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.

Sodium-Glucose Transport Protein 2 Inhibitor Use for Type 2 Diabetes and the Incidence of Acute Kidney Injury in Taiwan.

Importance: The association between sodium-glucose transport protein 2 inhibitor (SGLT2i) use and the incidence of acute kidney injury (AKI) remains controversial. The benefits of SGLT2i use in patients to reduce AKI requiring dialysis (AKI-D) and concomitant diseases with AKI as well as improve AKI prognosis have not yet been established. Objective: To investigate the association between SGLT2i use and AKI incidence in patients with type 2 diabetes (T2D). Design, Setting, and Participants: This nationwide retrospective cohort study used the National Health Insurance Research Database in Taiwan. The study analyzed a propensity score-matched population of 104 462 patients with T2D treated with SGLT2is or dipeptidyl peptidase 4 inhibitors (DPP4is) between May 2016 and December 2018. All participants were followed up from the index date until the occurrence of outcomes of interest, death, or the end of the study, whichever was earliest. Analysis was conducted between October 15, 2021, and January 30, 2022. Main Outcomes and Measures: The primary outcome was the incidence of AKI and AKI-D during the study period. AKI was diagnosed using International Classification of Diseases diagnostic codes, and AKI-D was determined using the diagnostic codes and dialysis treatment during the same hospitalization. Conditional Cox proportional hazard models assessed the associations between SGLT2i use and the risks of AKI and AKI-D. The concomitant diseases with AKI and its 90-day prognosis, ie, the occurrence of advanced chronic kidney disease (CKD stage 4 and 5), end-stage kidney disease, or death, were considered when exploring the outcomes of SGLT2i use. Results: In a total of 104 462 patients, 46 065 (44.1%) were female patients, and the mean (SD) age was 58 (12) years. After a follow-up of approximately 2.50 years, 856 participants (0.8%) had AKI and 102 (<0.1%) had AKI-D. SGLT2i users had a 0.66-fold risk for AKI (95% CI, 0.57-0.75; P < .001) and 0.56-fold risk of AKI-D (95% CI, 0.37-0.84; P = .005) compared with DPP4i users. The numbers of patients with AKI with heart disease, sepsis, respiratory failure, and shock were 80 (22.73%), 83 (23.58%), 23 (6.53%), and 10 (2.84%), respectively. SGLT2i use was associated with lower risk of AKI with respiratory failure (hazard ratio [HR], 0.42; 95% CI, 0.26-0.69; P < .001) and shock (HR, 0.48; 95% CI, 0.23-0.99; P = .048) but not AKI with heart disease (HR, 0.79; 95% CI, 0.58-1.07; P = .13) and sepsis (HR, 0.77; 95% CI, 0.58-1.03; P = .08). The 90-day AKI prognosis for the risk of advanced CKD indicated a 6.53% (23 of 352 patients) lower incidence in SGLT2i users than in DPP4i users (P = .045). Conclusions and Relevance: The study findings suggest that patients with T2D who receive SGLT2i may have lower risk of AKI and AKI-D compared with those who receive DPP4i.

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.

Anti-EMT and anti-fibrosis effects of protocatechuic aldehyde in renal proximal tubular cells and the unilateral ureteral obstruction animal model.

CONTEXT: Protocatechuic aldehyde (PCA) is a natural product that has various benefits for fibrosis. OBJECTIVE: This study evaluated the effects of PCA on renal fibrosis. MATERIALS AND METHODS: Epithelial-mesenchymal transition (EMT) was induced by 20 ng/mL transforming growth factor-ß1 (TGF-ß1), followed by treatment with 1 and 5 µM PCA, in the rat renal proximal tubular cell line NRK-52E. Cell viability, protein expression, and scratch wound-healing assays were conducted. Sprague-Dawley (SD) rats underwent unilateral ureteral obstruction (UUO) surgery for renal fibrosis indication and were treated with 50 and 100 mg/kg PCA for 14 days. RESULTS: The IC50 of PCA was appropriately 13.75 ± 1.91 µM in NRK-52E cells, and no significant difference at concentrations less than 5 µM. PCA ameliorated TGF-ß1-induced EMT, such as enhanced E-cadherin and decreased vimentin. Fibrotic markers collagen IV and α-smooth muscle actin (α-SMA) increased in TGF-ß1-induced NRK-52E. Moreover, PCA reduced TGF-ß1-induced migration in the wound-healing assay. Analysis of rat kidneys indicated that PCA reduced UUO-induced hydronephrosis (control: 15.11 ± 1.00%; UUO: 39.89 ± 1.91%; UUO + PCA50: 18.37 ± 1.61%; UUO + PCA100: 17.67 ± 1.39%). Protein level demonstrated that PCA not only decreased vimentin expression and enhanced E-cadherin expression, but inhibited UUO-induced collagen IV and α-SMA upregulation, indicating that it could mitigate EMT in a rat model of UUO-induced renal fibrosis. DISCUSSION AND CONCLUSIONS: This study suggested that PCA decreases TGF-ß1-induced fibrosis and EMT in vitro and in vivo. These findings demonstrate pharmacological effects of PCA and might be a potential strategy for the prevention of organ fibrosis in clinics.

Association of SGLT2 inhibitors with lower incidence of death in type 2 diabetes mellitus and causes of death analysis.

Sodium-glucose cotransporter 2 inhibitor (SGLT2i) potentially decrease all-cause and cardiovascular death, however, associations with non-cardiovascular death remain unclear. Therefore, we investigated SGLT2i associations with death and the cause of death. We used the Taiwanese National Health Institutes Research database linked to the National Register of Deaths (NRD). Incident type 2 diabetes mellitus (T2DM) patients and propensity score matched T2DM SGLT2i and Dipeptidyl peptidase 4 inhibitor (DPP4i) users were investigated. The index year was the SGLT2i or DPP4i prescription date from May 2016. Patients were followed-up until death or December 2018. Deaths verified by the NRD and grouped accordingly. Multiple Cox proportional hazards models were used. In total, 261,211 patients were included in the population; 47% of the patients were female and the average age was 62 years. The overall incidence of all-cause death was 8.67/1000 patient-years for SGLT2i and 12.41 for DPP4i users during follow-up. After adjusting for potential risk factors in the propensity score matched population, SGLT2i users were associated with lower risks of all-cause death, cardiovascular death, cancer death, and non-cancer, non-vascular death compared with DPP4i-users. For specific death causes, significantly lower death risks from heart disease, cerebrovascular disease, and accidents were associated with SGLT2i-use. SGLT2i benefits for T2DM patients were not different across subgroups. Compared with DPP4i-use, SGLT2i-use for T2DM was associated with lower disease and death risk.

Impact of peritoneal dialysis-related peritonitis on PD discontinuation and mortality: A population-based national cohort study.

BACKGROUND: The impact of peritoneal dialysis-associated peritonitis (PD peritonitis) on long-term outcomes is uncertain. This nationwide retrospective study was conducted in Taiwan to understand the incidence, risk factors and long-term outcomes of PD peritonitis. METHODS: A total of 11,202 incident adult peritoneal dialysis (PD) patients from 2000 to 2010 were collected from a Longitudinal Health Insurance Database and followed up until the end of 2011. Definition of peritonitis, the primary outcome, simultaneously met the diagnosis of peritonitis (International Classification of Diseases, Ninth Revision, Clinical Modification 567) and antibiotic use. Secondary outcomes included the impact of peritonitis on PD discontinuation and survival. Cox proportional hazards models with and without time-dependent variables were used to calculate hazard ratios (HRs) and 95% confidence intervals (CIs). RESULTS: There were 7634 peritonitis episodes in 4245 patients during the follow-up period. The overall incidence of peritonitis was 0.18 episodes per patient-year. Peritonitis-associated risk factors included older age, female gender, chronic heart failure, cerebrovascular disease, liver cirrhosis and lower monthly income. In an adjusted Cox hazard proportional regression with the time-dependent model, peritonitis patients had a higher risk of PD discontinuation (HR 2.71, 95% CI 2.52-2.92) and mortality (HR 1.68, 95% CI 1.57-1.81) compared to patients without peritonitis. The adjusted HRs for mortality increased with each prior episode: one episode, two episodes and more than two episodes (all p < 0.05). The adjusted HRs for PD discontinuation also increased with the frequency of peritonitis. These negative effects were greatest during the first year and persisted significantly after 5 years. In a sensitivity analysis in which peritonitis within 30 days of death or PD discontinuation was excluded, peritonitis patients still had significantly increased risk of PD discontinuation and mortality compared to patients without peritonitis. CONCLUSIONS: Although peritonitis incidence was low, our findings reveal that peritonitis carried acute and long-term sequelae of higher PD discontinuation and lower patient survival.

Association of Sodium-Glucose Transport Protein 2 Inhibitor Use for Type 2 Diabetes and Incidence of Gout in Taiwan.

Importance: The use of sodium-glucose transport protein 2 (SGLT2) inhibitors is currently a standard intervention in patients with type 2 diabetes (T2DM) and exerts favorable pleiotropic effects to consistently lower blood urate levels. However, to date, no association between SGLT2 inhibitor use and the incidence of gout have been established. Objective: To investigate whether prescribed SGLT2 inhibitors are associated with lower gout incidence in patients with T2DM. Design, Setting, and Participants: In a cohort study, all patients with incident T2DM in Taiwan National Health Institution databases between May 1, 2016, and December 31, 2018, were retrospectively analyzed. As a comparator, patients using dipeptidyl peptidase 4 (DPP4) inhibitors were included. A total of 47 905 individuals receiving an SGLT2 inhibitor and 183 303 receiving a DPP4 inhibitor were evaluated, along with 47 405 pairs of patients using an SGLT2 inhibitor or DPP4 inhibitor in 1:1 propensity score-matched analyses. Data analysis was conducted from April 1 to June 30, 2021. Main Outcomes and Measures: A gout diagnosis was based on the International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) and the International Statistical Classification of Diseases, Tenth Revision, Clinical Modification (ICD-10-CM). Multiple Cox proportional hazards regression models were used to calculate hazard ratios (HRs) and 95% CIs. Results: In total, 231 208 patients with T2DM were included in the population; 113 812 individuals (49.22%) were women, and the mean (SD) age was 61.53 (12.86) years. The overall gout incidence was 20.26 per 1000 patient-years for SGLT2 inhibitor users and 24.30 per 1000 patient-years for DPP4 inhibitor users. When potential risk factors were adjusted in the propensity score-matched population, use of SGLT2 inhibitors was associated with a lower risk of gout (HR, 0.89; 95% CI, 0.82-0.96) compared with DPP4 inhibitors, particularly for patients receiving dapagliflozin (HR, 0.86; 95% CI, 0.78-0.95). A sensitivity analysis, performed when a gout diagnosis was ascertained using the ICD-9-CM or ICD-10-CM code with gout-related medication, also showed a significantly lower risk for gout incidence of 15% with SGLT2 inhibitors (HR, 0.85; 95% CI, 0.74-0.97). Subgroup analysis indicated that SGLT2 inhibitor benefits in patients with T2DM to achieve a lower gout risk were not different across subgroups. Conclusions and Relevance: The findings of this study suggest that patients with T2DM who are receiving SGLT2 inhibitors may have a lower risk for gout compared with those receiving DPP4 inhibitors.

Evaluation of the Therapeutic Effects of Protocatechuic Aldehyde in Diabetic Nephropathy.

Diabetic nephropathy (DN) is one of the most severe chronic kidney diseases in diabetes and is the main cause of end-stage renal disease (ESRD). Protocatechuic aldehyde (PCA) is a natural product with a variety of effects on pulmonary fibrosis. In this study, we examined the effects of PCA in C57BL/KS db/db male mice. Kidney morphology, renal function indicators, and Western blot, immunohistochemistry, and hematoxylin and eosin (H&E) staining data were analyzed. The results revealed that treatment with PCA could reduce diabetic-induced renal dysfunction, as indicated by the urine albumin-to-creatinine ratio (db/m: 120.1 ± 46.1µg/mg, db/db: 453.8 ± 78.7 µg/mg, db/db + 30 mg/kg PCA: 196.6 ± 52.9 µg/mg, db/db + 60 mg/kg PCA: 163.3 ± 24.6 µg/mg, p < 0.001). However, PCA did not decrease body weight, fasting plasma glucose, or food and water intake in db/db mice. H&E staining data revealed that PCA reduced glomerular size in db/db mice (db/m: 3506.3 ± 789.3 µm2, db/db: 6538.5 ± 1818.6 µm2, db/db + 30 mg/kg PCA: 4916.9 ± 1149.6 µm2, db/db + 60 mg/kg PCA: 4160.4 ± 1186.5 µm2p < 0.001). Western blot and immunohistochemistry staining indicated that PCA restored the normal levels of diabetes-induced fibrosis markers, such as transforming growth factor-beta (TGF-ß) and type IV collagen. Similar results were observed for epithelial-mesenchymal transition-related markers, including fibronectin, E-cadherin, and α-smooth muscle actin (α-SMA). PCA also decreased oxidative stress and inflammation in the kidney of db/db mice. This research provides a foundation for using PCA as an alternative therapy for DN in the future.

Primary aldosteronism is associated with risk of urinary bladder stones in a nationwide cohort study.

We analyzed database from the Taiwan National Health Insurance to investigate whether primary aldosteronism (PA) increases the risk of bladder stones. This retrospective nationwide population-based cohort study during the period of 1998-2011 compared patients with and without PA extracted by propensity score matching. Cox proportional hazard models and competing death risk model were used to estimate the hazard ratios (HRs), sub-hazard ratios (SHRs) and corresponding 95% confidence intervals (CIs). There were 3442 patients with PA and 3442 patients without PA. The incidence rate of bladder stones was 5.36 and 3.76 per 1000 person-years for both groups, respectively. In adjusted Cox hazard proportional regression models, the HR of bladder stones was 1.68 (95% CI 1.20-2.34) for patients with PA compared to individuals without PA. Considering the competing risk of death, the SHR of bladder stones still indicates a higher risk for PA than a comparison cohort (SHR, 1.79; 95% CI 1.30-2.44). PA, age, sex, and fracture number were the variables significantly contributing to the formation of bladder stones. In conclusion, PA is significantly associated with risk of bladder stones.

EGR-1 plays a protective role in AMPK inhibitor compound C-induced apoptosis through ROS-induced ERK activation in skin cancer cells.

Skin cancer is caused by abnormal proliferation, gene regulation and mutation of epidermis cells. Compound C is commonly used as an inhibitor of AMP-activated protein kinase (AMPK), which serves as an energy sensor in cells. Recently, compound C has been reported to induce apoptotic and autophagic death in various skin cancer cell lines via an AMPK-independent pathway. However, the signaling pathways activated in compound C-treated cancer cells remain unclear. The present oligodeoxynucleotide-based microarray screening assay showed that the mRNA expression of the zinc-finger transcription factor early growth response-1 (EGR-1), which helps regulate cell cycle progression and cell survival, was significantly upregulated in compound C-treated skin cancer cells. Compound C was demonstrated to induce EGR-1 mRNA and protein expression in a time and dose-dependent manner. Confocal imaging showed that compound C-induced EGR-1 protein expression was localized in the nucleus. Compound C was demonstrated to activate extracellular signal-regulated kinase (ERK) phosphorylation. Inhibition of this compound C-induced ERK phosphorylation downregulated the mRNA and protein expression of EGR-1. In addition, removal of compound C-induced reactive oxygen species (ROS) not only decreased ERK phosphorylation, but also inhibited compound C-induced EGR-1 expression. A functional assay showed that knock down of EGR-1 expression in cancer cells decreased the survival rate while also increasing caspase-3 activity and apoptotic marker expression after compound C treatment. However, no difference in autophagy marker light chain 3-II protein expression was observed between compound C-treated control cells and EGR-1-knockdown cells. Thus, it was concluded that that EGR-1 may antagonize compound C-induced apoptosis but not compound C-induced autophagy through the ROS-mediated ERK activation pathway.

Imiquimod Accelerated Antitumor Response by Targeting Lysosome Adaptation in Skin Cancer Cells.

Lysosomal adaptation is a cellular physiological process in which the number and function of lysosomes are regulated at the transcriptional and post-transcriptional levels in response to extracellular and/or intracellular cues or lysosomal damage. Imiquimod (IMQ), a synthetic toll-like receptor 7 ligand with hydrophobic and weak basic properties, exhibits both antitumor and antiviral activity against various skin malignancies as a clinical treatment. Interestingly, IMQ has been suggested to be highly concentrated in the lysosomes of plasmacytoid dendritic cells, indicating that IMQ could modulate lysosome function after sequestration in the lysosome. In this study, we found that IMQ not only induced lysosomal membrane permeabilization and dysfunction but also increased lysosome biogenesis to achieve lysosomal adaptation in cancer cells. IMQ-induced ROS production but not lysosomal sequestration of IMQ was the major cause of lysosomal adaptation. Moreover, IMQ-induced lysosomal adaptation occurred through lysosomal calcium ion release and activation of the calcineurin/TFEB axis to promote lysosome biogenesis. Finally, depletion of TFEB sensitized skin cancer cells to IMQ-induced apoptosis in vitro and in vivo. In summary, a disruption of lysosomal adaptation might represent a therapeutic strategy for synergistically enhancing the cytotoxicity of IMQ in skin cancer cells.

Is combined peritoneal dialysis and hemodialysis redundant? A nationwide study from Taiwan.

BACKGROUND: Combined peritoneal dialysis (PD) and hemodialysis (HD) therapy (combined therapy) has numerous clinical benefits and should be emphasized for PD patients encountering technique failure. METHODS: This 12-year nationwide retrospective study was conducted to compare long-term outcomes (including admission and mortality risks) between combined therapy patients (combined group) and patients directly transferred from PD to HD (transfer group). RESULTS: All 12,407 incidental PD patients from 2000 to 2010 were enrolled and followed up until the end of 2011. A total of 688 patients in the combined group and 688 patients in the transfer group were selected after 1:1 frequency matching based on age, sex, and PD duration. The overall admission and mortality risks of the two groups were comparable in a Cox proportional hazards model (adjusted hazard ratio [HR] = 1.06 [95% confidence interval (CI) = 0.95-1.19] and 1.02 [95% CI = 0.80-1.30]), respectively). Compared with the transfer group, combined group patients with recent peritonitis or frequent hemodialysis (four HD sessions per month) had significantly higher risk of admission while combined group patients without peritonitis had significantly lower risk. The number of incidents in the combined group increased over time. On average, patients stayed on combined therapy for 2 years. CONCLUSIONS: Combined therapy (two HD sessions per month) is not redundant but a rational and cost-effective treatment, particularly for patients without recent peritonitis. Dialysis staff should be familiar with the advantages and disadvantages of combined therapy and consider it an essential part of integrated dialysis care.

Imiquimod-induced ROS production disrupts the balance of mitochondrial dynamics and increases mitophagy in skin cancer cells.

BACKGROUND: Mitochondrial homeostasis is a highly dynamic process involving continuous fission and fusion cycles and mitophagy to maintain mitochondrial functionality. Imiquimod (IMQ), a Toll-like receptor (TLR) 7 ligand, is used to treat various skin malignancies. IMQ also induces apoptotic and autophagic cell death in various cancers through a TLR7-independent pathway. OBJECTIVE: To investigate whether IMQ-induced ROS production is involved in mitochondrial dysfunction, mitochondrial fragmentation and mitophagy in skin cancer cells. METHODS: BCC/KMC-1, B16F10 and A375 skin cancer cells, AGS gastric cancer cells and primary human keratinocytes were treated with 50 µg/mL IMQ. After 4 h, ROS were detected by CM-H2DCFDA, DHE, and MitoSOX Red staining. After 24 h, cell viability and the mitochondrial membrane potential were evaluated by a CCK-8 assay and JC-1 staining, respectively. Oxygen consumption was assessed with an Oroboros instrument. Mitochondrial morphology and mitophagy were evaluated by MitoTracker and LysoTracker staining. Mitochondrial dynamics markers, including MFN-1, DRP-1 and OPA1, and mitophagy markers, including LC3, S65-phosphorylated ubiquitin, PINK1 and TOM20, were detected by immunoblotting. RESULTS: IMQ not only induced severe ROS production but also resulted in increased mitochondrial membrane potential loss, mitochondrial fission and mitophagy and decreased oxygen consumption in skin cancer cells compared with normal keratinocytes. Pretreatment with the antioxidant NAC reduced IMQ-induced ROS production and attenuated IMQ-induced mitochondrial fission and mitophagy in skin cancer cells. CONCLUSIONS: IMQ-induced ROS might be associated with mitochondrial dysfunction, mitochondrial fission and mitophagy in cancer cells. Alleviating IMQ-induced ROS production would reduce mitochondrial fission-to-fusion skewing and further reduce IMQ-induced mitophagy.

Atorvastatin-induced senescence of hepatocellular carcinoma is mediated by downregulation of hTERT through the suppression of the IL-6/STAT3 pathway.

Hepatocellular carcinoma (HCC), a hepatic malignancy, has a poor prognosis and contributes to cancer-related death worldwide. Cellular senescence is an anticancer therapeutic strategy that causes irreversible cell cycle arrest and enables immune-mediated clearance of cancer cells. Atorvastatin, an HMG-CoA reductase inhibitor, has been shown to inhibit tumor growth and induce apoptosis or autophagy in malignant tumors. However, whether atorvastatin can induce HCC cell senescence and the mechanisms involved are poorly understood. The effects of atorvastatin-induced senescence were examined in both HCC cells and mouse xenograft models. The phenomenon and mechanism of senescence were examined by cell cycle analysis, senescence-associated ß-galactosidase (SA-ß-gal) staining and western blotting in HCC cells, and HCC tissues from mice were analyzed by immunohistochemical (IHC) staining. We demonstrated that atorvastatin induced cell growth inhibition and G0/G1 phase cell cycle arrest, leading to senescence in HCC cells. Atorvastatin-induced senescence was independent of p53, p14, and p16, and atorvastatin not only decreased the secretion of IL-6, a major senescence-associated secretory phenotype (SASP) factor, and the phosphorylation of STAT3 but also inhibited the expression of hTERT, a catalytic subunit of telomerase. Supplementation with exogenous IL-6 reversed both atorvastatin-induced suppression of STAT3 phosphorylation and hTERT expression and atorvastatin-induced senescence. Overexpression of constitutively activated STAT3 rescued HCC cells from atorvastatin-induced hTERT suppression and senescence. Moreover, atorvastatin decreased tumor growth in mouse xenograft models. Consistent with these results, atorvastatin decreased the IL-6, p-STAT3, and hTERT levels and increased ß-gal expression in tumor sections. Taken together, these data indicate that atorvastatin can induce atypical cellular senescence in HCC cells to inhibit tumor growth, an effect mediated by downregulation of hTERT through suppression of the IL-6/STAT3 pathway.

Imiquimod Exerts Antitumor Effects by Inducing Immunogenic Cell Death and Is Enhanced by the Glycolytic Inhibitor 2-Deoxyglucose.

The induction of immunogenic cell death (ICD) in cancer cells triggers specific immune responses against the same cancer cells. Imiquimod (IMQ) is a synthetic ligand of toll-like receptor 7 that exerts antitumor activity by stimulating cell-mediated immunity or by directly inducing apoptosis. Whether IMQ causes tumors to undergo ICD and elicits a specific antitumor immune response is unknown. We demonstrated that IMQ-induced ICD-associated features, including the surface exposure of calreticulin and the secretion of adenosine triphosphate and HMGB1, were mediated by ROS and endoplasmic reticulum stress. In a B16F10 melanoma mouse model, vaccinating mice with IMQ-induced ICD cell lysate or directly injecting IMQ in situ reduced tumor growth that was mediated by inducing tumor-specific T-cell proliferation, promoting tumor-specific cytotoxic killing by CD8+ T cells, and increasing the infiltration of various immune cells into tumor lesions. The ICD-associated features were crucial in the induction of specific antitumor immunity in vivo. The glycolytic inhibitor 2-deoxyglucose enhanced IMQ-induced ICD-associated features and strengthened the antitumor immunity mediated by IMQ-induced ICD cell lysate in p53-mutant cancer cells, which were IMQ-resistant in vitro. We conclude that IMQ is an authentic ICD inducer and provide a concept connecting IMQ-induced cancer cell death and antitumor immune responses.

M2-like polarization of THP-1 monocyte-derived macrophages under chronic iron overload.

Macrophages are characterized by phenotypical and functional heterogeneity. In different microenvironments, macrophages can polarize into two types: classically activated macrophages (M1) or alternatively activated macrophages (M2). M1 macrophages are a well-known bacteriostatic macrophage, and conversely, M2 macrophages may play an important role in tumor growth and tissue remodeling. M1 macrophages have been reported to have high intracellular iron stores, while M2 macrophages contain lower intracellular iron. It has been well-described that disturbances of iron homeostasis are associated with altered immune function. Thus, it is important to investigate if chronic iron overload is capable of polarizing macrophages. Human monocytic leukemia THP-1 cells were maintained in culture medium that contained 100 µM ferrous sulfate heptahydrate (FeSO4) (I-THP-1) and differentiated into THP-1-derived macrophages (I-TDMs) by induction with phorbol 12-myristate 13-acetate (PMA). We characterized that I-TDMs not only enhanced the surface expression of CD163 and CD206 but also increased arginase and decreased iNOS protein expression. I-TDMs enhanced pSTAT6 expression and decreased pSTAT1 and NF-κB expressions. Furthermore, the gene expression profile of I-TDMs was comparable with M2 macrophages by performing human oligonucleotide DNA microarray analysis. Finally, functional assays demonstrated I-TDMs secreted higher levels of IL-10 but not M1 cytokines. Additionally, the conditional medium of I-TDMs had enhanced migration and increased invasion of A375 melanoma cells which was similar to the characteristics of tumor-associated macrophages. Taken together, we demonstrated that THP-1-derived macrophages polarized to a phenotype of M2-like characteristics when subjected to chronic iron overload.

Subcutaneous injection of recombinant heat shock protein 70 ameliorates atopic dermatitis skin lesions in a mouse model.

Atopic dermatitis (AD) is a chronic inflammatory skin disease and sometimes is a tough challenge for physicians. We previously reported that in Th2 environment, the production and secretion of thymic stromal lymphopoietin (TSLP) from human keratinocytes was inhibited by recombinant heat shock protein 70 (rHSP70). The present study assessed the therapeutic effectiveness of rHSP70 in a mouse model of AD. An experimental model of AD was reproduced by systemic sensitization and local epicutaneous challenge with ovalbumin (OVA). Treatment of rHSP70 was performed by subcutaneous administration. The levels of OVA-specific IgE, as well as cytokines, were detected by ELISA. Skin samples from patch areas were also taken for histologic examination. Injection of rHSP70 improved the histologic picture by reducing the thickness of epidermis and allergic inflammation. Skin sonography revealed rHSP70 ameliorated skin remodeling. rHSP70 also significantly decreased the protein expression of TSLP of skin from patch areas. Furthermore, in ex vivo studies also showed group of rHSP70 treatment decreased IL-13, RANTES, MIP-1ß and increased IFN-γ secreted from splenocytes stimulated with OVA. The rHSP70 intervention in the mouse model of AD reduced the skin expression of TSLP and attenuated the clinical appearance of OVA-induced AD mice. The effect was achieved by suppressed Th2 immune response in injected skin tissue and enhanced systemic Th1 immune response. These results suggest that rHSP70 have potential as a promising protein for the treatment of AD.

Intestinal microbiota profiling and predicted metabolic dysregulation in psoriasis patients.

OBJECTIVES: The intestinal microbiota has been known to involve in obesity and host immune response. We aimed to investigate the intestinal microbiota and potential genetic function in relation to clinical presentation in psoriasis patients. METHODS: Faecal microbiota and predicted genetic function inferred from high-throughput 16S ribosomal RNA sequencing were analysed between psoriasis (n = 32) and age-, gender- and body mass index (BMI)-matched non-psoriasis subjects (n = 64), from a referral medical centre. The correlation between altered microbiota and disease activity, arthritis and systemic anti-psoriatic drugs was also investigated. RESULTS: We observed a distinct faecal microbial community structure in psoriasis patients, with an increased abundance of phylum Firmicutes and decreased abundance of phylum Bacteroidetes, across different subgroup of subjects. Ruminococcus and Megasphaera, of the phylum Firmicutes, were the top-two genera of discriminant abundance in psoriasis. A number of functional genes and metabolic pathways involving bacterial chemotaxis and carbohydrate transport were predicted over-represented, whereas genes related to cobalamin and iron transport were predicted under-represented in faecal microbiota of psoriasis patients. CONCLUSIONS: The distinct faecal microbial composition in psoriasis might be associated with altered transport of carbohydrate, cobalamin and iron, as well as chemotaxis.

Imiquimod-induced autophagy is regulated by ER stress-mediated PKR activation in cancer cells.

BACKGROUND: Autophagy is a highly conserved cellular catabolic pathway for degradation and recycling of intracellular components in response to nutrient starvation or environmental stress. Endoplasmic reticulum (ER) homeostasis can be disturbed by physiological and pathological influences, resulting in accumulation of misfolded and unfolded proteins in the ER lumen, a condition referred to as ER stress. Imiquimod (IMQ), a Toll-like receptor (TLR) 7 ligand, possesses anti-tumor and anti-viral activities in vitro and in vivo. OBJECTIVE: IMQ has been reported to promote the apoptosis of THP-1-derived macrophages through an ER stress-dependent pathway. However, the role of ER stress in IMQ-induced autophagy is unknown. In this study, we investigated the relationship between ER stress and IMQ-induced autophagy. METHODS: The expression of LC3, P62, p-PERK, Grp78, p-elF2α and IRE1α proteins were determined by immunoblotting. The relationship between ER stress and IMQ-induced autophagy were analyzed by ER stress inhibitors, a PERK inhibitor and the genetic silencing of PERK. The role of double-strand RNA-dependent protein kinase (PKR) activation in IMQ-induced autophagy was assessed by inhibiting PKR and genetically silencing PKR. The IMQ-induced autophagy was evaluated by immunoblotting and EGFP-LC3 puncta formation. RESULTS: IMQ induced reactive oxygen species (ROS) production in cancer cells. Additionally, IMQ markedly induced ER stress via ROS production and increased autophagosome formation in a dose- and time-dependent manner in both TLR7/8-expressing and TLR7/8-deficient cancer cells. Pharmacological or genetic inhibition of ER stress dramatically reduced LC3-II expression and EGFP-LC3 puncta formation in IMQ-treated cancer cells. IMQ-induced autophagy was markedly reduced by depletion and/or inhibition of PKR, a downstream effector of ER stress. CONCLUSION: IMQ-induced autophagy is dependent on PKR activation, which is mediated by ROS-triggered ER stress. These findings might provide useful information for basic research and for the clinical application of IMQ.

Simvastatin-induced cell cycle arrest through inhibition of STAT3/SKP2 axis and activation of AMPK to promote p27 and p21 accumulation in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is characterized by a poor prognosis and is one of the leading causes of cancer-related death worldwide. Simvastatin, an HMG-CoA reductase inhibitor, which decreases cholesterol synthesis by inhibiting mevalonate pathways and is widely used to treat cardiovascular diseases. Simvastatin exhibits anticancer effects against several malignancies. However, the molecular mechanisms underlying the anticancer effects of simvastatin on HCC are still not well understood. In this study, we demonstrated simvastatin-induced G0/G1 arrest by inducing p21 and p27 accumulation in HepG2 and Hep3B cells. Simvastatin also promoted AMP-activated protein kinase (AMPK) activation, which induced p21 upregulation by increasing its transcription. Consistent with this finding, we found genetic silencing of AMPK reduced p21 expression; however, AMPK silencing had no effect on p27 expression in HCC cells. Simvastatin decreased Skp2 expression at the transcriptional level, which resulted in p27 accumulation by preventing proteasomal degradation, an effect mediated by signal transducer and activator of transcription 3 (STAT3) inhibition. Constitutive STAT3 activation maintained high-level Skp2 expression and lower level p27 expression and significantly prevented G0/G1 arrest in simvastatin-treated HCC cells. Mevalonate decreased simvastatin-induced AMPK activation and rescued phospho-STAT3 and Skp2 expression in HCC cells, which resulted in the prevention of G0/G1 arrest through inhibition of p21 and p27 accumulation. Moreover, simvastatin significantly decreased tumor growth in HepG2 xenograft mice. Consistently, we found that simvastatin also increased p21 and p27 expression in tumor sections by reducing Skp2 expression and inducing AMPK activation and STAT3 suppression in the same tumor tissues. Taken together, these findings are demonstrative of the existence of a novel pathway in which simvastatin induces G0/G1 arrest by upregulating p21 and p27 by activating AMPK and inhibiting the STAT3-Skp2 axis, respectively. The results identify novel targets that explain the beneficial anticancer effects of simvastatin treatment on HCC in vitro and in vivo.