Structure Elucidation and Enantioselective Total Synthesis of the Potent HMG-CoA Reductase Inhibitor FR901512 via Catalytic Asymmetric Nozaki−Hiyama Reactions

The structure elucidation and enantioselective total synthesis of the potent HMG-CoA reductase inhibitor FR901512 were accomplished. FR901512 was prepared in 15 steps from the commercially available 2-bromo-4-methylbenzaldehyde via FR901516 in 16.3% overall yield (89% average yield). The catalytic asymmetric Nozaki−Hiyama reactions developed by us proved their applicability and reliability through this work, enabling the concise, efficient, and protecting-group-free enantioselective total syntheses of these new statins

Studies on Catalytic Asymmetric Nozaki−Hiyama Propargylation

Catalytic asymmetric Nozaki−Hiyama propargylation with ligand 1c proceeds with good to excellent enantioselectivity. Tuning of ligand 1 dramatically changes the enantioselectivity, and we propose models A and B to explain the change and outcome of the enantioselectivity

Structure Elucidation and Enantioselective Total Synthesis of the Potent HMG-CoA Reductase Inhibitor FR901512 via Catalytic Asymmetric Nozaki−Hiyama Reactions

The structure elucidation and enantioselective total synthesis of the potent HMG-CoA reductase inhibitor FR901512 were accomplished. FR901512 was prepared in 15 steps from the commercially available 2-bromo-4-methylbenzaldehyde via FR901516 in 16.3% overall yield (89% average yield). The catalytic asymmetric Nozaki−Hiyama reactions developed by us proved their applicability and reliability through this work, enabling the concise, efficient, and protecting-group-free enantioselective total syntheses of these new statins

Asymmetric Catalysis of Nozaki−Hiyama Allylation and Methallylation with A New Tridentate Bis(oxazolinyl)carbazole Ligand

This work describes the development of a new tridentate ligand effective for the asymmetric catalysis of Nozaki−Hiyama allylation and methallylation. Various aldehydes were allylated or methallylated with good enantioselectivity (86−96%), and a key intermediate of calcitriol lactone synthesis was also obtained with excellent diastereoselectivity (97% de, 91%). The enantioselective reaction catalyzed by this Cr−ligand complex is applicable to a broad range of aldehydes and has great potential for natural product synthesis. Another remarkable feature of this ligand is the stability of the Cr−ligand complex which was recovered after the enantioselective reaction and recycled twice without diminishing the enantioselectivity and yield

Downregulation of AKT phosphorylation is important for induction of dormant status.

<p>A) Immunoblot of the AKT/mTORC1 or ERK/p38 MAPK pathway in AsPC-1 cells cultured in hypoxia. B) Immunoblot of AKT signaling and HIF-1α in AsPC-1 cells expressing vector control, AKT-wt, or AKT-3A (inactive). C) Cell cycle status of the cells at day 7 in hypoxia. Percentages of the cells in S phase are indicated above the plot and in the right graph. D) ATP turnover measured by adding inhibitor cocktail for glycolysis and oxidative phosphorylation. N1, normoxia 1 day; H1, hypoxia 1 day; H7, hypoxia 7 days. E, F) Viable cell number (E) and percent cell death (F) of AsPC-1 cells cultured in hypoxia. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p

The role of apoptosis in hypoxia-induced gene downregulation in MIN6 cells.

<p>(A) MIN6 cells were incubated at 5% O2 for 10 h and the expression levels of <i>Ddit3</i> mRNA were examined by qPCR (n = 4). (B, C) Annexin V-positive cell (B) and PI-positive cell death (C) ratios were evaluated by flow cytometric analysis (n = 6) after MIN6 cells were cultured at 5% O2 for 10 h. (D) Expression levels of <i>Pdx1</i>, <i>Neurod1</i>, <i>Wfs1</i>, and <i>Slc2a2</i> were examined by qPCR (n = 4) in the same conditions as in (A). (E) Ctrl MIN6 cells (gray bars) and CHOP knockdown MIN6 cells (black bars) were incubated either in normoxia (20% O₂) or in hypoxia (5% O₂) for 40 h and the expression levels of <i>Ddit3</i> mRNA were examined by qPCR (n = 3). (F) Ctrl MIN6 cells (gray bars) (n = 5) and CHOP knockdown MIN6 cells (black bars) (n = 5) were incubated either in normoxia (20% O₂) or in hypoxia (5% O₂). The PI-positive cell death ratio was evaluated by flow cytometric analysis. (G) Ctrl MIN6 cells (gray bars) and CHOP knockdown MIN6 cells (black bars) were incubated in the same conditions as in (E). Downregulation of mRNA levels (Δ mRNA levels) by 5% O₂. Δ mRNA levels indicate (1-expression levels at 5% O₂/expression levels at 20% O₂) ×100 (%). In qPCR analysis, the mRNA value of each gene was normalized to that of <i>Tbp</i>. The data are shown as the means ± S.E. (error bars) of values from each group. *, p<0.05; **, p<0.01; ***, p<0.001.</p

Dormancy of Cancer Cells with Suppression of AKT Activity Contributes to Survival in Chronic Hypoxia

<div><p>A hypoxic microenvironment in tumors has been recognized as a cause of malignancy or resistance to various cancer therapies. In contrast to recent progress in understanding the acute response of cancer cells to hypoxia, the characteristics of tumor cells in chronic hypoxia remain elusive. We have identified a pancreatic cancer cell line, AsPC-1, that is exceptionally able to survive for weeks under 1% oxygen conditions while most tested cancer cell lines die after only some days under these conditions. In chronic hypoxia, AsPC-1 cells entered a state of dormancy characterized by no proliferation, no death, and metabolic suppression. They reversibly switched to active status after being placed again in optimal culture conditions. ATP turnover, an indicator of energy demand, was markedly decreased and accompanied by reduced AKT phosphorylation. Forced activation of AKT resulted in increased ATP turnover and massive cell death <i>in vitro</i> and a decreased number of dormant cells <i>in vivo</i>. In contrast to most cancer cell lines, primary-cultured colorectal cancer cells easily entered the dormant status with AKT suppression under hypoxia combined with growth factor–depleted conditions. Primary colorectal cancer cells in dormancy were resistant to chemotherapy. Thus, the ability to survive in a deteriorated microenvironment by entering into dormancy under chronic hypoxia might be a common property among cancer cells. Targeting the regulatory mechanism inducing this dormant status could provide a new strategy for treating cancer.</p></div

Downregulation of AKT phosphorylation is necessary for induction of dormant status in primary colorectal cancer.

<p>A) CTOS growth was measured by size relative to day 0. C45 CTOS samples were cultured in medium with (GF+) or without (GF−) growth factors. B) Representative images of C45 CTOS cultured in indicated conditions. Scale bar  = 100 µm. C) Regrowth of CTOS in dormant status after re-oxygenation and exposure to growth factor–containing medium. D) Immunohistochemistry of C45 CTOS cultured in indicated conditions for 1 day. TUNEL staining was at day 14. Scale bar  = 50 µm. E) Immunoblot of AKT/mTORC1 signaling and HIF-1α in C45 CTOS cultured in indicated conditions.</p

Moderate Hypoxia Induces β-Cell Dysfunction with HIF-1–Independent Gene Expression Changes

<div><p>Pancreatic β-cell failure is central to the development and progression of type 2 diabetes. We recently demonstrated that β-cells become hypoxic under high glucose conditions due to increased oxygen consumption and that the pancreatic islets of diabetic mice but not those of control mice are moderately hypoxic. However, the impact of moderate hypoxia on β-cell number and function is unknown. In the present study, moderate hypoxia induced a hypoxic response in MIN6 cells, as evidenced by increased levels of HIF-1α protein and target genes. Under these conditions, a selective downregulation of <i>Mafa</i>, <i>Pdx1</i>, <i>Slc2a2</i>, <i>Ndufa5</i>, <i>Kcnj11</i>, <i>Ins1</i>, <i>Wfs1</i>, <i>Foxa2</i>, and <i>Neurod1</i>, which play important roles in β-cells, was also observed in both MIN6 cells and isolated pancreatic islets. Consistent with the altered expression of these genes, abnormal insulin secretion was detected in hypoxic MIN6 cells. Most of the hypoxia-induced gene downregulation in MIN6 cells was not affected by the suppression of HIF-1α, suggesting a HIF-1–independent mechanism. Moderate hypoxia also induced apoptosis in MIN6 cells. These results suggest that hypoxia is a novel stressor of β-cells and that hypoxic stress may play a role in the deterioration of β-cell function.</p></div

Forced activation of AKT reduces the inactive zone <i>in vivo</i>.

<p>A) Immunohistochemistry of xenotumors of AsPC-1 cells expressing control vector (upper) or AKT-mΔPH (lower). N, necrosis; scale bar  = 100 µm. B) Percent of BrdU-positive cells in the area distal or proximal to pimonidazole-positive zone. C) Width of pimonidazole-positive zone in tumors from vector or AKT-mΔPH; *<i>p</i><0.05, ***<i>p</i><0.001.</p