Fludarabine

Fludarabine efficiently inhibits the proliferation of RPMI 8226 cells with IC50 of 1.54 μg/mL. The IC50 of this compound against MM.1S and MM.1R cells is 13.48 μg/mL and 33.79 μg/mL, respectively. In contrast, U266 cells are resistant to this chemical with IC50 of 222.2 μg/mL. This compound treatment results in increased number of cells in the G1 phase of cell cycle, accompanied with a concomitant reduction of cells at the S phase of cell cycle in a time-dependent manner. It induces a cell cycle block and triggers apoptosis in MM cells. It triggers time-dependent cleavage of caspase-8, -9, and -3, -7, followed by PARP cleavage. It increases expression of Bax in a time-dependent fashion, while the expression of Bak doesn't change. After exposure to this chemical for 12 hours, RPMI 8226 cells shows a loss of membrane potential with 61.05% of the cells expressing low fluorescence of rhodamine 123 compared with 8.62% of cells in untreated control. [1] To enhance solubility, it is formulated as the monophosphate (F-ara-AMP, fudarabine), which is instantaneously and quantitatively dephosphorylated to the parent nucleoside upon intravenous infusion. Inside the cells rephosphorylation occurs which leads to fuoroadenine arabinoside triphosphate (F-ara-ATP), the major cytotoxic metabolite of F-ara-A. [2] It can also induce pro-inflammatory stimulation of monocytic cells, as evaluated by increased expression of ICAM-1 and IL-8 release. [3] This compound does not affect the growth of ovarian cancer cell lines, whereas it induces marked and dose-dependent inhibition of proliferation in melanoma cell lines. [4] It is an inhibitor of STAT1 that specifically reduces STAT1 without affecting other STAT family members[5]. In addition to cytoplasmic accumulation, repeated low-dose cisplatin (RLDC) induces HMGB1 expression, which is marked suppressed by STAT1 knockdown. Consistently, this chemical suppresses HMGB1 expression during RLDC treatment dose-dependently in RLDC-treat renal tubular cells[5].

After treated with Fludarabine or control, dexamethasone-sensitive (MM.1S) and -resistant (MM.1R) human MM cell lines, RPMI8226 and U266 cell lines (5 × 10⁵ cells) are washed twice in phosphate-buffered saline (PBS) and fixed with 70% ice-cold ethanol, then centrifuged and suspended in PBS containing 100 μg/mL RNase A. After incubated for 30 minutes at 37 ºC, samples are resuspended in 25 μg/mL propidium iodide. Flow cytometry is performed on a FACSCalibur automated system. Apoptosis is determined by Annexin V-FITC apoptosis detection kit, according to the manufacturer's instructions. For TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling) assay, cells are analyzed by flow cytometry using the in situ cell death detection kit.

Tumors treated with PBS grow rapidly to approximately 10-fold their initial volume in 25 day, whereas, the tumors in the Fludarabine at 40 mg/kg increase less than 5-fold. A significant antitumor effect of 40 mg/kg of this compound on RPMI8226 tumor growth is demonstrated. RPMI8226 tumors treated with 40 mg/kg of this chemical at day 10 increase apoptotic nuclei. This compound is effective in suppressing RPMI8226 myeloma xenografts in SCID mice. [1]