Effect of thalidomide in combination with gemcitabine on human pancreatic carcinoma SW-1990 cell lines in vitro and in vivo

Abstract

Pancreatic cancer is one of the most frequently occurring malignancies worldwide and it is the fourth most common cause of cancer-associated mortality in Western countries. Thalidomide (THD) plays an important role in tumor therapy, as it is able to promote early stage apoptosis and inhibit the process of angiogenesis. The present study evaluated the ability of the combination of THD and gemcitabine (GEM) to inhibit the growth of the pancreatic cancer SW-1990 cell line in vitro and in vivo. Early apoptosis in the SW-1990 cells was detected by the Annexin V/propidium iodide double staining method, the level of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) were detected by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. In addition, the expression of vascular endothelial growth factor in transplanted tumor tissue was measured by RT-PCR, immunohistochemistry and western blot analysis. Cluster of differentiation 34 positivity was considered to indicate the microvessel density. Subsequent to treatment with THD and GEM alone or in combination, it was found that the expression of Bax was upregulated, while the expression of Bcl-2 was downregulated, and the growth of SW-1990 cells and transplanted tumors in nude mice was evidently inhibited. The administration of THD in combination with GEM may demonstrate a potent antitumor effect that increases with increasing dose. The mechanism behind the antitumor effect may be associated with the inhibition of tumor angiogenesis and induction of the apoptosis pathway.

Keywords: angiogenesis; apoptosis; gemcitabine; pancreatic cancer; thalidomide.

Figure 1.

The inhibitory effect of THD on the growth of the pancreatic cancer SW-1990 cell line in vitro. (A) The SW-1990 cells were incubated with increasing concentrations of THD, ranging between 0 and 200 µg/ml, for 0, 24, 48 and 72 h. The cell counting kit 8 assay was then used to analyze the cell viability. (B) The growth inhibition exerted on SW-1990 cells by the treatments was calculated and the data are presented as the mean ± standard error. (C) The percentage of apoptotic and necrotic SW-1990 cells was analyzed using an Annexin V/propidium iodide assay. (D) Subsequent to treatment with the indicated concentrations of THD, the SW-1990 cells were harvested and the mRNA levels of Bcl-2 and Bax were analyzed by reverse transcription-polymerase chain reaction. (E) The expression of the Bcl-2 and Bax proteins in the SW-1990 cells was determined by western blotting. THD. thalidomide; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; Con, control.

Figure 2.

Inhibitory effect of THD combined with GEM on the growth of the pancreatic cancer SW-1990 cell line in vitro. (A) The SW-1990 cells were incubated with 50 µg/ml THD, 20 µmol/l GEM or a combination of the two for 0, 24, 48 and 72 h. A counting cell kit 8 assay was then used to analyze the viability of the cells. (B) The growth inhibition exerted on SW-1990 cells was calculated and the data are presented as the mean ± standard error. (C) The percentage of apoptotic and necrotic SW-1990 cells was analyzed using an Annexin V/propidium iodide assay. (D) Subsequent to treatment with the indicated concentrations of 50 µg/ml THD, 20 µmol/l GEM or a combination of the two, the SW-1990 cells were harvested and the levels of Bcl-2 and Bax mRNA were analyzed by reverse transcription-polymerase chain reaction. (E) The expression of the Bcl-2 and Bax proteins in the SW-1990 cells was determined by western blotting. THD, thalidomide; GEM, gemcitabine; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; Con, control.

Figure 3.

Inhibitory effect of combined treatment with THD and GEM on the growth of the pancreatic cancer SW-1990 cell line in vivo. (A) Gross morphology of xenografts in nude mice on the 28th day of treatment. (B) Changes in the volume of the xenograft in nude mice subsequent to treatment with THD and GEM. Compared with the NS-treated control, treatment with THD, GEM and a combination of the two demonstrated a significantly increased inhibition of tumor growth. (C) Four weeks after treatment with THD, GEM and the combination of the two, the tumor weights were evidently decreased compared with the NS-treated control treatment. The data are expressed as the mean ± standard error. (D) Subsequent to treatment with the indicated concentrations of 200 mg/kg THD, 50 mg/kg gemcitabine and combined treatment, the mRNA levels of Bcl-2, Bax and VEGF in nude mice xenografts were analyzed by reverse transcription-polymerase chain reaction. (E) The expression of the Bcl-2, Bax and VEGF proteins in nude mice xenografts was determined by western blotting. *P<0.05 vs. NS-treated mice. THD. thalidomide; GEM, gemcitabine; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; VEGF, vascular endothelial growth factor.

Figure 4.

Antiangiogenic effect of the administration of 200 mg/kg THD combined with 50 mg/kg GEM on a nude mouse xenograft model, evaluated by immunohistochemical analysis. (A) Immunohistochemical expression of vascular endothelial growth factor in paraffin sections that were probed and stained with the appropriate antibody and analyzed under light microscopy. Magnification, ×200. (B) Immunohistochemical detection of the expression of cluster of differentiation 34. Magnification, ×200. THD, thalidomide; GEM, gemcitabine.