|Year : 2015 | Volume
| Issue : 1 | Page : 195-198
Suppression effects of negative pressure on the proliferation and metastasis in human pancreatic cancer cells
Xiujiang Yang1, Bo Sun1, Haihang Zhu2, Ziting Jiang3
1 Department of Endoscopy, Fudan University, Shanghai Cancer Center; Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032, China
2 Department of Gastroenterology, Subei People's Hospital of Jiangsu Province, Yangzhou 225001, China
3 Department of Gastroenterology, Yangpu District Central Hospital, Yangpu District, Shanghai 200090, China
|Date of Web Publication||16-Apr-2015|
Department of Gastroenterology, Yangpu District Central Hospital, No. 450 Tengyue Road, Yangpu District, Shanghai 200090
Source of Support: None, Conflict of Interest: None
Background and Aims: The aim was to explore the effect of negative pressure on the proliferation and metastasis of human pancreatic cancer SW1990 cells.
Settings and Design: Three groups were conducted in the work: normal control group (NC group, 0 mm Hg), low negative pressure group (LN group, –300 mm Hg), and high negative pressure group (HN group, –600 mm Hg).
Materials and Methods: Cell morphological assay was conducted using an inverted Nikon TE2000-S microscope. Cell viability was assayed using cell counting kit-8 solution. Cell apoptosis was evaluated with flow cytometry. Cell migration was investigated using transwell assay.
Results: Compared to LN and HN groups, SW1990 cells in NC group grew quite well, showing a higher density. The NC group represented the highest cell viability. The HN group represented the lowest cell viability, which was lower than that of the LN group (P < 0.01). The apoptosis rate in NC group, LN group and HN group was 1.91% ±0.13%, 2.31% ± 0.06% and 15.22% ± 0.81%, respectively (P < 0.05). The average number of migration cells in NC group was 53.60 ± 4.14 (×200), which was decreased to 18.93 ± 3.67 and 11.07 ± 3.01 in LN group and HN group, respectively (P < 0.01).
Conclusion: The negative pressure shows suppression effects on the proliferation and metastasis of human pancreatic cancer SW1990 cells. It is indicated that negative pressure may be involved in the development of human pancreatic cancer by influencing cell biological characteristics.
Keywords: Metastasis, negative pressure, pancreatic cancer, proliferation
|How to cite this article:|
Yang X, Sun B, Zhu H, Jiang Z. Suppression effects of negative pressure on the proliferation and metastasis in human pancreatic cancer cells. J Can Res Ther 2015;11:195-8
|How to cite this URL:|
Yang X, Sun B, Zhu H, Jiang Z. Suppression effects of negative pressure on the proliferation and metastasis in human pancreatic cancer cells. J Can Res Ther [serial online] 2015 [cited 2019 Sep 21];11:195-8. Available from: http://www.cancerjournal.net/text.asp?2015/11/1/195/140802
| > Introduction|| |
Pancreatic cancer is one of the most lethal diseases in the world. Until date, pancreatic cancer remains the fourth leading cause of cancer deaths for both men and women.  Pancreatic cancer usually develops without early symptoms and most patients are diagnosed with metastatic cancer. , As a consequence of high metastasis rate, pancreatic cancer is very complex and invasive with extremely poor prognosis. , Tumor invasion and metastasis are very complicated processes. The movement of cancer cells into the surrounding tissues and vasculature is the first step in the spread of metastatic cancers.  Therefore, inhibiting proliferation and migration of cancer cells is one of the effective methods for cancer therapy. ,
Metastasis is a complex, multistep process responsible for >90% of cancer-related deaths. In addition to genetic and external environmental factors, the physical interactions of cancer cells with their microenvironment, as well as their modulation by mechanical forces, are key determinants for metastasis.  It has been reported that both positive and negative pressure show mechanical stress on cells and make different impacts on cells through different directions.  Kato et al. have studied the pressure-induced changes of biological membrane, and they found that high pressure induced membrane-damage to subcellular organelles. Environment pressure more than 200 MPa can result in membrane protein loosening, lipid bilayer breakage and cell death.  In addition, previous reports have indicated that mechanical stress may initiate signal transduction in vascular smooth muscle cells in vitro and in vivo, and Ras/rac1-p38 mitogen-activated protein kinase-nuclear factor kappaB (MAPK-NF)-kB signal pathway and CBF-1/RBP-JK depended notches1/3 signal pathway participate in smooth muscle cells apoptosis process mediated by mechanical stress. , Nevertheless, to the best of our knowledge, there is little report describing the effect of negative pressure on the human pancreatic cancer cells.
In the present work, we investigated the effect of negative pressure on the proliferation and metastasis of human pancreatic cancer cell line SW1990. A total of three groups were conducted in the work, including normal control group (NC group), low negative pressure group (LN group) and high negative pressure group (HN group). The morphological changes, cell viability, cell apoptosis, and cell migration of SW1990 cells in NC group, LN group and HN group were evaluated.
| > Materials and methods|| |
Human pancreatic cancer cell line SW1990 was purchased from Chinese Academy of Sciences (Shanghai, China). SW1990 cells were grown in RPMI1640 culture medium supplemented with 10% fetal bovine serum (FBS) (Gibco, CA, USA). Cultures were maintained in a humidified atmosphere of 5% CO 2 at 37°C.
Construction of negative pressure model
Vacuum extractor was used to set up negative pressure environment. Three groups were conducted in the work: NC group (0 mm Hg), LN pressure group (−300 mm Hg), and HN pressure group (−600 mm Hg). For LN and HN groups, cells were treated under corresponding negative pressure for 1 h and each time and twice daily. For NC group, cells were cultured under normal pressure. Cells were cultured for 3 days for each group.
Cell morphological assay
The cell morphological assay was conducted using an inverted Nikon TE2000-S (Nikon, Tokyo, Japan) microscope equipped with a ×60 immersion objective with Nomarski contrast according to the technique described previously.  Image analysis was performed using a LUCIAG image analyzer (Nikon, Tokyo, Japan) on a HP Workstation xw4100.
Cell viability assay
Log phase cells were suspended in complete medium to prepare cell suspension. Hemacytometer was used to determine cell count. Cells were seeded at a density of 10,000 cells in 100 μl per well into 96-well plates in triplicate for each group, and incubated for another 2-4 h (37°C, 5% CO 2 ). When cells were completely adhered to plates, 10 μl cell counting kit (CCK)-8 solution was added to each well, and the growth was continued for another 4 h. The optical density (OD) value for each well was read at wavelength 450 nm to determine the cell viability on a microplate reader (Multiskan, Thermo, USA).
Cell apoptosis assay by flow cytometry
Cell apoptosis was evaluated by annexin V-fluorescein isothiocyanate (FITC) and flow cytometry. Cells were grown at a density of 1 × 10 6 cells in six-well culture dishes and were treated with different concentrations of resveratrol (0-200 μm in dimethyl sulfoxide) for 24 h. After treatment, the cells were harvested, washed twice with pre-chilled PBS, and resuspended in ×1 binding buffer at a concentration of 1 × 10 6 cells/ml. Later 100 μl solution was mixed with 5 μL annexin V-FITC and 5 μL propidium iodide for 15 min, then 400 μL × 1 binding buffer was added. Analysis was carried out using a FC500 flow cytometer with CXP software (Beckman Coulter, Fullerton, CA, USA) within 1 h. The percentage of apoptotic cells was assessed by CXP software.
Cell migration assay with transwell
SW1990 cells were digested with 0.25% trypsin and suspended in RPMI 1640 culture medium to prepare cell suspension at a density of l × 10 6 /ml. The 500 μl RPMI1640 (containing 10% FBS) was added to lower chambers of the transwell pre-coated with matrigel matrix (BD, NJ, USA), and then 250 μl cell suspension was added to the upper chambers. The whole transwell was put in vacuum extractor for 1 h, and then was culture normally in a humidified atmosphere of 5% CO 2 at 37°C. After 12 h incubation, noninvaded cells were removed from the upper surface of the transwell membrane with a cotton swab. Invaded cells on the lower membrane surface were fixed in paraformaldehyde for 15 min, and stained with 500 μL Giemsa solution. Five randomly selected fields (×200) were photographed with Image-Pro Plus software (MD, USA), and then counted.
All data were expressed as mean ± standard deviation (X ± SD). Statistical analysis was performed using SPSS13.0 software (one-way ANOVA) (Chicago, USA). P <0.05 was considered to be statistically significant.
| > Results|| |
Morphological character of cells in each group
SW1990 cells in NC group were grown quite well and at a high density, in fusiform or polygon, forming cell colonies [Figure 1]a. Cells in the negative pressure groups [Figure 1]b and c proliferated very slowly, and morphological change could be found [Figure 1]b and c. As the negative pressure increased and time passed, more cells in the negative pressure groups became round, shrinking, floating and dead in the end [Figure 1]c.
|Figure 1: Morphological character of SW1990 cells in NC group (a), LN group (b), and HN group (c), respectively (×100). NC group=Normal control group, 0 mm Hg; LN group=Low negative pressure group, -300 mm Hg; HN group=High negative pressure group, -600 mm Hg|
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Negative pressure reduced cell viability
Cell Counting Kit-8 assay kit was used to test cell viability after 4 days' treatment. At the 4 th day, the OD value of HN group was 2.15 ± 0.05, whereas the OD value of LN group was 2.86 ± 0.04, which were lower than that of NC group (3.28 ± 0.04) [Figure 2]. The difference among these groups was statistically significant (P < 0.01). The NC group represented the highest cell viability. The HN group represented the lowest cell viability, which was lower than that of the LN group [Figure 2].
|Figure 2: Growth curves of SW1990 cells in NC group, LN group, and HN group, respectively. Triangle represents the NC group. Circle represents the LN group. Square represents the HN group. NC group: normal control group, 0 mm Hg; LN group=Low negative pressure group, -300 mm Hg; HN group=High negative pressure group, -600 mm Hg. *, P < 0.01, comparing with control group|
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Negative pressure induced cell apoptosis
The apoptosis rate in NC group, LN group and HN group was 1.91% ±0.13%, 2.31% ±0.06% and 15.22% ±0.81%, respectively [Figure 3]a-c. The difference among these groups was statistically significant ([Figure 3]d, P < 0.05). Obviously, negative pressure induced cell apoptosis in SW1990 cells.
|Figure 3: Apoptosis of SW1990 cells evaluated by annexin V-fluorescein isothiocyanate and flow cytometry in NC group (a), LN group (b), and HN group (c), respectively. NC group=Normal control group, 0 mm Hg; LN group=Low negative pressure group, -300 mm Hg; HN group=High negative pressure group, -600 mm Hg. #P < 0.05, comparing with control group|
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Negative pressure suppressed cell migration
Transwell assay was used to investigate the cell migration. Without negative pressure treatment, the average number of migration cells was 53.60 ± 4.14 in a ×200 view [Figure 4]a, which was decreased to 18.93 ± 3.67 in LN group [Figure 4]b. In HN group, the average number of migration cells was 11.07 ± 3.01, which was the lowest one among three groups [Figure 4]c. There was statistically significant difference among these groups ([Figure 4]d, P < 0.01).
|Figure 4: Migration of SW1990 cells in NC group (a), LN group (b) and HN group (c) observed under inverted microscope (×200). The blue dots represent cells, and the small holes with clear outline represent membrane (8.0 μm diameter). NC group=Normal control group, 0 mm Hg; LN group=Low negative pressure group, -300 mmHg; HN group=High negative pressure group, -600 mm Hg. *P < 0.01, comparing with control group|
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| > Discussion|| |
Pancreatic cancer is one of the most lethal diseases in the world with a high metastasis rate. Inhibiting proliferation and migration of cells is important for the treatment of pancreatic cancer. In the present work, we investigated the effect of negative pressure on the proliferation and metastasis of human pancreatic cancer cell line SW1990. Several key results were found in our study:
- Compared to LN and HN groups, SW1990 cells in NC group were grown quite well, showing a higher density
- SW1990 cells in HN and LN groups represented lower cell viability than that of the NC group
- The apoptosis rate of LN and HN groups was higher than that of the NC group
- The average number of migration cells in NC group was higher than that of LN and HN groups.
Pressure stress affects all levels of cellular physiology including metabolism, membrane physiology, transport, transcription and translation.  Environmental changes such as osmotic stress could affect the morphological changes of lipid vesicles.  Lipids are particularly sensitive to pressure effects, which are on average an order of magnitude more compressible than proteins.  Hydrostatic pressure has been used as a physical-chemical parameter for studying the energetics and phase behavior of membrane systems.  The membrane system changes caused by negative pressure may result in the morphological changes of SW1990 cells.
Compared with normal pressure, the negative pressure could inhibit the proliferation, cell viability, migration and invasion of SW1990 cells, while promote the apoptosis of SW1990 cells. Kim et al. found that significant overproduction of reactive oxygen species and reduction in cell viability were induced by plasma exposure on cancer cells.  In the case of (mechanosensitive channel of large conductance, with a large pore diameter and low ion selectivity), a low level of membrane tension below normal pressure would cause the membrane to rupture, thereby providing a useful "pressure relief valve" in the event of extreme osmotic swelling. , Li C et al. have demonstrated that mechanical stress induced apoptosis of vascular smooth muscle cells in vitro and in vein grafts.  Ras/rac1-p38 MAPK-NF-kB signal pathway and CBF-1/RBP-JK depended notches1/3 signal pathway participate in smooth muscle cells apoptosis process mediated by mechanical stress. , Unlike positive pressure, negative pressure has centripetal force on cells, the direction of which is opposite to that of cancer cells invasion and migration.  The proliferation, migration and invasion of tumor cells are continuous processes for cancer cell metastasis. Any part of this process being blocked can restrain tumors metastasis.  Therefore, the suppression of tumor cell movement may inhibit the metastasis of pancreatic cancer cells.
In the present work, the effect of negative pressure on the proliferation and metastasis of human pancreatic cancer cell line SW1990 was investigated. Compared with normal pressure, the negative pressure can inhibit the proliferation, cell viability, migration and invasion of SW1990 cells and promote the apoptosis of SW1990 cells. It is speculated that negative pressure can efficiently prevent development of human pancreatic cancer. The mechanism of negative pressure suppressing SW1990 cells may be resulted from the mechanical stimulation. Nevertheless, the evidence needs to be explored in our further work.
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