Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Jan 20:12:28.
doi: 10.1186/1471-2407-12-28.

Inhibition of STAT3 signaling and induction of SHP1 mediate antiangiogenic and antitumor activities of ergosterol peroxide in U266 multiple myeloma cells

Yun-Hee Rhee  1 , Soo-Jin Jeong, Hyo-Jeong Lee, Hyo-Jung Lee, Wonil Koh, Ji Hoon Jung, Sun-Hee Kim, Kim Sung-Hoon
Affiliations

Inhibition of STAT3 signaling and induction of SHP1 mediate antiangiogenic and antitumor activities of ergosterol peroxide in U266 multiple myeloma cells

Yun-Hee Rhee et al. BMC Cancer. .

Abstract

Background: Ergosterol peroxide (EP) derived from edible mushroom has been shown to exert anti-tumor activity in several cancer cells. In the present study, anti-angiogenic activity of EP was investigated with the underlying molecular mechanisms in human multiple myeloma U266 cells.

Results: Despite weak cytotoxicity against U266 cells, EP suppressed phosphorylation, DNA binding activity and nuclear translocalization of signal transducer and activator of transcription 3 (STAT3) in U266 cells at nontoxic concentrations. Also, EP inhibited phosphorylation of the upstream kinases Janus kinase 2 (JAK2) and Src in a time-dependent manner. Furthermore, EP increased the expression of protein tyrosine phosphatase SHP-1 at protein and mRNA levels, and conversely silencing of the SHP-1 gene clearly blocked EP-mediated STAT3 inactivation. In addition, EP significantly decreased vascular endothelial growth factor (VEGF), one of STAT3 target genes at cellular and protein levels as well as disrupted in vitro tube formation assay. Moreover, EP significantly suppressed the growth of U266 cells inoculated in female BALB/c athymic nude mice and immunohistochemistry revealed that EP effectively reduced the expression of STAT3 and CD34 in tumor sections compared to untreated control.

Conclusion: These findings suggest that EP can exert antitumor activity in multiple myeloma U266 cells partly with antiangiogenic activity targeting JAK2/STAT3 signaling pathway as a potent cancer preventive agent for treatment of multiple myeloma cells.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Ergosterol peroxide (EP) suppresses STAT3 activation in U266 cells. (A) Cells were treated with 25 μM EP for 0, 4, 8, 16 or 24 h. (B) Cells were treated with various concentrations of EP (0, 6.25, 12.5 or 25 μM) for 8 h. (C) SCC4, DU145 and MDA-MB-231 cells were treated with or without EP (25 μM) for 8 h. Whole cell extracts were prepared and subjected to Western blotting to determine level of phospho-STAT3 and STAT3. (D and E) Gel shift mobility assay was performed using nuclear extracts to examine the STAT3-DNA binding activity.
Figure 2
Figure 2
Ergosterol peroxide (EP) inhibits phosphorylation of JAK2 and Src in U266 cells. Cells were treated with 25 μM EP for 0, 4, 8, 16 or 24 h. Whole cell extracts were prepared and subjected to Western blotting to determine level of (A) phospho-JAK2 and JAK2, adn (B) phospho-Src and Src.
Figure 3
Figure 3
Ergosterol peroxide (EP) enhances expression of SHP-1 in U266 cells. (A) Cells were treated with 25 μM EP in the absence or presence of pervanadate (10, 25 or 50 μM) for 4 h. Whole cell extracts were prepared and subjected to Western blotting to determine level of phospho-STAT3 and STAT3. (B and C) Cells were treated with 25 μM EP for 0, 4, 8, 16 or 24 h (B) and various concentrations of EP (0, 6.25, 12.5 or 25 μM) for 4 h (C). Whole cell extracts were prepared and subjected to Western blotting to determine SHP-1 expressoin. (D) Cells were treated with 25 μM EP for 0, 4, 8, 16 or 24 h. RT-PCR was performed to analyze mRNA expression of SHP-1. (E) Cells were treated with 25 μM EP for 4 h (left), or treated with 25 μM EP for 1 h followed by washing PBS to remove EP and resuspension in fresh medium (right). Western blotting was performed for SHP-1. (F) Cells were transiently transfected with either SHP-1 or scrambled siRNA (50 nM) for 48 h and then treated with 25 μM EP for 4 h. Western blotting was performed for phospho-STAT3 and STAT3.
Figure 4
Figure 4
Ergosterol peroxide (EP) exerts anti-angiogenic activity in vitro. (A) U266 cells were treated with various concentrations of EP (0, 6.25, 12.5, 25 or 50 μM) for 24 h. Cytotoxic effect of EP was evaluated by MTT assay. (B) U266 cells were treated with 25 μM EP for 0, 4, 8, 16 or 24 h. Cell lysates were prepared and subjected to Western blotting for VEGF. (C and D) U266 cells were seeded at density of 1 × 106 cells/well onto 6-well plates and treated with 25 μM EP for 0, 4, 8, 16 or 24 h (C) and various concentrations of EP (0, 6, 12.5 or 25 μM) for 24 h (D). Levels of VEGF in the supernatants were measured by using a Quantikine VEGF ELISA kit. (E) For in vitro tube formation assay, HUVECs (3 × 104 cells/well) were seeded onto Matrigel coated 24-well plates and treated with VEGF (20 ng/ml) in the absence or presence of EP (0, 12.5 or 25 μM) for 6 h. Cells were fixed with 2% paraformaldehyde, stained with 2% crystal violet, and the number of tube was randomly counted in selected areas (left). Representative photographs of tube formation in cells treated with VEGF (20 ng/ml) in the absence or presence of 25 μM EP (right). Each experiment was repeated three times and all data were expressed as means ± S.D. **, p < 0.01 vs untreated control. (F) HUVECs were treated with VEGF (20 ng/ml) in the absence or presence of 25 μM EP. Western blotting was performed for phopho-STAT3 and STAT3.
Figure 5
Figure 5
Ergosterol peroxide (EP) exerts anti-tumor activity in vivo. Six-week-old female BALB/c athymic nude mice were subcutaneously injected in the flank area with 2 × 106 cells of U266 in 100 μl of matrigel mixed PBS. Five days after inoculation, mice (n = 5/group) were each given every 2-3 day intraperitonial (i.p.) injection of EP at 100 mg/kg in 2% tween-80 or bortezomib at 0.25 mg/kg in PBS. Control mice were administered the solvent vehicle. Tumor volumes were measured every other day with caliper. (A) Body weight was measured every other day after administration of EP or bortezomib. (B) Tumor volume was calculated according to the formula V = 0.25a2b, where a is the smallest superficial diameter and b is the largest superficial diameter. (C) Mice were killed on day 20 after cell inoculation, and tumors were immediately removed, fixed, embedded and sectioned at 4 μm for immunostaining of biomarkers. Representative photomicrographs of apoptosis detected by immunostaining of CD34, phospho-STAT3 and TUNEL at ×400, 200 and 100 of magnification, respectively. The sections were detected as DAB substrate staining (brown) and counterstained with Mayer's hematoxylin solution (right). Graphs show the CD34 index (angiogenesis), TUNEL index (apoptosis) and, pSTAT3 index in tumor sections. **p < 0.01 and ***p < 0.001 compared with vehicle-treated control mice (left).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Kim DS, Baek NI, Oh SR, Jung KY, Lee IS, Kim JH, Lee HK. Anticomplementary activity of ergosterol peroxide fromNaematoloma fasciculare and reassignment of NMR data. Arch Pharm Res. 1997;20(3):201–205. doi: 10.1007/BF02976145. - DOI - PubMed
    1. Piovano M, Guzman G, Garbarino JA, Chamy MC. The chemistry of Bacidia stipata (Lichens, Ascomycotina) Biochem System Ecol. 1999;27:663–664. doi: 10.1016/S0305-1978(98)00120-3. - DOI
    1. Arisawa M, Fujita A, Saga M, Fukumura H, Hayashi T, Shimizu M, Morita N. Three new lanostanoids from Ganoderma lucidum. J Nat Prod. 1986;49(4):621–625. doi: 10.1021/np50046a010. - DOI - PubMed
    1. Sgarbi DB, da Silva AJ, Carlos IZ, Silva CL, Angluster J, Alviano CS. Isolation of ergosterol peroxide and its reversion to ergosterol in the pathogenic fungus Sporothrix schenckii. Mycopathologia. 1997;139(1):9–14. doi: 10.1023/A:1006803832164. - DOI - PubMed
    1. Bok JW, Lermer L, Chilton J, Klingeman HG, Towers GH. Antitumor sterols from the mycelia of Cordyceps sinensis. Phytochemistry. 1999;51(7):891–898. doi: 10.1016/S0031-9422(99)00128-4. - DOI - PubMed

Publication types

MeSH terms