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

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.

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Effect of CRT overexpression and CRT downregulation on proliferation of HL-60 cells induced by DADS. Inhibition of CRT expression by siRNA and CRT overexpression by plasmid transfection in HL-60 cells were evaluated by (A) reverse transcription-polymerase chain reaction: 1, HL-60; 2, HL-60/CRT; 3, HL-60/CRT siRNA; 4, DADS+HL-60; 5, DADS+HL-60/CRT siRNA; 6, DADS+HL-60/CRT (*P<0.05 vs. control cells) and (B) western blot analysis: 1, HL-60; 2, HL-60/CRT; 3, HL-60/CRT siRNA; 4, DADS+HL-60; 5, DADS+HL-60/CRT siRNA; 6, DADS+HL-60/CRT. (C) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide analysis in CRT-overexpressing and CRT-downregulated HL-60 cells, with and without DADS treatment. *P<0.05 vs. control cells. CRT, calreticulin; DADS, diallyl disulfide; siRNA, small interfering RNA; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
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f2-ol-0-0-4850: Effect of CRT overexpression and CRT downregulation on proliferation of HL-60 cells induced by DADS. Inhibition of CRT expression by siRNA and CRT overexpression by plasmid transfection in HL-60 cells were evaluated by (A) reverse transcription-polymerase chain reaction: 1, HL-60; 2, HL-60/CRT; 3, HL-60/CRT siRNA; 4, DADS+HL-60; 5, DADS+HL-60/CRT siRNA; 6, DADS+HL-60/CRT (*P<0.05 vs. control cells) and (B) western blot analysis: 1, HL-60; 2, HL-60/CRT; 3, HL-60/CRT siRNA; 4, DADS+HL-60; 5, DADS+HL-60/CRT siRNA; 6, DADS+HL-60/CRT. (C) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide analysis in CRT-overexpressing and CRT-downregulated HL-60 cells, with and without DADS treatment. *P<0.05 vs. control cells. CRT, calreticulin; DADS, diallyl disulfide; siRNA, small interfering RNA; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Mentions: The present study then analyzed the functional role of CRT in DADS-induced effects on HL-60 cells using CRT overexpression and CRT downregulation mediated by siRNA. The RT-PCR and western blot analyses confirmed that CRT knockdown by RNA interference was ~70%. In the DADS+siRNA-treated group, CRT expression was further inhibited (P=0.027). Notably, upon treatment of untransfected and CRT-overexpressing cells with 1.25 mg·l−1 DADS for 48 h, CRT levels were significantly reduced compared with the corresponding untreated cells (P=0.019), indicating that DADS could downregulate CRT expression in HL-60 cells (Fig. 2A and B).


Involvement of calreticulin in cell proliferation, invasion and differentiation in diallyl disulfide-treated HL-60 cells
Effect of CRT overexpression and CRT downregulation on proliferation of HL-60 cells induced by DADS. Inhibition of CRT expression by siRNA and CRT overexpression by plasmid transfection in HL-60 cells were evaluated by (A) reverse transcription-polymerase chain reaction: 1, HL-60; 2, HL-60/CRT; 3, HL-60/CRT siRNA; 4, DADS+HL-60; 5, DADS+HL-60/CRT siRNA; 6, DADS+HL-60/CRT (*P<0.05 vs. control cells) and (B) western blot analysis: 1, HL-60; 2, HL-60/CRT; 3, HL-60/CRT siRNA; 4, DADS+HL-60; 5, DADS+HL-60/CRT siRNA; 6, DADS+HL-60/CRT. (C) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide analysis in CRT-overexpressing and CRT-downregulated HL-60 cells, with and without DADS treatment. *P<0.05 vs. control cells. CRT, calreticulin; DADS, diallyl disulfide; siRNA, small interfering RNA; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2-ol-0-0-4850: Effect of CRT overexpression and CRT downregulation on proliferation of HL-60 cells induced by DADS. Inhibition of CRT expression by siRNA and CRT overexpression by plasmid transfection in HL-60 cells were evaluated by (A) reverse transcription-polymerase chain reaction: 1, HL-60; 2, HL-60/CRT; 3, HL-60/CRT siRNA; 4, DADS+HL-60; 5, DADS+HL-60/CRT siRNA; 6, DADS+HL-60/CRT (*P<0.05 vs. control cells) and (B) western blot analysis: 1, HL-60; 2, HL-60/CRT; 3, HL-60/CRT siRNA; 4, DADS+HL-60; 5, DADS+HL-60/CRT siRNA; 6, DADS+HL-60/CRT. (C) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide analysis in CRT-overexpressing and CRT-downregulated HL-60 cells, with and without DADS treatment. *P<0.05 vs. control cells. CRT, calreticulin; DADS, diallyl disulfide; siRNA, small interfering RNA; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Mentions: The present study then analyzed the functional role of CRT in DADS-induced effects on HL-60 cells using CRT overexpression and CRT downregulation mediated by siRNA. The RT-PCR and western blot analyses confirmed that CRT knockdown by RNA interference was ~70%. In the DADS+siRNA-treated group, CRT expression was further inhibited (P=0.027). Notably, upon treatment of untransfected and CRT-overexpressing cells with 1.25 mg·l−1 DADS for 48 h, CRT levels were significantly reduced compared with the corresponding untreated cells (P=0.019), indicating that DADS could downregulate CRT expression in HL-60 cells (Fig. 2A and B).

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.


Related in: MedlinePlus