Treatment of Retinoblastoma 1-Intact Hepatocellular Carcinoma With Cyclin-Dependent Kinase 4/6 Inhibitor Combination Therapy.

BACKGROUND AND AIMS: Synthetic cyclin-dependent kinase (CDK) 4/6 inhibitors exert antitumor effects by forcing RB1 in unphosphorylated status, causing not only cell cycle arrest but also cellular senescence, apoptosis, and increased immunogenicity. These agents currently have an indication in advanced breast cancers and are in clinical trials for many other solid tumors. HCC is one of promising targets of CDK4/6 inhibitors. RB family dysfunction is often associated with the initiation of HCC; however, this is revivable, as RB family members are not frequently mutated or deleted in this malignancy. APPROACH AND RESULTS: Loss of all Rb family members in transformation related protein 53 (Trp53)-/- mouse liver resulted in liver tumor reminiscent of human HCC, and re-expression of RB1 sensitized these tumors to a CDK4/6 inhibitor, palbociclib. Introduction of an unphosphorylatable form of RB1 (RB7LP) into multiple liver tumor cell lines induced effects similar to palbociclib. By screening for compounds that enhance the efficacy of RB7LP, we identified an I kappa B kinase (IKK)ß inhibitor Bay 11-7082. Consistently, RB7LP expression and treatment with palbociclib enhanced IKKα/ß phosphorylation and NF-κB activation. Combination therapy using palbociclib with Bay 11-7082 was significantly more effective in hepatoblastoma and HCC treatment than single administration. Moreover, blockade of IKK-NF-κB or AKT pathway enhanced effects of palbociclib on RB1-intact KRAS Kirsten rat sarcoma viral oncogene homolog mutated lung and colon cancers. CONCLUSIONS: In conclusion, CDK4/6 inhibitors have a potential to treat a wide variety of RB1-intact cancers including HCC when combined with an appropriate kinase inhibitor.

Cytotoxic activity of dimeric acridone alkaloids derived from Citrus plants towards human leukaemia HL-60 cells.

OBJECTIVES: Acridone alkaloids from Citrus and their derivatives show various kinds of biological activity. However, the anticancer activities of dimeric acridone alkaloids with unique structures and the molecular mechanism of these effects are poorly understood. METHODS: We investigated the cytotoxicity effects of dimeric acridone alkaloids isolated from Marsh grapefruit on human myeloid leukaemia HL-60 cells. KEY FINDINGS: Of the six dimeric acridone alkaloids tested, citbismine-E, the most potent, dose- and time-dependently decreased HL-60 cell viability by inducing apoptosis. The treatment of HL-60 cells with citbismine-E yielded a significant increase in levels of intracellular reactive oxygen species (ROS). Citbismine-E lowered the mitochondrial membrane potential and increased the activities of caspase-9 and -3. In addition, citbismine-E-induced apoptosis, decrease in mitochondrial membrane potential and caspase activation were significantly alleviated by pretreatment of the cells with antioxidant N-acetylcysteine (NAC). Citbismine-E induced intrinsic caspase-dependent apoptosis through ROS-mediated c-Jun N-terminal kinase activation. Citbismine-E-induced production of oxidative stress biomarkers, malondialdehyde and 8-hydroxy-2'-deoxyguanosine was also attenuated by pretreatment with NAC. CONCLUSIONS: Citbismine-E is a powerful cytotoxic agent against HL-60 cells that acts by inducing mitochondrial dysfunction-mediated apoptosis through ROS-dependent JNK activation. Citbismine-E also induced oxidative stress damage via ROS-mediated lipid peroxidation and DNA damage in HL-60 cells.

Undifferentiated State Induced by Rb-p53 Double Inactivation in Mouse Thyroid Neuroendocrine Cells and Embryonic Fibroblasts.

Retinoblastoma tumor suppressor protein (RB) is inactivated more frequently during tumor progression than during tumor initiation. However, its exact role in controlling the malignant features associated with tumor progression is poorly understood. We established in vivo and in vitro models to investigate the undifferentiated state induced by Rb inactivation. Rb heterozygous mice develop well-differentiated thyroid medullary carcinoma. We found that additional deletion of Trp53, without change in lineage, converted these Rb-deficient tumors to a poorly differentiated type associated with higher self-renewal activity. Freshly prepared mouse embryonic fibroblasts (MEFs) of Rb(-/-) ; Trp53(-/-) background formed stem cell-like spheres that expressed significant levels of embryonic genes despite of lacking the ability to form colonies on soft agar or tumors in immune-deficient mice. This suggested that Rb-p53 double inactivation resulted in an undifferentiated status but without carcinogenic conversion. We next established Rb(-/-) ; N-ras(-/-) MEFs that harbored a spontaneous carcinogenic mutation in Trp53. These cells (RN6), in an Rb-dependent manner, efficiently generated spheres that expressed very high levels of embryonic genes, and appeared to be carcinogenic. We then screened an FDA-approved drug library to search for agents that suppressed the spherogenic activity of RN6 cells. Data revealed that RN6 cells were sensitive to specific agents including ones those are effective against cancer stem cells. Taken together, all these findings suggest that the genetic interaction between Rb and p53 is a critical determinant of the undifferentiated state in normal and tumor cells.

Methyl galbanate, a novel inhibitor of nitric oxide production in mouse macrophage RAW264.7 cells.

It is well known that inflammation is associated with various neurodegenerative diseases, such as Parkinson's disease and Alzheimer's disease. An inflammatory mediator, nitric oxide (NO), is produced by inducible NO synthase (iNOS) in microglia and seems to be one of the possible causes of neurodegeneration. Several natural and synthetic compounds which exert anti-inflammatory effects by inhibiting NO production have been reported to date. The aim of this work was to investigate whether any of the 6 terpenoid coumarins (methyl galbanate, galbanic acid, farnesiferol A, badrakemone, umbelliprenin, and aurapten) isolated from Ferula szowitsiana DC. have inhibitory activity against NO production in RAW264.7 mouse macrophage cells stimulated with lipopolysaccharide (LPS) and interferon-γ (IFN-γ). Of the 6 terpenoid coumarins tested, methyl galbanate significantly decreased NO production in LPS/IFN-γ-stimulated RAW264.7 cells. In the presence of methyl galbanate, LPS/IFN-γ-induced iNOS mRNA expression was significantly decreased to 52% of the level found with LPS/IFN-γ stimulation alone. Methyl galbanate slightly attenuated COX-2 mRNA expression. Using the RAW264.7-tsAM5NE co-culture system, we showed that methyl galbanate protected neuronally differentiated tsAM5NE cells from NO-induced cell death by inhibiting the production of NO. Our finding suggests that methyl galbanate may be useful for developing a new drug against neurodegenerative diseases.