A H2AX–CARP-1 Interaction Regulates Apoptosis Signaling Following DNA Damage

Cell Cycle and Apoptosis Regulatory Protein (CARP-1/CCAR1) is a peri-nuclear phosphoprotein that regulates apoptosis via chemotherapeutic Adriamycin (doxorubicin) and a novel class of CARP-1 functional mimetic (CFM) compounds. Although Adriamycin causes DNA damage, data from Comet assays revealed that CFM-4.16 also induced DNA damage. Phosphorylation of histone 2AX (γH2AX) protein is involved in regulating DNA damage repair and apoptosis signaling. Adriamycin or CFM-4.16 treatments inhibited cell growth and caused elevated CARP-1 and γH2AX in human breast (HBC) and cervical cancer (HeLa) cells. In fact, a robust nuclear or peri-nuclear co-localization of CARP-1 and γH2AX occurred in cells undergoing apoptosis. Knock-down of CARP-1 diminished γH2AX, their co-localization, and apoptosis in CFM-4.16- or Adriamycin-treated cells. We found that CARP-1 directly binds with H2AX, and H2AX interacted with CARP-1, but not CARP-1 (Δ600–652) mutant. Moreover, cells expressing CARP-1 (Δ600–652) mutant were resistant to apoptosis, and had diminished levels of γH2AX, when compared with cells expressing wild-type CARP-1. Mutagenesis studies revealed that H2AX residues 1–35 harbored a CARP-1-binding epitope, while CARP-1 amino acids 636–650 contained an H2AX-interacting epitope. Surface plasmon resonance studies revealed that CARP-1 (636–650) peptide bound with H2AX (1–35) peptide with a dissociation constant (Kd) of 127 nM. Cells expressing enhanced GFP (EGFP)-tagged H2AX (1–35) peptide or EGFP-tagged CARP-1 (636–650) peptide were resistant to inhibition by Adriamycin or CFM-4.16. Treatment of cells with transactivator of transcription (TAT)-tagged CARP-1 (636–650) peptide resulted in a moderate, statistically significant abrogation of Adriamycin-induced growth inhibition of cancer cells. Our studies provide evidence for requirement of CARP-1 interaction with H2AX in apoptosis signaling by Adriamycin and CFM compounds

Efficient Drug Delivery of Paclitaxel Glycoside: A Novel Solubility Gradient Encapsulation into Liposomes Coupled with Immunoliposomes Preparation

Although the encapsulation of paclitaxel into liposomes has been extensively studied, its significant hydrophobic and uncharged character has generated substantial difficulties concerning its efficient encapsulation into the inner water core of liposomes. We found that a more hydrophilic paclitaxel molecule, 7-glucosyloxyacetylpaclitaxel, retained tubulin polymerization stabilization activity. The hydrophilic nature of 7-glucosyloxyacetylpaclitaxel allowed its efficient encapsulation into the inner water core of liposomes, which was successfully accomplished using a remote loading method with a solubility gradient between 40% ethylene glycol and Cremophor EL/ethanol in PBS. Trastuzumab was then conjugated onto the surface of liposomes as immunoliposomes to selectively target human epidermal growth factor receptor-2 (HER2)-overexpressing cancer cells. In vitro cytotoxicity assays revealed that the immunoliposomes enhanced the toxicity of 7-glucosyloxyacetylpaclitaxel in HER2-overexpressing cancer cells and showed more rapid suppression of cell growth. The immunoliposomes strongly inhibited the tumor growth of HT-29 cells xenografted in nude mice. Notably, mice survived when treated with the immunoliposomes formulation, even when administered at a lethal dose of 7-glucosyloxyacetylpaclitaxel in vivo. This data successfully demonstrates immunoliposomes as a promising candidate for the efficient delivery of paclitaxel glycoside

miR-181a initiates and perpetuates oncogenic transformation through the regulation of innate immune signaling

The majority of high grade serous ovarian cancers originate from fallopian tube secretory epithelial cells (FTSECs). Here the authors show that miR-181a drives oncogenic transformation in FTSECs through the cooperative inhibition of the tumor suppressor RB1 and of STING, resulting in genomic instability and suppression of intrinsic interferon signaling

<span style="font-variant: small-caps">A H2AX–CARP-1 </span>Interaction Regulates Apoptosis Signaling Following DNA Damage

Cell Cycle and Apoptosis Regulatory Protein (CARP-1/CCAR1) is a peri-nuclear phosphoprotein that regulates apoptosis via chemotherapeutic Adriamycin (doxorubicin) and a novel class of CARP-1 functional mimetic (CFM) compounds. Although Adriamycin causes DNA damage, data from Comet assays revealed that CFM-4.16 also induced DNA damage. Phosphorylation of histone 2AX (&#947;H2AX) protein is involved in regulating DNA damage repair and apoptosis signaling. Adriamycin or CFM-4.16 treatments inhibited cell growth and caused elevated CARP-1 and &#947;H2AX in human breast (HBC) and cervical cancer (HeLa) cells. In fact, a robust nuclear or peri-nuclear co-localization of CARP-1 and &#947;H2AX occurred in cells undergoing apoptosis. Knock-down of CARP-1 diminished &#947;H2AX, their co-localization, and apoptosis in CFM-4.16- or Adriamycin-treated cells. We found that CARP-1 directly binds with H2AX, and H2AX interacted with CARP-1, but not CARP-1 (&#916;600&#8315;652) mutant. Moreover, cells expressing CARP-1 (&#916;600&#8315;652) mutant were resistant to apoptosis, and had diminished levels of &#947;H2AX, when compared with cells expressing wild-type CARP-1. Mutagenesis studies revealed that H2AX residues 1&#8315;35 harbored a CARP-1-binding epitope, while CARP-1 amino acids 636&#8315;650 contained an H2AX-interacting epitope. Surface plasmon resonance studies revealed that CARP-1 (636&#8315;650) peptide bound with H2AX (1&#8315;35) peptide with a dissociation constant (Kd) of 127 nM. Cells expressing enhanced GFP (EGFP)-tagged H2AX (1&#8315;35) peptide or EGFP-tagged CARP-1 (636&#8315;650) peptide were resistant to inhibition by Adriamycin or CFM-4.16. Treatment of cells with transactivator of transcription (TAT)-tagged CARP-1 (636&#8315;650) peptide resulted in a moderate, statistically significant abrogation of Adriamycin-induced growth inhibition of cancer cells. Our studies provide evidence for requirement of CARP-1 interaction with H2AX in apoptosis signaling by Adriamycin and CFM compounds

Anticancer efficacy of different gPTX formulations with repeated administration in HT-29 cells tumor-bearing mice.

<p>gPTX-IL (open circle with line), gPTX-L (open triangle), gPTX-L with trastuzumab (open square), trastuzumab (closed square), CEP-IL (closed circle), CEP-L (closed triangle), or PBS (cross) was intravenously injected at day 0, 10, and 20. The dose of each administration was 150 mg/kg gPTX. A and B, Changes in tumor volume. C, Changes in body weight. D, Survival curves. Data are presented as the mean ± S.D. The changes of tumor volume and body weight in the mice administered with gPTX-L do not show S.D. after day 10. The <i>P</i> value shown compared with gPTX-IL and gPTX-L with trastuzumab treated group at day 43 (n = 4). *, <i>P</i><0.05.</p

Cytotoxicity of different gPTX formulations by the MTT assay.

<p>A, The IC₅₀ values after drug exposure for 72 h are shown. B, The IT₅₀ values at IC₁₀₀ are shown. The data are presented as the mean ± S.D. for three independent experiments. *, <i>P</i><0.05. **, <i>P</i><0.01. ***, <i>P</i><0.005. ****, <i>P</i><0.001. NSD, no significant difference.</p

Solubility of PTX and gPTX in different solvents.

<p>* <i>Solubility of H₂O was referred as described previously</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107976#pone.0107976-Mandai1" target="_blank">[19]</a>.</p><p>** <i>CEP consists of Cremophor EL and ethanol in PBS (12∶12∶76 volume %)</i>.</p><p>Solubility of PTX and gPTX in different solvents.</p

Schematic illustration of remote loading with a solubility gradient.

<p>CEP-encapsulated liposomes (CEP-L) and gPTX dissolved in 40% EG were mixed and incubated. gPTX was encapsulated into liposomes under a solubility gradient.</p