mRNA transfection by a Xentry-protamine cell-penetrating peptide is enhanced by TLR antagonist E6446

<div><p>Messenger RNA (mRNA) transfection is a developing field that has applications in research and gene therapy. Potentially, mRNA transfection can be mediated efficiently by cell-penetrating peptides (CPPs) as they may be modified to target specific tissues. However, whilst CPPs are well-documented to transfect oligonucleotides and plasmids, mRNA transfection by CPPs has barely been explored. Here we report that peptides, including a truncated form of protamine and the same peptide fused to the CPP Xentry (Xentry-protamine; XP), can transfect mRNAs encoding reporter genes into human cells. Further, this transfection is enhanced by the anti-malarial chloroquine (CQ) and the toll-like receptor antagonist E6446 (6-[3-(pyrrolidin-1-yl)propoxy)-2-(4-(3-(pyrrolidin-1-yl)propoxy)phenyl]benzo[d]oxazole), with E6446 being >5-fold more potent than CQ at enhancing this transfection. Finally, E6446 facilitated the transfection by XP of mRNA encoding the cystic fibrosis transmembrane regulator, the protein mutated in cystic fibrosis. As such, these findings introduce E6446 as a novel transfection enhancer and may be of practical relevance to researchers seeking to improve the mRNA transfection efficiency of their preferred CPP.</p></div

mRNA transfection by protamine-containing CPPs is enhanced by certain concentrations of TLR antagonist E6446.

<p><b>(a)</b> Fluorescence imaging of RFP expression (red) in human gastric cancer (AGS) cells 24 h after treatment with RFP mRNA mixed with Xentry, truncated human protamine, or XP. <b>(b)</b> Fluorescence imaging of RFP expression (red) in AGS cells 24 h after treatment with RFP mRNA mixed with either truncated human protamine or XP. <b>(c, d)</b> Fluorescence imaging and quantification of EGFP expression (green) in human lung (A549), gastric (AGS), liver (HepG2), colon (HT-29), and breast (MCF7) cancer cell lines 24 h after treatment with EGFP mRNA mixed with XP and 0–40 μM E6446. Cell counts following transfection were also quantified <b>(e)</b>. Cell nuclei are stained blue with DAPI; scale bars = 100 μm. Data are shown as mean ± 95% confidence interval and are representative of 4+ independent experiments.</p

Comparing E6446 and CQ for their ability to enhance XP-mediated transfection of EGFP mRNA.

<p>Fluorescence imaging of EGFP expression (green) in A549, AGS, and HepG2 human cancer cell lines 24 h after treatment with EGFP mRNA mixed with XP, E6446 (0–20 μM), and CQ (0–100 μM). Quantified for each cell line and treatment condition are the percentages of cells expressing EGFP (<i>top graph in each panel</i>) and the cell counts relative to vehicle-treated controls (<i>bottom graph in each panel</i>). Data are shown as mean ± 95% confidence interval and are representative of 4+ independent experiments. Cell nuclei are stained blue with DAPI. Veh = vehicle; scale bars = 100 μm.</p

Liposome-enabled bufalin and doxorubicin combination therapy for trastuzumab-resistant breast cancer with a focus on cancer stem cells

Breast cancer stem cells (BCSCs) play a key role in therapeutic resistance in breast cancer treatments and disease recurrence. This study aimed to develop a combination therapy loaded with pH-sensitive liposomes to kill both BCSCs and the okbulk cancer cells using trastuzumab-sensitive and resistant human epidermal growth factor receptor 2 positive (HER2+) breast cancer cell models. The anti-BCSCs effect and cytotoxicity of all-trans retinoic acid, salinomycin, and bufalin alone or in combination with doxorubicin were compared in HER2+ cell line BT-474 and a validated trastuzumab-resistant cell line, BT-474R. The most potent anti-BCSC agent was selected and loaded into a pH-sensitive liposome system. The effects of the liposomal combination on BCSCs and bulk cancer cells were assessed. Compared with BT-474, the aldehyde dehydrogenase positive BCSC population was elevated in BT-474R (3.9 vs. 23.1%). Bufalin was the most potent agent and suppressed tumorigenesis of BCSCs by ∼50%, and showed strong synergism with doxorubicin in both BT-474 and BT-474R cell lines. The liposomal combination of bufalin and doxorubicin significantly reduced the BCSC population size by 85%, and inhibited both tumorigenesis and self-renewal, although it had little effect on the migration and invasiveness. The cytotoxicity against the bulk cancer cells was also enhanced by the liposomal combination than either formulation alone in both cell lines (p + breast cancer.</p

Additional file 8: Figure S7. of ZFAS1: a long noncoding RNA associated with ribosomes in breast cancer cells

Effect of cycloheximide, a translational inhibitor, on the abundance of ZFAS1 and GAS5. Relative expression of genes was measured by qPCR using total RNA extracted from each cell. 18S and 28S rRNA transcripts were used to normalise the expression of ZFAS1 and GAS5. The Y axis represents the fold change relative to time 0. The X axis shows treatment time. Error bars are SEM of three biological replicates, p values were calculated using Student’s t test. (PDF 136 kb

Additional file 14: Figure S11. of ZFAS1: a long noncoding RNA associated with ribosomes in breast cancer cells

The presence of ZFAS1, 18S and 28S expression were assessed by qPCR using fractions derived from dissociated ribosomes (Fig. 4i). Red bars show the samples derived from the peak of the graph in Fig. 4Ai for 40S and 60S subunit. (PDF 235 kb

Additional file 2: Figure S2. of ZFAS1: a long noncoding RNA associated with ribosomes in breast cancer cells

Analyses of ZFAS1 in breast cancer samples derived from TCGA. A(i) Expression of ZFAS1 in normal breast (n = 113) and breast cancer (n = 1069) samples. (ii) Expression of ZFAS1 by tumour subtype based on PAM50 classification. ZFAS1 is more highly expressed in normal tissues compared to basal and HER2 breast cancer subtypes. (iii) Expression of ZFAS1 in ER+ (n = 601) and ER- (n = 179) breast cancer samples. Unpaired Student’s t-test showed that ZFAS1 was differentially expressed according to estrogen status. (B) Kaplan-Meier plot generated from http://www.oncolnc.org/ of TCGA breast cancer data set. High expressers are those 50 % of patients with the highest ZFAS1 expression, and low expressers are those 50 % of patients with the lowest ZFAS1 expression. High expressers of ZFAS1 do not show altered survival up to 6000 days. (C) Gene expression of candidate ribosomal proteins by tumour subtype based on PAM50 classification. Unpaired student’s t-test relative to normal tissue samples was used to calculate P values. (PDF 475 kb

Additional file 9: Table S2. of ZFAS1: a long noncoding RNA associated with ribosomes in breast cancer cells

List of primers used in these experiments. The sequences are 5′ to 3′. (DOC 43 kb

Additional file 7: Figure S6. of ZFAS1: a long noncoding RNA associated with ribosomes in breast cancer cells

Effect of puromycin, a translational inhibitor, on the abundance of ZFAS1 and GAS5. Relative expression of genes was measured by qPCR using total RNA extracted from each cell. 18S and 28S rRNA transcripts were used to normalise the expression of ZFAS1 and GAS5. The Y axis represents the fold change relative to time 0. The X axis shows treatment time. Error bars are SEM of three biological replicates, p values were calculated using Student’s t test. (PDF 172 kb

Additional file 13: Figure S10. of ZFAS1: a long noncoding RNA associated with ribosomes in breast cancer cells

Primer efficiency of ZFAS1 and ZNFX1. A) The primer efficiency test for the primers used in the experiments. B) Slopes of standard curve indicate PCR efficiency for ZFAS1 and ZNFX1 primers sets. The X axis represent the log of dilution. Y axis shows Ct values. C) Correlation between the expression of ZFAS1 and ZNFX1 in two different primer sets in panel of cell lines (PDF 317 kb