Anti-Tumor Effects of <em>Ganoderma lucidum</em> (Reishi) in Inflammatory Breast Cancer in <em>In Vivo</em> and <em>In Vitro</em> Models

<div><p>The medicinal mushroom <i>Ganoderma lucidum</i> (Reishi) was tested as a potential therapeutic for Inflammatory Breast Cancer (IBC) using <i>in vivo</i> and <i>in vitro</i> IBC models. IBC is a lethal and aggressive form of breast cancer that manifests itself without a typical tumor mass. Studies show that IBC tissue biopsies overexpress E-cadherin and the eukaryotic initiation factor 4GI (eIF4GI), two proteins that are partially responsible for the unique pathological properties of this disease. IBC is treated with a multimodal approach that includes non-targeted systemic chemotherapy, surgery, and radiation. Because of its non-toxic and selective anti-cancer activity, medicinal mushroom extracts have received attention for their use in cancer therapy. Our previous studies demonstrate these selective anti-cancer effects of Reishi, where IBC cell viability and invasion, as well as the expression of key IBC molecules, including eIF4G is compromised. Thus, herein we define the mechanistic effects of Reishi focusing on the phosphoinositide-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway, a regulator of cell survival and growth. The present study demonstrates that Reishi treated IBC SUM-149 cells have reduced expression of mTOR downstream effectors at early treatment times, as we observe reduced eIF4G levels coupled with increased levels of eIF4E bound to 4E-BP, with consequential protein synthesis reduction. Severe combined immunodeficient mice injected with IBC cells treated with Reishi for 13 weeks show reduced tumor growth and weight by ∼50%, and Reishi treated tumors showed reduced expression of E-cadherin, mTOR, eIF4G, and p70S6K, and activity of extracellular regulated kinase (ERK1/2). Our results provide evidence that Reishi suppresses protein synthesis and tumor growth by affecting survival and proliferative signaling pathways that act on translation, suggesting that Reishi is a potential natural therapeutic for breast and other cancers.</p> </div

Reishi decreases EIF4F complex levels and protein synthesis in IBC cells.

<p>A. SUM-149 and MCF10A cells were treated with vehicle (0mg/mL, SV or MV) or 0.5mg/mL Reishi (SR or MR) for 24 hours before lysis. Western blot analyses were completed for total eIF4G, eIF4A, eIF4E, and 4E-BP1 obtained from m7GTP pull-down lysates and whole cell lysates. B. Graph represents quantification of eIF4F complex as in Dumstorf et al., 2010 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057431#pone.0057431-Dumstorf1" target="_blank">[25]</a>, where eIF4G normalized to eIF4E is divided by 4E-BP1 normalized to eIF4E [(eIF4G/eIF4E)/(4E-BP1/eIF4E)]. Number of biological replicates (n) varies among experiments (SUM-149; n = 3, MCF10A; n = 1). Columns show means ± SEM of integrated density units, shown relative to vehicle controls. Reishi significantly reduces eIF4F complex assembly at *<i>P</i><0.02 in IBC SUM-149 cells. C. 1×10⁵ cells (SUM-149 and MCF10A) were seeded per well in a six well plate and treated with vehicle or 0.5 mg/mL Reishi for 24 hours. The treatment was removed and the cells were re-incubated for 30 minutes in methionine/cysteine - free DMEM. L-[³⁵S] Methionine and L-[³⁵S] Cysteine (2 mCi/mL) +2% FBS was then added to the cultures. Total cell lysates prepared in NP-40 lysis buffer were analyzed for incorporated radioactivity in trichloroacetic acid precipitates. Data are expressed as means ± SEM of duplicate determinations. Experiment was repeated three times. Reishi significantly (*<i>P</i><0.05) reduces protein synthesis by 48%.</p

Reishi reduces tumor growth, tumor weight, and proliferative and mesenchymal marker expression.

<p>1.5×10⁶ cells/100µL of SUM-149 cells, were injected into the mammary fat pad of severe combined immunodeficient (SCID) mice. One week following injection, mice were orally gavaged with vehicle, (n = 11) or 28 mg/kg BW Reishi (n = 11) daily for a period of 13 weeks. A. Mice weights were recorded weekly. There were no differences in body weights of mice that received Reishi compared to animals that received vehicle control. B. Tumor volume was recorded weekly using caliper measurements, and measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057431#s2" target="_blank">materials and methods</a>. C. Average tumor volume measurements per week from mice treated with vehicle or Reishi were normalized relative to the average tumor volume measurements from mice treated with vehicle or Reishi obtained at week one. Reishi significantly reduces tumor growth by 58% (<i>P</i><0.02). D. Tumor weights were obtained at the end of the study. Columns show means ± SEM. Reishi significantly (*<i>P</i><0.05) reduces tumor weight by 45%. E. Tumors were excised on the 13^th week post Reishi, fixed in 10% formalin and embedded in paraffin before immunostaining with antibodies against Ki-67 and vimentin. Reishi treated tumors show reduced size, lower Ki-67 and Vimentin protein expression.</p

Reishi decreases the expression of PI3K/AKT signaling pathway genes and of mTORC1 effectors.

<p>A. Total SUM-149 cell RNA extraction was performed from three different experimental plates treated with 0 mg/mL (n = 3/vehicle) or 0.5 mg/mL Reishi (n = 3/treatment) for 3 hours. RT² PCR arrays designed to profile the expression of PI3K/AKT pathway-specific genes were used according to manufacturer’s instructions (SA Biosciences). Volcano plot shows the effects on gene expression analyzed at −1.4≥1.4 log₂-fold change (dashed lines). Down-regulated genes are to the left of the vertical black line while up-regulated genes are to the right. Statistically significant (<i>P</i><0.05) regulated genes are above the horizontal black line. B. SUM-149 cells were grown in 5% FBS media for 24 hours prior to treatment with vehicle (0 mg/mL) or Reishi extract (0.5 mg/mL) for 2, 4, and 6 hours before lysis. Equal amount of protein from each sample was used for Western blot analysis with antibodies against mTORC1 effector proteins. C. Columns represent means ±SEM of integrated density units of protein, normalized to β-actin levels and shown relative to vehicle controls (without Reishi treatment). Statistically significant differences are shown at *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.0001.</p

<i>In vivo</i> effects of Reishi on the expression of PI3K/AKT/mTOR pathway genes.

<p>Only genes that demonstrated −1.3>1.3-log₂ fold difference and P<0.05 from RT² PCR arrays are shown.</p

<i>In vitro</i> effects of 0.5 mg/mL Reishi on the expression of PI3K/AKT/mTOR pathway genes.

<p>Only genes that demonstrated −1.4>1.4 log₂-fold difference and P<0.05 from RT² PCR arrays are shown.</p

A list of the genes and primers used in the Real Time RT-PCR analysis.

<p>Real time PCR primers were designed using the websites: <a href="http://www.idtdna.com" target="_blank">www.idtdna.com</a>, <a href="http://www.basic.northwestern.edu/biotools/oligocalc.html" target="_blank">www.basic.northwestern.edu/biotools/oligocalc.html</a>, <a href="http://blast.ncbi.nlm.nih.gov/Blast" target="_blank">http://blast.ncbi.nlm.nih.gov/Blast</a>. cgi, and synthesized at Sigma-Genosys (St. Louis, MI).</p