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Enhancing effects of indirubin on the arsenic disulfide-induced apoptosis of human diffuse large B-cell lymphoma cells.

Wang L, Li X, Liu X, Lu K, Chen NA, Li P, Lv X, Wang X - Oncol Lett (2015)

Bottom Line: The qPCR results revealed that indirubin alone had no enhancing effect upon the Bax/Bcl-2 mRNA expression ratio and caspase-3 mRNA expression.However, the combination of indirubin and As2S2 yielded enhancing effects.The enhancing effect was due, in part, to the induction of the mitochondrial apoptotic pathway, which involves the cleavage of Bax.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology, Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, P.R. China ; Department of Hematology, Taian City Central Hospital, Tai'an, Shandong 271000, P.R. China.

ABSTRACT

The aim of the present study was to investigate the indirubin-enhanced effects of arsenic disulfide (As2S2) on the proliferation and apoptosis of diffuse large B-cell lymphoma (DLBCL) cells in order to identify an optimum combination therapy. The human DLBCL cells, LY1 and LY8, were treated with different concentrations of indirubin for 24, 48 and 72 h. Next, the cells were treated with 10 μM As2S2 or a combination of 10 μM As2S2 and 20 μM indirubin for 48 h. Cell proliferation inhibition was detected using cell counting kit-8 and cell apoptosis was determined using flow cytometry. The expression levels of Bcl-2, Bcl-2-associated X protein (Bax) and caspase-3 were analyzed by quantitative polymerase chain reaction (qPCR) and western blotting. The DLBCL cell viability exhibited no significant changes at 24, 48 or 72 h with increasing indirubin concentration. In addition, the apoptotic rates of the LY1 and LY8 cells demonstrated no noticeable effects at 48 h with increasing indirubin concentration. Following treatment with the combination of indirubin and As2S2, the inhibitory and apoptotic rates of the cells were notably increased compared with those of the As2S2-treated group. The qPCR results revealed that indirubin alone had no enhancing effect upon the Bax/Bcl-2 mRNA expression ratio and caspase-3 mRNA expression. Western blot analysis revealed that indirubin alone had an enhancing effect upon the Bax/Bcl-2 protein ratio and procaspase-3 protein expression. In addition, the results demonstrated that the 21-KDa Bax protein was proteolytically cleaved into an 18-KDa Bax in the DLBCL cells treated with the combination of indirubin and As2S2. Indirubin alone did not inhibit proliferation or induce the apoptosis of the LY1 and LY8 cells. However, the combination of indirubin and As2S2 yielded enhancing effects. Therefore, the results of the present study demonstrated that with regard to antitumor activities, As2S2 served as the principal drug, whereas indirubin served as the adjuvant drug. The enhancing effect was due, in part, to the induction of the mitochondrial apoptotic pathway, which involves the cleavage of Bax.

No MeSH data available.


Related in: MedlinePlus

Effects of indirubin and arsenic disulfide (As2S2), alone or in combination, on the levels of B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax) and caspase-3 protein in diffuse large B-cell lymphoma cells. (A) LY1 and (B) LY8 cells were treated with 10 μM As2S2 and 20 μM indirubin, alone or in combination, for 48 h. Western blotting was used to analyze whole cell lysates for Bax, Bcl-2 and caspase-3. β-actin expression was used as an internal control. The relative density of caspase-3 and the Bax/Bcl-2 ratio in (C) LY1 and (D) LY8 cells was calculated from three separate experiments. *P<0.05 and **P<0.01 vs. untreated control group.
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f5-ol-09-04-1940: Effects of indirubin and arsenic disulfide (As2S2), alone or in combination, on the levels of B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax) and caspase-3 protein in diffuse large B-cell lymphoma cells. (A) LY1 and (B) LY8 cells were treated with 10 μM As2S2 and 20 μM indirubin, alone or in combination, for 48 h. Western blotting was used to analyze whole cell lysates for Bax, Bcl-2 and caspase-3. β-actin expression was used as an internal control. The relative density of caspase-3 and the Bax/Bcl-2 ratio in (C) LY1 and (D) LY8 cells was calculated from three separate experiments. *P<0.05 and **P<0.01 vs. untreated control group.

Mentions: The protein expression levels of Bax, Bcl-2 and caspase-3 were investigated. The protein levels following treatment are shown in Fig. 5. Subsequent to treatment for 48 h, the expression of Bcl-2 was markedly downregulated, whereas the expression of Bax was notably upregulated in the LY1 and LY8 cells of the combination group compared with the control. Furthermore, the Bax/Bcl-2 ratio was significantly increased. Our previous study demonstrated that following exposure to 10 μM As2S2, the levels of the 21-KDa Bax protein and the 18-KDa Bax cleavage protein were markedly increased (21). Western blot analysis also revealed that following treatment, the levels of the 21-KDa Bax protein and the cleaved 18-KDa Bax protein were elevated in the combination group. By contrast, procaspase-3 expression was evidently reduced.


Enhancing effects of indirubin on the arsenic disulfide-induced apoptosis of human diffuse large B-cell lymphoma cells.

Wang L, Li X, Liu X, Lu K, Chen NA, Li P, Lv X, Wang X - Oncol Lett (2015)

Effects of indirubin and arsenic disulfide (As2S2), alone or in combination, on the levels of B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax) and caspase-3 protein in diffuse large B-cell lymphoma cells. (A) LY1 and (B) LY8 cells were treated with 10 μM As2S2 and 20 μM indirubin, alone or in combination, for 48 h. Western blotting was used to analyze whole cell lysates for Bax, Bcl-2 and caspase-3. β-actin expression was used as an internal control. The relative density of caspase-3 and the Bax/Bcl-2 ratio in (C) LY1 and (D) LY8 cells was calculated from three separate experiments. *P<0.05 and **P<0.01 vs. untreated control group.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4356417&req=5

f5-ol-09-04-1940: Effects of indirubin and arsenic disulfide (As2S2), alone or in combination, on the levels of B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax) and caspase-3 protein in diffuse large B-cell lymphoma cells. (A) LY1 and (B) LY8 cells were treated with 10 μM As2S2 and 20 μM indirubin, alone or in combination, for 48 h. Western blotting was used to analyze whole cell lysates for Bax, Bcl-2 and caspase-3. β-actin expression was used as an internal control. The relative density of caspase-3 and the Bax/Bcl-2 ratio in (C) LY1 and (D) LY8 cells was calculated from three separate experiments. *P<0.05 and **P<0.01 vs. untreated control group.
Mentions: The protein expression levels of Bax, Bcl-2 and caspase-3 were investigated. The protein levels following treatment are shown in Fig. 5. Subsequent to treatment for 48 h, the expression of Bcl-2 was markedly downregulated, whereas the expression of Bax was notably upregulated in the LY1 and LY8 cells of the combination group compared with the control. Furthermore, the Bax/Bcl-2 ratio was significantly increased. Our previous study demonstrated that following exposure to 10 μM As2S2, the levels of the 21-KDa Bax protein and the 18-KDa Bax cleavage protein were markedly increased (21). Western blot analysis also revealed that following treatment, the levels of the 21-KDa Bax protein and the cleaved 18-KDa Bax protein were elevated in the combination group. By contrast, procaspase-3 expression was evidently reduced.

Bottom Line: The qPCR results revealed that indirubin alone had no enhancing effect upon the Bax/Bcl-2 mRNA expression ratio and caspase-3 mRNA expression.However, the combination of indirubin and As2S2 yielded enhancing effects.The enhancing effect was due, in part, to the induction of the mitochondrial apoptotic pathway, which involves the cleavage of Bax.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology, Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, P.R. China ; Department of Hematology, Taian City Central Hospital, Tai'an, Shandong 271000, P.R. China.

ABSTRACT

The aim of the present study was to investigate the indirubin-enhanced effects of arsenic disulfide (As2S2) on the proliferation and apoptosis of diffuse large B-cell lymphoma (DLBCL) cells in order to identify an optimum combination therapy. The human DLBCL cells, LY1 and LY8, were treated with different concentrations of indirubin for 24, 48 and 72 h. Next, the cells were treated with 10 μM As2S2 or a combination of 10 μM As2S2 and 20 μM indirubin for 48 h. Cell proliferation inhibition was detected using cell counting kit-8 and cell apoptosis was determined using flow cytometry. The expression levels of Bcl-2, Bcl-2-associated X protein (Bax) and caspase-3 were analyzed by quantitative polymerase chain reaction (qPCR) and western blotting. The DLBCL cell viability exhibited no significant changes at 24, 48 or 72 h with increasing indirubin concentration. In addition, the apoptotic rates of the LY1 and LY8 cells demonstrated no noticeable effects at 48 h with increasing indirubin concentration. Following treatment with the combination of indirubin and As2S2, the inhibitory and apoptotic rates of the cells were notably increased compared with those of the As2S2-treated group. The qPCR results revealed that indirubin alone had no enhancing effect upon the Bax/Bcl-2 mRNA expression ratio and caspase-3 mRNA expression. Western blot analysis revealed that indirubin alone had an enhancing effect upon the Bax/Bcl-2 protein ratio and procaspase-3 protein expression. In addition, the results demonstrated that the 21-KDa Bax protein was proteolytically cleaved into an 18-KDa Bax in the DLBCL cells treated with the combination of indirubin and As2S2. Indirubin alone did not inhibit proliferation or induce the apoptosis of the LY1 and LY8 cells. However, the combination of indirubin and As2S2 yielded enhancing effects. Therefore, the results of the present study demonstrated that with regard to antitumor activities, As2S2 served as the principal drug, whereas indirubin served as the adjuvant drug. The enhancing effect was due, in part, to the induction of the mitochondrial apoptotic pathway, which involves the cleavage of Bax.

No MeSH data available.


Related in: MedlinePlus