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 . 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 , resveratrol  and capsaicin  as potent anti-cancer agents for multiple myeloma treatment. We also found that genipin  and icariside II  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 . 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 . Especially, STAT3 is constitutively activated in many human cancers, including prostate cancer , breast cancer , squamous cell carcinoma of the head and neck (SCCHN) , nasopharyngeal carcinoma and multiple myelomas . Thus, STAT3 is considered as a valuable therapeutic target molecule for cancer treatment.
Ergosterol peroxide (EP) is a steroid derivative isolated from medicinal mushroom . 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 . Kobori and colleagues reported that EP suppressed inflammatory response in RAW264.7 macrophages and growth of HT29 colon adenocarcinoma cells . 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 . 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 , SHP-2 , PTEN , SOCS-1  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 . Wei and colleagues also reported that overexpression of constitutively activated mutant STAT3 sufficiently increased VEGF expression and tumor angiogenesis in vivo . 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 . Consistently, in the present study, EP significantly prevented VEGF-induced phosphorylation of STAT3 as well as VEGF-mediated tube formation in HUVECs, indicating anti-angiogenic 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