Abstract

A dried powder from basidiomycetous fungi, Ganoderma lucidum, has been used in East Asia in therapies for several different diseases, including cancer. However, the molecular mechanisms involved in the biological actions of Ganoderma are not well understood. We have recently demonstrated that phosphatidylinositol 3-kinase (PI 3-kinase) and nuclear factor-j B (NF-jB) regulate motility of highly invasive human breast cancer cells by the secretion of urokinase-type plasminogen activator (uPA). In this study, we investigated the effect of G. lucidum on highly invasive breast and prostate cancer cells. Here we show that spores or dried fruiting body of G. lucidum inhibit constitutively active transcription factors AP-1 and NF-jB in breast MDA-MB-231 and prostate PC-3 cancer cells. Furthermore, Ganoderma inhibition of expression of uPA and uPA receptor (uPAR), as well secretion of uPA, resulted in the suppression of the migration of MDA-MB-231 and PC-3 cells. Our data suggest that spores and unpurified fruiting body of G. lucidum inhibit invasion of breast and prostate cancer cells by a common mechanism and could have potential thera- peutic use for cancer treatment.

Ó 2002 Elsevier Science (USA). All rights reserved.
Keywords: Ganoderma lucidum; Cancer; MDA-MB-231; PC-3; Cell migration; NF-jB; AP-1; uPA; uPAR

The fungi Ganoderma lucidum (Reishi, Mannentake, or Lingzhi) has been used for centuries in East Asia to cure various human diseases such as hepatitis, hepa- topathy, hypertension, nephritis, bronchitis, and cancers [1–3]. Its dried powder was especially popular as a cancer chemotherapy agent in the Imperial Court of ancient China [4]. Some of the triterpenes, such as ganoderic and lucidic acids, recently isolated from Ganoderma demonstrated cytotoxicity against mouse sarcoma and mouse lung carcinoma cells in vitro [5]. Intraperitoneal administration of water-soluble poly- saccharides isolated from Ganoderma inhibited growth of sarcoma-180 solid tumors in mice [6,7]. In addition, polysaccharides from Ganoderma also potentiated

* Corresponding author. Fax: 1-317-962-7468.E-mail address: dsliva@clarian.org (D. Sliva).

production of cytokines, which subsequently suppressed proliferation of HL-60 and U937 leukemic cell lines [8]. Tumor invasion and metastasis are multifaceted processes involving cell adhesion, proteolytic degrada- tion of tissue barriers, and cell migration [9]. The uro- kinase-type plasminogen activator uPA and the uPA receptor (uPAR) play a crucial role in cancer metastasis (for review, see [10]). uPA is a serine protease that cleaves the extracellular matrix and is also involved in cell adhesion and migration [11]. Therefore, uPA can stimulate cell migration directly through its proteolytic activity by activating transforming growth factor-b(TGF-b) and fibroblast growth factor (FGF) [12,13], or alternatively, uPA lacking proteolytic activity stimu- lated cell migration directly through interaction with uPAR [14,15]. In addition, antibodies preventing bind- ing of uPA to uPAR inhibited uPA-induced cell

0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S0006-291X(02)02496-8

604 D. Sliva et al. / Biochemical and Biophysical Research Communications 298 (2002) 603–612

Cell culture. The human breast cancer cell line MDA-MB-231 and human prostate cancer cell line PC-3 were obtained from ATCC (Manassas, VA). MDA-MB-231 was maintained in DulbeccoÕs modi- fied EagleÕs medium (DMEM) and PC-3 cells were maintained in F-12 medium containing penicillin (50U/ml), streptomycin (50U/ml), and 10% fetal bovine serum (FBS). Media and supplements came from GIBCO BRL (Grand Island, NY). FBS was obtained from Hyclone (Logan, UT).

Cell migration assay. MDA-MB-231 and PC-3 cells were harvested and incubated for 4 h and for 24 h, respectively, with either GS or GFB from Ganoderma, as indicated in the text. Chemokinesis was assessed in Transwell chambers in DMEM for MDA-MB-231 cells or in the F- 12mediumcontaining10%FBSforPC-3cells,aspreviouslydescribed [33]. After fixing and staining, we counted the number of migrating cells from at least four random fields using a microscope at 20magnifi- cations [33]. Data points represent the average SD of individual filters within one representative experiment repeated at least twice.

Cell viability. MDA-MB-231 and PC-3 cells were harvested and incubated with Ganoderma for 4 and 24 h, respectively, as described for the cell migration assay. Cell viability was determined with trypan blue stain according to the standard protocol [34]. Data points represent the average SD in one representative experiment repeated at least twice.

DNA transfection and chloramphenicol acetyltransferase (CAT) assay. Both MDA-MB-231 and PC-3 cells were transfected with either NF-jB-CAT or AP-1-CAT reporter constructs and b-galactosidase expression vector pCH110, as previously described [31]. Twenty-four hours after transfection, cells were treated with either GS or GFB for an additional 24 h at 37 °C, as indicated in the text. CAT assays were performed as described [31]. Data points represent the average SD of 3–6 independent transfection experiments.

Gel electrophoretic mobility shift assay (GEMSA). MDA-MB-231 and PC-3 cells were treated for 24 h with GS and GFB, and nuclear extracts were prepared as described previously [35]. A GEMSA was performed with 32P-labeled AP-1 and NF-jB according to the stan- dard protocol [31]. Oligonucleotide probes containing consensus se- quences for AP-1 and NF-jB were purchased from Promega (Madison, WI). Supershift analyses were performed with anti-NF-jB p50 antibodies (Upstate Biotechnology, Lake Placid, NY).

Expression of IjBa and uPA secretion. MDA-MB-231 and PC-3 cells were treated for 24 h with GS and GFB, and whole cell extracts were prepared [36]. IjBa (inhibitor jBa) and actin expression were determined in the extracts (25 lg) by Western blot analysis [31]. The secretion of uPA was determined after treatment with GS and GFB for 24h in concentrated DMEM from MDA-MB-231 cells or concen- tratedF-12mediumfromPC-3cellsasdescribed[31].

Immunohistochemistry. MDA-MB-231 and PC-3 cells were seeded and grown in a Chamber Slide System (Lab-Tek II, Nalgene Nunc). After 24 h of treatment with GS and GFB, the cells were washed twice with phosphate-buffered saline (PBS) and air-dried without chemical fixation at room temperature. Samples were washed for 30 min in PBS and incubated for 15min in a moisture box with 25ll of uPA (Ce- darlane, Ontario, Canada) and uPAR (American Diagnostica, Green- wich, CT) antibody, respectively. After washing with PBS (3 10 min), the samples were incubated for 15min with 25ll anti-mouse IgG conjugated to fluorescein isothiocyanate (FITC) (Protos ImmunoRe- search, San Francisco, CA). The samples were subsequently rinsed three times with PBS. After the last wash, slides were reacted with propidium iodide (50 lg/ml) for 15 min. Finally, the slides were washed three times (10min) and coverslipped. The expression of uPA and uPAR and the nuclei staining were detected by epi-illumination in a Leitz DMRB microscope. The expressions of uPA and uPAR were semiquantitatively evaluated for the intensity of immunoreactivity

D. Sliva et al. / Biochemical and Biophysical Research Communications 298 (2002) 603–612 605

using the following scale: strong, moderate, weak, and very weak. uPA and uPAR expressions were statistically analyzed by comparing control to GS- and GFB-treated samples, respectively. Statistical analysis was performed using the Cochran–Mantel–Haenszel v2 method [37] with thestatisticalsoftwareSASversion8.2(SASInstitute,Cary,NC).

Results

Ganoderma inhibits migration of highly invasive human breast and prostate cancer cells

We have recently demonstrated that cell migration of highly invasive and chemotherapy-resistant human breast cancer cells MDA-MB-231 is dependent on con- stitutively active AP-1 and NF-jB and secretion of uPA [31,32]. Because extracts from Ganoderma have been used for cancer treatment in Asian countries for centu- ries [4], we wanted to elucidate the mechanisms involved in its antitumor activities. The morphology of Gano- derma samples was confirmed by microscopy (Fig. 1A). To assess the role of Ganoderma on the metastatic motility of human breast cancer cells, we preincubated MDA-MB-231 cells for 1 h with spores (GS) and fruit- ing body (GFB) and determined cell motility after an additional 3 h under standard cell migration assay con- ditions [33]. As seen in Fig. 1B, both spores and fruiting body (0.5–2.5 mg/ml) significantly inhibited cell motility of breast cancer cells in a dose-dependent manner.

To determine whether Ganoderma also inhibits mo- tility of highly invasive prostate cancer cells, we prein- cubated PC-3 cells with GS and GFB (0.5–2.5mg/ml) for 1 h and determined cell migration after an additional 23 h of incubation (although prostate cancer cells PC-3 are highly invasive they need longer incubation time in migration assays than the highly invasive breast cancer cells MDA-MB-231). The cell motility of Fig. 1C shows that both spores and fruiting body significantly inhibited constitutive migration of PC-3 cells. Therefore, Gano- derma was able to suppress constitutive migration of the highly invasive breast cancer cells MDA-MB-231 as well as the highly invasive prostate cancer cells PC-3.

To determine whether the inhibition of cell motility is caused by the cytotoxic effect of Ganoderma, we incu- bated both MDA-MB-231 and PC-3 cells with GS and GFB under the same conditions as the cell migration assay, and cell viability was determined with trypan blue stain. Spores or fruiting body did not demonstrate any cytotoxicity for either MDA-MB-231 or PC-3 cells at the highest used concentration, 2.5mg/ml (data not shown).

Ganoderma suppresses constitutively active AP-1 and NF-jB in breast and prostate cancer cells

Constitutive DNA-binding activity of NF-jB has been reported previously in primary breast cancers [22],

and we have recently demonstrated that inhibition of PI 3-kinase and protein kinase C (PKC) suppresses con- stitutive transactivation of NF-jB and AP-1 in the highly invasive breast cancer cells MDA-MB-231

606 D. Sliva et al. / Biochemical and Biophysical Research Communications 298 (2002) 603–612

[31,32]. To examine whether Ganoderma can inhibit transactivation of NF-jB and AP-1, we transiently transfected MDA-MB-231 cells with either NF-jB-CAT or AP-1-CAT reporter gene plasmids and treated the cells with spores and fruiting body for 24 h. As seen in Fig. 2A, treatment with both spores and fruiting body inhibited constitutive activation of NF-jB in a dose- dependent manner (0.5–2.5 mg/ml). We also observed the same effect on the constitutive activation of AP-1 where increased concentration of spores and fruiting body inhibited AP-1 in MDA-MB-231 cells (Fig. 2B). Because we have recently shown that the activity of NF-jB and AP-1 was suppressed only at the transactivation level without the changes in DNA-binding activity in MDA-MB-231 cells [31,32], we were interested to see whether Ganoderma inhibits constitutive NF-jB and AP-1 activity by the same mechanism. Nuclear extracts from MDA-MB-231 cells treated with spores or fruiting body for 24h were subjected to gel-shift analysis with either NF-jB and AP-1-labeled probes; the specificity of NF-jB and AP-1 binding in MDA-MB-231 cells was confirmed previously by competitive and supershift gel analysis [31,32]. As seen in Fig. 2C, pretreatment of spores and fruiting body at a concentration of 2.5 mg/ml

markedly reduced the constitutive DNA-binding activ- ity of NF-jB. However, the constitutive DNA-binding activity of AP-1 was not affected by Ganoderma treat- ment (Fig. 2D). Therefore, the decrease in the constit- utive NF-jB activity in a reporter gene assay by Ganoderma reflects the suppression of the NF-jB DNA binding, whereas constitutive AP-1 activity is suppressed at the transactivation level without changing AP-1 DNA-binding.

Constitutive NF-jB activity was recently reported in androgen-independent prostate tumor cell lines [29]. In light of this finding, we wanted to examine the effect of Ganoderma on NF-jB and AP-1 activity in the highly invasive prostate cancer cells PC-3. Gel-shift analysis confirmed constitutive NF-jB DNA-binding activity in nuclear extracts from PC-3 cells, the specificity of which was determined by competitive and supershift assays with anti-NF-jB p50 antibodies (Fig. 3A). In addition, nuclear extracts from PC-3 cells demonstrated the high constitutive DNA-binding activity of AP-1 (Fig. 3B). To examine whether Ganoderma employs the same mech- anism of inhibition of migration in both breast and prostate cancer cells, we transfected PC-3 cells with both NF-jB-CAT and AP-1-CAT reporter gene plasmids

D. Sliva et al. / Biochemical and Biophysical Research Communications 298 (2002) 603–612 607

and treated the cells with spores and fruiting body for 24 h as described above. As seen in Fig. 3C, both spores and fruiting body inhibited the constitutive activation of NF-jB in PC-3 cells in a dose-dependent manner (0.5–2.5 mg/ml). The constitutive activation of AP-1 was also inhibited by treatment with spores and fruiting body (Fig. 3D). Gel-shift analysis demonstrated that the DNA binding of NF-jB in PC-3 cells was also reduced by the treatment with spores and fruiting body of Ganoderma (Fig. 3E). Nevertheless, the DNA-binding activity of AP-1 was not affected by the same treatment with spores or fruiting body (Fig. 3F). Therefore,

Ganoderma inhibited both DNA binding and transac- tivation of NF-jB, whereas the inhibition of AP-1 occurred only at the transactivation level. We can con- clude from this that the same mechanism of inhibition is employed by both breast and prostate cancer cells.

Inhibition of migration and NF-jB activation by Gano- derma is independent of IjBa

NF-jB can be inhibited by sequestering in the cyto- plasm through assembly with the inhibitory protein IjBa [38] or by the alternative mechanism, independent

608 D. Sliva et al. / Biochemical and Biophysical Research Communications 298 (2002) 603–612

of IjBa [39]. Furthermore, we have recently demon- strated that although overexpression of IjBa suppressed migration of MDA-MB-231 cells, the inhibition of NF-jB transactivation by PI 3-kinase and PKC inhibitors was process independent of IjBa [31,32]. Therefore, we investigated whether Ganoderma would inhibit consti- tutive activation of NF-jB by increasing the levels of IjBa and by sequestering NF-jB in cytoplasm. MDA- MB-231 cells were treated with spores (0.5–2.5mg/ml) and fruiting body (0.5–2.5mg/ml) of Ganoderma for 24h and whole cell extracts were subjected to SDS- PAGE. Western blot analysis with anti-IjBa antibody revealed that treatment with spores or fruiting body of Ganoderma did not increase the levels of IjBa (not shown). As expected, we observed the same behavior in highly invasive prostate cancer cells, where treatment with spores or fruiting body of Ganoderma also did not increase the levels of IjBa in PC-3 cells (not shown). Altogether, these data clearly demonstrated that Ganoderma inhibits migration and constitutive NF-jB activation in breast and prostate cancer cells by a distinct signaling mechanism independent of IjBa.

Ganoderma suppresses constitutive secretion of uPA from breast and prostate cancer cells

We have previously demonstrated that highly inva- sive breast cancer cells constitutively secrete uPA and that this secretion can be suppressed by the inhibition of PI 3-kinase and PKC [31,32]. To examine the effect of Ganoderma on uPA secretion from breast cancer cells, we treated MDA-MB-231 cells with spores (0.5–2.5 mg/ ml) and fruiting body (0.5–2.5mg/ml) of Ganoderma and concentrated media were subjected to Western blot analysis with anti-uPA antibody. As seen in Fig. 4A, both spores and fruiting body markedly inhibited se- cretion of uPA in a dose-dependent manner. Because uPA expression usually correlates with the aggressive phenotype of prostate cancer cells [40], we were inter- ested in whether the highly invasive prostate cancer cells PC-3 also secrete uPA and whether Ganoderma inhibits this secretion. PC-3 cells were treated and analyzed as described above for MDA-MB-231 cells. Western blot analysis demonstrated that PC-3 cells secrete uPA and the treatment with spores and fruiting body clearly suppressed the uPA secretion from PC-3 cells in a dose- dependent manner (Fig. 4B). Therefore, Ganoderma inhibited secretion of uPA from both breast and pros- tate cancer cells.

Inhibition of expression of uPA and uPAR by Ganoderma in breast and prostate cancer cells

As mentioned above, the expression of uPA and uPAR is controlled by transcription factors AP-1 and NF-jB [24–26], and we have demonstrated that Gano-

derma inhibits both AP-1 and NF-jB in MDA-MB-231 and PC-3 cells. To examine the effect of Ganoderma on the expression of uPA and uPAR, we treated MDA-MB- 231 cells with spores or fruiting body (2.5 mg/ml) for 24 h and immunostained the cells with anti-uPA antibodies. As seen in Fig. 5, both spores and fruiting body of Ganoderma significantly reduced expression of uPA in MDA-MB-231 cells (Fig. 5A, a–c, Table 1). Immuno- staining with anti-uPAR antibodies demonstrated that uPAR expression was also decreased in MDA-MB-231 cells treated with both spores and fruiting body (Fig. 5A, d–f, Table 1). Similar to the results obtained with MDA- MB-231 cells, the expression of uPA and uPAR in PC-3 cells was also inhibited by spores and fruiting body (Fig. 5B, Table 2). Taken together, these data show that Ganoderma inhibited expression of uPA and uPAR in both highly invasive breast and prostate cancer cells.

Discussion

We have recently demonstrated that constitutively active AP-1 and NF-jB control secretion of uPA and that inhibition of AP-1 and NF-jB suppressed cell

D. Sliva et al. / Biochemical and Biophysical Research Communications 298 (2002) 603–612 609

Table 1
Expressions of uPA and uPAR in MDA-MB-231 cells

Immunostaining (%) uPA uPAR ControlN1⁄412GSN1⁄47GFBN1⁄47ControlN1⁄49GSN1⁄47GFBN1⁄47

Strong 50.0 0 0 55.6 0 0

Moderate Weak Very weakp-valuea

50.0 0
0 57.1 0 42.9

p < 0:01

0 44.4 14.3 0 85.7 0
p < 0:01

0 42.9 57.1

p < 0:01

0 14.3 85.7

p < 0:01

Expressions of uPA and uPAR in MDA-MB-231 cells (treated as described in Fig. 5A) were detected by the intensity of uPA and uPAR by immunostaining. The intensity of the immunohistochemical reactivity was evaluated semiquantitatively as described in Materials and methods because more than 95% of the cells per well showed similar characteristics and no significant variability was identified between cells within the same well.Thedatafrom7–12independentsampleswerestatisticallyanalyzedbytheCochran–Mantel–Haenszelv2methodasdescribedunderMaterials and methods.

a Each treatment was compared separately to control using the Mantel–Haenszel v2 test.

motility and uPA secretion of highly invasive breast cancer cells [31,32]. In the present study, we examined the effect of the old Asian medicinal mushroom G. lucidum on highly invasive breast and prostate cancer cells. Here we show that both spores and fruiting body

of Ganoderma inhibit constitutive activation of AP-1 and NF-jB. Furthermore, Ganoderma inhibits ex- pression of uPA and uPAR as well as secretion of uPA and cell migration of MDA-MB-231 and PC-3 cells.

610 D. Sliva et al. / Biochemical and Biophysical Research Communications 298 (2002) 603–612Table 2

Expressions of uPA and uPAR in PC-3 cells
Immunostaining (%) uPA uPAR

Control N 1⁄4 13 GS N 1⁄4 7 GFB N 1⁄4 10 Control N 1⁄4 9 GS N 1⁄4 7 GFB N 1⁄4 7

Strong 69.20077.800

Moderate Weak Very weakp-valuea

30.8 0
0 57.1 0 42.9

p < 0:01

0 22.2 50.0 0 50.0 0
p < 0:01

0 71.4 28.6

p < 0:01

0 42.9 57.1

p < 0:01

Expressions of uPA and uPAR in PC-3 cells (treated as described in Fig. 5B) were quantified and statistically analyzed in 7–13 independent samples as described in Table 1.

a Each treatment was compared separately to control using the Mantel–Haenszel v2 test.

Although Ganoderma has been used in Asian medi- cine for centuries to treat and prevent various diseases and its purified spores or whole fruiting body are dis- tributed as a dietary supplements, the biological effects of Ganoderma remain elusive. The antitumor effects of Ganoderma were suggested to be caused by triterpenes [5], polysaccharides [6,7], and immunomodulatory pro- teins [41] and by the mechanisms involving inhibition of post-translational modification of the Ras oncoprotein [42], inhibition of DNA polymerase [4], or the stimula- tion of cytokine production [8]. Recently, the mitogen activated kinase (MAP) kinase signaling pathway, acti- vated by Ganoderma extracts, was identified in the in- duction of neuronal differentiation and prevention of apoptosis [43]. In our study, we show that Ganoderma inhibited augmented cell motility of highly invasive breast and prostate cancer cells by inhibiting constitu- tively active AP-1 and NF-jB signaling. The inhibition of AP-1 and NF-jB by the Asian mushroom Gano- derma is of particular interest because recent studies suggest that AP-1 and NF-jB are potential targets for cancer treatment [44,45]. In addition, inhibition of NF-jB by proteasome inhibitor PS-341 is considered in clinical studies to be an adjuvant to systemic chemo- therapy of cancer [46]. In our experiments, we used purified spores or whole fruiting body of Ganoderma, which are available as dietary supplements, and we found no significant difference in biological activity be- tween the spores and the whole fruiting body of Gano- derma. The dose used, 0.5–2.5 mg/ml spores or fruiting body of Ganoderma per 1 million cancer cells, markedly inhibited migration as well as constitutive activation of AP-1 and NF-jB in breast and prostate cancer cells. The recommended dose, based on data from clinical studies with other fungi, should be 5–10 g fruiting body per day, with a linear enhancement in efficacy expected up to 30 g/day [47].

Here we show the inhibition of NF-jB in the DNA- binding activity and reporter gene assays in both breast and prostate cancer cells. In our previous studies [31,32] we demonstrate that inhibition of PI 3-kinase and PKC suppresses NF-jB in breast cancer cells only at the transactivation level. Therefore, Ganoderma probably

employs a specific mechanism for NF-jB inhibition in- dependent of the PI 3-kinase and PKC pathways. Al- though constitutive AP-1 transactivation was inhibited by Gonaderma in the reporter gene assay in both breast and prostate cancer cells, the DNA-binding activity of AP-1 was not changed. The inhibition of AP-1 at the transactivation level can be explained by the different mechanisms of NF-jB and AP-1 activation. Because NF-jB has been shown to induce transcription of AP-1 regulated genes through the interaction of NF-jB with AP-1 [30], it is possible that Ganoderma inhibits DNA binding and transactivation of NF-jB, which in turns inhibits AP-1 only at the transactivation level without changing the DNA-binding activity of AP-1.

The activity of NF-jB is controlled by NF-jB in- hibitors, IjBs, a family of proteins that bind to NF-jB dimers, hiding their nuclear localization sequence and resulting in cytoplasmic retention of NF-jB [38,48]. In the present study, we show that although Ganoderma inhibits NF-jB, the levels of jBa are not changed, suggesting that NF-jB activity is regulated by a distinct pathway not involving IjB. Recently, the NF-jB acti- vation pathway, separate from IjB degradation, was described in PI 3-kinase- and Akt-dependent signal transduction pathways [39,49,50]. In addition, our pre- vious data demonstrate that inhibition of PI 3-kinase in MDA-MB-231 cells also suppresses cell migration through NF-jB pathway independent of IjBa degra- dation [31].

A compelling body of evidence has accumulated showing that uPA and uPAR play an important role in cancer metastasis. The correlation between uPA and uPAR concentrations and poor prognosis is especially in agreement with the idea that uPAR-bound uPA at the surface of cancer cells is necessary for invasion by cancer cells and metastasis [10]. In addition, we have recently reported that uPA secretion is responsible for cell migration of highly invasive breast cancer cells and AP-1- and NF-jB-mediated secretion of uPA [31,32]. As shown above, Ganoderma inhibits AP-1 and NF-jB in both breast and prostate cancer cells. Therefore, we examined the effect of Ganoderma on uPA ex- pression and secretion from MDA-231 and PC-3

D. Sliva et al. / Biochemical and Biophysical Research Communications 298 (2002) 603–612 611

cancer cells. Our data demonstrated that spores and Referencesfruiting body of Ganoderma markedly decreased ex-

pression and secretion of uPA from breast and pros- tate cancer cells. In addition, the expression of uPA receptor was also decreased after treatment with Ganoderma. As mentioned above, both uPA and uPAR contain AP-1 and NF-jB DNA binding se- quences in their promoter regions. Therefore, Gano- derma is able to suppress expression of both uPA and uPAR in highly invasive breast and prostate cancer cells, suggesting that common mechanism can be employed to inhibit cancer metastasis.

In the last few years, the use of herbal therapies in alternative medicine has been increasing, and although the number of cancer patients using herbal dietary supplements is not exactly known, there is evidence of the increasing use of dietary supplements in cancer treatment [51]. Ganoderma lucidum is one of the herbs in the herbal mixture PC–SPES, which showed activity against hormone-refractory disease in two prostate cancer patients [52]. Extracts of PC–SPES demon- strated estrogenic effects [53] and decreased growth of hormone-sensitive as well hormone-insensitive prostate cancer cells [54]. Our data clearly demonstrate that Ganoderma inhibited transcription factors AP-1 and NF-jB, followed by the suppression of uPA and uPAR expression, and resulting in the inhibition of cell mo- tility of highly invasive breast and prostate cancer cells. These data suggest GanodermaÕs anticancer biological activity in the herbal mixture PC–SPES. Although different biologically active compounds isolated from Ganoderma demonstrate anticancer activities [5–8], here we show that whole nonfractionated fruiting body or spores of G. lucidum can suppress cancer cell mi- gration by inhibiting a specific signaling pathway. Further study will be required to isolate the most active components from Ganoderma and to identify their optimal ratio with the highest inhibitory activity against cancer cells.

In summary, our data demonstrate that G. luciduminhibited the cell motility of highly invasive breast and prostate cancer cells by suppressing uPA and uPAR. Therefore, Ganoderma in the form of dietary supple- ment can be considered as an additional therapeutic intervention for the inhibition of metastasis of highly invasive breast and prostate cancers.

Acknowledgments

We thank Dr. Karen Spear for editing the manuscript, Ed Bri- zendine for statistical analysis, and Denise Lyons and Tatiana Valachovicova for technical assistance. This work was supported by grants from the Methodist Cancer Center to D.S. and F.P.L., Amer- ican Heart Association Grant 0051661Z to C.L., and grants from Showalter Foundation to C.L. and D.S.

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