Triptolide induces cytosolic translocation of lysosomal hydrolases and mitochondrial permeabilization in MCF-7 cells

Triptolide is a Chinese herb that has been shown to induce apoptosis in various tumor cells. We have previously demonstrated that triptolide induces lysosomal-mediated apoptosis in MCF-7 breast cancer cells. These findings are significant because MCF-7 cells lack caspase-3, a key executioner caspase, causing them to be resistant to chemotherapeutics. In the present study, we examine whether triptolide can induce apoptosis by targeting lysosomes and mitochondria. The effects of triptolide on lysosomal membrane integrity, subcellular localization of cathepsin B, mitochondrial localization, and mitochondrial membrane permeabilization in MCF-7 cells were assessed via fluorescence microscopy. Acridine orange staining demonstrated that triptolide caused rupture of lysosomal membranes. This effect on disruption of the lysosomal membrane was confirmed by immunofluorescent detection of cathepsin B in the cytosol. MitoTracker Green staining revealed mitochondria limited to the cytosol in control cells while mitochondria were observed in nuclear regions in experimental cells. Triptolide caused depolarization of the mitochondrial membrane, as assessed by JC-1 staining. Taken together, our results demonstrate for the first time in MCF-7 cells that triptolide induces apoptosis by lysosomal- and mitochondrial-dependent pathways. Our study provides a  mechanism that may be used to develop novel breast cancer therapies wherein triptolide sensitizes resistant breast cancer cells to cell death

Relevance of iPSC-derived human PGC-like cells at the surface of embryoid bodies to prechemotaxis migrating PGCs

Pluripotent stem cell-derived human primordial germ cell-like cells (hPGCLCs) provide important opportunities to study primordial germ cells (PGCs). We robustly produced CD38+ hPGCLCs [∼43% of FACS-sorted embryoid body (EB) cells] from primed-state induced pluripotent stem cells (iPSCs) after a 72-hour transient incubation in the four chemical inhibitors (4i)-naïve reprogramming medium and showed transcriptional consistency of our hPGCLCs with hPGCLCs generated in previous studies using various and distinct protocols. Both CD38+ hPGCLCs and CD38− EB cells significantly expressed PRDM1 and TFAP2C, although PRDM1 mRNA in CD38− cells lacked the 3′-UTR harboring miRNA binding sites regulating mRNA stability. Genes up-regulated in hPGCLCs were enriched for cell migration genes, and their promoters were enriched for the binding motifs of TFAP2 (which was identified in promoters of T, NANOS3, and SOX17) and the RREB-1 cell adhesion regulator. In EBs, hPGCLCs were identified exclusively in the outermost surface monolayer as dispersed cells or cell aggregates with strong and specific expression of POU5F1/OCT4 protein. Time-lapse live cell imaging revealed active migration of hPGCLCs on Matrigel. Whereas all hPGCLCs strongly expressed the CXCR4 chemotaxis receptor, its ligand CXCL12/SDF1 was not significantly expressed in the whole EBs. Exposure of hPGCLCs to CXCL12/SDF1 induced cell migration genes and antiapoptosis genes. Thus, our study shows that transcriptionally consistent hPGCLCs can be readily produced from hiPSCs after transition of their pluripotency from the primed state using various methods and that hPGCLCs resemble the early-stage PGCs randomly migrating in the midline region of human embryos before initiation of the CXCL12/SDF1-guided chemotaxis

Triptolide Induces Lysosomal-Mediated Programmed Cell Death in MCF-7 Breast Cancer Cells

Background: Breast cancer is a major cause of death; in fact, it is the most common type, in order of the number of global deaths, of cancer in women worldwide. This research seeks to investigate how triptolide, an extract from the Chinese herb Tripterygium wilfordii Hook F, induces apoptosis in MCF-7 human breast cancer cells. Accumulating evidence suggests a role for lysosomal proteases in the activation of apoptosis. However, there is also some controversy regarding the direct participation of lysosomal proteases in activation of key apoptosis-related caspases and release of mitochondrial cytochrome c. In the present study, we demonstrate that triptolide induces an atypical, lysosomal-mediated apoptotic cell death in MCF-7 cells because they lack caspase-3. Methods: MCF-7 cell death was characterized via cellular morphology, chromatin condensation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric cell growth inhibition assay and the expression levels of proapoptotic proteins. Acridine orange and LysoTracker® staining were performed to visualize lysosomes. Lysosomal enzymatic activity was monitored using an acid phosphatase assay and western blotting of cathepsin B protein levels in the cytosolic fraction, which showed increased enzymatic activity in drug-treated cells. Results: These experiments suggest that triptolide-treated MCF-7 cells undergo atypical apoptosis and that, during the early stages, lysosomal enzymes leak into the cytosol, indicating lysosomal membrane permeability. Conclusion: Our results suggest that further studies are warranted to investigate triptolide\u27s potential as an anticancer therapeutic agent

Technical adequacy of bisulfite sequencing and pyrosequencing for detection of mitochondrial DNA methylation: Sources and avoidance of false-positive detection

The existence of cytosine methylation in mammalian mitochondrial DNA (mtDNA) is a controversial subject. Because detection of DNA methylation depends on resistance of 5’-modified cytosines to bisulfite-catalyzed conversion to uracil, examined parameters that affect technical adequacy of mtDNA methylation analysis. Negative control amplicons (NCAs) devoid of cytosine methylation were amplified to cover the entire human or mouse mtDNA by long-range PCR. When the pyrosequencing template amplicons were gel-purified after bisulfite conversion, bisulfite pyrosequencing of NCAs did not detect significant levels of bisulfite-resistant cytosines (brCs) at ND1 (7 CpG sites) or CYTB (8 CpG sites) genes (CI95 = 0%-0.94%); without gel-purification, significant false-positive brCs were detected from NCAs (CI95 = 4.2%-6.8%). Bisulfite pyrosequencing of highly purified, linearized mtDNA isolated from human iPS cells or mouse liver detected significant brCs (~30%) in human ND1 gene when the sequencing primer was not selective in bisulfite-converted and unconverted templates. However, repeated experiments using a sequencing primer selective in bisulfite-converted templates almost completely (< 0.8%) suppressed brC detection, supporting the false-positive nature of brCs detected using the non-selective primer. Bisulfite-seq deep sequencing of linearized, gel-purified human mtDNA detected 9.4%-14.8% brCs for 9 CpG sites in ND1 gene. However, because all these brCs were associated with adjacent non-CpG brCs showing the same degrees of bisulfite resistance, DNA methylation in this mtDNA-encoded gene was not confirmed. Without linearization, data generated by bisulfite pyrosequencing or deep sequencing of purified mtDNA templates did not pass the quality control criteria. Shotgun bisulfite sequencing of human mtDNA detected extremely low levels of CpG methylation (<0.65%) over non-CpG methylation (<0.55%). Taken together, our study demonstrates that adequacy of mtDNA methylation analysis using methods dependent on bisulfite conversion needs to be established for each experiment, taking effects of incomplete bisulfite conversion and template impurity or topology into consideration

Shotgun bisulfite sequencing of human iPSC mtDNA.

<p>(A) Ratios of copy numbers of human mtDNA and nuclear DNA were determined by qPCR of mtDNA and nuclear DNA marker genes (Mean±SD, triplicated assays). Numbers show fold enrichment compared to total DNA. (B) CpG methylation of mtDNA-encoded genes (box plot; boxes and whiskers indicate quartiles and minimum/maximum values, horizontal lines in boxes indicate median). Numbers at the top indicate <i>p</i>-values of 2-tailed t-test against unmethylated lambda DNA. (C, D) Cytosine methylation of mtDNA (C) and unmethylated lambda DNA (D). Percentage of cytosine methylation in the CpG and non-CpG contexts is shown with red and blue dots, respectively. Deep sequencing read coverage at cytosines is shown with green dots. In panel (C), locations of mtDNA-encoded genes and D-loop are indicated at the top, where positions of tRNA genes are shown with vertical bars without gene names.</p

Enrichment of human and mouse mtDNA over nuclear DNA.

<p>Ratios of copy numbers of human (A) or mouse (B) mtDNA and nuclear DNA were determined by qPCR of mtDNA and nuclear DNA marker genes (Mean±SD, triplicated assays). Numbers show fold enrichment compared to total DNA.</p

Detection of bisulfite-resistant cytosines in purified, linearized human mtDNA by bisulfite pyrosequencing using converted template-selective (A9515) and unselective (hND1) sequencing primers.

<p>Ratios of brCs were determined by bisulfite pyrosequencing (Mean±SD, triplicated assays). (A) The A9515 sequencing primer, which was highly selective to bisulfite-converted DNA, interrogated three CpG sites (CpG #3–5) whereas non-selective sequencing primer hND1 interrogated all these CpG sites plus two additional CpG sites (CpG #1 and 2). (B) Positive control assay was performed using <i>in vitro</i> partially methylated NCAs templates. High CpG methylation levels at three CpG sites (CpG #3–5) were detected using A9515 sequencing primer (CpG sites #1 and #2 were out of the assay coverage using this sequencing primer). hND1 sequencing primer detected high CpG methylation at all five CpG sites (CpG #1–5).</p

Published studies on methylation of mammalian mtDNA determined using bisulfite pyrosequencing or deep sequencing.

<p>Published studies on methylation of mammalian mtDNA determined using bisulfite pyrosequencing or deep sequencing.</p

False-positive detection of bisulfite-resistant cytosines (brCs) in the negative control amplicons (NCAs) of mtDNA by bisulfite pyrosequencing.

<p>BrCs were detected in gel-purified or unpurified, bisulfite-converted PCR amplicons by pyrosequencing for 15 CpG sites: 5 in human ND1, 5 in human CYTB, 2 in mouse ND1, and 3 in mouse CYTB. The <i>p</i>-value was calculated using paired t-test (two-tails). The lines show mean ± 95% Confidence Intervals.</p

Targeted bisulfite deep sequencing of purified, linearized human mtDNA using hND1 primer.

<p>(A) Methylation of nine CpG sites in the mitochondrial ND1 gene determined based on the C/T SNP ratio. CpG sites #1 - #5 (shown in bold) correspond to the same names of CpG sites shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192722#pone.0192722.g004" target="_blank">Fig 4</a>. (B) Deep sequencing coverage tracks showing C/T SNPs at CpG sites #0 - #8 indicated in panel A. CpG and non-CpG cytosines are indicated by red and blue arrows, respectively. Pileup coverages of C/T SNPs are indicated with brown C (unconverted) and green T (converted) bars, respectively.</p