METTL16 promotes liver cancer stem cell self-renewal via controlling ribosome biogenesis and mRNA translation.

BACKGROUND: While liver cancer stem cells (CSCs) play a crucial role in hepatocellular carcinoma (HCC) initiation, progression, recurrence, and treatment resistance, the mechanism underlying liver CSC self-renewal remains elusive. We aim to characterize the role of Methyltransferase 16 (METTL16), a recently identified RNA N6-methyladenosine (m6A) methyltransferase, in HCC development/maintenance, CSC stemness, as well as normal hepatogenesis. METHODS: Liver-specific Mettl16 conditional KO (cKO) mice were generated to assess its role in HCC pathogenesis and normal hepatogenesis. Hydrodynamic tail-vein injection (HDTVi)-induced de novo hepatocarcinogenesis and xenograft models were utilized to determine the role of METTL16 in HCC initiation and progression. A limiting dilution assay was utilized to evaluate CSC frequency. Functionally essential targets were revealed via integrative analysis of multi-omics data, including RNA-seq, RNA immunoprecipitation (RIP)-seq, and ribosome profiling. RESULTS: METTL16 is highly expressed in liver CSCs and its depletion dramatically decreased CSC frequency in vitro and in vivo. Mettl16 KO significantly attenuated HCC initiation and progression, yet only slightly influenced normal hepatogenesis. Mechanistic studies, including high-throughput sequencing, unveiled METTL16 as a key regulator of ribosomal RNA (rRNA) maturation and mRNA translation and identified eukaryotic translation initiation factor 3 subunit a (eIF3a) transcript as a bona-fide target of METTL16 in HCC. In addition, the functionally essential regions of METTL16 were revealed by CRISPR gene tiling scan, which will pave the way for the development of potential inhibitor(s). CONCLUSIONS: Our findings highlight the crucial oncogenic role of METTL16 in promoting HCC pathogenesis and enhancing liver CSC self-renewal through augmenting mRNA translation efficiency.

Quantifying cell viability through organelle ratiometric probing.

Detecting cell viability is crucial in research involving the precancerous discovery of abnormal cells, the evaluation of treatments, and drug toxicity testing. Although conventional methods afford cumulative results regarding cell viability based on a great number of cells, they do not permit investigating cell viability at the single-cell level. In response, we rationally designed and synthesized a fluorescent probe, PCV-1, to visualize cell viability under the super-resolution technology of structured illumination microscopy. Given its sensitivity to mitochondrial membrane potential and affinity to DNA, PCV-1's ability to stain mitochondria and nucleoli was observed in live and dead cells, respectively. During cell injury induced by drug treatment, PCV-1's migration from mitochondria to the nucleolus was dynamically visualized at the single-cell level. By extension, harnessing PCV-1's excellent photostability and signal-to-noise ratio and by comparing the fluorescence intensity of the two organelles, mitochondria and nucleoli, we developed a powerful analytical assay named organelle ratiometric probing (ORP) that we applied to quantitatively analyze and efficiently assess the viability of individual cells, thereby enabling deeper insights into the potential mechanisms of cell death. In ORP analysis with PCV-1, we identified 0.3 as the cutoff point for assessing whether adding a given drug will cause apparent cytotoxicity, which greatly expands the probe's applicability. To the best of our knowledge, PCV-1 is the first probe to allow visualizing cell death and cell injury under super-resolution imaging, and our proposed analytical assay using it paves the way for quantifying cell viability at the single-cell level.

Quantifying cell viability through organelle ratiometric probing.

Detecting cell viability is crucial in research involving the precancerous discovery of abnormal cells, the evaluation of treatments, and drug toxicity testing. Although conventional methods afford cumulative results regarding cell viability based on a great number of cells, they do not permit investigating cell viability at the single-cell level. In response, we rationally designed and synthesized a fluorescent probe, PCV-1, to visualize cell viability under the super-resolution technology of structured illumination microscopy. Given its sensitivity to mitochondrial membrane potential and affinity to DNA, PCV-1's ability to stain mitochondria and nucleoli was observed in live and dead cells, respectively. During cell injury induced by drug treatment, PCV-1's migration from mitochondria to the nucleolus was dynamically visualized at the single-cell level. By extension, harnessing PCV-1's excellent photostability and signal-to-noise ratio and by comparing the fluorescence intensity of the two organelles, mitochondria and nucleoli, we developed a powerful analytical assay named organelle ratiometric probing (ORP) that we applied to quantitatively analyze and efficiently assess the viability of individual cells, thereby enabling deeper insights into the potential mechanisms of cell death. In ORP analysis with PCV-1, we identified 0.3 as the cutoff point for assessing whether adding a given drug will cause apparent cytotoxicity, which greatly expands the probe's applicability. To the best of our knowledge, PCV-1 is the first probe to allow visualizing cell death and cell injury under super-resolution imaging, and our proposed analytical assay using it paves the way for quantifying cell viability at the single-cell level.

Two-photon photodynamic ablation of tumour cells using an RGD peptide-conjugated ruthenium(ii) photosensitiser.

An RGD-peptide conjugated ruthenium(ii) complex has been developed, which functions as a two-photon absorption (TPA) photodynamic therapy (PDT) agent for ablating tumours by selectively targeting the mitochondria of integrin αvß3-rich tumour cells. This approach offers a new and effective design and application for tumour-targeting metallo-anticancer drugs via two-photon PDT.

Correction: Synthesis, characterization and anticancer mechanism studies of fluorinated cyclometalated ruthenium(ii) complexes.

Correction for 'Synthesis, characterization and anticancer mechanism studies of fluorinated cyclometalated ruthenium(ii) complexes' by Ya Wen et al., Dalton Trans., 2020, DOI: .

Synthesis, characterization and anticancer mechanism studies of fluorinated cyclometalated ruthenium(ii) complexes.

The drug-resistance of cancer cells has become a major obstacle to the development of clinical drugs for chemotherapy. In order to overcome cisplatin-resistance, seven cyclometalated ruthenium(ii) complexes were synthesized with a varying degree of fluorine substitution, for use as anticancer agents. A cytotoxicity assay testified that the complexes possessed a more cytotoxic effect than cisplatin towards the cisplatin-resistant cell line A549R. The number of fluorine atoms regulated the lipophilicity of the complexes, but the relationship was not linear. Ru1 containing one fluorine atom had the highest lipophilicity and the best therapeutic effect. The complexes enter cells through an energy-dependent pathway and then localize in the nuclei and mitochondria. The complexes induced nuclear dysfunction by the inhibition of DNA replication as well as mitochondrial dysfunction by the loss of membrane potential. The damage to these vital organelles leads to cell apoptosis via the caspase 3/7 pathway. Our results indicated that the modulation of the number of fluorine atoms in therapeutic agents can have a profound effect and Ru1 is a complex with a high potential as a drug for the treatment of cisplatin-resistant cancer.

Nucleus-targeting ultrasmall ruthenium(iv) oxide nanoparticles for photoacoustic imaging and low-temperature photothermal therapy in the NIR-II window.

Nucleus-targeting NPs based on RuO2 (RuO2NPs) were developed by controlling the size and the surface charge of nanoparticles (NPs). This study not only demonstrates a facile approach for the fabrication of ultrasmall CS-RuO2NPs with good biocompatibility and excellent photothermal properties but also their unique potential for the nucleus-targeted low-temperature PTT.

Targeted photoredox catalysis in cancer cells.

Hypoxic tumours are a major problem for cancer photodynamic therapy. Here, we show that photoredox catalysis can provide an oxygen-independent mechanism of action to combat this problem. We have designed a highly oxidative Ir(III) photocatalyst, [Ir(ttpy)(pq)Cl]PF6 ([1]PF6, where 'ttpy' represents 4'-(p-tolyl)-2,2':6',2''-terpyridine and 'pq' represents 3-phenylisoquinoline), which is phototoxic towards both normoxic and hypoxic cancer cells. Complex 1 photocatalytically oxidizes 1,4-dihydronicotinamide adenine dinucleotide (NADH)-an important coenzyme in living cells-generating NAD• radicals with a high turnover frequency in biological media. Moreover, complex 1 and NADH synergistically photoreduce cytochrome c under hypoxia. Density functional theory calculations reveal π stacking in adducts of complex 1 and NADH, facilitating photoinduced single-electron transfer. In cancer cells, complex 1 localizes in mitochondria and disrupts electron transport via NADH photocatalysis. On light irradiation, complex 1 induces NADH depletion, intracellular redox imbalance and immunogenic apoptotic cancer cell death. This photocatalytic redox imbalance strategy offers a new approach for efficient cancer phototherapy.

The stepwise photodamage of organelles by two-photon luminescent ruthenium(ii) photosensitizers.

Ru(ii) polypyridyl complexes, containing a morpholine moiety, and possessing two-photon absorption properties and pH dependent singlet oxygen production were used for stepwise lysosomes-to-mitochondria photodamage of cancer cells.

Mitochondria-targeted Ir@AuNRs as bifunctional therapeutic agents for hypoxia imaging and photothermal therapy.

Ir(iii) complex modified gold nanorods, Ir@AuNRs, were developed as mitochondria-targeted theranostic nanoagents. Their hypoxia imaging and photothermal therapeutic properties were demonstrated in vitro and in vivo.

Fabrication of red blood cell membrane-camouflaged Cu2-xSe nanoparticles for phototherapy in the second near-infrared window.

Cu2-xSe nanoparticles (Cu2-xSeNPs) were camouflaged with a red blood cell membrane (RBC) to create nanoparticles with improved biocompatibility, longer blood retention times, excellent absorption properties, superior photothermal conversion efficiency (67.2%) and singlet oxygen production capabilities for the synergistic photothermal and photodynamic therapy of cancer in the second near-infrared (NIR-II) window.

A viscosity-sensitive iridium(iii) probe for lysosomal microviscosity quantification and blood viscosity detection in diabetic mice.

Abnormal levels of biological viscosity are closely associated with some diseases and malfunction (e.g. cancer, diabetes). Sensitive viscosity probes are of significant importance in disease diagnosis and remain an unmet need. In this research, we present a novel viscosity-sensitive dinuclear Ir(iii) complex (1), which freely rotates and shows weak phosphorescence in a low-viscosity environment, while in viscous media, both the phosphorescence intensity and lifetime are enhanced significantly. 1 can further be applied for phosphorescence lifetime imaging (PLIM) and measure lysosomal microviscosity with high accuracy and reliability. Interestingly, this PLIM property can distinguish between tumorous and nontumorous cells. Importantly, the probe shows much stronger phosphorescence in the fresh blood of diabetic mice than that of normal mice. This work offers a potential efficient probe for diagnosing viscosity related diseases.

A mitochondria-targeting photothermogenic nanozyme for MRI-guided mild photothermal therapy.

Iridium complexes were used as a mitochondria-targeting agent to modify the surface of a photothermogenic nanozyme Fe3O4 nanoparticles (Ir@Fe3O4 NPs). Upon NIR irradiation, Ir@Fe3O4 NPs increase the localized temperature to 42 °C which accelerates the catalysis of ˙OH production from H2O2 resulting in excellent mild photothermal therapy.

Tracking mitochondrial dynamics during apoptosis with phosphorescent fluorinated iridium(iii) complexes.

Mitochondria are the control centers of apoptosis. To study mitochondrial dynamics during apoptosis, four phosphorescent fluorinated iridium(iii) complexes (Ir1-Ir4) were designed and synthesized. The complexes' emission maxima, phosphorescent quantum yields, and phosphorescent lifetimes are tuned by the degree of fluorination of the ligand. The complexes exhibit excellent photostability and low (photo)cytotoxicity in HeLa cells. As the complexes are cationic and lipophilic, they localize in mitochondria and enter cells through an energy-independent pathway. In comparison with commercially available mitochondrial trackers (e.g., MTR), Ir1-Ir4 exhibit high specificity to mitochondria even in fixed cells. Due to these outstanding properties, the complexes were successfully used to track mitochondrial dynamics during apoptosis.

A NIR phosphorescent osmium(ii) complex as a lysosome tracking reagent and photodynamic therapeutic agent.

A novel near infrared (NIR) phosphorescent osmium complex (Os1) was developed for lysosome tracking and photodynamic therapy. Owing to its NIR photophysical properties, cellular imaging ability and phototoxicity, it has advantages over its ruthenium analogue (Ru1).

Extending the Excitation Wavelength of Potential Photosensitizers via Appendage of a Kinetically Stable Terbium(III) Macrocyclic Complex for Applications in Photodynamic Therapy.

The development of viable photodynamic therapy protocols is often hindered by photosensitizers that require high-energy UV irradiation that has limited potential for clinical use due to its low tissue penetration. Herein, we report a strategy for extending the excitation wavelength of potential photosensitizers via the covalent attachment of a terbium(III)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetate complex (DO3A-Tb). The method was systematically demonstrated with a series of polycyclic aromatic hydrocarbons (naphthalene, phenanthrene, anthracene, pyrene, and fluoranthene) to prepare six new complexes (Tb1-Tb6) with bathochromic shifts that extended into the visible region. Determination of their quantum yields for singlet oxygen (1O2) production at 350 and 420 nm showed significant enhancements from the parent molecule in all cases. Cell viability studies on cervical cancer cells (HeLa) and noncancerous MRC-5 cells showed no measurable cytotoxicity for all complexes prior to light irradiation. However, after irradiation at 420 nm (20 min, 9.27 J cm-2), Tb3-Tb6 were phototoxic to HeLa cells with IC50 values between 14.3-32.3 µM. Cell morphological studies and fluorescence microscopy with live/dead cell stains confirmed these findings. In addition, these complexes were highly stable in human blood plasma, with no significant degradation observed after 96 h at 37 °C. This excellent phototoxicity profile and high stability in blood plasma, coupled with the moderately lipophilic nature of the complexes, favorably indicate the potential of DO3A-Tb as a heavy atom-bearing moiety for modification of potential photosensitizers into ideal phototherapeutic drug candidates with longer excitation wavelengths for in vivo application.

Crossfire for Two-Photon Photodynamic Therapy with Fluorinated Ruthenium (II) Photosensitizers.

Synergistic photodynamic therapy (PDT) that combines photosensitizers (PSs) to attack different key sites in cancer cells is very attractive. However, the use of multiple PSs may increase dark cytotoxicity. Additionally, realizing the multiple vein passage of several PSs through dosing could be a challenge in clinical treatment. To address these issues, a novel strategy that enables a single PS to ablate two key sites (i.e., cytomembranes on the outside and mitochondria on the inside) of cancer cells synergistically was proposed. Five new fluorinated ruthenium (II) complexes (Ru1-Ru5), which possessed excellent two-photon properties and good singlet oxygen quantum yields, were designed and synthesized. When incubated with HeLa cells, the complexes were observed on the cytomembranes at first. With an extension of the treatment time, both the cytomembranes and mitochondria were lit up by the complexes. Under two-photon laser irradiation, the mitochondria and cytomembranes were ablated simultaneously, and the HeLa cells were destroyed effectively by the complexes, whether the cells were in a monolayer or in multicellular spheroids. With the largest phototoxicity index under the two-photon laser, Ru4 was used for two-photon PDT of in vivo xenograft tumors and successfully inhibited the growth of the tumors. Our results emphasized that the strategy of attacking two key sites with a single PS is an efficient method for PDT.

Enhancing the photothermal stability and photothermal efficacy of AuNRs and AuNTs by grafting with Ru(ii) complexes.

Gold nanorods (AuNRs) and nanostars (AuNTs) were widely applied in photothermal cancer therapy recently. However, due to the photothermal effect, naked AuNRs and AuNTs easily melt into gold spheres. This drawback results in loss of the characteristic near-infrared (NIR) surface plasmon resonance (SPR) and limits their therapeutic applications. In this paper, we reported that ruthenium(ii) complex-functionalized AuNRs (AuNRs@Ru) and AuNTs (AuNTs@Ru) exhibit higher photothermal stability and photothermal efficiency than naked AuNRs and AuNTs. AuNRs@Ru and AuNTs@Ru maintain the morphology and NIR SPR absorption of gold nanoparticles upon 0.25 W cm-2 laser irradiation, which is lower than the maximal permissible exposure of skin as per ANSI regulation (0.33 W cm-2 at 808 nm). Further photothermal therapy studies on three-dimensional (3D) HeLa spheroids and an in vivo tumor model show that AuNRs@Ru and AuNTs@Ru are more effective for the photothermal destruction of tumors than AuNRs and AuNTs.

Two-photon photodynamic ablation of tumor cells by mitochondria-targeted iridium(iii) complexes in aggregate states.

By integrating targeting, imaging and treatment, organelle-targeted photodynamic therapy (PDT) has been reported to be an effective strategy for cancer therapy. However, targeting leads to the accumulation of photosensitizers (PSs) in the targeted organelles, which leads to a reduction in 1O2 generation and fluorescence quenching, especially for the lipophilic mitochondria-targeted PSs. Moreover, because PSs always need exposure to light for a specific period, photobleaching is difficult to avoid. To address these issues, two iridium(iii) complexes with aggregation-induced two-photon emission (AITPE) characteristics were developed. With lipophilicity, the complexes aggregated in water and targeted mitochondria. Owing to their impressive 1O2 production quantum yields and excellent two-photon properties in the aggregate states, the complexes were successfully used for mitochondria-targeted two-photon PDT in monolayer cells and multicellular spheroids. Our results highlighted that the use of a PS with aggregation enhanced 1O2 generation and fluorescence is an effective solution for aggregation in organelle-targeted PDT.

Synthesis, characterization and biological evaluation of labile intercalative ruthenium(ii) complexes for anticancer drug screening.

DNA binding and DNA transcription inhibition is regarded as a promising strategy for cancer chemotherapy. Herein, chloro terpyridyl Ru(ii) complexes, [Ru(tpy)(N^N)Cl](+) (Ru1, N^N = 2,2'-bipyridine; Ru2, N^N = 3-(pyrazin-2-yl)-as-triazino[5,6-f]acenaphthylene; Ru3, N^N = 3-(pyrazin-2-yl)-as-triazino[5,6-f]phenanthrene; Ru4, N^N = 3-(pyrazin-2-yl)-as-triazino[5,6-f]pyrene) were prepared as DNA intercalative and covalent binding anticancer agents. The chloro ligand hydrolysis slowly and the octanol and water partition coefficient of Ru2-Ru4 were between 0.6 and 1.2. MALDI-TOF mass, DNA gel electrophoresis confirmed covalent and intercalative DNA binding modes of Ru2-Ru4, while Ru1 can only bind DNA covalently. As a result, Ru2-Ru4 exhibited stronger DNA transcription inhibition activity, higher cell uptake efficiency and better anticancer activity than Ru1. Ru4 was the most toxic complex toward all cancer cells which inhibited DNA replication and transcription. AO/EB, Annexin V/PI, nuclear staining, JC-1 assays further confirmed that Ru2-Ru4 induced cancer cell death by an apoptosis mechanism.