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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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Related in: MedlinePlus

Reishi decreases EIF4F complex levels and protein synthesis in IBC cells.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 [25], 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 *P<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 (*P<0.05) reduces protein synthesis by 48%.
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pone-0057431-g002: Reishi decreases EIF4F complex levels and protein synthesis in IBC cells.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 [25], 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 *P<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 (*P<0.05) reduces protein synthesis by 48%.

Mentions: Reishi reduced 4E-BP1 phosphorylation at early timepoints (Figure 1B) and eIF4G levels by 24 hours post-treatment [9]. As the hypophosphorylation of 4E-BP1 increases the binding of 4E-BP1 to eIF4E [24], thereby diminishing eIF4F complex levels, we sought to determine whether Reishi would increase this association using m7GTP cap analog beads to capture eIF4E and to pull down associated proteins as described in [25]. Accordingly, we found that at 24 hours post-Reishi treatment the amount of 4E-BP1 bound to eIF4E increases in SUM-149 cells (Figure 2A). To quantify this, we normalized the levels of co-captured 4E-BP1 and eIF4G to eIF4E and then divided the normalized eIF4G values by the normalized 4E-BP1 values after m7GTP co-capture in MCF10A and SUM-149 cells (Figure 2B). eIF4F translation initiation complex assembly levels in SUM-149 cells, are significantly (∼60%) reduced in Reishi treated cells (P<0.02). Interestingly, this effect was not observed in Reishi treated non-cancerous mammary epithelial MCF10A cells, or in IBC cells treated with or without Reishi for 4 (data not shown) or 6 hours (figure S3). Thus, disruption of eIF4F translation initiation complex levels, unlike downregulation of mTOR, required an extended period of treatment.


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 EIF4F complex levels and protein synthesis in IBC cells.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 [25], 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 *P<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 (*P<0.05) reduces protein synthesis by 48%.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585368&req=5

pone-0057431-g002: Reishi decreases EIF4F complex levels and protein synthesis in IBC cells.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 [25], 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 *P<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 (*P<0.05) reduces protein synthesis by 48%.
Mentions: Reishi reduced 4E-BP1 phosphorylation at early timepoints (Figure 1B) and eIF4G levels by 24 hours post-treatment [9]. As the hypophosphorylation of 4E-BP1 increases the binding of 4E-BP1 to eIF4E [24], thereby diminishing eIF4F complex levels, we sought to determine whether Reishi would increase this association using m7GTP cap analog beads to capture eIF4E and to pull down associated proteins as described in [25]. Accordingly, we found that at 24 hours post-Reishi treatment the amount of 4E-BP1 bound to eIF4E increases in SUM-149 cells (Figure 2A). To quantify this, we normalized the levels of co-captured 4E-BP1 and eIF4G to eIF4E and then divided the normalized eIF4G values by the normalized 4E-BP1 values after m7GTP co-capture in MCF10A and SUM-149 cells (Figure 2B). eIF4F translation initiation complex assembly levels in SUM-149 cells, are significantly (∼60%) reduced in Reishi treated cells (P<0.02). Interestingly, this effect was not observed in Reishi treated non-cancerous mammary epithelial MCF10A cells, or in IBC cells treated with or without Reishi for 4 (data not shown) or 6 hours (figure S3). Thus, disruption of eIF4F translation initiation complex levels, unlike downregulation of mTOR, required an extended period of treatment.

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