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. 2008 Sep;99(9):1820-6.
doi: 10.1111/j.1349-7006.2008.00872.x. Epub 2008 Jun 9.

Cantharidin induces apoptosis of human multiple myeloma cells via inhibition of the JAK/STAT pathway

Morihiko Sagawa  1 , Tomonori Nakazato, Hideo Uchida, Yasuo Ikeda, Masahiro Kizaki
Affiliations

Cantharidin induces apoptosis of human multiple myeloma cells via inhibition of the JAK/STAT pathway

Morihiko Sagawa et al. Cancer Sci. 2008 Sep.

Abstract

Multiple myeloma is an incurable B-cell malignancy requiring new therapeutic strategies in clinical settings. Interleukin (IL)-6 signaling pathways play a critical role in the pathogenesis of multiple myeloma. The traditional Chinese medicine cantharidin (CTD) has been shown to inhibit cellular proliferation and induce apoptosis of various cancer cells. The aim of this study was to investigate the possibility of CTD as a novel therapeutic agent for the patients with multiple myeloma. We investigated the in vitro effects of CTD for its antimyeloma activity, and further examined the molecular mechanisms of CTD-induced apoptosis. CTD inhibited the cellular growth of human myeloma cell lines as well as freshly isolated myeloma cells in patients. Cultivation with CTD induced apoptosis of myeloma cells in a cell-cycle-independent manner. Treatment with CTD induced caspase-3, -8, and -9 activities, and it was completely blocked by each caspase inhibitor. We further examined the effect of CTD on the IL-6 signaling pathway in myeloma cells, and found that CTD inhibited phosphorylation of STAT3 at tyrosine 705 residue as early as 1 h after treatment and down-regulated the expression of the antiapoptotic bcl-xL protein. STAT3 directly bound and activated the transcription of bcl-xL gene promoter, resulting in the induction of the expression of bcl-xL in myeloma cells. The essential role of STAT3 in CTD effects was confirmed by transfection with the constitutively active and dominant negative form of STAT3 in U266 cells. In conclusion, we have demonstrated that CTD is a promising candidate to be a new therapeutic agent in signal transduction therapy.

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Figures

Figure 1
Figure 1
Cantharidin (CTD) induced apoptosis of myeloma cells. (a) Chemical structure of CTD. (b,c) Various myeloma cells (RPMI‐8226, U266, and IM9) were treated with various concentrations (0–10 µM) of CTD for 24 h (b), and were treated with 5 µM of CTD for the indicated time (0–48 h) (c). Cell viability was assessed by MTS assay. Results are expressed as the mean ± SD of three different experiments. The unpaired Student's t‐test was used to evaluate statistical significance (*P < 0.01). (d) Detection of apoptotic cells by annexin V–propidium iodine (PI) double‐staining. U266 cells were treated with 5 µM of CTD for the indicated times, stained with annexin V and PI, and then analyzed by flow cytometry. Similar results were obtained in three independent experiments. (e) Morphological changes of CTD‐treated U266 cells. Cells were cultured with 5 µM of CTD for 24 h, and cytospin slides were then prepared and stained with Giemsa. Original magnification, 1000×. (f) Agarose gel electrophoresis demonstrating DNA fragmentation in CTD‐treated U266 cells. (g) Cell‐cycle analysis. U266 cells were treated with 5‐µM CTD for the indicated times, and then stained with PI. The DNA content was analyzed by flow cytometry. Sub‐G1 DNA content refers to the portion of apoptotic cells. Similar results were obtained in three independent experiments. (h) CD138‐positive B cells from normal peripheral blood samples showed that CTD treatment for 24 h and 48 h at 5 µM did not induce apoptosis.
Figure 1
Figure 1
Cantharidin (CTD) induced apoptosis of myeloma cells. (a) Chemical structure of CTD. (b,c) Various myeloma cells (RPMI‐8226, U266, and IM9) were treated with various concentrations (0–10 µM) of CTD for 24 h (b), and were treated with 5 µM of CTD for the indicated time (0–48 h) (c). Cell viability was assessed by MTS assay. Results are expressed as the mean ± SD of three different experiments. The unpaired Student's t‐test was used to evaluate statistical significance (*P < 0.01). (d) Detection of apoptotic cells by annexin V–propidium iodine (PI) double‐staining. U266 cells were treated with 5 µM of CTD for the indicated times, stained with annexin V and PI, and then analyzed by flow cytometry. Similar results were obtained in three independent experiments. (e) Morphological changes of CTD‐treated U266 cells. Cells were cultured with 5 µM of CTD for 24 h, and cytospin slides were then prepared and stained with Giemsa. Original magnification, 1000×. (f) Agarose gel electrophoresis demonstrating DNA fragmentation in CTD‐treated U266 cells. (g) Cell‐cycle analysis. U266 cells were treated with 5‐µM CTD for the indicated times, and then stained with PI. The DNA content was analyzed by flow cytometry. Sub‐G1 DNA content refers to the portion of apoptotic cells. Similar results were obtained in three independent experiments. (h) CD138‐positive B cells from normal peripheral blood samples showed that CTD treatment for 24 h and 48 h at 5 µM did not induce apoptosis.
Figure 2
Figure 2
Effects of cantharidin (CTD) on caspase activation. (a) U266 cells were cultured with 5‐µM CTD for 6 or 24 h, and analyzed for activation of caspase‐3 by flow cytometry. (b) Western blot analysis of caspase‐3, ‐8, ‐9, and Bid proteins. Protein levels were detected by Western blot analysis using antibodies against caspase‐3, ‐8,‐9, and Bid. β‐Actin was used to confirm that equal amounts of protein were in each lane. (c) Effects of caspase inhibitors on CTD‐treated U266 cells. Cells were preincubated with each caspase inhibitor (a synthetic pan‐caspase inhibitor; 20‐µM Z‐VAD‐FMK, caspase‐8 inhibitor; 20‐µM Z‐IETD‐FMK, and caspase‐9 inhibitor; 20‐µM Z‐LEHD‐FMK) for 2 h before the addition of 5‐µM CTD. Results represent the percentage relative to each control culture. Values are given as mean ± SD of a triplicate experiment. The unpaired Student's t‐test was used to evaluate statistical significance (*P < 0.05). The similar results were also shown in RPMI‐8226, and IM9 cells.
Figure 3
Figure 3
(a) Flow cytometric analysis of the mitochondria transmembrane potential (MMP) using DioC6 intensity. U266 cells were cultured with or without 5 µM of cantharidin (CTD) for 3 h (termed as 3 h and 0 h in the figure, respectively), and DioC6 fluorescence was analyzed by flow cytometry. (b) Expression of CD95 (Fas) in CTD‐treated U266 cells. Cells were treated with or without 5 µM of CTD for 3 h (termed as 3 h and 0 h in the figure, respectively), and Fas expression was determined by flow cytometry using anti‐Fas IgG‐fluorescein‐5‐isothiocyanate (FITC) antibody. Anti‐mouse IgG1‐FITC antibody was used as an isotype control (termed as control in both figures).
Figure 4
Figure 4
Cantharidin (CTD) induced apoptosis through blocking the JAK/STAT signaling pathway. U266 cells were cultured with 5‐µM CTD for the indicated times. (a) Western blotting with antibodies against phospho‐STAT3 and STAT3 was performed. (b) Expression of apoptosis‐associated proteins (bcl‐xL, bcl‐2, and mcl‐1). Re‐blotting with β‐actin revealed that equal amounts of protein were present in each lane (a–b). Similar results were obtained in three independent experiments.
Figure 5
Figure 5
Down‐regulation of in vitro DNA‐binding activity of STAT3 by cantharidin (CTD). (a) mRNA expression of bcl‐xL in CTD‐treated U266 cells. CTD repressed bcl‐xL mRNA expression. β‐Actin was used as an internal control. (b) Chromatin immunoprecipitation (Ch‐IP) assay using U266 cells was performed by using STAT3 antibody. DNA from control U266 cells or U266 cells treated with 5‐µM CTD for 24 h was amplified by polymerase chain reaction with the primer set to the bcl‐xL binding site. (c) STAT3 levels in U266 pEF‐BOS (mock‐transfected cells), U266 pEF‐HA‐STAT3D (constitutively active form of STAT3‐transfected cells), and U266 pEF‐HA‐STAT3F (dominant negative form of STAT3‐transfected cells). Anti‐HA‐HRP antibody was used to confirm the insertion. (d) Induction of apoptosis by CTD (5 µM) in U266 pEF‐BOS (control), U266 pEF‐HA‐STAT3D, and U266 pEF‐HA‐STAT3F cells. The unpaired Student's t‐test was used to evaluate statistical significance.
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