Low-dose imiquimod induces melanogenesis in melanoma cells through an ROS-mediated pathway.

BACKGROUND: Melanogenesis is the process of melanin maturation which not only protects skin from UV radiation but also plays an important role in antigenicity of melanomas. Imiquimod (IMQ) is a toll-like receptor 7 (TLR7) agonist that exhibits antiviral and anticancer activity. OBJECTIVE: To explore whether IMQ could induce melanogenesis in melanoma cells. METHODS: The mouse melanoma cell line B16F10, the mouse immortalized melanocyte Melan-A, and human melanoma cell lines MNT-1, C32 and A375 were utilized in this study. The pigmented level was observed by the centrifuged cell pellet. The intracellular and extracellular melanin levels were examined in the absorbance in NaOH-extracted cell lysate and cell-cultured medium, respectively. The expression of melanogenesis related proteins was examined by immunoblotting. The intracellular cyclic AMP amount was evaluated by the cAMP Glo assay kit. The activity of phosphodiesterase 4B (PDE4B) was investigated by CREB reporter assay with overexpressed PDE4B or not. RESULTS: We demonstrated that a low dose of IMQ could trigger melanogenesis in B16F10 cells. IMQ induced microphthalmia-associated transcription factor (MITF) nuclear translocation, upregulated the expression of melanogenesis-related proteins, increased tyrosinase (TYR) activity, and led to pigmentation in B16F10 cells. Next, we found that IMQ-induced melanogenesis was activated by excessive intracellular cAMP accumulation, which was regulated through IMQ-mediated PDE4B inhibition. Finally, IMQ-induced ROS production was found to be involved in melanogenesis by its control of PDE4B activity. CONCLUSIONS: Low dose of IMQ could activate melanogenesis through the ROS/PDE4B/PKA pathway in melanoma cells.

Heat shock protein 70 protects the lungs from hyperoxic injury in a neonatal rat model of bronchopulmonary dysplasia.

Hyperoxia plays a significant role in the pathogenesis of lung injury, such as bronchopulmonary dysplasia (BPD), in premature infants or newborns. BPD management aims to minimize further injury, provide an optimal environment to support growth and recovery. In clinic neonatal care, we need a new therapy for BPD. Heat shock protein 70 (Hsp70) inhibit cell apoptosis and promote cell repair allowing cells to survive lethal injury. We hypothesized that Hsp70 could be used to prevent hyperoxia related BPD in the neonatal rat model through its anti-apoptotic and anti-inflammatory effects. In this study, we explored the effect of Hsp70 on hyperoxia-induced lung injury using neonatal rats. Neonatal Wistar rats were delivered naturally at full term of gestation and were then pooled and randomly assigned to several groups to receive heat stimulation (41°C for 20 min) or room temperature conditions. The Hsp70 group received recombinant Hsp70 intraperitoneally (200 µg/kg, daily). All newborn rats were placed under hyperoxic conditions (85% oxygen) for 21 days. Survival rates in both heat-hyperoxia and Hsp70-hyperoxia groups were higher than those in the hyperoxia group (p < 0.05). Both endogenous and exogenous Hsp70 could reduce early apoptosis of alveolar cells under hyperoxia. Additionally, there were less macrophage infiltration in the lung of the Hsp70 groups (p < 0.05). Heat stress, heat shock proteins, and exogenous recombinant Hsp70 significantly increased the survival rate and reduced pathological hyperoxia induced lung injuries in the development of BPD. These results suggest that treating hyperoxia-induced lung injury with Hsp70 may reduce the risk of developing BPD.

Imiquimod-induced ROS production causes lysosomal membrane permeabilization and activates caspase-8-mediated apoptosis in skin cancer cells.

BACKGROUND: Lysosomal cell death is induced by lysosomal membrane permeabilization (LMP) and the subsequent release of lysosomal proteolytic enzymes, including cathepsins (CTSs), which results in mitochondrial dysfunction and apoptosis. Imiquimod (IMQ), a synthetic TLR7 ligand, has both antiviral and antitumor activity against various skin malignancies in clinical treatment. Previously, we demonstrated IMQ not only caused lysosomal dysfunction but also triggered lysosome biogenesis to achieve lysosomal adaptation in cancer cells. OBJECTIVE: To determine whether lysosomes are involved in IMQ-induced apoptosis. METHODS: The human skin cancer cell lines BCC, A375 and mouse melanoma cell line B16F10 were used in all experiments. Cell death was determined by the Cell Counting Kit-8 (CCK-8) assay and DNA content assay. Protein expression was determined by immunoblotting. Caspase-8 activity was assessed using a fluorescence caspase-8 kit and determined by flow cytometry and confocal microscopy. RESULTS: IMQ not only induced lysosome damage but also abrogated lysosome function in skin cancer cells. IMQ-induced caspase-8 activation contributed to the processes of lysosomal cell death. Moreover, the use of ROS scavengers significantly abolished caspase-8 activation and inhibited IMQ-induced LMP. Additionally, pharmacological inhibition of CTSD not only abrogated caspase-8 activation but also rescued IMQ-induced cell death. Finally, lysosome-alkalizing agents enhanced the cytotoxicity of IMQ in vitro and in vivo. CONCLUSIONS: IMQ-induced ROS accumulation promotes LMP, releases CTSs into the cytosol, stimulates caspase-8 activation and finally causes lysosomal cell death. Lysosomal cell death and the CTSD/caspase-8 axis may play a crucial role in IMQ-induced cell death.

Using a neonatal rat model to explore the therapeutic potential of coenzyme Q10 in prematurity under hyperoxia.

Hyperoxia, is often used in preterm supportive care, leading to high oxygen exposure in neonates. Coenzyme Q10 (CoQ10) is a free radical scavenger that has been studied in older children but never be investigated for its role in preterm care. We hypothesize that the administration of exogenous CoQ10 would raise serum concentrations of CoQ10 and mitigate the adverse effects of hyperoxia on the organs by reducing oxygen-free radicals and inflammation. The aim of this study was to evaluate the effects of oxidative stress, inflammatory response, and survival in neonatal rats after CoQ10 treatment. Neonatal rats delivered from four pregnant Wistar rats were randomly divided into four groups: (a) control, (b) CoQ10, (c) hyperoxia (O2 group), and (d) treatment (CoQ10 + O2 ) groups. The dose of CoQ10 injected was 30 mg/kg. The CoQ9, CoQ10, cytokines, oxidative stress, and antioxidant enzyme activity were measured. Tissue samples were histologically examined and mortality was monitored for 16 days. The level of CoQ9 significantly increased in the liver, kidney, and plasma, while the level of CoQ10 significantly increased in most organ tissues in the CoQ10 + O2 group. Additionally, CoQ10 decrease oxidative stress in the liver, increase antioxidant enzyme activity in the heart, kidney, and brain, and reverse an inclined level of hematopoietic growth factors. However, CoQ10 had no effect on inflammation, organ damage, or mortality. Therefore, the use of CoQ10 in potential adjuvant therapy for neonatal hyperoxia requires further research.

Synergistic Anticancer Effects of Gemcitabine with Pitavastatin on Pancreatic Cancer Cell Line MIA PaCa-2 in vitro and in vivo.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy with an overall 5-year survival rate of 9.3%, and this malignancy is expected to become the second leading cause of cancer-related death by 2030. Gemcitabine resistance develops within weeks of PDAC patient's chemotherapeutic initiation. Statins, including pitavastatin, have been indicated to have anticancer effects in numerous human cancer cell lines. Thus, in this study, we hypothesized that a combination of gemcitabine and pitavastatin may have a greater anticancer effect than gemcitabine alone on the human pancreatic carcinoma cell line MIA PaCa-2. METHODS: The anticancer effects of gemcitabine with pitavastatin were evaluated using human MIA PaCa-2 cell line in vitro and in vivo Balb/c murine xenograft tumor model. Cell viability was assessed with CCK-8, and cell migration was stained by crystal violet. Cell cycle distribution, apoptosis and mitochondrial membrane potential were examined by flow cytometry. Activation of drug transporters (hENTs, hCNTs), intracellular drug activating (dCK) and inhibition of inactivating enzymes (RRMs) pathways were assessed by Western blotting analysis. Molecular mechanisms and signaling pathways of apoptosis, necrosis and autophagy also were assessed by Western blotting. RESULTS: We observed that gemcitabine and pitavastatin synergistically suppressed the proliferation of MIA PaCa-2 cells through causing sub-G1 and S phase cell cycle arrest. Activation of apoptosis/necrosis was confirmed by annexin V/propidium iodide double staining, which showed increasing levels of active caspase 3, cleaved poly(ADP-ribose) polymerase and the RIP1-RIP3-MLKL complex. Moreover, gemcitabine-pitavastatin-mediated S phase arrest downregulated cyclin A2/CDK2 and upregulated p21/p27 in MIA PaCa-2 cells. Furthermore, this combination improved drug cellular metabolism pathway, mitochondria function and activated autophagy as part of the cell death mechanism. In vivo, gemcitabine-pitavastatin effectively inhibited tumor growth in a nude mouse mode of Mia PaCa-2 xenografts without observed adverse effect. CONCLUSION: Combined gemcitabine-pitavastatin may be an effective novel treatment option for pancreatic cancer.