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, 16 (8), 19387-400

15,16-Dihydrotanshinone I From the Functional Food Salvia Miltiorrhiza Exhibits Anticancer Activity in Human HL-60 Leukemia Cells: In Vitro and in Vivo Studies

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15,16-Dihydrotanshinone I From the Functional Food Salvia Miltiorrhiza Exhibits Anticancer Activity in Human HL-60 Leukemia Cells: In Vitro and in Vivo Studies

Jun-Jen Liu et al. Int J Mol Sci.

Abstract

15,16-Dihydrotanshinone I (DHTS) is extracted from Salvia miltiorrhiza Bunge which is a functional food in Asia. In this study, we investigated the apoptotic effect of DHTS on the human acute myeloid leukemia (AML) type III HL-60 cell line. We found that treatment with 1.5 μg/mL DHTS increased proapoptotic Bax and Bad protein expressions and activated caspases-3, -8, and -9, thus leading to poly ADP ribose polymerase (PARP) cleavage and resulting in cell apoptosis. DHTS induced sustained c-Jun N-terminal kinase (JNK) phosphorylation and Fas ligand (FasL) expression. The anti-Fas blocking antibody reversed the DHTS-induced cell death, and the JNK-specific inhibitor, SP600125, inhibited DHTS-induced caspase-3, -8, -9, and PARP cleavage. In a xenograft nude mice model, 25 mg/kg DHTS showed a great effect in attenuating HL-60 tumor growth. Taken together, these results suggest that DHTS can induce HL-60 cell apoptosis in vitro and inhibit HL-60 cell growth in vivo; the underlying mechanisms might be mediated through activation of the JNK and FasL signal pathways.

Keywords: 15,16-dihydrotanshinone I; Fas ligand; acute myeloid leukemia; apoptosis; c-Jun N-terminal kinase.

Figures

Figure 1
Figure 1
Effects of 15,16-dihydrotanshinone I (DHTS) on the cell viability and cytotoxicity of human HL-60 and K562 leukemia cells. (A) HL-60 cells were treated with various concentrations of DHTS for 24 h. Cell numbers and cytotoxicity were measured by counting viable cells using an MTT assay and lactate dehydrogenase (LDH) release, respectively; (B) K562 cells were treated with various concentrations of DHTS for 24 h. Cell numbers was measured by counting viable cells using an MTT assay; and (C) Cells were treated with various concentrations of DHTS for 24 h, and apoptotic cells were determined by FACS using the Annexin V-Alexa Fluor488 Apoptosis Assay Kit. Data are expressed as the mean ± S.D. of three independent experiments. * p < 0.05, compared to the control.
Figure 2
Figure 2
Effects of 15,16-dihydrotanshinone I (DHTS) on activation of caspases in human HL-60 promyelocytic leukemia cells. Cells were treated with (A) various concentrations of DHTS for 24 h or (B) 1.5 μg/mL DHTS for different time periods, and total cellular proteins were collected to determine protein expressions by Western blotting. CF, cleaved form; CF-casp-3, cleaved form of caspase-3; CF-casp-8, cleaved form of caspase-8; CF-casp-9, cleaved form of caspase-9. Relative intensities of CF-PARP and CF-caspases were quantified using ImageJ, normalized versus internal controls α-tubulin, and shown below the band pictures. Three independent experiments were performed and representative results are shown.
Figure 3
Figure 3
Effects of 15,16-dihydrotanshinone I (DHTS) on Bad and Bax expressions in human HL-60 promyelocytic leukemia cells. Cells were treated with (A) various concentrations of DHTS for 24 h or (B) 1.5 μg/mL DHTS for different time periods, and total cellular proteins were collected to determine protein expressions by Western blotting; (C) Total cellular lysate were collected from the cells treated with 1.5 μg/mL DHTS for 24 h, and incubated with or without alkaline phosphatase (ALP) in tube, and then determined protein expressions by Western blotting; and (D) Cells were transfected with Bad siRNA and/or Bax siRNAs by electroporation and then treated with 1.0 μg/mL DHTS for 24 h. Cell numbers was measured by counting viable cells using an MTT assay (left panel), and total cell lysates were used to detect the Bad and Bax protein levels by Western blotting (right panel). Arrowhead, a shifted band of Bad or Bax. Data are expressed as the mean ± S.D. of three independent experiments. * p < 0.05, compared to the column 2.
Figure 4
Figure 4
Effects of 15,16-dihydrotanshinone I (DHTS) on FasL and nuclear factor (NF)-κB expressions and mitogen-activated protein kinase (MAPK) phosphorylation in human HL-60 promyelocytic leukemia cells. (A,B) Cells were treated with various concentrations of DHTS for 24 h or 1.5 μg/mL DHTS for different time periods. (A) Total RNA was collected to determine FasL mRNA expression by an RT-PCR; (B) Total cellular proteins were collected to determine FasL protein expression by Western blotting; (C) Cells were treated with 1.0 μg/mL DHTS and 10 μg/mL anti-Fas blocking antibody for 24 h. Viable cell numbers were measured by an MTT assay. Data are expressed as the mean ± S.D. of three independent experiments. * p < 0.05, compared to the column 3; and (D) Cells were treated with 1.5 μg/mL DHTS for different time periods. Total cellular proteins were collected to determine protein expressions by Western blotting. p-ERK, phosphorylated extracellular signal-regulated kinase; p-JNK, phosphorylated c-Jun N-terminal kinase; p-p38, phosphorylated p38.
Figure 4
Figure 4
Effects of 15,16-dihydrotanshinone I (DHTS) on FasL and nuclear factor (NF)-κB expressions and mitogen-activated protein kinase (MAPK) phosphorylation in human HL-60 promyelocytic leukemia cells. (A,B) Cells were treated with various concentrations of DHTS for 24 h or 1.5 μg/mL DHTS for different time periods. (A) Total RNA was collected to determine FasL mRNA expression by an RT-PCR; (B) Total cellular proteins were collected to determine FasL protein expression by Western blotting; (C) Cells were treated with 1.0 μg/mL DHTS and 10 μg/mL anti-Fas blocking antibody for 24 h. Viable cell numbers were measured by an MTT assay. Data are expressed as the mean ± S.D. of three independent experiments. * p < 0.05, compared to the column 3; and (D) Cells were treated with 1.5 μg/mL DHTS for different time periods. Total cellular proteins were collected to determine protein expressions by Western blotting. p-ERK, phosphorylated extracellular signal-regulated kinase; p-JNK, phosphorylated c-Jun N-terminal kinase; p-p38, phosphorylated p38.
Figure 5
Figure 5
Effects of a c-Jun N-terminal kinase (JNK) inhibitor on 15,16-dihydrotanshinone I (DHTS)-induced cleavages of poly ADP ribose polymerase (PAPR) and caspases in human HL-60 promyelocytic leukemia cells. Cells were pretreated with the JNK inhibitor, SP600125, for 1 h, and treated with various concentrations of DHTS for 24 h. Total cellular proteins were collected to determine protein expressions by Western blotting. SP, SP600125; CF, cleaved form; CF-casp-3, cleaved form of caspase-3; CF-casp-8, cleaved form of caspase-8; CF-casp-9, cleaved form of caspase-9. Relative intensities of CF-PARP and CF-caspases were quantified using ImageJ, normalized versus internal controls α-tubulin, and shown below the band pictures. Three independent experiments were performed and representative results are shown.
Figure 6
Figure 6
Effects of 15,16-dihydrotanshinone I (DHTS) on HL-60 tumor xenografts in nude mice. HL-60 cells were subcutaneously injected between the scapulas of athymic nude mice, and the mice received an i.p. injection of 12.5 or 25 mg/kg DHTS a day for a week. (A) Body weight and (B) tumor weight were measured at the end of the experiment. Values were obtained in five samples and are presented as the mean ± S.D. * p < 0.05 versus the control.

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