Inducing apoptosis using chemical treatment and acidic pH, and detecting it using the Annexin V flow cytometric assay v1

Cell death is a key component of mammalian physiology, and can happen as a result of structural damage, or actively as a sequence of programmed cellular processes known as apoptosis. Pathogenic alterations in apoptosis occur in a number of diseases, including cancer, viral infections, autoimmune diseases, immunodeficiencies and degenerative conditions. Developing accurate and reproducible laboratory methods for inducing and detecting apoptosis is vital for research into these conditions. A number of methods are employed to detect cell death, including DNA fragmentation, the TUNEL assay, and electron microscopy although each has its limitations. Flow cytometry allows for the distinction between live, early apoptotic, late apoptotic and necrotic cells. In this protocol we successfully induce apoptosis using chemical treatment and treatment with low pH in solid tumour cell lines, and have optimized detection using the Annexin V apoptosis assay.

Reduction of Rapid Proliferating Tumour Cell Lines by Inhibition of the Specific Glycine Transporter GLYT1

Studies have highlighted the relevance of extracellular glycine and serine in supporting high growth rates of rapidly proliferating tumours. The present study analysed the role of the specific glycine transporter GLYT1 in supplying glycine to cancer cells and maintaining cell proliferation. GLYT1 knockdown in the rapidly proliferating tumour cell lines A549 and HT29 reduced the number of viable cells by approximately 30% and the replication rate presented a decrease of about 50% when compared to cells transfected with control siRNA. In contrast, when compared to control, GLYT1 siRNA had only a minimal effect on cell number of the slowly proliferating tumour cell line A498, reducing the number of viable cells by 7% and no significant difference was observed when analysing the replication rate between GLYT1 knockdown and control group. When utilising a specific GLYT1 inhibitor, ALX-5407, the doubling time of rapidly proliferating cells increased by about 8 h presenting a significant reduction in the number of viable cells after 96 h treatment when compared to untreated cells. Therefore, these results suggest that GLYT1 is required to maintain high proliferation rates in rapidly proliferating cancer cells and encourage further investigation of GLYT1 as a possible target in a novel therapeutic approach.

Synthesis and Biological Application of Isosteviol-Based 1,3-Aminoalcohols

Starting from isosteviol, a series of diterpenoid 1,3-aminoalcohol derivatives were stereoselectively synthesised. The acid-catalysed hydrolysis and rearrangement of natural stevioside gave isosteviol, which was transformed to the key intermediate methyl ester. In the next step, Mannich condensation of diterpenoid ketone, paraformaldehyde, and secondary amines resulted in the formation of 1,3-aminoketones with different stereoselectivities. During the Mannich condensation with dibenzylamine, an interesting N-benzyl → N-methyl substituent exchange was observed. Reduction of 1,3-aminoketones produced diastereoisomeric 1,3-aminoalcohols. Alternatively, aminoalcohols were obtained via stereoselective hydroxy-formylation, followed by oxime preparation, reduction, and finally, reductive alkylation of the obtained primary aminoalcohols. An alternative 1,3-aminoalcohol library was prepared by reductive amination of the intermediate 3-hydroxyaldehyde obtained from isosteviol in two-step synthesis. Cytotoxic activity of compounds against human tumour cell lines (A2780, SiHa, HeLa, MCF-7 and MDA-MB-231) was investigated. In our preliminary study, the 1,3-aminoalcohol function and N-benzyl substitution seemed to be essential for the reliable antiproliferative activity. To extend their application, a diterpenoid condensed with 2-phenylimino-1,3-thiazine and -1,3-oxazine was also attempted to prepare, but only formation of thioether intermediate was observed.

Effect of SFX-01 on proliferation of human glioblastoma cell lines

Glioblastoma (GBM) is the most aggressive and lethal of brain tumours and there is a clear need for novel therapies. SFX-01, a stabilised formu of sulforaphane (SFN) that is complexed with α-cyclodextrin, is being investigated as a potential anticancer drug. And as part of this investigation we have determined its effect on the proliferation of a series of tumour cell lines. Growth-inhibitory concentrations (IC50 values) of SFX-01 and reference SFN were highly correlated but showed a divergence at higher drug concentrations that is likely to reflect drug binding to α-cyclodextrin complexes. Because SFN is redox-sensitive, we also compared the effects of these drugs on four GBM lines that had been derived and cultured under physiological oxygen (5%) conditions. Comparisons were also made using a three-dimensional spheroid model, used to mimic the in vivo state of glioblastomas and to simulate barriers to drug delivery in vivo. SFX-01 and reference SFN and inhibited proliferation of four GBM cell lines in monolayers as well as in spheroids; α-cyclodextrin alone had no significant effect. Our results are consistent with the hypothesis that SFN is liberated from SFX-01 at concentrations sufficient to inhibit cell growth. We envision that SFX-01 will have improved pharmacokinetic properties in vivo, warranting further pre-clinical investigation and clinical development.

Nanobody-armed T cells endow CAR-T cells with cytotoxicity against lymphoma cells

Background Taking advantage of nanobodies (Nbs) in immunotherapy, we investigated the cytotoxicity of Nb-based chimeric antigen receptor T cells (Nb CAR-T) against lymphoma cells. Methods CD19 Nb CAR-T, CD20 Nb CAR-T, and Bispecific Nb CAR-T cells were generated by panning anti-human CD19- and CD20-specific nanobody sequences from a natural Nb-expressing phage display library, integrating Nb genes with a lentiviral cassette that included other CAR elements, and finally transducing T cells that were expanded under an optimization system with the above generated CAR lentivirus. Prepared Nb CAR-T cells were cocultured with tumour cell lines or primary tumour cells for 24 h or 5 days to evaluate their biological function. Results The nanobodies that we selected from the natural Nb-expressing phage display library had a high affinity and specificity for CD19 and CD20. CD19 Nb CAR-T, CD20 Nb CAR-T and Bispecific Nb CAR-T cells were successfully constructed, and these Nb CAR-T cells could strongly recognize Burkitt lymphoma cell lines (Raji and Daudi), thereby leading to activation, enhanced proliferation, and specific killing of target cells. Furthermore, similar results were obtained when using patient samples as target cells, with a cytotoxicity of approximately 60%. Conclusions Nanobody-based CAR-T cells can kill both tumour cell lines and patient-derived tumour cells in vitro, and Nb-based CAR-T cells may be a promising therapeutic strategy in future immunotherapy.

Interrogation of cancer gene dependencies reveals novel paralog interactions of autosome and sex chromosome encoded genes

Genetic networks are characterized by extensive buffering. During tumour evolution, disruption of these functional redundancies can create de novo vulnerabilities that are specific to cancer cells. In this regard, paralog genes are of particular interest, as the loss of one paralog gene can render tumour cells dependent on a remaining paralog. To systematically identify cancer-relevant paralog dependencies, we searched for candidate dependencies using CRISPR screens and publicly available loss-of-function datasets. Our analysis revealed >2,000 potential candidate dependencies, several of which were subsequently experimentally validated. We provide evidence that DNAJC15-DNAJC19, FAM50A-FAM50B and RPP25-RPP25L are novel cancer relevant paralog dependencies. Importantly, our analysis also revealed unexpected redundancies between sex chromosome genes. We show that chrX- and chrY- encoded paralogs, as exemplified by ZFX-ZFY, DDX3X-DDX3Y and EIF1AX-EIF1AY, are functionally linked so that tumour cell lines from male patients with Y-chromosome loss become exquisitely dependent on the chrX-encoded gene. We therefore propose genetic redundancies between chrX- and chrY- encoded paralogs as a general therapeutic strategy for human tumours that have lost the Y-chromosome.