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MSX2 overexpression inhibits gemcitabine-induced caspase-3 activity in pancreatic cancer cells.

AIM
To evaluate the effect of MSX2 on gemcitabine-induced caspase-3 activation in pancreatic cancer cell line Panc-1.


METHODS
Using V5-tagged MSX2 expression vector, stable transfectant of MSX2 was generated from Panc-1 cells (Px14 cells). Cell viability under gemcitabine administration was determined by MTT assay relative to control cell line (empty-vector transfected Panc-1 cells; P-3EV cells). Hoechst staining was used for the detection of apoptotic cell. Activation of caspase-3 was assessed using Western blotting analysis and direct measurement of caspase-3 specific activities.


RESULTS
MSX2 overexpression in Panc-1 cells resulted in decreased gemcitabine-induced caspase-3 activation and increased cell viability under gemcitabine treatment in Px14 cells.


CONCLUSION
MSX2 exerts repressive effects on gemcitabine-induced apoptotic pathway. This novel apoptosis-regulating function of MSX2 may provide a new therapeutic target for pancreatic cancer.


INTRODUCTION
Pancreatic cancer is known as a cancer with poor prognosis. Surgical resection of pancreatic cancer is available only in 15-20% of all patients [1] , while medical approaches, such as chemotherapy or radiation, have no cure. The resistance of pancreatic cancer to chemotherapeutic agents is one of the serious problems in clinical situation. Gemcitabine (2',2'-dif luoro-2'-deoxycytidine) is a widely used chemotherapeutic agent for unresectable pancreatic cancer treatment, and its administration triggers apoptosis in pancreatic cancer cell line [2] . On the other hand, previous report showed that acquired gemcitabine resistance was accompanied by altered expression of apoptosis regulatory genes [3] . The mechanisms how cancer cells evade apoptotic signals are beginning to come to light, but its upstream regulators are not fully understood as yet.
We have previously demonstrated that overexpression of homeobox gene MSX2 accelerated proliferation of pancreatic cancer cell lines, combined with epithelialmesenchymal transition (EMT)-like phenotypic changes in vitro (Satoh et al, submitted). In addition, forced expression of MSX2 increased anchorage-independent growth of pancreatic cancer cells, indicating enhanced aggressive biological behavior. Recent studies have demonstrated that MSX2 is a downstream target of the Wnt signaling pathway in several cancer cell lines [4,5] . The upregulation of Wnt signaling is reported in various cancer cell lines, and its target genes are closely related to cancer cell growth and survival [6][7][8][9] . At this point of view, we hypothesized that MSX2 might affect apoptotic signaling pathway, which leads to the chemoresistance of pancreatic cancer cells.
In this study, we generated MSX2-overexpressing pancreatic cancer cell line Px14, and this cell line revealed resistance to the gemcitabine treatment. When cells were treated with gemcitabine, the caspase-3 activation was significantly upregulated in empty vector-transfected control cells, but not in Px14 cells compared to basal control cells. Taken together, MSX2 might act as a negative regulator of apoptosis in this cell line. This new upstream regulator of apoptotic signaling pathway may provide a novel therapeutic target of chemotherapy-resistant pancreatic cancer. supplemented with 100 mL/L fetal bovine serum (FBS), and incubated at 37 °C in a humidified atmosphere containing 50 mL/L CO2 in air.

Chemical substances
Gemcitabine (Gemzar; Eli Lilly Co., Indianapolis, IN, USA) was dissolved in phosphate buffered saline (PBS) at various concentrations of 0.1, 1, 10, 100 μg/mL and 1 mg/mL as stock solutions. In cell culture, vehicle and these stock solutions were used in 1:100 dilutions.

Plasmids, gene transduction and expression
Expression vector, pcDNA3.1-MSX2V5, was generated as described previously (Satoh et al submitted). Panc-1 cells were plated in six-well plates and cultured until reaching a subconfluent state. Cells were transfected with 1 μg of pcDNA3.1-V5His (empty vector) or pcDNA3.1-MSX2V5 using FuGENE6 transfection reagent (Roche Diagnostics, Basel, Switzerland) in normal growth medium. Three days later, cells were plated on 10-cm dishes and cultured until reaching a confluent state. After G418 selection, clones were subjected to Western blot analysis with a specific anti-V5 antibody (Invitrogen, Carlsbad, CA, USA).

Cell proliferation and cell viability assays
For cell proliferation assay, 6 000 cells were seeded per well in triplicate in 96-well plates in normal growth media. After 24-h (d1) and 72-h (d3) incubation, cell proliferation assay was performed using cell proliferation ELISA, BrdU (5-bromo-2-deoxyuridine) kit (Roche Diagnostics) according to the manufacturer's instructions. Cell proliferation ratio at d3 was normalized by that of d1. For cell viability assay, 10 000 cells were seeded per well in triplicate in 96-well plates in normal growth media. After 24-h incubation, cells were incubated with gemcitabine at various concentrations. After 48-h incubation, cell viability was measured by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were treated with MTT solution at a concentration o f 0 . 5 m g / m L f o r 2 h , a n d t h e n s o l u b i l i z e d i n dimethylsulfoxide (DMSO). Color reaction was measured by a spectrometer at a wavelength of 570 nm. Each experiment was repeated at least twice.

Detection of apoptotic cells
Apoptosis was determined by staining with Hoechst. Cells were plated at 1×10 4 on culture slide (Becton Dickinson, Franklin Lakes, NJ, USA) and allowed to adhere to the slide overnight. Gemcitabine (1 μg/mL) was added and incubated for an additional 24 h. The cells were fixed with 40 g/L paraformaldehyde for 20 min, followed by Hoechst 33342 (Calbiochem-Novabiochem, La Jolla, CA, USA) staining in these cells for 30 min. Then the cells were analyzed under a fluorescence microscope (Leica, Cambridge, UK). Between the incubations, the specimens were washed thrice with PBS. Chromatin condensation, nuclear shrinkage, and nucleosomal fragmentation were considered to be morphological markers of apoptosis.

Measurement of caspase-3 activities
APOCYTO Caspase-3 Colorimetric Assay Kit (MBL, Nagoya, Japan) was used for the detection of caspase-3 activity following the manufacturer's protocol. The subconfluent state cells in 10-cm dishes were harvested after 24-h incubation with gemcitabine (1 µg/mL), and subjected to caspase-3 activity detection.

Statistical analysis
The unpaired t-test and one-way ANOVA were used for statistical comparison. Calculations were made with the help of Microsoft Excel computer software (Microsoft, Redmond, WA, USA). P<0.05 was considered statistically significant.

Forced MSX2 expression in human pancreatic cancer cell line Panc-1
To evaluate whether MSX2 expression correlates with gemcitabine resistance in pancreatic cancer cells, we generated MSX2 stable transfectant cell line. As described previously (Hamada et al, submitted), V5-tagged MSX2 expression vector was transfected in Panc-1 cells. MSX2expressing clone (Px14) was selected and subjected to further analysis ( Figure 1A). Panc-1 cells transfected with empty vector were maintained with the culture media containing G418, and used as control cell line (P-3EV).
BrdU incorporation assay showed no significant difference in cell growth between two cell lines, but there was a tendency of slightly increased proliferation ratio in Px14 cells (P = 0.074, Figure 1B) relative to control cells (P-3EV cells), which was compatible to our previous report (Satoh et al, submitted).

Decrease of gemcitabine sensitivity in Panc-1 cells by MSX2 overexpression
Gemcitabine administration at a concentration of 10 µg/mL thus we examined caspase-3 activation in P-3EV and Px14 cells. Gemcitabine administration at a concentration of 1 µg/mL clearly induced active (cleaved) form of caspase-3 in P-3EV cells, but not in Px14 cells ( Figure  3A). Caspase-3 activation is a critical event in apoptosis induction; therefore, we hypothesized that gemcitabine resistance in Px14 cells might be due to suppressed caspase-3 activity. To evaluate this hypothesis, we conducted direct measurements of caspase-3 activities in P-3EV cells and Px14 cells, with or without gemcitabine treatment. The basal activities of caspase-3 in these cells were not significantly different ( Figure 3B). However, gemcitabine treatment at a concentration of 1 µg/mL significantly increased caspase-3 activity in P-3EV cells but not in Px14 cells as compared with its basal activity in control cells.

DISCUSSION
Even in pancreatic cancer cells, apoptotic signal does exist, but the signal is considered to be overwhelmed by inhibitor of apoptosis [10] . In our experiment, gemcitabine treatment induced apoptotic changes in P-3EV cells, whereas this effect was attenuated by MSX2 overexpression. Studies have shown that the ability of evasion from apoptosis is one of the critical steps for tumor progression. For example, interference of X-linked inhibitor of apoptosis (XIAP) expression in MDA-MB-231 mammary cancer cell line increases sensitivity to several chemotherapeutic agents [11] . The expression level of survivin in pancreatic cancer tissue is significantly associated with the reduction of the apoptotic index of tumor cells [12] and is also reported to be correlated with the prognosis of patients [13] , for 48 h decreased the viability of P-3EV cells by 60%, whereas that of Px14 cells only by 30% (Figure 2A). Px14 cells also depicted gemcitabine resistance at concentrations of 1 and 0.1 µg/mL (Figure 2A). Since Px14 cells were thought to be resistant to gemcitabine cytotoxicity, we analyzed gemcitabine-induced morphological alteration in P-3EV cells and Px14 cells. Gemcitabine-treated P-3EV cells at a concentration of 1 µg/mL for 24 h showed obvious apoptotic characteristics such as nuclear fragmentation and chromatin condensation, while these morphological changes were unremarkable in Px14 cells ( Figure 2B).

Gemcitabine-induced caspase-3 activation in P-3EV cells and Px14 cells
Gemcitabine treatment led P-3EV cells to apoptotic state,