Shuntaro Tsukamoto

67-kDa laminin receptor increases cGMP to induce cancer-selective apoptosis

The 67-kDa laminin receptor (67LR) is a laminin-binding protein overexpressed in various types of cancer, including bile duct carcinoma, colorectal carcinoma, cervical cancer, and breast carcinoma. 67LR plays a vital role in growth and metastasis of tumor cells and resistance to chemotherapy. Here, we show that 67LR functions as a cancer-specific death receptor. In this cell death receptor pathway, cGMP initiated cancer-specific cell death by activating the PKCδ/acid sphingomyelinase (PKCδ/ASM) pathway. Furthermore, upregulation of cGMP was a rate-determining process of 67LR-dependent cell death induced by the green tea polyphenol (-)-epigallocatechin-3-O-gallate (EGCG), a natural ligand of 67LR. We found that phosphodiesterase 5 (PDE5), a negative regulator of cGMP, was abnormally expressed in multiple cancers and attenuated 67LR-mediated cell death. Vardenafil, a PDE5 inhibitor that is used to treat erectile dysfunction, significantly potentiated the EGCG-activated 67LR-dependent apoptosis without affecting normal cells and prolonged the survival time in a mouse xenograft model. These results suggest that PDE5 inhibitors could be used to elevate cGMP levels to induce 67LR-mediated, cancer-specific cell death.

Green tea polyphenol EGCG induces lipid-raft clustering and apoptotic cell death by activating protein kinase Cδ and acid sphingomyelinase through a 67 kDa laminin receptor in multiple myeloma cells

EGCG [(-)-epigallocatechin-3-O-gallate], the major polyphenol of green tea, has cancer chemopreventive and chemotherapeutic activities. EGCG selectively inhibits cell growth and induces apoptosis in cancer cells without adversely affecting normal cells; however, the underlying molecular mechanism in vivo is unclear. In the present study, we show that EGCG-induced apoptotic activity is attributed to a lipid-raft clustering mediated through 67LR (67 kDa laminin receptor) that is significantly elevated in MM (multiple myeloma) cells relative to normal peripheral blood mononuclear cells, and that aSMase (acid sphingomyelinase) is critical for the lipid-raft clustering and the apoptotic cell death induced by EGCG. We also found that EGCG induces aSMase translocation to the plasma membrane and PKCδ (protein kinase Cδ) phosphorylation at Ser664, which was necessary for aSMase/ceramide signalling via 67LR. Additionally, orally administered EGCG activated PKCδ and aSMase in a murine MM xenograft model. These results elucidate a novel cell-death pathway triggered by EGCG for the specific killing of MM cells.

Green Tea Polyphenol EGCG Sensing Motif on the 67-kDa Laminin Receptor

Background We previously identified the 67-kDa laminin receptor (67LR) as the cell-surface receptor conferring the major green tea polyphenol (–)-epigallocatechin-3-O-gallate (EGCG) responsiveness to cancer cells. However, the underlying mechanism for interaction between EGCG and 67LR remains unclear. In this study, we investigated the possible role of EGCG-67LR interaction responsible for its bioactivities. Methodology/Principal Findings We synthesized various peptides deduced from the extracellular domain corresponding to the 102-295 region of human 67LR encoding a 295-amino acid. The neutralizing activity of these peptides toward EGCG cell-surface binding and inhibition of cancer cell growth were assayed. Both activities were inhibited by a peptide containing the 10-amino acid residues, IPCNNKGAHS, corresponding to residues 161-170. Furthermore, mass spectrometric analysis revealed the formation of a EGCG-LR161-170 peptide complex. A study of the amino acid deletion/replacement of the peptide LR161-170 indicated that the 10-amino acid length and two basic amino acids, K166 and H169, have a critical role in neutralizing EGCG’s activities. Moreover, neutralizing activity against the anti-proliferation action of EGCG was observed in a recombinant protein of the extracellular domain of 67LR, and this effect was abrogated by a deletion of residues 161-170. These findings support that the 10 amino-acid sequence, IPCNNKGAHS, might be the functional domain responsible for the anti-cancer activity of EGCG. Conclusions/Significance Overall, our results highlight the nature of the EGCG-67LR interaction and provide novel structural insights into the understanding of 67LR-mediated functions of EGCG, and could aid in the development of potential anti-cancer compounds for chemopreventive or therapeutic uses that can mimic EGCG-67LR interactions.

Green tea extract containing a highly absorbent catechin prevents diet-induced lipid metabolism disorder.

We investigated the effects of extracts of Benifuuki (a tea cultivar that contains methylated catechins such as epigallocatechin-3-O-(3-O-methyl) gallate (EGCG3”Me)) in mice fed a high-fat/high-sucrose (HF/HS) diet. This tea cultivar was then compared with an extract of Yabukita (a popular tea cultivar that lacks methylated catechins). For 6 weeks, C57BL/6J mice were fed either HF/HS diet with or without tea extracts from tea cultivars, which contained almost identical ingredients except for methylated catechins (i.e., Yabukita (0.2% and 1%) or Benifuuki (0.2% and 1%) extract powders). Supplementation with Benifuuki 0.2% markedly lowered plasma levels of TG and NEFAs compared with mice supplemented with Yabukita 0.2%. The diet containing Benifuuki 1% decreased adipose tissue weights, liver TG, and expression of lipogenic genes in the liver. These results suggested that Benifuuki had much greater lipid-lowering effects than Yabukita. Taken together, these data suggest that methylated catechins direct the strong lipid-lowering activity of Benifuuki.

Epigallocatechin-3-O-gallate up-regulates microRNA-let-7b expression by activating 67-kDa laminin receptor signaling in melanoma cells

MicroRNAs (miRNAs) are non-coding RNAs involved in various biological processes by regulating their target genes. Green tea polyphenol (−)-epigallocatechin-3-O-gallate (EGCG) inhibits melanoma tumor growth by activating 67-kDa laminin receptor (67LR) signaling. To examine the effect of EGCG on miRNA expression in melanoma cells, we performed miRNA microarray analysis. We showed that EGCG up-regulated miRNA-let-7b expression through 67LR in melanoma cells. The EGCG-induced up-regulation of let-7b led to down-regulation of high mobility group A2 (HMGA2), a target gene related to tumor progression. 67LR-dependent cAMP/protein kinase A (PKA)/protein phosphatase 2A (PP2A) signaling pathway activation was involved in the up-regulation of let-7b expression induced by EGCG. These findings provide a basis for understanding the mechanism of miRNA regulation by EGCG.

67-kDa Laminin Receptor-dependent Protein Phosphatase 2A (PP2A) Activation Elicits Melanoma-specific Antitumor Activity Overcoming Drug Resistance

Background: Green tea polyphenol (−)-epigallocatechin-3-O-gallate (EGCG) inhibits melanoma proliferation in a cancer-specific manner through 67-kDa laminin receptor (67LR). Results: Identified protein phosphatase 2A (PP2A) as a critical downstream factor of 67LR. Conclusion: Targeting 67LR/PP2A elicits activation of tumor suppressor Merlin and inhibition of mTOR pathway overcoming drug resistance. Significance: 67LR/PP2A may be a promising therapeutic target for melanomas. The Ras/Raf/MEK/ERK pathway has been identified as a major, druggable regulator of melanoma. Mutational activation of BRAF is the most prevalent genetic alteration in human melanoma, resulting in constitutive melanoma hyperproliferation. A selective BRAF inhibitor showed remarkable clinical activity in patients with mutated BRAF. Unfortunately, most patients acquire resistance to the BRAF inhibitor, highlighting the urgent need for new melanoma treatment strategies. Green tea polyphenol (−)-epigallocatechin-3-O-gallate (EGCG) inhibits cell proliferation independently of BRAF inhibitor sensitivity, suggesting that increased understanding of the anti-melanoma activity of EGCG may provide a novel therapeutic target. Here, by performing functional genetic screening, we identified protein phosphatase 2A (PP2A) as a critical factor in the suppression of melanoma cell proliferation. We demonstrated that tumor-overexpressed 67-kDa laminin receptor (67LR) activates PP2A through adenylate cyclase/cAMP pathway eliciting inhibitions of oncoproteins and activation of tumor suppressor Merlin. Activating 67LR/PP2A pathway leading to melanoma-specific mTOR inhibition shows strong synergy with the BRAF inhibitor PLX4720 in the drug-resistant melanoma. Moreover, SET, a potent inhibitor of PP2A, is overexpressed on malignant melanoma. Silencing of SET enhances 67LR/PP2A signaling. Collectively, activation of 67LR/PP2A signaling may thus be a novel rational strategy for melanoma-specific treatment.

Phosphodiesterase 5 inhibitor acts as a potent agent sensitizing acute myeloid leukemia cells to 67-kDa laminin receptor-dependent apoptosis

(−)‐Epigallocatechin‐3‐O‐gallate (EGCG), a polyphenol in green tea, induces apoptosis in acute myeloid leukemia (AML) cells without affecting normal cells. In this study, we observed that cGMP acts as a cell death mediator of the EGCG‐induced anti‐AML effect through acid sphingomyelinase activation. EGCG activated the Akt/eNOS axis, a well‐known mechanism in vascular cGMP upregulation. We also observed that a major cGMP negative regulator, phosphodiesterase 5, was overexpressed in AML cells, and PDE5 inhibitor, an anti‐erectile dysfunction drug, synergistically enhanced the anti‐AML effect of EGCG. This combination regimen killed AML cells via overexpressed 67‐kDa laminin receptors.

Oxygen partial pressure modulates 67-kDa laminin receptor expression, leading to altered activity of the green tea polyphenol, EGCG

(−)‐Epigallocatechin‐3‐O‐gallate (EGCG) exhibits anti‐tumor activity mediated via the 67‐kDa laminin receptor (67LR). In this study, we found that 67LR protein levels are reduced by exposure to low O2 levels (5%), without affecting the expression of HIF‐1α. We also found that EGCG‐induced anti‐cancer activity is abrogated under low O2 levels (5%) in various cancer cells. Notably, treatment with the proteasome inhibitor, prevented down‐regulation of 67LR and restored sensitivity to EGCG under 5% O2. In summary, 67LR expression is highly sensitive to O2 partial pressure, and the activity of EGCG can be regulated in cancer cells by O2 partial pressure.

FOXO3 is essential for CD44 expression in pancreatic cancer cells

Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal types of cancer and the 5-year survival rate is only 5%. Several studies have suggested that cancer stem cells (CSCs) are thought to be involved in recurrence and metastasis and so it is essential to establish an approach targeting CSCs. Here we have demonstrated that cyclic guanosine monophosphate (cGMP) suppressed CD44 expression and the properties of CSCs in PDAC. Microarray analysis suggested that cGMP inhibited Forkhead box O3 (FOXO3), which is known as a tumor suppressor. Surprisingly, our data demonstrated that FOXO3 is essential for CD44 expression and the properties of CSCs. Our data also indicated that patients with high FOXO3 activation signatures had poor prognoses. This evidence suggested that cGMP induction and FOXO3 inhibition could be ideal candidates for pancreatic CSC.

Vardenafil, a clinically available phosphodiesterase inhibitor, potentiates the killing effect of EGCG on CLL cells

helped to conceptualize the structure of the study. YZ contributed to the execution of the research. VJ helped with data analysis. As chief of the department, WH helped conceptualize the study and provided essential reagents or tools. MD acted in an advisory role, designed the research study and contributed essential reagents or tools. The ‘Lymphoma Research Foundation’ and the ‘European Mantle Cell Lymphoma Network’ supported this work. DH and MD have a patent pending on obinutuzumab combination treatment. WH and MD have received speaker’s honoraria and support of investigatorinitiated trials from Roche and are members of the scientific advisory board for Roche. MW, GH, YZ, VJ declare no potential conflict of interest. We thank the ‘Helmholtz Zentrum M€ unchen – German Research Centre for Environmental Health’ for providing research facilities. We thank Dr. Christian Klein, Roche Glycart AG (Schlieren, Switzerland) for discussion and providing obinutuzumab (GA101) and Dr. Helmut Burtscher and Ute Baer, Roche Diagnostics GmbH (Penzberg, Germany) for mRNA profiling experiments. Daniel A. Heinrich Marc Weinkauf Grit Hutter Yvonne Zimmermann Vindi Jurinovic Wolfgang Hiddemann Martin Dreyling Medizinische Klinik und Poliklinik IV, Klinikum der Universit€at M€ unchen, Helmholtz Zentrum München – German Research Center for Environmental Health, Medizinische Klinik und Poliklinik III, Klinikum der Universit€at M€ unchen, and Institut für medizinische Informationsverarbeitung, Biometrie und Epidemiologie, LudwigMaximilians-Universit€at, München, Germany E-mail: daniel.heinrich@med.uni-muenchen.de