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

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.

STAT proteins originally discovered as latent cytoplasmic transcription factors [11] are involved in a variety of cellular processes such as cell proliferation, differentiation and apoptosis [12,13]. Of the STAT proteins, STAT3 is often constitutively activated in many human cancer cells including multiple myeloma, leukemia, lymphoma, and solid tumors [14,15]. Upon activation, STAT3 undergoes phosphorylation-induced homodimerization, leading to nuclear translocation, DNA binding and subsequent gene transcription. STAT3 phosphorylation is mediated through the activation of non-receptor protein tyrosine kinases Janus activated kinases (JAKs) and c-Src [16].
STAT3 participates in oncogenesis through up-regulation of genes encoding apoptosis inhibitors such as bclx L , bcl-2, and survivin [15]. Also, recent studies reported the evidences that STAT3 is involved in the regulation of angiogenesis through modulation of VEGF, a key regulator of angiogenesis [17][18][19]. In this regards, we investigated anti-angiogenic effect of EP in association with JAK2/STAT3 pathway and STAT3 related VEGF expression in U266 cells in vitro and mouse xenograft model.

Isolation of ergosterol peroxide (EP)
EP was isolated according to the Krzyczkowski's method as previously described [20].

Cytotoxicity assay
The cytotoxic effect of EP was evaluated by 3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. U266 cells were seeded onto 96-well microplate at a density of 1 × 10 4 cells/well and treated with various concentrations of EP (0, 6.25, 12.5, 25 or 50 μM) for 24 h. The culture medium was removed after centrifuging the plate and MTT solution (1 mg/ml) was added to each well until formazan was constituted. MTT lysis solution (20% SDS and 50% dimethylfermamide) was added to dissolve formazan and optical density (OD) was measured using microplate reader (Tecan Austria GmbH, Grödig, Austria) at 570 nm. Cell viability was calculated as a percentage of viable cells in EP-treated group versus untreated control by following equation.
Enzyme-linked immunosorbent assay (ELISA) for VEGF U266 cells were seeded onto 6-well plates at a density of 1 × 10 6 cells/well and treated with 25 μM EP. The VEGF levels in the supernatant were measured by using a Quantikine VEGF ELISA kit (R&D systems, Minneapolis, MN) according to manufacturer's protocols.

In vitro tube formation assay
Tube formation assay was performed on Matrigel (Becton Dickinson Labware, Bedford, MA) as described previously [23]. In brief, human umbilical vein endothelial cells (HUVECs) (3 × 10 4 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, 10 or 25 μM) for 6 h. Cells were fixed with 2% paraformaldehyde and stained with 2% crystal violet. Tube formation was observed under an Axiovert S 100 light microscope (Carl Zeiss, Weimar, Germany) and counted in randomly selected areas using NIH Scion image program.

Mouse xenograft model
Six-week-old female athymic nude mice were purchased from Jung Ang lab animal (Seoul, Republic of Korea) and maintained under conventional conditions. U266 cells (2 × 10 6 cells) were mixed with Matrigel (Becton Dickinson, 50%, in 100 μl) and injected subcutaneously on the right flank of the mice. After 5 days of inoculation, the mice (n = 5/group) were given intraperitonial (i.p) injection of EP at 100 mg/Kg in 2% tween-80, or bortezomib i.p. 0.25 mg/kg in PBS every 2 or 3 days. Control mice were administered the solvent vehicle. Tumor volume was measured every other day with caliper and calculated according to the formula; V = 0.25a 2 b, where a is the smallest superficial diameter and b is the largest superficial diameter.

Statistical analysis
All values were expressed as means ± S.D. Data were analyzed by one-way analysis of variance (ANOVA) and by student's t-test (Sigma plot ® , San Rafael, CA, USA).

Ergosterol peroxide suppresses STAT3 activation in U266 cells
STAT3 is implicated in cell survival and growth in multiple myeloma cells [14]. In light of this event, we examined whether EP can modulate the constitutive activation of STAT3 in U266 cells. EP significantly inhibited the constitutive activation of STAT3 in timeand dose-dependent manners ( Figure 1A and 1B, respectively). Tyrosine phosphorylation of STATs causes their dimerization and translocation to the nucleus, where they bind to DNA and regulate gene transcription [22]. Furthermore, we evaluated whether EP can affect on constitutive STAT3 activation in other cancer cell lines. SCC4 (head and neck squamous cell carcinoma), DU145 (prostate cancer), and MDA-MB-231 (breast cancer) cells expressing constitutive STAT3 were treated with or without EP. As shown in Figure 1C, EP inhibited STAT3 activation without affecting total STAT3 level. Consistent with the results of Western blotting in Figure  1A and 1B, gel shift assay showed that EP clearly inhibited the STAT3 DNA-binding activity in time-and dose-dependent manners ( Figure 1D and 1E, respectively).

Ergosterol peroxide suppresses JAK2 and Src in U266 cells
STAT3 is activated by soluble tyrosine kinases JAKs [24]. Effect of EP was measured on the activation of JAK2 by Western blotting. EP suppressed the phosphorylation of JAK2 in a time-dependent manner ( Figure  2A), while it did not change the level of JAK1 (data not shown). The Src is another tyrosine kinase family to activate STAT3 signaling [16]. EP also decreased the phosphorylation of Src in a time-dependent manner ( Figure 2B). These results indicate that EP inactivates the upstream kinases JAK2 and Src of STAT3 in U266 cells.

Ergosterol peroxide enhances expression of SHP-1 in U266 cells
Non-transmembrane protein tyrosine phosphatases (PTPs) play important roles in the negative regulation of the JAK/STAT signaling [25]. Thus, we investigated whether PTPs are involved in EP-mediated STAT3 inactivation using a PTP inhibitor sodium pervanadate. As shown in Figure 3A, pervanadate reversed EP-induced STAT3 inactivation in a dose-dependent manner, indicating the important role of PTP(s) in EP induced STAT3 inactivation in U266 cells.
Since SHP-1 is a PTP expressed most abundantly in hematopoietic cells [26,27], the effect of EP on the expression of SHP-1 was examined. EP enhanced SHP-1 protein expression in time-and dose-dependent manners ( Figure 3B and 3C). Consistently, EP increased the expression of SHP-1 at the mRNA level in a timedependent manner ( Figure 3D). Conversely, EPmediated SHP-1 was reduced in a time course by the removal of EP containing media, indicating that SHP-1 expression by EP is reversible ( Figure 3E). Furthermore, blocking SHP-1 using its specific siRNA clearly abolishes the ability of EP to inhibit STAT3 while scrambled siRNA had no effect on the expression (Figure 3F). Taken together, these results showed evidence that SHP-1 plays a critical role in the suppression of STAT3 phosphorylation by EP.

Ergosterol peroxide (EP) exerts anti-angiogenic activity in vitro
Cytotoxicity of EP against U266 cells were determined by MTT assay. U266 cells were treated with various concentrations of EP (0, 6.25, 12.5, 25 or 50 μM) for 24 h. As shown in Figure 4A, EP had no significant cytotoxic effect in U266 cells. STAT3 contributes to angiogenic regulation by inducing VEGF expression [28,29]. Thus, VEGF protein expression was analyzed at the cellular and protein levels in EP-treated cells. EP suppressed protein expression of VEGF in a timedependent manner by Western blotting ( Figure 4B).
Consistently, secretion of VEGF was significantly inhibited by EP treatment in time-and dose-dependent manners by ELISA ( Figure 4C and 4D, respectively).
To further confirm the anti-angiogenic activity of EP, in vitro tube formation assay was conducted using HUVECs treated either with or without VEGF in the absence or presence of EP. As shown in Figure 4E, no tube formation was observed in untreated cells, while clear tube formation was exhibited in VEGF-treated control. Notably, EP treatment inhibited VEGF-induced tube formation in a dose-dependent manner. Then, we tested whether EP can affect VEGF-mediated STAT3 activation in HUVECs. As shown in Figure 4F, VEGF treatment clearly enhanced phosphorylation of STAT3 in HUVECs, which was supported by Ebrahem's evidences [30]. In contrast, total STAT3 levels were not significantly changed by the treatment of VEGF and/or EP ( Figure 4F).

Ergosterol peroxide exerts anti-tumor activity in vivo
To confirm the anti-tumor efficacy of EP, U266 cells were subcutaneously inoculated into BALB/c athymic nude mice in the flank area, and starting after 5 days injection, the mice were administered EP or bortezomib (a positive control) every 2-3 day with intraperitonial (i. p.) injection. Tumor growth was monitored every other day for 20 days. Tumor volumes in EP-or bortezomibtreated groups were decreased compared with untreated control ( Figure 5A) without significant body weight loss in EP-treated group ( Figure 5B). Consistently, immunohistochemistry showed that EP treatment reduced the expression of phospho-STAT3 and angiogenesis marker CD34 compared to untreated control ( Figure 5C), implying the antitumor effect of EP is associated with regulation of angiogenesis and STAT3 in vitro and in vivo. In addition, TUNEL positive cells for apoptosis were effectively increased in EP-treated tumor sections. However, considering that EP treatment for 48 h or 72 h exerted significant cytotoxicity against U266 cells, we can postulate that EP can exert antitumor effects via antiangiogenic activity at low concentration and apoptotic activity at higher concentration.

Discussion
Multiple myeloma, also called plasma cells myeloma, is characterized by accumulation of secretory plasma cells. For the treatment of multiple myeloma, various chemotherapeutic agents such as vincristine, carmustine, mephalan, cyclophosphamide, doxorubicin, prednisone and dexamethasone have been used in combination or either drug alone [31]. However, many multiple myeloma patients frequently had no response to these agents and their prolonged exposure induced toxicity even in normal cells. Recently, phytochemicals are considerably advocated as rich sources of anti-cancer agents that deserve more rigorous and valuable investigations. Regarding this issue, several studies suggested phytochemicals such as curcumin [14], resveratrol [32] and capsaicin [33] as potent anti-cancer agents for multiple myeloma treatment. We also found that genipin [34] p-JAK2   and icariside II [35] could be applied for multiple myeloma therapy by inducing apoptosis and targeting the signaling molecules such as STAT3. The STAT proteins were identified for the last decade as latent cytoplasmic transcription factors in response to all cytokine driven signaling [15]. Seven mammalian STAT proteins such as STAT1, 2, 3, 4, 5a, 5b and 6 act as multifunctional mediators to regulate various cellular processes such as cell proliferation, differentiation, angiogenesis, and apoptosis [36]. Especially, STAT3 is constitutively activated in many human cancers, including prostate cancer [37], breast cancer [38], squamous cell carcinoma of the head and neck (SCCHN) [39], nasopharyngeal carcinoma and multiple myelomas [40]. Thus, STAT3 is considered as a valuable therapeutic target molecule for cancer treatment.
Ergosterol peroxide (EP) is a steroid derivative isolated from medicinal mushroom [41]. Several studies reported anti-cancer activity of EP in various types of cancer cells. For instance, Russo and colleagues reported that EP attenuated cell growth and induced apoptosis in human prostate cancer LNCaP and DU-145 cells [42]. Kobori and colleagues reported that EP suppressed inflammatory response in RAW264.7 macrophages and growth of HT29 colon adenocarcinoma cells [43]. Also, Chen and colleagues suggested that EP from the fermentation mycelia of Ganoderma lucidum cultivated in the medium exerted the cytotoxic effect against Hep 3B cells [44]. In the present study, we found that EP exerts anti-cancer activity through the inhibition of angiogenesis by targeting the STAT3 signaling pathway in U266 cells in vitro and in vivo.
EP suppressed constitutive activation of STAT3 in U266, SCC4, DU145 and MDA-MB-231 cells. EP also inhibited the STAT3-DNA binding activity and the nuclear translocation of STAT3, suggesting that EP can prevent STAT3 activation at the transcriptional level. The inhibitory effect of EP on STAT3 activation was partly associated with the inhibition of upstream kinases JAK2 and Src by EP treatment. Furthermore, protein tyrosine phosphatases (PTPs) are known to be implicated in STAT3 signaling, including SHP-1 [45], SHP-2 [46], PTEN [47], SOCS-1 [48] and so on. Our results revealed that EP remarkably enhanced the expression of SHP-1 at levels of protein and mRNA. In contrast, EP had no significant effect on other PTPs such as SHP-2 and PTEN (data not shown). To confirm the significance of SHP-1, we utilized PTP inhibitor pervanadate or SHP-1 siRNA to block the expression of SHP-1. As expected, EP failed to inhibit STAT3 activation in the presence of pervanadate or SHP-1 siRNA, supporting that SHP-1 plays a critical role in dephoshophorylating STAT3 by EP in U266 cells.
Recently, Niu and colleagues reported that constitutive activity of STAT3 up-regulated VEGF expression and tumor angiogenesis [49]. Wei and colleagues also reported that overexpression of constitutively activated mutant STAT3 sufficiently increased VEGF expression and tumor angiogenesis in vivo [50]. In contrast, dominant negative STAT3 mutant inhibited VEGF expression as well as angiogenesis. Additionally, it is of interest that STAT3 activation by its upstream Src regulates VEGF mediated angiogenesis and conversely blocking STAT3 inhibits Src-induced VEGF expression [51]. Consistently, in the present study, EP significantly prevented VEGFinduced phosphorylation of STAT3 as well as VEGFmediated tube formation in HUVECs, indicating antiangiogenic activity of EP by inhibiting STAT3 phosphorylation. Furthermore, we confirmed anti-tumor effect of EP in mouse xenograft tumor model. Consistent with the results of in vitro experiments, EP decreased U266 tumor growth as well as suppressed the expression levels of phospho-STAT3 and CD34 by immunohistochemistry. Although there are evidences that EP induces apoptosis at concentrations of 12.5-50 μM in LNCaP and DU-145 cells for 72 h [41,42,52], in the current study, EP did not show any cytotoxicity against U266 cells for 24 h up to 50 μM. Actually, immunohistochemistry revealed that TUNEL positive cells were increased in EP treated tumor sections, implying that EP can exert anti-angiogenic activity at nontoxic concentrations and possibly induce apoptosis only after long term culture or at high doses. Thus, mechanism and pharmacokinetic studies with EP are still required in vitro and in vivo to elucidate the relationship between its anti-angiogenic and apoptotic activities at different doses in the near future

Conclusions
Our findings demonstrate that EP can exert antitumor activity in multiple myeloma U266 cells by anti-angiogenic activity targeting JAK2/STAT3 signaling pathway as a potent anti-cancer agent for multiple myeloma treatment.