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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 reduces PI3K/AKT/mTOR and MAPK pathway gene and protein expression.A. RT2 PCR array designed to profile the expression of PI3K/AKT pathway-specific genes was performed using 500 ng of tumor extracted RNA, according to manufacturer’s instructions (SA Biosciences). Volcano plot show effects on gene expression analyzed at −1.3≥1.3 log2-fold change (dashed line). Down-regulated genes are to the left of the vertical black line while up-regulated genes are to the right. Statistically significant regulated genes are above the horizontal black line at P<0.05. B. Equal amount of protein from each sample was used for western blot analysis with antibodies against key IBC proteins. Each lane depicts a representative tumor lysate from a different mouse of either vehicle or Reishi treatment. C. Quantification was done using integrated density units, normalized to β-actin and relative to vehicle. Columns show means ± SEM. Reishi downregulates the expression of key IBC proteins in vivo. *P<0.05, **P<0.01.
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pone-0057431-g004: Reishi reduces PI3K/AKT/mTOR and MAPK pathway gene and protein expression.A. RT2 PCR array designed to profile the expression of PI3K/AKT pathway-specific genes was performed using 500 ng of tumor extracted RNA, according to manufacturer’s instructions (SA Biosciences). Volcano plot show effects on gene expression analyzed at −1.3≥1.3 log2-fold change (dashed line). Down-regulated genes are to the left of the vertical black line while up-regulated genes are to the right. Statistically significant regulated genes are above the horizontal black line at P<0.05. B. Equal amount of protein from each sample was used for western blot analysis with antibodies against key IBC proteins. Each lane depicts a representative tumor lysate from a different mouse of either vehicle or Reishi treatment. C. Quantification was done using integrated density units, normalized to β-actin and relative to vehicle. Columns show means ± SEM. Reishi downregulates the expression of key IBC proteins in vivo. *P<0.05, **P<0.01.

Mentions: Total RNA was extracted from tumor lysates and PI3K/AKT/mTOR PCR arrays were conducted to determine Reishi effects on genes involved in this pro-survival pathway. As shown in Figure 4A, Reishi reduced the expression of 64% of the genes in the PCR array. Reishi significantly reduced the expression of 5 genes, including the eukaryotic initiation factor 4B and ribosomal protein S6 kinase, 70 kDa, polypeptide 1 (EIF4B, RPS6KB1), gap junction protein alpha 1, 43 kDa (GJA1), the pro-invasion gene encoding the p21 protein (cdc42/Rac)-activated kinase 1 (PAK1), and pyruvate dehydrogenase kinase, isozyme 1 (PDK1) (Table 3), while it increased the expression of the nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (NFKBIA). Additional genes affected by Reishi that show strong statistical tendencies are listed in Table S2. Moreover, to assess Reishi anti-IBC effects in vivo, we examined the expression of various proteins in tumor lysates. First we assessed the effects of Reishi on key IBC proteins. As shown in Figures 4B & 4C, Reishi reduced the expression of IBC biomarker, E-cadherin, and two proteins in which their mRNAs are translated in an IRES-dependent manner, p120-catenin, and c-myc. Next, we examined the in vivo effects of Reishi on mTOR signaling proteins, where Reishi significantly reduced the expression of mTOR, p70S6K, and eIF4G. However, the total expression or activation of Akt was not affected by the treatment (figure S4). Because loss of mTOR function has an impact on MAPK activation status [26], we verified if Reishi activates the MAPK pathway. Herein we show that Reishi reduces the expression of RAS, and of p-ERK1/2 without affecting total ERK1/2 levels (Figure 4B, 4C). These results provide evidence that the various compounds found in Reishi, which have yet to be isolated, have an inhibitory anti-cancer effect manifested by reduced tumor growth, gene expression, protein synthesis and concomitant inhibition of the mTOR and MAPK pathways showing relevant therapeutic implications in IBC. This study provides compelling reason to pursue further purification and isolation of these compounds.


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 reduces PI3K/AKT/mTOR and MAPK pathway gene and protein expression.A. RT2 PCR array designed to profile the expression of PI3K/AKT pathway-specific genes was performed using 500 ng of tumor extracted RNA, according to manufacturer’s instructions (SA Biosciences). Volcano plot show effects on gene expression analyzed at −1.3≥1.3 log2-fold change (dashed line). Down-regulated genes are to the left of the vertical black line while up-regulated genes are to the right. Statistically significant regulated genes are above the horizontal black line at P<0.05. B. Equal amount of protein from each sample was used for western blot analysis with antibodies against key IBC proteins. Each lane depicts a representative tumor lysate from a different mouse of either vehicle or Reishi treatment. C. Quantification was done using integrated density units, normalized to β-actin and relative to vehicle. Columns show means ± SEM. Reishi downregulates the expression of key IBC proteins in vivo. *P<0.05, **P<0.01.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585368&req=5

pone-0057431-g004: Reishi reduces PI3K/AKT/mTOR and MAPK pathway gene and protein expression.A. RT2 PCR array designed to profile the expression of PI3K/AKT pathway-specific genes was performed using 500 ng of tumor extracted RNA, according to manufacturer’s instructions (SA Biosciences). Volcano plot show effects on gene expression analyzed at −1.3≥1.3 log2-fold change (dashed line). Down-regulated genes are to the left of the vertical black line while up-regulated genes are to the right. Statistically significant regulated genes are above the horizontal black line at P<0.05. B. Equal amount of protein from each sample was used for western blot analysis with antibodies against key IBC proteins. Each lane depicts a representative tumor lysate from a different mouse of either vehicle or Reishi treatment. C. Quantification was done using integrated density units, normalized to β-actin and relative to vehicle. Columns show means ± SEM. Reishi downregulates the expression of key IBC proteins in vivo. *P<0.05, **P<0.01.
Mentions: Total RNA was extracted from tumor lysates and PI3K/AKT/mTOR PCR arrays were conducted to determine Reishi effects on genes involved in this pro-survival pathway. As shown in Figure 4A, Reishi reduced the expression of 64% of the genes in the PCR array. Reishi significantly reduced the expression of 5 genes, including the eukaryotic initiation factor 4B and ribosomal protein S6 kinase, 70 kDa, polypeptide 1 (EIF4B, RPS6KB1), gap junction protein alpha 1, 43 kDa (GJA1), the pro-invasion gene encoding the p21 protein (cdc42/Rac)-activated kinase 1 (PAK1), and pyruvate dehydrogenase kinase, isozyme 1 (PDK1) (Table 3), while it increased the expression of the nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (NFKBIA). Additional genes affected by Reishi that show strong statistical tendencies are listed in Table S2. Moreover, to assess Reishi anti-IBC effects in vivo, we examined the expression of various proteins in tumor lysates. First we assessed the effects of Reishi on key IBC proteins. As shown in Figures 4B & 4C, Reishi reduced the expression of IBC biomarker, E-cadherin, and two proteins in which their mRNAs are translated in an IRES-dependent manner, p120-catenin, and c-myc. Next, we examined the in vivo effects of Reishi on mTOR signaling proteins, where Reishi significantly reduced the expression of mTOR, p70S6K, and eIF4G. However, the total expression or activation of Akt was not affected by the treatment (figure S4). Because loss of mTOR function has an impact on MAPK activation status [26], we verified if Reishi activates the MAPK pathway. Herein we show that Reishi reduces the expression of RAS, and of p-ERK1/2 without affecting total ERK1/2 levels (Figure 4B, 4C). These results provide evidence that the various compounds found in Reishi, which have yet to be isolated, have an inhibitory anti-cancer effect manifested by reduced tumor growth, gene expression, protein synthesis and concomitant inhibition of the mTOR and MAPK pathways showing relevant therapeutic implications in IBC. This study provides compelling reason to pursue further purification and isolation of these compounds.

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