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Morphocumin coordinated RuII–p-cymene complex acts as a type-I photosensitizer to inhibit growth of CSC enriched 3D-spheroids of oral squamous carcinoma downregulating stemness genes (cMYC, SOX2, OCT4) with no systemic toxicity to zebrafish embryos in the dark.

Eight new ruthenium(II) complexes of N,N-chelating pyrazolylbenzimidazole ligands of the general formula [RuII(p-cym)(L)X]+ [where the ligand L is 2-(1H-pyrazol-1-yl)-1H-benzo[d]imidazole (L1) substituted at the 4 position of the pyrazole ring by Cl (L2), Br (L3), or I (L4) and X = Cl- and I-] were synthesized and characterized using various analytical techniques. Complexes 1 and 3 were also characterized by single-crystal X-ray crystallography, and they crystallized as a monoclinic crystal system in space groups P21/n and P21/c, respectively. The complexes display good solution stability at physiological pH 7.4. The iodido-coordinated pyrazolylbenzimidazole ruthenium(II) p-cymene complexes (2, 4, 6, and 8) are more resistant toward hydrolysis and have less tendency to form monoaquated complexes in comparison to their chlorido analogues (1, 3, 5, and 7). The halido-substituted 2-(1H-pyrazol-1-yl)-1H-benzo[d]imidazole ligands, designed as organic-directing molecules, inhibit vascular endothelial growth factor receptor 2 (VEGFR2) phosphorylation. In addition, the ruthenium(II) complexes display a potential to bind to DNA bases. The cytotoxicity profile of the complexes (IC50 ca. 9-12 μM for 4-8) against the triple-negative breast cancer cells (MDA-MB-231) show that most of the complexes are efficient. The lipophilicity and cellular accumulation data of the complexes show a good correlation with the cytotoxicity profile of 1-8. The representative complexes 3 and 7 demonstrate the capability of arresting the cell cycle in the G2/M phase and induce apoptosis. The inhibition of VEGFR2 phosphorylation with the representative ligands L2 and L4 and the corresponding metal complexes 3 and 7 in vitro shows that the organic-directing ligands and their complexes inhibit VEGFR2 phosphorylation. Besides, L2, L4, 3, and 7 inhibit the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and proto-oncogene tyrosine-protein kinase (Src), capable of acting downstream of VEGFR2 as well as independently. Compounds L2, L4, 3, and 7 have a lesser effect on ERK1/2 and more prominently affect Src phosphorylation. We extended the study for L2 and 3 in the Tg(fli1:gfp) zebrafish model and found that L2 is more effective in vivo compared to 3 in inhibiting angiogenesis.

Background: Stem cell-like properties in cancer cells are found to be responsible for its aggressive behaviour. However, this has not been studied with respect to the bimodal NOTCH-pathway-activity status, found in oral cancer. Methods:Oral-SLCCs were enriched in 3D-spheroids. Constitutively-active and inactive status of NOTCH-pathway was achieved by genetic or pharmacological approaches. RNA sequencing and real-time PCR was performed for gene expression studies. in vitro cytotoxicity assessments were performed by AlamarBlue assay and in vivoeffects were studied by xenograft growth in zebrafish embryo. The t tests were performed to estimate statistical significance of the study. Results: Here, we have demonstrated the stochastic plasticity on NOTCH-activity axis; maintaining both NOTCH-active and inactive states of oral stem-like cancer cells (Oral-SLCCs). While Oral-SLCCs with inactive NOTCH-pathway status showed higher proliferation and aggressive tumor growth, the Cisplatin refraction was associated with active-status of NOTCH-pathway; suggesting the crucial role of plasticity on NOTCH-axis. The differentially expressed genes between NOTCH-pathway active and inactive clones clearly suggested the upregulation of JAK-STAT signaling in subset of Oral-SLCCs with lower NOTCH-pathway activity status. Confirming the function; the 3D-spheroids generated by oral-SLCCs with lower NOTCH-activity-status displayed significantly higher sensitivity to JAK-selective drugs, Ruxolitinib or Tofacitinib and siRNA mediated downregulation of tested partners STAT 3 and 4. Therefore, we adopted the strategy of synthetic lethality, where Oral-SLCCs were reprogrammed to maintain the inactive status of NOTCH-pathway by exposure to γ-secretase inhibitors, LY411575 or RO4929097 followed by targeting with JAK-inhibitors, Ruxolitinib or Tofacitinib. This resulted in a very significant inhibition in viability of 3D-spheroids as well as xenograft formation in Zebrafish embryos; whereas inhibition of either of these pathway alone were largely ineffective. Conclusion: We have demonstrated the stochastic cellular plasticity on NOTCH-activity axis. Study revealed for the first time that NOTCH-HES and JAK-STAT pathways may act as synthetic lethal pair, and as novel targets against diverse states of stemness in oral cancer. Therefore, we have provided the rational for sequential combination of NOTCH and JAK inhibitors as possible therapeutic strategy against aggressive oral cancer.

Correction for ‘A photoactive lysosome targeting RuII complex downregulates stemness genes in oral squamous cell carcinoma’ by Souryadip Roy et al., Inorg. Chem. Front., 2022, 9, 5840–5852, https://doi.org/10.1039/D2QI01079H.