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ORIGINAL ARTICLE
Year : 2016  |  Volume : 12  |  Issue : 5  |  Page : 1-4

Gemcitabine inhibits proliferation and induces apoptosis in human pancreatic cancer PANC-1 cells


1 Department of Oncology, Xinxiang Central Hospital of Henan Province, Henan 453000, PR China
2 Department of Endoscopy, Xinxiang Central Hospital of Henan Province, Henan 453000, PR China
3 Department of Oncology, 1st Affiliated Hospital of Fujian Medical University, Fuzhou 350004, PR China

Date of Web Publication7-Oct-2016

Correspondence Address:
Tian Guo-Fang
Department of Oncology, Xinxiang Central Hospital of Henan Province, Xinxiang, Henan 453000
PR China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.191615

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 > Abstract 


Aim: The aim of the study is to investigate the underlying molecular mechanisms by which gemcitabine (gem) inhibits proliferation and induces apoptosis in human pancreatic cancer PANC-1 cells in vitro.
Materials and Methods: After PANC-1 cells had been treated by indicated concentration (0, 5, and 25 mg/L) of gem for 48 h, cell proliferation was evaluated by 3'-(4, 5 dimethyl-thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay; cell morphology was observed by transmission electron microscopy; Expression of c-IAP2 and Bcl-2 proteins was analyzed by Western blot; the activity of caspase-3 and -9 was detected by spectrophotometry.
Results: Gem significantly inhibited cell proliferation and could induce apoptosis of human pancreatic cancer PANC-1 cells, with a dose-dependent manner. Western blot analysis showed that gem significantly reduced c-IAP2 and Bcl-2 proteins expression level (P < 0.05). Spectrophotometric assay showed that gem significantly increased caspase-3 and -9 activity in PANC-1 cells.
Conclusion: Gem could induce apoptosis of human pancreatic cancer PANC-1 cells, probably through downregulating c-IAP2 and Bcl-2 expression levels, and at the same time activating caspase-3 and -9.

Keywords: Apoptosis, gemcitabine, pancreatic cancer, proliferation


How to cite this article:
Yong-Xian G, Xiao-Huan L, Fan Z, Guo-Fang T. Gemcitabine inhibits proliferation and induces apoptosis in human pancreatic cancer PANC-1 cells. J Can Res Ther 2016;12:1-4

How to cite this URL:
Yong-Xian G, Xiao-Huan L, Fan Z, Guo-Fang T. Gemcitabine inhibits proliferation and induces apoptosis in human pancreatic cancer PANC-1 cells. J Can Res Ther [serial online] 2016 [cited 2018 Aug 16];12:1-4. Available from: http://www.cancerjournal.net/text.asp?2016/12/5/1/191615

Gui Yong-Xian and Li Xiao-Huan contributed equally to this work.





 > Introduction Top


Pancreatic cancer is one of the most common types of solid carcinoma in clinic. With high malignancy and unnoticeable morbidity, distant metastasis will occur even in early stage.[1] Moreover, most patients are in terminal when they are diagnosed.[2],[3] Chemotherapy is an important treatment for advanced pancreatic cancer. Cytotoxicity of chemotherapeutics which induces cancer cell apoptosis is one of the main mechanisms of chemotherapeutics to inhibit cancer cell growth and finally promote its death.[4] Gemcitabine (gem) is a kind of cytotoxic fluoride nucleoside antimetabolite.[5] It is a soluble analog of deoxycytidine, which plays an antitumor role by repressing the activity of the ribonucleotide reductase, thus inhibiting DNA synthesis and blocking cell proliferation and division.[6] Previous studies showed that gem could induce cancer cell apoptosis, but the exact mechanisms have not yet been clear.[7] In this study, we evaluated the molecular mechanisms by which gem induces cell apoptosis in human pancreatic cancer cell line PANC-1 by molecular biology experiments and electron microscope technology.


 > Materials and Methods Top


Materials

Reagents

Gem was from Eli Lilly and company. 3'-(4, 5 dimethyl-thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) reagents were from Sigma. c-IAP2 monoclonal antibody was from R and D company. Bcl-2 antibody, Annexin v-FITC Apoptosis Detection Kit, and Caspase-3 and -9 Activity Assay Kits were from Beyotime Institute of Biotechnology, Shanghai, China.

Key instruments

Water jacket type CO2 incubator, models for 3111, was purchased from the American Thermo Forma Company; transmission electron microscope, models for Tecnai G2-20, was purchased from Holland FEI company; spectrophotometry, Multiscan ELISA reader, was purchased from Molecular Devices Company, USA.

Cell culture

Human pancreatic cancer PANC-1 cells were purchased from Cell Bank, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. It was cultured in RPMI 1640 medium (Hyclone) with 10% serum (Hyclone) in a humidified 50 ml/L CO2 atmosphere at 37°C.

Methods

3'-(4, 5 dimethyl-thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay

The cytotoxic effect of gem on the cells was determined by MTT assay.[8] PANC-1 cells in logarithmic phase were made to monoplast suspension, and living cell counts were determined over 98% by trypan blue staining. Then, cells were seeded at a density of 2000 cells per well in 96-well plates and were allowed to grow overnight. After treatment with indicated concentration (0, 5, and 25 mg/L) of gem, each with four replicates for 48 h, cell viability was determined by MTT assay. After incubation, 20 µl of MTT buffer (5 g/L) was added to each well and incubated for 4 h. Then, medium was carefully removed; dimethyl sulfoxide was added to each well and was shaken for 10 min to fully split cells until all the cellular crystals melted. The absorbance (A) was read at a wavelength of 570 nm in a plate reader. Mean value was taken, and cell viability from the control group was defined as 100%. The proliferating inhibition ratio (IR) was plotted against mean values which were calculated using the following equation: IR (%) = (1 − A570 [treated]/A570 [control]) ×100%.

Electron microscopy

PANC-1 cells in logarithmic phase were trypsinized and made to monoplast suspension. Then, they were seeded at a density of 4 × 107 in 50 ml culture flask, each containing 4.5 ml medium. After incubation at 37°C in a humidified atmosphere of 5% CO2 in air with 95% humidity for 12 h, gem was added to make a final concentration of 0, 5, and 25 mg/L.[9] Forty-eight hours later, cells of each group were collected and centrifugated at 1500 rpm for 10 min. After removal of supernatant, cells were washed twice with PBS and adjusted to 1 × 106 per group. After another centrifuge, cells were fixed in suspension with 2.5% glutaraldehyde-1% paraformaldehyde intermixture precooling at 4°C. Then, they were postfixed with 1% osmium tetroxide. Subsequently, the cells were dehydrated, embedded, and solidified according to the usual methods. The ultrathin sections were stained with 3% uranyl acetate, followed by lead citrate staining and examined using a Tecnai G2–20 transmission electron microscope.

Western blotting

To reveal the mechanism of the apoptotic effect of gem, Western blotting was done for apoptotic-related proteins such as c-IAP2 and Bcl-2. Briefly, after treatment with indicated doses of gem for 48 h, cells were collected and lysed with TRIzol for extracting total protein. The protein lysates were separated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gel and transferred to a nitrocellulose membrane. The membranes were soaked in blocking buffer (50 g/L bovine serum albumin dissolved in tris-buffered saline) for 1 h. To probe for c-IAP2 and Bcl-2, membranes were incubated overnight at 4°C with relevant antibodies (1:200 dilution), followed by appropriate horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence detection. β-actin (1:400 dilution) purchased from Zhongshan Golden Bridge Biotechnology (Beijing, China) was used as an internal control. Semi-quantification analysis of Western blottings was done by Image-pro plus 6.0 software (http://www.mediacy.com).

Measurement of caspase-3, -9 activity

After treatment with gem for 48 h, cells were lysed to measure the activity of caspase-3 and -9 using Caspase-3 and -9 Activity Assay Kits according to the manufacturer's instructions, at the spectrophotometry Multiscan ELISA reader.

Statistics analysis

For the statistical analysis of data, we used SPSS 11.5 software (http://www-01.ibm.com/software/analytics/spss/). Data are expressed as mean ± standard deviation of at least three independent experiments. Student's t-test was employed to determine the statistical significance of the difference between different experimental and control groups. P < 0.05 value was defined as statistically significant.


 > Results Top


Gemcitabine inhibits proliferation in human pancreatic cancer PANC-1 cells

To investigate whether gem has growth inhibition effect, we performed MTT assay after treatment with gem in PANC-1 cells in vitro. We observed that gem significantly inhibited cell proliferation, with a half maximal (50%) inhibitory concentration of 5.79 µg/ml. The proliferating IR at 25 mg/L of gem was significantly higher than that of 5 mg/L, with statistical difference (P < 0.05).

Gemcitabine induces apoptosis of pancreatic cancer PANC-1 cells

Next, we determined the other possible function of gem in PANC-1 cells. Transmission electron microscopy photographing of the normal PANC-1 cells showed large nucleus, obvious euchromatin, visible heterochromatin which scattered in nuclear or in the nuclear membrane adjacent place in small pieces, hypertrophy nucleolus which focuses on the edge, obvious nucleosome and rough endoplasmic reticulum [Figure 1]a. While after treatment with gem, microscopic views of the PANC-1 cells demonstrated that the morphology of the cells changed. It acted as cell apoptosis, with increasing electron density of the cytoplasm and nucleus, smaller volume, concentrated nuclear chromatin, narrowing nuclear type which was divided into several small pieces, each with a nuclear membrane around. Besides, apoptosome was visible, and small pieces of chromatin were along with a little cytoplasm, with cell membrane outside. Apoptosome was separated with the cell and was free in the outer cell mass [Figure 1]b and [Figure 1]c.
Figure 1: Transmission electron microscopy images (×5000) of PANC-1 cells treated with indicated concentration of gem: 0 mg/L (a), 5 mg/L (b), 25 mg/L (c)

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Gemcitabine downregulates expression of c-IAP2 and Bcl-2

To further explain the observed phenotype by gem treatment in PANC-1 cells, we started searching for the gem-regulated proteins. We determined a few proteins that play important roles in regulating cell apoptosis in PANC-1 cells. We found that treating PANC-1 cells with gem could lead to a dose-dependent dramatic decrease of antiapoptotic c-IAP2 and Bcl-2 (P < 0.05) [Figure 2], suggesting that c-IAP2 and Bcl-2 might be involved in gem-mediated survival of the cells.
Figure 2: Different concentrations of gemcitabine induced downregulation of c-IAP2 and Bcl-2 proteins expression by Western blotting

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Caspase-3, -9 activity is responsible for cytotoxicity induced by gemcitabine in PANC-1 cells

Next, we examined whether gem treatment could impact on caspase-3 and -9 activity. As shown in [Table 1], after exposure to indicated concentrations of gem (5 and 25 mg/L) in PANC-1 cells, the activity of cellular caspase-3 and -9 significantly increased (P < 0.05), with a dose-dependent manner.
Table 1: Impaired activity of caspases in PANC-1 cells after treatment with gemcitabine

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 > Discussion Top


Pancreatic cancer is one of the common malignant tumors of digestive tract, about 90% of which were originated from the epithelium of the gland in ductal adenocarcinoma.[2] It appears an increasing rate of morbidity and mortality in recent years. Gem is a novel efficient and cell cycle specific antimetabolic drug, which majorly acting on tumor cells in DNA synthesis phase, namely S phase.[10],[11] Under certain conditions, gem could prevent cells progressing from G1 phase to S phase.[12] Gem is one of the commonly used chemotherapy drugs in human pancreatic cancer. Clinical studies have reported that the objective clinical efficiency is at around 30% when curing advanced pancreatic cancer patients by gem alone or along with oxaliplatin, cisplatin, or gonow. Moreover, single drug application or combinations of drugs achieve almost similar therapeutic effect.[13] While a meta-analysis of 19 literature shows that gem combined with other drugs could notably improve the clinical efficiency of treating pancreatic cancer, in which combined treatment group significantly improves 1-year survival.[14] Although the clinical efficiency of chemotherapy strategies of gem alone or combination therapy is definite, whether gem could inhibit proliferation or induce apoptosis is unknown. In addition, the exact mechanism is still unclear.[15]

In this study, we take human pancreatic cancer PANC-1 cells as our research object. We used molecular biological experiment and transmission electron microscopy technology to explore biological phenotypes such as cell proliferation and apoptosis of PANC-1 cells upon treated by different concentrations of gem. Our results demonstrated that gem significantly inhibits cell growth and it also induced apoptosis of PANC-1 cells. Through transmission electron microscopy, we found that after exposure to gem for 48 h, apoptosome appeared and apoptosis percentage increased obviously, morphologically characterized by increasing electron density of cytoplasm and nucleus, smaller volume, concentrated chromatin, and narrower nuclear shape. Recently, Hong-Tao et al.[16] employed human pancreatic cancer PC-2 cells as research target. They found that gem could significantly promote PC-2 cell apoptosis at a concentration-dependent manner after treating PC-2 cells with gem (10–100 mg/L, with a concentration gradient of 10 mg/L). At the same time, the ratio of G0/G1 phase increased significantly at the treated group, which was in line with our results. We also showed that different concentrations of gem could inhibit pancreatic cancer cell proliferation and induce its apoptosis. Besides, our investigations indicated that the molecular mechanisms might involve downregulating apoptosis inhibitor c-IAP2 and Bcl-2 and activating caspase-3 and -9.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
 > References Top

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Ying-Wen S, Hai-Yan D, Lu-Ming L. A comparison between gemcitabine-based combination chemotherapy and gemcitabine alone on advanced pancreatic cancer: A meta-analysis in phase III clinical trials. China Oncol 2008;18:913-7.  Back to cited text no. 14
    
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    Figures

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