Knockdown of PARP1 Inhibits β-Lapachone-Mediated Reductions in AGS Cell Proliferation and Migration

Research Article

Austin Med Sci. 2019; 4(1): 1033.

Knockdown of PARP1 Inhibits β-Lapachone-Mediated Reductions in AGS Cell Proliferation and Migration

Wang C1*, Li C4*, Jin W2, Lin Z3, Quan X2,5# and Shen X2#

1Department of Interventional Medicine, Jilin Cancer Hospital, China

2Department of Oncology, Yanbian University Affiliated Hospital, China

3Cancer Research Center and Department of Pathology, Yanbian University Medical College, China

4Department of Gastrology, Yanbian University Affiliated Hospital, China

5Department of Postdoctoral Programme, Yanbian University Affiliated Hospital, Yanji, Jilin, China *Chu Wang and Chenghao Li contributed equally to this work

*Corresponding author: Xionghu Shen and Xianglan Quan, Department of Oncology, Yanbian University Affiliated Hospital, Yanji, Jilin, China

Received: July 01, 2019; Accepted: August 14, 2019; Published: August 21, 2019


Background: β-lapachone selectively induces apoptotic cell death in a variety of human cancer cells in vitro and in vivo. However, the mechanism by which β-lapachone promotes cell death in gastric cancer is not fully understood. The present study aimed to investigate the role and molecular mechanism of PARP1 in the β-lapachone-mediated killing of the human gastric carcinoma cell line AGS.

Methods: AGS cells were transfected with non-specific or target-specific small interfering RNAs (siRNAs) using riboFECTTM CP. After transfection, each of the 4 groups was treated with 4 μmol/l β-lapachone, and western blotting was used to detect changes in the protein expression of the PARP1 cleavage product. Variance analysis was used to compare the results of the 4 treatment groups, and the most stably transfected cell lines were selected. The best siRNA was selected for subsequent cell transfections, and a negative control group with NC-siRNA was also established.

Results: AGS cell proliferation and colony-forming ability were detected by the MTT and colony formation assays, respectively, whereas AGS cell migration was detected by the scratch assay. Knockdown of PARP1 via siRNA transfection did not change the cells’ morphology. The expression of cleaved PARP1 protein was significantly reduced in cells with PARP1 knockdown. Treatment with β-lapachone markedly abolished the proliferative, colony-forming and migratory abilities of the control AGS cells but did not affect AGS cells with PARP1 knockdown.

Conclusion: Our novel findings suggest that PARP1 has an important role in the β-lapachone-mediated killing of gastric cancer cells.

Keywords: Gastric cancer; PARP1; β-lapachone


In China, gastric cancer has the second highest incidence among malignant tumors. Gastric cancer has a high rate of metastasis and high mortality with reported metastasis and recurrence rates of 33% after radical gastrectomy [1]. A recent study shows that treatment with radiotherapy and chemotherapy can result in drug resistance due to the survival of solid tumors in a hypoxic environment; this ultimately leads to treatment failure [2]. Recently, some drugs, including β-lapachone, streptonigrin and deoxynyboquinones (DNQs), have been developed to specifically target tumor cells that can survive in hypoxic conditions [3]. β-lapachone is primarily extracted from the bark of red sandalwood trees from Central and South America; additionally, it possesses antibacterial, antifungal, and antitumor properties and has extensive biological activity [5]. Nevertheless, β-lapachone can not only inhibit the activity of cancer cells but also induce tumor cell apoptosis [4]. Moreover, β-lapachone can enhance NQO1-mediated toxicity in cells and exert anti-cancer activities [5].

The PARP family contains 18 subtypes, with PARP1 as the predominantly expressed subtype. PARP1 was first extracted from the nuclei of liver cells in 1963 by a French group led by Mandel. The important role of PARP1 in DNA damage monitoring, DNA repair, transcriptional regulation, chromatin modification and so on has attracted the attention of many scholars. In addition, PARP1 is highly expressed in breast cancer, liver cancer, gastric cancer, lymphoma, colon cancer, ovarian cancer and other malignant tumor cells and plays a key role in tumor progression [6-8]; furthermore, PARP1 is also involved in cell death pathways [9,10]. Previous studies reported that β-lapachone induces apoptosis in gastric cancer cells via the classical caspase pathway, and one study observed that PARP1 was overactivated and cleaved during the study [11]. It remains unclear, however, whether PARP1 is involved in the β-lapachone-mediated killing of gastric cancer cells and the mechanism by which this occurs. In this study, we observed that knocking down PARP1 with siRNA transfection did not change the cells’ morphology. The expression of cleaved PARP1 protein was significantly reduced in AGS cells with PARP1 knockdown. Furthermore, treatment with β-lapachone markedly abolished the proliferative, colony-forming and migratory abilities of AGS cells but did not affect cells with PARP1 knockdown.

Materials and Methods

Cell cultures

AGS cells were supplied by the Cancer Research Center of Yanbian University and cultured in a humidified atmosphere (37°C) containing 5% CO2 in Dulbecco’s modified Eagle medium (DMEM) with RPMI 1640 (Gibco, Thermo Fisher Scientific, China) supplemented with 10% FBS (Gibco), Standard Serum Supplementation, and 4500 mg/L glucose. The growth of AGS cells was observed under a microscope until the AGS cells were adherent, after which the cells were treated promptly.

siRNA transfection

pAGS cells were transfected with non-targeting or targetspecific small interfering RNA (siRNA) using riboFECTTM CP (Guangzhou Rui Bo Biological Technology). siRNAs targeting PARP1 were designed and synthesized by Guangzhou Ruibo Biotechnology. Three unique PARP1 siRNA sequences were constructed as follows: 1, 5’-GGAGGAAGGTATCAACAAA-3’; 2, 5’-GGGCAAGCACAGTGTCAAA-3’; and 3, 5’-GGAACAAGGATGAAGTGAA-3’. The NC-siRNA sequence was 5’-TTCTCCGAACGTGTCACGT-3’. AGS cells were suspended in complete medium, seeded in 6-well plates at 8 x 105 cells / well, and divided into the following 4 groups: PARP1-siRNA 1 transfection group, PARP1-siRNA 2 transfection group, PARP1-siRNA 3 transfection group and NC-siRNA (negative control) group. When the AGS cells were 30 to 50% confluent, the siRNA mixtures were diluted and prepared with riboFECTTM CP, which were then added to the cell culture medium and mixed. The plates were incubated at 37°C in an atmosphere containing 5% CO2, and the infection rate was assessed under a microscope 48 h after transfection. Then, 4 μmol/ L β-lapachone was added to each group for subsequent western blotting.

Western blot analysis

Cells from the 4 treatment groups were lysed and processed to produce protein extracts that were subjected to SDS-PAGE, membrane transfer, antibody hybridization and ECL chemiluminescence detection to detect the expression of the PARP1 cleavage product. A primary antibody for cleaved PARP1 was used, and quantitative data were analyzed using NIH ImageJ software.

MTT assay

For the MTT assay, siRNA-transfected cells were incubated with 3-(4,5-dimethylhioazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT (50 μg/well) for 4 hrs and then treated with dimethylsulfoxide (100 μl/well). The absorbance (A570-A630) of each well was measured by using a microplate reader (Molecular Devices, Sunnyvale, CA, USA).

Colony formation assay

Cells in the logarithmic growth phase and that had been treated with β-lapachone were digested with trypsin. After the cells were dissociated and centrifuged, they were resuspended in cell culture medium. The cell suspension was serially diluted and seeded in culture dishes at a density of 5000 cells/dish. The culture dishes were gently shaken to evenly distribute the cells and were placed in an incubator for 2 weeks. Cell growth was observed periodically until colonies were visible to the naked eye, at which point the supernatant was discarded, and 2 ml of anhydrous formaldehyde per well as added for 15 min to fix the cells. Next, GIMSA staining was performed on the cells for 10 ~ 30 min, after which the residual dye was washed off, and the plate was dried at room temperature. The culture dish was inverted on transparent stage, and the colonies were counted either at a low magnification microscopy or with the naked eye to calculate the colony formation rate.

Scratch assay

The bottom of a 6-well plate was marked with a pen and ruler to draw a straight line with the average width of 0.5 ~ 1 cm. Cells from the PARP1-siRNA and NC-siRNA transfection groups were seeded into the 6-well plate. When the cells reached 90% confluence, scratches were carefully made through the monolayer with a 10-μl pipette tip to avoid tilt. After the cells were washed three times with PBS and then administered serum-free medium, they were treated with 4 μmol/L β-lapachone and placed in an incubator (5% CO2, 37°C). Photographs of the marks were taken at 0, 12, 24, and 48 h after scratching, and the distance between the scratches was measured under a microscope.

Statistical analyses

The data were analyzed by SPSS 18.0 statistical software. The data were expressed as x±s. Using analysis of variance, the experimental data between the two groups were compared by t-test. All experiments were repeated 3 times, and p‹0.05 indicated statistically significant differences.


Transfection efficiency of AGS gastric cancer cell lines

AGS cells were transfected with siRNA for 48 hours. The morphology of the cells (round, adherent growth) as observed under a microscope showed no significant changes compared with the control group, indicating that silencing PARP1 has no significant effect on cell morphology and growth (data not shown).

Western blot of the PARP1 cleavage protein

To understand the effect of silencing PARP1 after on the killing effects of β-lapachone, we treated AGS cells that were transfected with siRNA with 4 μmol/L β-lapachone and assessed PARP1 expression via western blotting. Compared with the control group, the three PARP1 siRNA groups showed significant reductions in the expression of PARP1 cleavage protein (Figure 1, p‹0.05). Cells transfected with PARP1-siRNA1 exhibited an 89% decrease (Figure 1, p‹0.01); PARP1-siRNA2, a 73% decrease (Figure 1, p‹0.05), and PARP1- siRNA3, an 83% decrease (Figure 1, p‹0.05). These data indicate that PARP1-siRNA 1 was the most effective sequence to silence PARP1 gene expression and was thus used for all subsequent experiment