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Involvement of calreticulin in cell proliferation, invasion and differentiation in diallyl disulfide-treated HL-60 cells

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ABSTRACT

Diallyl disulfide (DADS) has shown potential as a therapeutic agent in various cancers. Previously, calreticulin (CRT) was found to be downregulated in differentiated HL-60 cells treated with DADS. The present study investigated the role of CRT proteins in DADS-induced proliferation, invasion and differentiation in HL-60 cells. The present study demonstrated that DADS treatment significantly changed the morphology of HL-60 cells and caused the significant time-dependent downregulation of CRT. Small interfering RNA (siRNA)-mediated knockdown of CRT expression significantly inhibited proliferation, decreased invasion ability, increased the expression of cluster of differentiation (CD)11b and reduced the expression of CD33 in DADS-treated HL-60 cells. DADS also significantly affected cell proliferation, invasion and differentiation in CRT-overexpressed HL-60 cells. Nitroblue tetrazolium (NBT) reduction assays showed decreased NBT reduction activity in the CRT overexpression group and increased NBT reduction in the CRT siRNA group. Following treatment with DADS, the NBT reduction abilities in all groups were increased. In conclusion, the present study clearly demonstrates the downregulation of CRT during DADS-induced differentiation in HL-60 cells and indicates that CRT is involved in cell proliferation, invasion and differentiation in DADS-treated HL-60 cells.

No MeSH data available.


Downregulation of CRT during DADS-induced differentiation in HL-60 cells. (A) Morphological changes of HL-60 cells after 1.25 mg/l DADS treatment for 48 h (Wright-Giemsa staining; magnification, ×100). (B) CRT messenger RNA levels in HL-60 cells were decreased by 1.25 mg/l DADS treatment at 24, 48 and 72 h, as determined by reverse transcription-quantitative polymerase chain reaction (relative to β-actin). *P<0.05 vs. control cells. (C) Representative western blot analysis of CRT expression using GAPDH as the internal control. (D) Quantification of CRT expression in western blot analyses. Values are mean ± standard deviation from three independent experiments. *P<0.05 vs. control cells. CRT, calreticulin; DADS, diallyl disulfide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
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f1-ol-0-0-4850: Downregulation of CRT during DADS-induced differentiation in HL-60 cells. (A) Morphological changes of HL-60 cells after 1.25 mg/l DADS treatment for 48 h (Wright-Giemsa staining; magnification, ×100). (B) CRT messenger RNA levels in HL-60 cells were decreased by 1.25 mg/l DADS treatment at 24, 48 and 72 h, as determined by reverse transcription-quantitative polymerase chain reaction (relative to β-actin). *P<0.05 vs. control cells. (C) Representative western blot analysis of CRT expression using GAPDH as the internal control. (D) Quantification of CRT expression in western blot analyses. Values are mean ± standard deviation from three independent experiments. *P<0.05 vs. control cells. CRT, calreticulin; DADS, diallyl disulfide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Mentions: As shown in Fig. 1A, the treatment of HL-60 cells with 1.25 mg/l DADS for 48 h induced cells to differentiate into granulocyte-like cells. The cytomorphoses of the DADS-treated cells were clearly varied from the controls. RT-PCR confirmed the significant downregulation of CRT messenger RNA (mRNA) levels in HL-60 cells following treatment with 1.25 mg/l DADS in a time-dependent manner, compared with untreated HL-60 cells (P=0.042; Fig. 1B). Western blot analysis was consistent with the RT-PCR results, showing a decrease in CRT protein levels in HL-60 cells treated with 1.25 mg/l DADS in a time-dependent manner (P=0.021; Fig. 1C and D).


Involvement of calreticulin in cell proliferation, invasion and differentiation in diallyl disulfide-treated HL-60 cells
Downregulation of CRT during DADS-induced differentiation in HL-60 cells. (A) Morphological changes of HL-60 cells after 1.25 mg/l DADS treatment for 48 h (Wright-Giemsa staining; magnification, ×100). (B) CRT messenger RNA levels in HL-60 cells were decreased by 1.25 mg/l DADS treatment at 24, 48 and 72 h, as determined by reverse transcription-quantitative polymerase chain reaction (relative to β-actin). *P<0.05 vs. control cells. (C) Representative western blot analysis of CRT expression using GAPDH as the internal control. (D) Quantification of CRT expression in western blot analyses. Values are mean ± standard deviation from three independent experiments. *P<0.05 vs. control cells. CRT, calreticulin; DADS, diallyl disulfide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4998039&req=5

f1-ol-0-0-4850: Downregulation of CRT during DADS-induced differentiation in HL-60 cells. (A) Morphological changes of HL-60 cells after 1.25 mg/l DADS treatment for 48 h (Wright-Giemsa staining; magnification, ×100). (B) CRT messenger RNA levels in HL-60 cells were decreased by 1.25 mg/l DADS treatment at 24, 48 and 72 h, as determined by reverse transcription-quantitative polymerase chain reaction (relative to β-actin). *P<0.05 vs. control cells. (C) Representative western blot analysis of CRT expression using GAPDH as the internal control. (D) Quantification of CRT expression in western blot analyses. Values are mean ± standard deviation from three independent experiments. *P<0.05 vs. control cells. CRT, calreticulin; DADS, diallyl disulfide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Mentions: As shown in Fig. 1A, the treatment of HL-60 cells with 1.25 mg/l DADS for 48 h induced cells to differentiate into granulocyte-like cells. The cytomorphoses of the DADS-treated cells were clearly varied from the controls. RT-PCR confirmed the significant downregulation of CRT messenger RNA (mRNA) levels in HL-60 cells following treatment with 1.25 mg/l DADS in a time-dependent manner, compared with untreated HL-60 cells (P=0.042; Fig. 1B). Western blot analysis was consistent with the RT-PCR results, showing a decrease in CRT protein levels in HL-60 cells treated with 1.25 mg/l DADS in a time-dependent manner (P=0.021; Fig. 1C and D).

View Article: PubMed Central - PubMed

ABSTRACT

Diallyl disulfide (DADS) has shown potential as a therapeutic agent in various cancers. Previously, calreticulin (CRT) was found to be downregulated in differentiated HL-60 cells treated with DADS. The present study investigated the role of CRT proteins in DADS-induced proliferation, invasion and differentiation in HL-60 cells. The present study demonstrated that DADS treatment significantly changed the morphology of HL-60 cells and caused the significant time-dependent downregulation of CRT. Small interfering RNA (siRNA)-mediated knockdown of CRT expression significantly inhibited proliferation, decreased invasion ability, increased the expression of cluster of differentiation (CD)11b and reduced the expression of CD33 in DADS-treated HL-60 cells. DADS also significantly affected cell proliferation, invasion and differentiation in CRT-overexpressed HL-60 cells. Nitroblue tetrazolium (NBT) reduction assays showed decreased NBT reduction activity in the CRT overexpression group and increased NBT reduction in the CRT siRNA group. Following treatment with DADS, the NBT reduction abilities in all groups were increased. In conclusion, the present study clearly demonstrates the downregulation of CRT during DADS-induced differentiation in HL-60 cells and indicates that CRT is involved in cell proliferation, invasion and differentiation in DADS-treated HL-60 cells.

No MeSH data available.