SPARC silencing inhibits the growth of acute myeloid leukemia transformed from myelodysplastic syndrome via induction of cell cycle arrest and apoptosis

Abstract

Secreted protein acidic and rich in cysteine (SPARC) plays key roles in erythropoiesis; haploinsufficiency of SPARC is implicated in the progression of the 5q- syndrome. However, the role of SPARC in other subtypes of myelodysplastic syndrome (MDS) is not fully understood, particularly in the del(5q) type with a complex karyotype, which has a high risk to transform into acute myeloid leukemia (AML). In the present study, we investigated the role of SPARC in the proliferation and apoptosis of SKM-1 cells, an acute myeloid leukemia cell line transformed from an MDS cell line. SKM-1 cells were infected with SPARC-RNAi-LV or NC-GFP-LV lentivirus. Apoptosis and cell cycle profiling were assessed by flow cytometry, and cell proliferation was evaluated by MTS assay. The mRNA and protein expression levels of SPARC, p53, caspase-3, caspase-9 and Fas were detected by RT-PCR, real-time PCR and western blot assay. The SPARC shRNA constructed by us led to a significant reduction in SPARC expression in SKM-1 cells. SPARC knockdown inhibited the proliferation of SKM-1 cells by inducing cell cycle arrest at the G1/G0 phase and apoptosis. SPARC knockdown elevated the expression of p53, caspase-9, caspase-3 and Fas at both the mRNA and protein levels. SPARC silencing inhibited the growth of AML transformed from MDS by activating p53-induced apoptosis and cell cycle arrest. These data indicate that SPARC acts as an oncogene in transformed MDS/AML and is a potential therapeutic target in MDS/AML.

SPARC ectopic overexpression inhibits growth and promotes programmed cell death in acute myeloid leukemia transformed from myelodysplastic syndrome cells, alone and in combination with Ara-C treatment

“…Of note, these results differ from the recent findings in AML patients and cells where it has been shown that an increased level of SPARC expression either ectopically in vitro, or when detected in ex vivo primary leukemic blasts from AML patients, contributes to leukemia growth in vitro and aggressive disease in vivo (18). By contrast, the results from this and a previous study (22), exhibit proliferation inhibition and increased cell death after SPARC overexpression or silencing in the AML/MDS SKM-1 cell line. These contradicting results of SPARC involvement in the biology of different cell types, underscores the pleiotropic function and effects of SPARC protein in the cell life cycle and its complex role in disease where its involvement is unique sometimes promoting growth and survival, whereas it may also lead to inhibition of proliferation and apoptosis.…”

“…Further association of SPARC expression to MDS treatment management has yet to be established. In a previous study (22), we developed a lentiviral vector carrying a short hairpin RNA silencer for the SPARC gene and we employed this RNA interfering technology to knock down SPARC expression in SKM-1 cells. SPARC knockdown inhibited the proliferation of SKM-1 cells by inducing cell cycle arrest at the G1/G0 phase and apoptosis through p53 elevation of caspase-9 and -3 and Fas at the mRNA and protein levels.…”

“…The construction of a recombinant lentivirus vector-expressing SPARC gene has been described in a previous study (22). Briefly, SPARC cDNA was amplified by means of reverse transcription polymerase chain reaction (RT-PCR), using SPARC sequence-specific primers (Table I) and was subcloned into the lentiviral vector pGC-GV to develop a recombinant lentiviral construct designated as pGC-GV-SPARC (Jikai Biomedical Corp., Shanghai, China).…”

“…SKM-1 cells express a relatively high endogenous level of SPARC mRNA (22). The expression of SPARC protein was multiplied ectopically in SKM-1 cells, to create a suitable in vitro model for the SPARC-overexpression experiments.…”

SPAG6 silencing inhibits the growth of the malignant myeloid cell lines SKM-1 and K562 via activating p53 and caspase activation-dependent apoptosis

“…Cells were harvested, washed twice with ice-cold PBS and resuspended in binding buffer containing 1 µl of AnnexinV-PE and 5 µl of 7-AAD (KeyGen Biotech, Shanghai, China) (15). All specimens were determined by flow cytometry using CellQuest software (BD Biosciences) after incubation for 15 min at room temperature in a light-protected area.…”