The PtIV prodrug strategy has emerged as an excellent alternative to tackle the problems associated with conventional PtII drug therapy. However, there is a lack of tools to study how this new class of PtIV drugs are processed at the cellular level. Herein, we report the first ratiometric probe for cisplatin detection and use it to investigate PtIV anticancer complexes in biological systems. The probe was able to distinguish between cisplatin and its PtIV derivatives, allowing us to probe the intracellular reduction of PtIV prodrug complexes. The correlation between the amount of active PtII species available after intracellular reduction of PtIV complexes and their cytotoxicity and the role glutathione plays in the reduction of PtIV complexes were investigated.
Highly cytotoxic AuI-dithiocarbamate complexes were designed to induce severe integrative stress in ovarian cancer cells, leading to the surface exposure of calreticulin, which is a first step in the activation of immune system.
Toll-like receptor 10 (TLR10) is the only orphan receptor whose natural ligand and function are unknown among the 10 human TLRs. In this study, to test whether TLR10 recognizes some known TLR ligands, we established a stable TLR10 knockdown human monocytic cell line THP-1 using TLR10 short hairpin RNA lentiviral particle and puromycin selection. Among 60 TLR10 knockdown clones that were derived from each single transduced cell, six clones were randomly selected, and then one of those clones, named E7, was chosen for the functional study. E7 exhibited approximately 50% inhibition of TLR10 mRNA and protein expression. Of all the TLRs, only the expression of TLR10 changed significantly in this cell line. Additionally, phorbol 12-myristate 13-acetate-induced macrophage differentiation of TLR10 knockdown cells was not affected in the knockdown cells. When exposed to TLR ligands, such as synthetic diacylated lipoprotein (FSL-1), lipopolysaccharide (LPS), and flagellin, significant induction of proinflammatory cytokine gene expression including Interleukin-8 (IL-8), Interleukin-1 beta (IL-1β), Tumor necrosis factor-alpha (TNF-α) and Chemokine (C–C Motif) Ligand 20 (CCL20) expression, was found in the control THP-1 cells, whereas the TLR10 knockdown cells exhibited a significant reduction in the expression of IL-8, IL-1β, and CCL20. TNF-α was the only cytokine for which the expression did not decrease in the TLR10 knockdown cells from that measured in the control cells. Analysis of putative binding sites for transcription factors using a binding-site-prediction program revealed that the TNF-α promoter does not have putative binding sites for AP-1 or c-Jun, comprising a major transcription factor along with NF-κB for TLR signaling. Our results suggest that TLR10 is involved in the recognition of FSL-1, LPS, and flagellin and TLR-ligand-induced expression of TNF-α does not depend on TLR10.
Mitochondria have emerged as important targets for cisplatin in cancer therapy. Apart from cisplatin, anticancer Pt complexes based on similar scaffolds have also been developed to target mitochondria. Yet cellular processing of cisplatin or these mitochondria‐targeting Pt analogues remained unexplored, largely due to a lack of tools capable of probing these Pt drugs within an intracellular environment. We developed the first mitochondria‐targeted fluorescent probe for real‐time monitoring of Pt accumulation in mitochondria. We applied the probe to investigate mitochondria as cellular targets for Pt drug complexes and uncovered two distinct pathways whereby these Pt complexes could be delivered to mitochondria after cell entry.
The PtIV prodrug strategy has emerged as an excellent alternative to tackle the problems associated with conventional PtII drug therapy. However, there is a lack of tools to study how this new class of PtIV drugs are processed at the cellular level. Herein, we report the first ratiometric probe for cisplatin detection and use it to investigate PtIV anticancer complexes in biological systems. The probe was able to distinguish between cisplatin and its PtIV derivatives, allowing us to probe the intracellular reduction of PtIV prodrug complexes. The correlation between the amount of active PtII species available after intracellular reduction of PtIV complexes and their cytotoxicity and the role glutathione plays in the reduction of PtIV complexes were investigated.
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