are presented as the mean ± standard deviation. The t-test was used for data analysis. *P < 0.05, **P < 0.01

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Western blot analyses for Bax, Bcl2, and Caspase-3

To investigate the 125I-induced cellular apoptosis promoted by GEM, we analyzed the expression of mitochondrial pathway-related proteins, including Bax, Bcl2, and Caspase-3 [Figure 4]. The level of Caspase-3 was increased by 125I, GEM, and combined 125I and GEM treatment. As expected, the level of Caspase-3 was the highest in cells treated by combined 125I and GEM (4 Gy, C vs. GEM, t = 16.34, P < 0.05; C vs.125I, t = 11.53, P < 0.05;125I vs.125I+GEM, t = 13.11, P < 0.01). Moreover, the Bax/Bcl2 ratio was increased by either 125I or GEM, which was more so by the combination (4 Gy, C vs. GEM, t = 16.34, P < 0.05; C vs.125I, t = 11.53, P < 0.05;125I vs.125I+GEM, t = 13.54, P < 0.05).
Figure 4: Cells were treated with 125I, GEM or concurrent 125I and GEM. At a dose of 4 Gy, Western blot was used to analyse caspase-3 expression and the Bax/Bcl2 ratio in different treatment groups (a). The level of Caspase-3 was significant higher in the group of GEM+125I (b) and Bax/BCL-2 ratio was lower in the group of GEM+125I (c). All experiments were performed in triplicate, and the data are presented as the mean±SD. The T test was used for data analysis. *P < 0.05, **P < 0.01

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 > Discussion Top


125I seed implantation has recently become an effective treatment for advanced pancreatic cancer in China.[11],[12],[13],[14],[15],[16] As radiotherapy, the complications of 125I seed implantation were related to dose or dose rate.[17] A cardinal principle of 125I seed implantation was to use the minimal dose, at which tumor cells would be killed without damaging neighboring normal cells. Enhancing the radio-sensitivity might be a resultful method to decrease the total dose and prevent radiological complications. GEM, superior to fluorouracil, has been widely proved as effective chemotherapy for many solid tumors, which has become a standard treatment for locally advanced and metastatic pancreatic cancer.[4] Although 125I or GEM had been considered as feasible treatment for pancreatic cancer, their combined effect has not been investigated yet. The study aimed to evaluate the combined effect of 125I and GEM on PANC-1 cells and explore the underlying molecular mechanism of combined treatment.

The study results indicated that combination of 125I and GEM in treating PANC-1 cells brought about better anti-proliferation effect than that of single-treatment [Figure 2]. The proliferation assay suggested that the cells treated with combined 125I and sensitizing concentration GEM were inhibited more significantly than cells treated with 125I or GEM alone, which strongly revealed that GEM could enhance the radio-sensitivity of PANC-1 cells to 125I. In additon, the inhibition was noticeably greater with the increased radiation dose. To further investigate the anti-proliferation effect, cell cycle test was performed. According to the study, the ratio of G2/M stage was higher in 125I group compared to that of in control group. In GEM group, G2/M stage was lower while G1 arrest appeared. Interestingly, in the combined group, cell cycle showed G1 arrest, which indicated that GEM could make such a decided impact on cell cycle of PANC-1 cells, even in sensitizing concentration. Although it was reported that cells at G2/M were more sensitive to radiation, GEM, caused G1 stage arrest, could also enhance the radiosensitivity of PANC-1 cells to 125I.[18]

We also suggested that GEM could increase 125I-induced apoptosis of PANC-1 cells. At first, Annexin V-FITC/PI assay was performed to explore whether combined treatment could cause more apoptosis cells than single-treatment. As reported, the apoptosis rate of cells treated with combined GEM and 125I was significantly higher than that of in GEM or 125I group.[19] To further testify the combined effect of GEM and 125I, TUNEL assay was performed. In [Figure 3]b, more positive cells were shown in the combined treatment group, which supported the conclusion that GEM could increase 125I-induced cellular apoptosis.

As reported, after radiation treatment, p53 would be triggered by ataxia telangiectasia mutated and DNA-PKcs, which could lead to the activities of various mitochondrial-related proteins.[20] It was known to us that Bax, Bcl-2, and Caspase-3 were mitochondrial apoptosis pathway related factors. The apoptosis could be induced by up-regulating the expression of Bax gene and down-regulating the expression of Bcl-2 gene and anti-apoptosis. In the present study, the combined treatment could obviously increase the expression level of Bax and decrease expression level of Bcl-2 in PANC-1 cells. The ratio of Bax and Bcl-2 was increased, which also manifested that GEM could increase the 125I-induced apoptosis of PANC-1 cells. However, we only investigate the mitochondrial-related apoptosis pathway, without including other potential apoptosis pathways, such as ER stress, which needed to be detect.[21]

In China,125I radio-active seeds implantation combined with GEM for advanced pancreatic cancer had been widely regarded as a feasible, safe, and effective treatment, which reached a high success rate and no serious complications.[22] A study reported that 125I seeds implantation could produce obvious pain relief and high local control rates.[23] More importantly, it was reported that 125I seeds implantation combined with a regional arterial infusion of GEM for late-stage pancreatic cancer achieved significant symptom relief and high-local control rate, indicating that combined effect of 125I and GEM was superior to single-treatment.[24]


 > Conclusion Top


The combined treatment of 125I and GEM induced stronger anti-proliferation effect than single-treatment, due to the cell cycle arrest and more cellular apoptosis on PANC-1 cells. The increased Bax/Bcl-2 ratio may lead to enhanced apoptosis. Based on these results, the study may contribute to explain one of the molecular bases for the combined effects of 125I and GEM on PANC-1 cells.

Financial support and sponsorship

  1. National Natural Science Foundation of China; Grant number: 61671276
  2. Natural Science Foundation of Shandong province; Grant number: 2014ZRE27479.
  3. Nature Science Foundation for Training of Shandong Province and the grant number; ZR2018PH032.


Conflicts of interest

There are no conflicts of interest.



 
 > References Top

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Wang J, Chai S, Zheng G, Jiang Y, Ji Z, Guo F, et al. Expert consensus statement on computed tomography-guided 125I radioactive seeds permanent interstitial brachytherapy. J Cancer Res Ther 2018;14:12-7.  Back to cited text no. 14
    
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Liang Y, Wang Z, Zhang H, Gao Z, Zhao J, Sui A, et al. Three-dimensional-printed individual template-guided 125I seed implantation for the cervical lymph node metastasis: A dosimetric and security study. J Cancer Res Ther 2018;14:30-5.  Back to cited text no. 16
    
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Ai KX, Zheng Q, Xia Y, Huang XY, Zou JH, Yan J, et al. Treatment of late-staged pancreatic carcinoma with implantation of 125I seeds in combination with regional arterial infusion chemotherapy. Zhonghua Wai Ke Za Zhi 2007;45:27-9.  Back to cited text no. 24
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]



 

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