Developing an Anticancer Platinum(II) Compound Based on the Uniqueness of Human Serum Albumin

To develop the next-generation Pt drug with remarkable activity and low toxicity to maximally inhibit tumor growth, we optimized a Pt(II) thiosemicarbazone compound (C4) with remarkable cytotoxicity to SK-N-MC cells and then constructed a new human serum albumin–C4 (HSA–C4) complex delivery system. The in vivo results showed that C4 and the HSA–C4 complex have remarkable therapeutic efficiency and almost no toxicity; they induced apoptosis and inhibited tumor angiogenesis. This system showed potential as a practical Pt drug. This study could pave the way for developing next-generation dual-targeted Pt drugs and achieving their targeting therapy for cancer

Developing an Anticancer Platinum(II) Compound Based on the Uniqueness of Human Serum Albumin

To develop the next-generation Pt drug with remarkable activity and low toxicity to maximally inhibit tumor growth, we optimized a Pt(II) thiosemicarbazone compound (C4) with remarkable cytotoxicity to SK-N-MC cells and then constructed a new human serum albumin–C4 (HSA–C4) complex delivery system. The in vivo results showed that C4 and the HSA–C4 complex have remarkable therapeutic efficiency and almost no toxicity; they induced apoptosis and inhibited tumor angiogenesis. This system showed potential as a practical Pt drug. This study could pave the way for developing next-generation dual-targeted Pt drugs and achieving their targeting therapy for cancer

Palmitate (PA) treatment induced NR4A3 expression and unfolded protein response (UPR) activation in MIN6 cells.

<p>(A, B) NR4A3 mRNA levels in response to (A) different doses of PA and (B) a fixed PA dose at a series of time points. (C, D) <i>Chop</i> mRNA levels in response to (C) different doses of PA and (D) a fixed PA dose at a series of time points. Relative mRNA levels of NR4A3 and Chop were determined with real-time quantitative PCR. (E, F) Spliced XBP1 (sXBP1) mRNA formation in response to (E) different doses of PA and (F) a fixed PA dose at different time points. Two forms of XBP1 (a UPR molecule) were detected with reverse transcription PCR. (G) NR4A3 protein profile in response to a fixed PA dose at a series of time points assayed with western blotting. (H) Semi-quantitative analyses of NR4A3 protein in response to a fixed PA dose at a series of time points. Data are shown as means ± S.E. (n = 4). * p<0.05, ** p<0.01 vs. con. Con is either (A, C, E) the vehicle (0.5% bovine serum albumin [BSA]) or (B, D, F– H) the average basal level in 0.5% BSA at a series of time points.</p

Insulin secretion and mRNA assay in MIN6 cells after infection with equivalent titer of adenovirus.

<p>(A, B) Analysis of insulin secretion after glucose stimulation assayed by radioimmunoassay (RIA). (A) MIN6 cells were treated with 0.5 µM thapsigargin (TG) or DMSO (vehicle control) for 1 h, 0.5 mM palmitate (PA) or 0.5% BSA (vehicle control) for 12 h, and the supernatants were assayed for insulin protein level (n = 3). (B) MIN6 cells were infected with a series of double dilutions of Ad-NR4A3 adenovirus (adenovirus encoding NR4A3). Additional Ad-GFP (control adenovirus expressing GFP only) was used for complementary infection in order to ensure each infection had an equal virus titer. Post-infection, levels of secreted insulin were assayed by RIA (n = 4). Western blotting showed NR4A3 protein expression gradually decreasing, and insulin secretion level increasing accordingly. (C) mRNA levels of two insulin genes (<i>Ins1</i> and <i>Ins2</i>) were determined with reverse transcription PCR in MIN6 cells infected with Ad-NR4A3/Ad-GFP. (D) Semi-quantitative analyses of <i>Ins1</i> and <i>Ins2</i> mRNA levels in different adenovirus-infected MIN6 cells (normalized to beta actin) (n = 6). Data are shown as mean ± S.E. # p<0.05; **, ## p<0.01 vs. the control group or Ad-GFP infection only.</p

Elevation of NR4A3 Expression and Its Possible Role in Modulating Insulin Expression in the Pancreatic Beta Cell

<div><p>Background</p><p>NR4A3/NOR-1 is a member of the NR4A orphan nuclear receptor subfamily, which contains early response genes that sense and respond to a variety of stimuli in the cellular environment. The role of NR4A3 in insulin expression in pancreatic beta cells remains unknown.</p><p>Methods</p><p>Dynamic changes in NR4A3 were examined in a pancreatic beta-cell line, MIN6, treated with thapsigargin (TG), palmitate (PA), tunicamycin (TM), and dithiothreitol (DTT), chemicals that produce cell stress and even apoptosis. We exploited virus infection techniques to induce expression of NR4A3 or three deletion mutants, and determined expression of insulin and insulin regulatory genes in MIN6 cells.</p><p>Results</p><p>TG and PA, two endoplasmic reticulum (ER) stress inducers, were able to induce unfolded protein response (UPR) activation and elevation of NR4A3 expression in MIN6 cells, whereas TM and DTT, two other ER stress inducers, were able to induce UPR activation but not NR4A3 elevation. MIN6 cells over-expressing NR4A3 protein after adenoviral infection exhibited reduced transcription of the insulin genes <i>Ins1</i> and <i>Ins2</i>, and reduced insulin protein secretion, which were negatively correlated with NR4A3 expression levels. Functional analysis of different deletion mutants of NR4A3 showed that deleting the activation domain AF1 or the DNA-binding domain abolished the down-regulation of insulin transcription by NR4A3 in MIN6 cells, indicating that this down-regulative role was closely related to the NR4A3 trans-activation activity. Over-expression of NR4A3 in MIN6 cells resulted in reduced mRNA transcription of the insulin positive-regulation genes, <i>Pdx1</i> and <i>NeuroD1</i>.</p><p>Conclusion</p><p>Some ER stress inducers, such as TG or PA, are able to elevate NR4A3 expression in MIN6 cells, while others, such as TM or DTT, are not. Over-expression of NR4A3 in MIN6 cells results in down-regulation of insulin gene transcription and insulin secretion. NR4A3 reduces insulin gene expression by modulating the expression of <i>Pdx1</i> and <i>NeuroD1</i>.</p></div

A model for possible role of NR4A3 in releasing pancreatic beta cells ER stress.

<p>Upon expression and activation of NR4A3 induced by factors such as long-chain free fatty acids (FFA) and thapsigargin (TG), which lead to ER stress, unfolded protein response (UPR) activation, and even apoptosis, this orphan nuclear receptor decreases insulin expression, which indirectly releases the burden of ER.</p

Possible roles of NR4A3 in modulating the expression of insulin genes in MIN6 cell lines.

<p>(A) Diagram of constructed wild-type and deletion forms of NR4A3 cDNA. (B) Verification by western blotting MIN6 cell lines stably over-expressing NR4A3 or its deletion forms. (C) mRNA levels of NR4A3 or two insulin genes (<i>Ins1</i> and <i>Ins2</i>) detected with reverse transcription PCR. Each image is representative of at least three experiments. (D) Semi-quantitative analyses of <i>Ins1</i> and <i>Ins2</i> mRNA levels normalized to beta actin in various stable cell lines (n = 5). ** <i>P</i><0.01 vs. the control cells. Data are representative of three clone lines. Con, cell line transfected with vector encoding GFP; N, cell line expressing the wild-type NR4A3; ΔA, cell line expressing the 2–288 amino acid (aa) deletion of AF1 (activation function-1 domain); ΔD, cell line expressing the 292–364 aa deletion of the DNA binding domain (DBD); ΔL, cell line expressing the 398–626 aa deletion of the ligand binding domain (LBD). The C terminal of all exogenous genes was HA-tagged to facilitate identification with western blotting.</p

Tunicamycin or dithiothreitol treatment resulted in UPR activation but no NR4A3 increase in MIN6 cells.

<p>(A, B) Relative mRNA levels of NR4A3 and <i>Chop</i>, respectively, determined with real-time quantitative PCR in response to different doses of tunicamycin (TM) in MIN6 cells. (D, E) Relative mRNA levels of NR4A3 and <i>Chop</i>, respectively, determined with real-time quantitative PCR in response to different doses of dithiothreitol (DTT) in MIN6 cells. (C, F) Spliced XBP1 (sXBP1) mRNA formation in response to different (C) TM or (D) DTT doses, respectively. Two forms of XBP1 (a UPR molecule) was detected with reverse transcription PCR. Data are shown as mean ± S.E. (n = 4). * p<0.05, ** p<0.01 vs. con (vehicle control group).</p