Broad in vitro efficacy of plant-derived betulinic acid against cell lines derived from the most prevalent human cancer types

Betulinic acid (BA) is a widely available plant-derived triterpene with reported activity against cancer cells of neuroectodermal origin and leukaemias. Treatment with BA was shown to protect mice against transplanted human melanoma and led to tumor regression. In contrast, cells from healthy tissues were resistant to BA and toxic side-effects in animals were absent. These findings have raised interest in the chemotherapeutical anti-cancer potential of BA. A comprehensive assessment of the efficacy of BA against the clinically most important cancer types is currently lacking. Therefore, we tested the in vitro sensitivity of broad cell line panels derived from lung, colorectal, breast, prostate and cervical cancer, which are the prevalent cancer types characterized with highest mortalities in woman and men. Multiple assays were used in order to allow a reliable assessment of anti-cancer efficacy of BA. After 48 h of treatment with BA, cell viability as assessed with MTT and cell death as measured with propidium iodide exclusion showed clear differences in sensitivity between cell lines. However, in all cell lines tested colony formation was completely halted at remarkably equal BA concentrations that are likely attainable in vivo. Our results substantiate the possible application of BA as a chemotherapeutic agent for the most prevalent human cancer type

SPI-CI and SPI-6 cooperate in the protection from effector cell-mediated cytotoxicity

Tumors have several mechanisms to escape from the immune system. One of these involves expression of intracellular anticytotoxic proteins that modulate the execution of cell death. Previously, we have shown that the serine protease inhibitor (serpin) SPI-6, which inactivates the cytotoxic protease granzyme B (GrB), is capable of preventing cytotoxic T lymphocyte (CTL)-mediated apoptosis. Despite its potent antiapoptotic activity, SPI-6 does not prevent membranolysis induced by cytotoxic lymphocytes. We now provide evidence that several colon carcinoma cell lines do resist membranolysis and that this protection is dependent on SPI-6 but also requires expression of a closely related serpin called SPI-CI (serine protease inhibitor involved in cytotoxicity inhibition). Expression of SPI-CI is absent from normal colon but observed in placenta, testis, early during embryogenesis, and in cytotoxic lymphocytes. SPI-CI encodes a chymotrypsin-specific inhibitor and irreversibly interacts with purified granzyme M. Moreover, SPI-CI can protect cells from purified perforin/GrM-induced lysis. Our data therefore indicate that SPI-CI is a novel immune escape molecule that acts in concert with SPI-6 to prevent cytotoxic lymphocyte-mediated killing of tumor cell

Rapid Generation of Marker-Free <i>P</i>. <i>falciparum</i> Fluorescent Reporter Lines Using Modified CRISPR/Cas9 Constructs and Selection Protocol

<div><p>The CRISPR/Cas9 system is a powerful genome editing technique employed in a wide variety of organisms including recently the human malaria parasite, <i>P</i>. <i>falciparum</i>. Here we report on further improvements to the CRISPR/Cas9 transfection constructs and selection protocol to more rapidly modify the <i>P</i>. <i>falciparum</i> genome and to introduce transgenes into the parasite genome without the inclusion of drug-selectable marker genes. This method was used to stably integrate the gene encoding GFP into the <i>P</i>. <i>falciparum</i> genome under the control of promoters of three different <i>Plasmodium</i> genes (<i>calmodulin</i>, <i>gapdh</i> and <i>hsp70</i>). These genes were selected as they are highly transcribed in blood stages. We show that the three reporter parasite lines generated in this study (GFP@<i>cam</i>, GFP@<i>gapdh</i> and GFP@<i>hsp70</i>) have <i>in vitro</i> blood stage growth kinetics and drug-sensitivity profiles comparable to the parental <i>P</i>. <i>falciparum</i> (NF54) wild-type line. Both asexual and sexual blood stages of the three reporter lines expressed GFP-fluorescence with GFP@<i>hsp70</i> having the highest fluorescent intensity in schizont stages as shown by flow cytometry analysis of GFP-fluorescence intensity. The improved CRISPR/Cas9 constructs/protocol will aid in the rapid generation of transgenic and modified <i>P</i>. <i>falciparum</i> parasites, including those expressing different reporters proteins under different (stage specific) promoters.</p></div

Drug-sensitivity and growth rate of asexual blood stages of three <i>P</i>. <i>falciparum</i> reporter lines (GFP@<i>cam</i>, GFP@<i>gapdh</i>, GFP@<i>hsp70</i>).

<p>A. Sensitivity to the drugs BSD and WR99210 as determined by flow cytometry in standard 72 h cultures in 96 well plates. Cultures of infected red blood cells (RBC) were incubated with different drug concentrations (in triplicate) and after 72 h samples were stained with the DNA-specific dye, Hoechst 33258, to determine parasitemia (% of infected RBC) by flow cytometry. Dot plots are shown of uninfected RBC (control, upper panel) selected using Forward Scatter parameter (FSC-A) and from cultures with the lowest and highest drug concentration (G1: infected RBC). Parasite survival is defined as the percentage of infected RBC in drug-treated wells divided by the percentage of infected RBC in non-treated wells multiplied by 100. IC₅₀ values WR99210 (nM): (NF54 <i>Pf</i>WT) 0.16; (GFP@<i>cam</i>) 0.25; (GFP@<i>gapdh</i>) 0.27; (GFP@<i>hsp70</i>) 0.19. IC₅₀ values BSD (μg/ml): (NF54 <i>Pf</i>WT) 0.48; (GFP@<i>cam</i>) 0.34; (GFP@<i>gapdh</i>) 0.54; (GFP@<i>hsp70</i>) 0.48. B. The growth rate of asexual blood stages in cultures maintained in the semi-automated culture system for a period of 5 days. Cultures were initiated with a parasitemia of 0.5%.</p

GFP-expression in blood stages of three reporter <i>P</i>. <i>falciparum</i> parasite lines (GFP@<i>cam</i>, GFP@<i>gapdh</i>, GFP@<i>hsp70</i>).

<p>A. Fluorescence microscopy of different blood stages. R: rings; T: trophozoites; ES: early schizonts; LS: late schizonts; G: gametocytes. Nuclei were stained with the DNA-specific dye Hoechst 33342. All pictures were recorded with standardized exposure/gain times to visualize differences in fluorescence intensity (GFP 0.7 s; Hoechst 0.136 s; bright field 0.62 s (1x gain)). In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168362#pone.0168362.s003" target="_blank">S3</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168362#pone.0168362.s005" target="_blank">S5</a> Figs. the complete set of microscope images are shown. B. Fluorescence intensity of rings (16 h), (late) trophozoites (30 h) and schizonts (42 h) as determined by flow cytometry. Infected red blood cells (RBC) were stained with the DNA-specific dye Hoechst 33258 to distinguish infected RBC from uninfected RBC and rings (Gate G1), trophozoites (Gate G2) from schizonts (Gate G3). Left side panels show dot plots of both Hoechst fluorescence intensity (DNA content) and GFP fluorescence intensity. Right side panels show GFP fluorescence intensity from parasites with either G1 and G2 (black) gate or G3 (red). MFI: mean fluorescence intensity and the black- (rings and trophozoites) and red- (schizonts) bar show the region selected to calculate the MFI.</p

Schematic representation of improved CRISPR/Cas9 plasmids and selection protocol.

<p>Parasites are transfected with two plasmids (Cas9 construct and sgRNA/donor construct). The Cas9 construct contains the <i>bsd</i> selectable marker. The sgRNA/donor construct contains a fusion of the positive selectable marker h<i>dhfr</i> and the negative selectable marker y<i>fcu</i> genes and two homology regions (HR1 and HR2) that target a gene of interest (GOI) and introduce the donor DNA by homologous recombination. Double positive selection using both BSD and WR99210 is applied from day (d) 1 resulting in the selection of parasites that contain both plasmids within a period of 3 weeks (w). After positive selection, cultures are maintained 2–4 days without drug before negative selection is applied using 5-FC to select parasites free of episomal plasmid DNA. Parasites are genotyped by diagnostic PCR for integration of the donor DNA followed by cloning of the parasites by limiting dilution (w6). Clones are genotyped for the correct genotype by diagnostic PCR and Southern analysis. This transfection and selection protocol can result in the generation of cloned mutant parasites within a period of 11 weeks.</p

Generation of three <i>P</i>. <i>falciparum</i> reporter lines (GFP@<i>cam</i>, GFP@<i>hsp70</i>, GFP@<i>gapdh</i>) expressing GFP under control of different promoters.

<p>A. Schematic of the different sgRNA/donor constructs generated to introduce the GFP expression cassettes into the <i>P</i>. <i>falciparum</i> (<i>Pf</i>) <i>230p</i> gene locus. <i>Pf230p</i> homology regions (HR1, HR2) used to introduce the donor DNA (i.e. <i>gfp</i> expression cassettes), location of primers (p) and sizes of restriction fragments (S: <i>Spe</i>I, X: <i>Xho</i>I; in red) and PCR amplicons (in black) are indicated. Primer sequences (shown in black and bold) are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168362#pone.0168362.s006" target="_blank">S1 Table</a>. Note that the GFP expression cassette from GFP@<i>cam</i> and GFP@<i>gapdh</i> was cloned in the same orientation whereas that the GFP expression cassette form GFP@<i>hsp70</i> was cloned in the reverse orientation. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168362#pone.0168362.g001" target="_blank">Fig 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168362#pone.0168362.s001" target="_blank">S1 Fig</a>. for details of the drug selectable marker and sgRNA sequences. This Figure is not shown to scale. B. Diagnostic (first 3 lanes) and long-range (LR-) PCR confirming correct integration of the GFP-expression cassettes into the <i>Pf230p</i> locus. Integration PCR of cloned parasites of GFP@<i>cam</i> (clone 1; primers p23/p24; expected size: 2538bp), GFP@<i>hsp70</i> (clone 5; primers p25/p26; expected size: 1622bp) and GFP@<i>gapdh</i> (clone 7; primers p23/p24; expected size: 2538bp). LR-PCR: GFP@<i>cam</i> (primers p23/p28; expected size: 4522bp), GFP@<i>hsp70</i> (primers p23/28; expected size: 4861bp) and GFP@<i>gapdh</i> (primers p30/p26; expected size: 5095bp); size of expected products shown in black and in bold in Fig 2A. Control PCR: <i>P</i>. <i>falciparum lisp2</i> gene (primers p21/p22; expected size: 5383bp); GFP: <i>gfp</i> gene (primers p24/p27; expected size: 606bp). C. Diagnostic Southern analysis confirms correct integration of the GFP-expression cassettes in the cloned lines of GFP@<i>cam</i>, GFP@<i>hsp70</i> and GFP@<i>gapdh</i>. <i>P</i>. <i>falciparum</i> NF54 (wild type WT) DNA, transfected parasite DNA after positive and negative selection (Uncloned; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168362#pone.0168362.g001" target="_blank">Fig 1</a>) and DNA from the different cloned lines was digested with <i>Spe</i>I and/or <i>Xho</i>I. The digested DNA fragments hybridized to probes recognizing either HR1 (GFP@<i>hsp70;</i> expected size: 2604bp) or HR2 (GFP@<i>cam;</i> expected size: 3764bp and GFP@<i>gapdh;</i> expected size: 4821bp) of the <i>Pf230p</i> target locus. In red are indicated the clones that have the correct genotype; absence of both plasmid and WT DNA (clone 1 and 5 for <i>GFP@cam;</i> clone 4 and 5 for <i>GFP@hsp70;</i> and clone 4 and 7 for <i>GFP@gapdh)</i>. As controls sgRNA/donor plasmid (Plasmid) DNA was digested and hybridised with a probe recognizing <i>ampicillin</i> (amp) of the donor DNA plasmid; *indicates probe used.</p