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Anti-tumor effects of Ganoderma lucidum (reishi) in inflammatory breast cancer in in vivo and in vitro models.

Suarez-Arroyo IJ, Rosario-Acevedo R, Aguilar-Perez A, Clemente PL, Cubano LA, Serrano J, Schneider RJ, Martínez-Montemayor MM - PLoS ONE (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Universidad Central del Caribe, School of Medicine, Bayamón, Puerto Rico, United States of America.

ABSTRACT
The medicinal mushroom Ganoderma lucidum (Reishi) was tested as a potential therapeutic for Inflammatory Breast Cancer (IBC) using in vivo and in vitro 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.

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Reishi decreases the expression of PI3K/AKT signaling pathway genes and of mTORC1 effectors.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 (P<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 *P<0.05, **P<0.01, ***P<0.0001.
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pone-0057431-g001: Reishi decreases the expression of PI3K/AKT signaling pathway genes and of mTORC1 effectors.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 (P<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 *P<0.05, **P<0.01, ***P<0.0001.

Mentions: Using the established IBC cell model, SUM-149 cells, we previously published that Reishi selectively reduced cancer cell viability and invasion [9]. To test whether Reishi treatment affects the expression of genes specifically involved in the PI3K/AKT/mTOR pathway, we performed PI3K/AKT signaling RT2 Profiler PCR arrays in SUM-149 cells treated with vehicle or 0.5 mg/mL Reishi for 3 hours. As shown in Figure 1A, Reishi reduced the expression of most of the genes assayed in this signaling pathway. Table 2 depicts genes in which the expression was significantly affected by Reishi treatment and that display a −1.4≥1.4 Log2 fold change. Of the genes that were statistically different (P<0.05), 19/21 were downregulated in expression by Reishi, including AKT1, CCND1, EIF4GI, MAPK1, and HRAS. The two genes that were significantly upregulated in expression were JUN and FOS by 1.7 and 1.4 fold, respectively. In addition, there were 10 additional genes that showed tendencies for downregulation by Reishi, depicted in Table S1. Because Reishi reduced the expression of CCND1, we also assessed the expression of additional cell cycle regulatory genes at pre-cell cycle (3 and 6 hours) and at post-cell cycle hours (24 and 48 hours) in SUM-149 cells treated with vehicle or 0.5 mg/mL. Although Reishi modulated the expression of these genes at various time points, Reishi significantly reduced the expression of CCNA2 and CCNB2 after 48 hours of treatment by −3.5 and −5.0 fold, respectively (figure S1). The modulatory effects of Reishi on cell cycle progression in IBC cells are consistent with its downregulation of mTOR signaling and the activation (reduced phosphorylation) of 4E-BP1.


Anti-tumor effects of Ganoderma lucidum (reishi) in inflammatory breast cancer in in vivo and in vitro models.

Suarez-Arroyo IJ, Rosario-Acevedo R, Aguilar-Perez A, Clemente PL, Cubano LA, Serrano J, Schneider RJ, Martínez-Montemayor MM - PLoS ONE (2013)

Reishi decreases the expression of PI3K/AKT signaling pathway genes and of mTORC1 effectors.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 (P<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 *P<0.05, **P<0.01, ***P<0.0001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585368&req=5

pone-0057431-g001: Reishi decreases the expression of PI3K/AKT signaling pathway genes and of mTORC1 effectors.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 (P<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 *P<0.05, **P<0.01, ***P<0.0001.
Mentions: Using the established IBC cell model, SUM-149 cells, we previously published that Reishi selectively reduced cancer cell viability and invasion [9]. To test whether Reishi treatment affects the expression of genes specifically involved in the PI3K/AKT/mTOR pathway, we performed PI3K/AKT signaling RT2 Profiler PCR arrays in SUM-149 cells treated with vehicle or 0.5 mg/mL Reishi for 3 hours. As shown in Figure 1A, Reishi reduced the expression of most of the genes assayed in this signaling pathway. Table 2 depicts genes in which the expression was significantly affected by Reishi treatment and that display a −1.4≥1.4 Log2 fold change. Of the genes that were statistically different (P<0.05), 19/21 were downregulated in expression by Reishi, including AKT1, CCND1, EIF4GI, MAPK1, and HRAS. The two genes that were significantly upregulated in expression were JUN and FOS by 1.7 and 1.4 fold, respectively. In addition, there were 10 additional genes that showed tendencies for downregulation by Reishi, depicted in Table S1. Because Reishi reduced the expression of CCND1, we also assessed the expression of additional cell cycle regulatory genes at pre-cell cycle (3 and 6 hours) and at post-cell cycle hours (24 and 48 hours) in SUM-149 cells treated with vehicle or 0.5 mg/mL. Although Reishi modulated the expression of these genes at various time points, Reishi significantly reduced the expression of CCNA2 and CCNB2 after 48 hours of treatment by −3.5 and −5.0 fold, respectively (figure S1). The modulatory effects of Reishi on cell cycle progression in IBC cells are consistent with its downregulation of mTOR signaling and the activation (reduced phosphorylation) of 4E-BP1.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Universidad Central del Caribe, School of Medicine, Bayamón, Puerto Rico, United States of America.

ABSTRACT
The medicinal mushroom Ganoderma lucidum (Reishi) was tested as a potential therapeutic for Inflammatory Breast Cancer (IBC) using in vivo and in vitro 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.

Show MeSH
Related in: MedlinePlus