Background
Ergosterol Peroxide (EP), 5α, 8α-epidioxy-22E-ergosta-6, 22-dien-3β-ol, is found in plants [
1], lichens [
2] and mushrooms such as
Ganoderma lucidum [
3],
Sporothrix schenckii [
4] and
Cordyceps sinensis [
5]. Despite various biological effects of EP such as immunosuppressive [
6‐
8], anti-viral [
9], anti-inflammatory [
10] and anti-tumor [
5,
10] activities, the underlying molecular mechanisms for anti-cancer activity of EP still remain unclear.
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 bcl-x
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‐
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.
Methods
Isolation of ergosterol peroxide (EP)
EP was isolated according to the Krzyczkowski's method as previously described [
20].
Cell culture
U266 (multiple myeloma), SCC4 (head and neck squamous cell carcinoma), DU145 (prostate cancer), and MDA-MB-231 (breast cancer) cells were obtained from American Type Culture Collection (ATCC) (Rockville, MD) and maintained in RPMI 1640 supplemented with 10% FBS and antibiotics. Human umbilical vein endothelial cells (HUVECs) were isolated from fresh human umbilical cord vein by collagenase treatment as described previously [
21]. The cells were maintained in in EBM-2 containing 2% FBS, 0.04% hydrocortisone, 0.4% hFGF-B, 0.1% VEGF, 0.1% IGF-1, 0.1% ascorbic acid, 0.1% hEGF, 1% GA-1000, and 1% Heparin (Walkersville, MD)
Cytotoxicity assay
The cytotoxic effect of EP was evaluated by 3-(4, 5-dimethylthiazol-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.
Western blotting
Cells lysates were prepared using lysis buffer [50 mM Tris (pH 7.4), 300 mM NaCl, 5 mM EDTA (pH 8.0), 0.5% Triton X-100, 1 mM aprotinin, 1 mM leupeptin, 1 mM pepstatin, 10 mM iodoacetamide, 2 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM NaVO4] and centrifuged at 14, 000 × g for 10 min at 4°C. Protein samples were collected, separated onto 10-12% SDS-PAGE gels and electrotransferred to a nitrocellulose membrane. The membranes were blocked in 5% nonfat skim milk, probed with primary antibodies for STAT3, phospho-STAT3Y705, JAK2, phospho-JAK2Y1007/1008, Src, phospho-SrcY416 (Cell Signaling, Danvers, MA), SHP-1, VEGF (Santa Cruz Biotechnology, Santa Cruz, CA) and β-actin (Sigma, St. Louis, MO) at 4°C for overnight, and then exposed to HRP-conjugated secondary antibodies at room temperature for 2 h. Protein expression was detected by using enhanced chemiluminescence (ECL) kit (Amersham Pharmacia, Piscataway, NJ).
Electrophoretic mobility shift assay (EMSA) for STAT3-DNA binding
The STAT3-DNA binding was analyzed by electrophoretic mobility shift assay (EMSA) using a
32P-labeled high-affinity sis-inducible element (hSIE) probe (5'-CTTCATTTCCCGTAAATCCCTAAAGCT-3' and 5'-AGCTTTAGGGATTTACGG GAAA TGA-3') as previously described [
22]. Briefly, nuclear extracts were incubated with the hSIE probe and the protein-DNA complexes were separated onto 5% native polyacrylamide gels. The gels were dried, and the radioactive bands were quantitated with Storm 820 and Imagequant software (Amersham Pharmacia, Piscataway, NJ).
RT-PCR analysis
Total RNA was extracted by using Trizol reagent (Invitrogen) according to the manufacturer's instructions. cDNA was synthesized from 1 μg of total RNA and subjected to PCR reaction by using Superscript One Step reverse transcription-PCR (RT-PCR) kit (Invitrogen). The PCR conditions were 30 cycles of 94°C for 15 s, 55°C for 30 s, and 72°C for 1 min. The primer sequences were as follows: SHP-1 (forward primer 5'-AAT GCG TCC CAT ACT GGC CCG A-3'; reverse primer 5'-CCC GCA GTT GGT CAC AGA GT-3'), and GAPDH (forward primer 5'-TCA CCA TCT TCC AGG AGC GA-3'; reverse primer 5'-CAC AAT GCC GAA GTG GTG GT-3').
siRNA transfection
siRNA oligonucleotides for SHP-1 (SantaCruz biotechnology, SantaCruz, CA) were transfected by using LipofectAMINE transfection reagent (Invitrogen, Carlsbad, CA) according to manufacturer's protocols.
Enzyme- linked immunosorbent assay (ELISA) for VEGF
U266 cells were seeded onto 6-well plates at a density of 1 × 106 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.
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 × 106 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.
Immunohistochemistry
The animals were sacrificed 20 days after U266 inoculation, and tumors were immediately removed, fixed in 4% paraformaldehyde, paraffin-embedded, and sectioned at 4 μm. Immunohistochemical staining for phospho-STAT3(Cell Signaling Technology, Danvers, MA), CD 34 (microvessel marker, Abcam, Boston, MA) and TUNEL (Calbiochem, Darmstadt, Germany) was performed and detected by DAB substrate staining (brown).
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).
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] 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 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
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
YHR SJJ and SHK SA designed the research studies; YHR, HJeL and HJuL carried out the experiments; YHR SJJ and SHK analyzed and interpreted the data; YHR SJJ and SHK wrote the draft of the manuscript. All authors read and approved the final manuscript.