Antitumor effects with apoptotic death in human promyelocytic leukemia HL‐60 cells and suppression of leukemia xenograft tumor growth by irinotecan HCl

Abstract

Irinotecan HCl (CPT-11) is an anticancer prodrug, but there is no available information addressing CPT-11-inhibited leukemia cells in in vitro and in vivo studies. Therefore, we investigated the cytotoxic effects of CPT-11 in promyelocytic leukemia HL-60 cells and in vivo and tumor growth in a leukemia xenograft model. Effects of CPT-11 on HL-60 cells were determined using flow cytometry, immunofluorescence staining, comet assay, real-time PCR, and Western blotting. CPT-11 demonstrated a dose- and time-dependent inhibition of cell growth, induction of apoptosis, and cell-cycle arrest at G0/G1 phase in HL-60 cells. CPT-11 promoted the release of AIF from mitochondria and its translocation to the nucleus. Bid, Bax, Apaf-1, caspase-9, AIF, Endo G, caspase-12, ATF-6b, Grp78, CDK2, Chk2, and cyclin D were all significantly upregulated and Bcl-2 was down-regulated by CPT-11 in HL-60 cells. Induction of cell-cycle arrest by CPT-11 was associated with changes in expression of key cell-cycle regulators such as CDK2, Chk2, and cyclin D in HL-60 cells. To test whether CPT-11 could augment antitumor activity in vivo, athymic BALB/c(nu/nu) nude mice were inoculated with HL-60 cells, followed by treatment with either CPT-11. The treatments significantly inhibited tumor growth and reduced tumor weight and volume in the HL-60 xenograft mice. The present study demonstrates the schedule-dependent antileukemia effect of CPT-11 using both in vitro and in vivo models. CPT-11 could potentially be a promising agent for the treatment of promyelocytic leukemia and requires further investigation.

Sulforaphane‐induced apoptosis in human leukemia HL‐60 cells through extrinsic and intrinsic signal pathways and altering associated genes expression assayed by cDNA microarray

“…HL‐60 cells (2 × 10 5 cells/well) were plated onto 12‐well plates and SFN was added at final concentrations of 0, 6, 7, 8, 9 and 10 μM for 24 and 48 h. At the end of incubation, cells were harvested and total viability was measured by flow cytometric methods described elsewhere (Chen et al, ).…”

“…HL‐60 cells (2 × 10 5 cells/well) in 12‐well plates were incubated with 0, 6, 7, 8, 9, and 10 μM of SFN for 24 and 48 h. Cells from each treatment were harvested, washed, fixed in 70% ethanol at −20°C, and then resuspended in PBS containing 40 μg/mL of PI, 0.1 mg/mL RNase, and 0.1% Triton X‐100 in a dark room for 30 min. They were then analyzed for cell‐cycle distribution and sub‐G1 phase (apoptosis) by a flow cytometer (FACS Calibur, Becton‐Dickinson, San Jose, CA, USA) as described previously (Chen et al ). CellQuest (Becton‐Dickinson) and ModFit LT software (Verity Software House Inc., USA) were used for calculating cell‐cycle distribution.…”

“…HL‐60 cells (2 × 10 5 cells/well) were placed in 12‐well plate and were incubated with or without 8 μM SFN for 0, 24, and 48 h. At the end of incubation, cells were harvested, washed, and resuspended in 25 μL of 10 μM substrate solution (PhiPhiLux and CaspaLux kit, OncoImmunin, Inc. Gaithersburg, MD, USA) before being incubated at 37°C for 60 min. Cells were washed and caspase activities were analyzed by flow cytometry as described previously (Chen et al ).…”

The ethanol extraction of prepared Psoralea corylifolia induces apoptosis and autophagy and alteres genes expression assayed by cDNA microarray in human prostate cancer PC‐3 cells

“…The transfer membrane was stained with primary antibodies anti‐Bax, ‐Bcl‐2, ‐Bid, ‐Fas‐L, ‐Fas, and p53 (Cell Signaling Technology, Beverly, MA) for apoptosis analysis and anti‐LC3 and ‐p62 (Cell Signaling Technology, Beverly, MA) for autophagy. Then the samples were washed and the membrane was incubated with peroxidase‐conjugated anti‐mouse, ‐rabbit, and ‐goat IgG (immunoglobulin G) (Santa Cruz Biotechnology) at room temperature for 1 h. Subsequently, the proteins were visualized, and chemiluminescence signals were enhanced using ECL (electrochemiluminescence) detection (Millipore) and then the “Image J” software was used to quantify the intensities of each protein band . The β‐actin was used to normalize the values shown in Figure .…”

“…Then the samples were washed and the membrane was incubated with peroxidase-conjugated anti-mouse, -rabbit, and -goat IgG (immunoglobulin G) (Santa Cruz Biotechnology) at room temperature for 1 h. Subsequently, the proteins were visualized, and chemiluminescence signals were enhanced using ECL (electrochemiluminescence) detection (Millipore) and then the "Image J" software was used to quantify the intensities of each protein band. 11,19 The b-actin was used to normalize the values shown in Figure 2 2.8 | cDNA microarray assay for genes expression in PCE treated PC-3 cells RNA extraction: PC-3 cells were incubated with and without 250 lg/ mL PCE for 48 h and then cells were scraped and collected by…”