CAL-101 (Idelalisib)

Idelalisib (CAL-101) is not sensitive to other PI3K class I subunits including p110α, p110β, and p110γ. It specifically blocks FcϵR1 p110δ-mediated CD63 expression with an EC50 of 8 nM in primary basophil. This compound exhibits greater activity in B-cell acute lymphoblastic leukemia (B-ALL) and chronic lymphocytic leukemia (CLL) cells compared with acute myeloid leukemia (AML) and myeloproliferative neoplasm (MPN) cells. It produces the reduction in pAktS473, pAktT308, and the downstream target S6 in SU-DHL-5, KARPAS-422 and CCRF-SB cells with EC50 of 0.1 to 1.0 μM. [1]

It induces selective cytotoxicity in CLL cells independent of IgVH mutational status or interphase cytogenetics, primarily through a caspase-dependent mechanism. The compound induces cytotoxicity preferentially to CLL cells compared with normal B cells, without producing cytotoxicity in other hematopoietic cells, compared to LY294002. It lacks direct cytotoxic potential to T cells and nature killer (NK) cells. It can inhibit production of inflammatory cytokines, such as IL-6, IL-10, TNF-α, and IFN-γ, and activation-induced cytokines, such as CD40L. It also antagonizes CD40L-mediated CLL cell survival. [2]

It induces an accumulation of cells in G1 and a decrease in the S-phase population in L1236 and L591 cells, which indicates this compound as a novel strategy for the treatment of hodgkin lymphoma (HL). [3]

• PI3K assay

  PI3K assay is preformed on whole-cell lysates from CLL or normal B cells. A PI3K ELISA assay is performed. Briefly, whole-cell extracts are added to a mixture of PI(4,5)P2 substrate and reaction buffer containing adenosine triphosphate (ATP) and allowed to incubate at room temperature. The reaction is stopped by adding PI(3,4,5)P3 detector mixed with EDTA (ethylenediaminetetraacetic acid) and allowed to incubate at room temperature for 1 hour. After this time, the mixture is transferred from each well to a PI3K ELISA plate and allowed to incubate 1 hour. Plates are washed and then incubated with secondary detector for 30 minutes. Plates are washed again, and 3,3′,5,5′-tetramethylbenzidine solution is added for 5 minutes at which time H2SO4 is added to stop all reactions. Plates are read at 450 nm on a Labsystems 96-well plate reader.

• 세포주

  CLL B cells or healthy volunteer T cells or NK cells

• 농도

  0.01-100 μM

• 배양 시간

  48 hours

• 방법

  MTT assays are performed to determine cytotoxicity. 1 × 10⁵ cells are incubated with Idelalisib (CAL-101). MTT reagent is then added, and plates are incubated for an additional 20 hours before washing in phosphate-buffered saline. DMSO is added, and absorbance is measured by spectrophotometry at 540 nm in a Labsystems plate reader. Cell viability is also measured at various time points with the use of annexin/PI flow cytometry. Data are analyzed. At least 104 cells are counted for each sample. Results are expressed as the percentage of total positive cells over untreated control. Experiments examining caspase-dependent apoptosis included the addition of 100 μM Z-VAD. Experiments examining survival signals include the addition of 1 μg/mL CD40L, 800 U/mL IL-4, 50 ng/mL BAFF, 20 ng/mL TNF-α, or coculturing on fibronectin or stromal (HS-5 cell line) coated plates. Stromal coculture is done by plating a 75-cm² flask (80%-100% confluent) per 6-well plate 24 hours before the addition of CLL cells.