Abstract. Studies conducted in our lab have indicated that thalidomide cytotoxicity in the KG-1a human acute myelogenous leukemia (AML) cell line was enhanced by combining it with arsenic trioxide. The current investigation was conducted in order to evaluate the effect of thalidomide either alone or in combination with arsenic trioxide on the release of tumor necrosis factor-α (TNF-α) and vascular endothelial growth factor (VEGF) from this cell line in an attempt to clarify its possible cytotoxic mechanism(s). Human AML cell line KG-1a was used in this study. The cells were cultured for 48 h in the presence or absence of thalidomide (5 mg/l), and or arsenic trioxide (4 µΜ). The levels of TNF-α and VEGF in the supernatant were determined by ELISA. Results obtained indicate that the levels of TNF-α in the supernatant of KG-1a cell cultures incubated with thalidomide, arsenic trioxide, or combination were statistically lower than those observed in the supernatant of control cells (2.89, 5.07, 4.15 and 16.88 pg/ml, respectively). However, the levels of VEGF in the supernatant of thalidomide-treated cells were statistically higher than those in the supernatant of control cells (69.61 vs. 11.48 pg/l). Arsenic trioxide, whether alone or in combination with thalidomide, did not produce any statistically significant difference in the levels of VEGF as compared to the control or thalidomide-treated cell supernatant. These findings indicate that thalidomide and the arsenic trioxide inhibition of TNF-α production by KG-1a cells may play an important role in their cytotoxic effect.

Abstract. Studies have shown that thalidomide exerts modest activity as a single agent in the therapy of acute myeloid leukemia (AML). The present investigation was conducted to test the hypothesis that the cytotoxic effect of thalidomide is enhanced when properly combined with other chemotherapeutic agents. The human AML cell line KG-1a was used in this study. Cells were cultured for 48 h in the presence or absence of thalidomide, arsenic trioxide and a combination of the two substances. Results obtained indicate that thalidomide at concentrations of 1, 2 and 5 mg/l produced a dose-dependent cytotoxic effect and at 5 mg/ml resulted in late apoptosis in 49.39% of the total cell population (as compared to 5.35% in the control cells). When the cells were incubated with arsenic trioxide alone (4 µM), late apoptosis was detected in 16.97% of the total cell population. However, when cells were incubated with a combination of thalidomide (5 mg/l) and arsenic trioxide (4 µM), late apoptosis was noted to be 80.6% in the total cell population. This percentage of late apoptosis was statistically significant from that observed when cells were incubated with thalidomide alone. These findings clearly indicate that arsenic trioxide enhances the cytotoxic effects of thalidomide.

4165 Although the exact mechanism of action of thalidomide is still unknown, it has been suggested that it may affect TNF-α in addition to its possible anti-angiogenic and immunomodulatory properties. Studies conducted in our lab have indicated that thalidomide cytotoxicity in KG-1a human acute myelogenous leukemia cell line was enhanced by combining it with arsenic trioxide. So, the current investigation was conducted in order to evaluate the effect of thalidomide either alone or in combination with arsenic trioxide on the release of TNF-α and VEGF from this cell line in an attempt to clarify its possible cytotoxic mechanism. Human acute myelogenous leukemia cell line KG-1a (obtained from American Type Culture Collection, ATCC) grown in complete medium containing Iscove's modified Dulbecco's medium and fetal bovine albumin were used in this study. The cells were cultured for 48 hours in a 12 well-culture plates in duplicates at a concentration of 2×106 cells/ml in the presence or absence of thalidomide (5 mg/L) [Tocris bioscience, Ellisville, Mo] and or arsenic trioxide (4 μM) [Sigma-Aldrich, Inc., St. Louis, MO]. Cells were harvested by centrifugation and the levels of TNF-α and VEGF in the supernatant were determined by ELISA using the Quantikine TNF-α and VEGF kits respectively (R&D Systems, Minneapolis, MN). Results obtained indicate that the levels of TNF-α in the supernatant of KG-1a cell cultures incubated with both thalidomide and arsenic trioxide, whether alone or in combination were statistically lower than those observed in the supernatant of control cells (2.89 pg/ml in thalidomide treated cells supernatant, 5.07 pg/ml in arsenic trioxide treated cells supernatant and 4.15 pg/ml in case of the combined treatment with thalidomide and arsenic trioxide versus 16.88 pg/ml in the supernatant of control cells, p<0.05). However, the levels of VEGF in the supernatant of thalidomide treated cells were statistically higher than those in the supernatant of control cells (69.61 pg/ml versus 11.48 pg/ml, p< 0.001). Of note, that arsenic trioxide whether alone or in combination with thalidomide did not produce any statistically significant difference in the levels of VEGF as compared to control or thalidomide treated cell supernatant. These findings clearly indicate that both thalidomide and arsenic trioxide inhibition of TNF-α production by KG-1a cells may play an important role in their cytotoxic effect. However the increase in VEGF levels in the supernatants of thalidomide treated KG-1a cells may reflect a compensatory mechanism of cells which have survived thalidomide cytotoxicity (Supported by NIH grant RR03020). Disclosures: No relevant conflicts of interest to declare.