Sirtuin 6 ameliorates arthritis through modulating cyclic AMP-responsive element binding protein/CCN1/cyclooxygenase 2 pathway in osteoblasts.

INTRODUCTION: CCN1 is an immediate-early gene product pivotal for arthritis progression. We have previously shown that sirtuin 6 (SIRT6) inhibited hypoxia-induced CCN1 expression in osteoblasts. Herein we examined the contribution of cyclic AMP-responsive element binding protein (CREB)/CRE to this suppressive action and the influence of CCN1 on cyclooxygenase (COX) 2 synthesis. MATERIALS AND METHODS: MC3T3-E1 murine osteoblasts were cultured under normoxia (21% oxygen) or hypoxia (2% oxygen). Expressions of CCN1, phospho-CREB (Ser133), COX2 and relevant kinases were assessed by Western blot. SIRT6 was overexpressed in cultured osteoblasts and arthritic joints by a lentiviral-based technique. Activities of CCN1 gene promoter constructs were examined by luciferase reporter assay. Interaction between CREB and CCN1 promoter was assessed by chromatin immunoprecipitation (ChIP). Collagen-induced arthritis (CIA) was established in 20 rats to evaluate the effects of SIRT6 therapy on osteoblastic expressions of phospho-CREB, CCN1 and COX2. RESULTS: SIRT6 suppressed hypoxia-enhanced CCN1 expression and CREB phosphorylation. Attenuation of calcium/calmodulin-dependent protein kinase II (CaMKII) may be responsible for SIRT6-induced CREB inhibition. CRE at - 286 bp upstream of the ATG start codon was essential for CCN1 expression under hypoxia and SIRT6 reduced hypoxia-stimulated CREB/CRE interaction. Forced expression of CREB rescued SIRT6-suppressed CCN1 synthesis. CCN1 induced COX2 expression in osteoblasts. In rat CIA, the therapeutic effect of SIRT6 was accompanied by decreases in osteoblastic expressions of phospho-CREB, CCN1 and COX2. CONCLUSION: Our study indicated that the benefits of SIRT6 to inflammatory arthritis and bone resorption are at least partially derived from its modulation of CREB/CCN1/COX2 pathway in osteoblasts.

Calreticulin Expression Controls Cellular Redox, Stemness, and Radiosensitivity to Function as a Novel Adjuvant for Radiotherapy in Neuroblastoma.

Radiotherapy (RT) is currently only used in children with high-risk neuroblastoma (NB) due to concerns of long-term side effects as well as lack of effective adjuvant. Calreticulin (CALR) has served distinct physiological roles in cancer malignancies; nonetheless, impact of radiation on chaperones and molecular roles they play remains largely unknown. In present study, we systemically analyzed correlation between CALR and NB cells of different malignancies to investigate potential role of CALR in mediating radioresistance of NB. Our data revealed that more malignant NB cells are correlated to lower CALR expression, greater radioresistance, and elevated stemness as indicated by colony- and neurospheroid-forming abilities and vice versa. Of note, manipulating CALR expression in NB cells of varying endogenous CALR expression manifested changes in not only stemness but also radioresistant properties of those NB cells. Further, CALR overexpression resulted in greatly enhanced ROS and led to increased secretion of proinflammatory cytokines. Importantly, growth of NB tumors was significantly hampered by CALR overexpression and was synergistically ablated when RT was also administered. Collectively, our current study unraveled a new notion of utilizing CALR expression in malignant NB to diminish cancer stemness and mitigate radioresistance to achieve favorable therapeutic outcome for NB.

A Single Nucleotide Polymorphism rs1010816 Predicts Sorafenib Therapeutic Outcomes in Advanced Hepatocellular Carcinoma.

Sorafenib is currently a targeted agent widely used in the treatment of advanced hepatocellular carcinoma (aHCC). However, to date there is still a lack of a reliable marker capable of predicting sorafenib therapeutic responses. Here, we conducted a genome-wide association study (GWAS) to identify candidate single-nucleotide polymorphism outcome predictors in aHCC patients. A total of 74 real-world sorafenib-treated aHCC patients were enrolled for GWAS and outcome analysis. GWAS showed that rs1010816 (p = 2.2 × 10-7) was associated with sorafenib therapeutic response in aHCC patients. Kaplan-Meier analysis indicated that the "TT" genotype was significantly associated with a favorable therapeutic response but not significantly associated with overall survival (OS). Univariate followed by multivariate Cox proportional hazard analysis showed that ascites, main portal vein thrombosis, lower platelet count, lower total sorafenib doses, higher PALBI score in model A and higher ALBI grade in model B were significantly associated with a shorter OS. Subgroup analysis showed that only in alcoholic aHCC patients treated by sorafenib, rs1010816 "TT" genotype was significantly associated with longer OS (p = 0.021). Sorafenib had a favorable therapeutic outcome in alcoholic aHCC patients carrying rs1010816 "TT" genotype.

CRNDE acts as an epigenetic modulator of the p300/YY1 complex to promote HCC progression and therapeutic resistance.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common primary liver malignancies worldwide. The long-term prognosis for HCC remains extremely poor, with drug resistance being the major underlying cause of recurrence and mortality. The lncRNA colorectal neoplasia differentially expressed (CRNDE) is an epigenetic mediator and plays an important role to drive proliferation and drug resistance in HCC. However, CRNDE as an epigenetic regulator with influences sorafenib resistance in HCC is unclear. Thus, we explore the potential of targeting the CRNDE/p300/YY1 axis as a novel therapeutic strategy to overcome sorafenib resistance of HCC. METHOD: Detection of the expression level of CRNDE and EGFR in clinical specimens of HCC. CRNDE, EGFR, p300, and YY1expression were altered in HCC cells through transfection with different plasmids, and cell proliferation, migration, invasion, and sorafenib resistance were subsequently observed. Immunoprecipitation, chromatin immunoprecipitation, re-chromatin immunoprecipitation, site-directed mutagenesis, RNA Immunoprecipitation, immune fluorescence, qRT-PCR, and western blotting were performed to uncover the mechanisms of CRNDE regulation. The xenograft nude mice model was used to investigate the tumor growth and sorafenib resistance. RESULTS: In this study, we showed that CRNDE expression is significantly positively correlated with that of epidermal growth factor receptor (EGFR) in clinical specimens of HCC and induces proliferation and sorafenib resistance of HCC via EGFR-mediated signaling. Mechanistically, CRNDE stabilized the p300/YY1 complex at the EGFR promoter and simultaneously enhanced histone H3K9 and H3K27 acetylation, which serve as markers of relaxed chromatin. EGFR was positively upregulated by the epigenetic complex, p300/YY1, in a manner dependent on CRNDE expression, leading to enhanced tumor cell proliferation and sorafenib resistance. Furthermore, C646, a p300 inhibitor, suppressed EGFR transcriptional activity by decreasing chromatin relaxation and YY1 binding, which effectively reduced proliferation/sorafenib resistance and prolonged overall survival. CONCLUSION: Our collective findings support the potential of targeting the CRNDE/p300/YY1 axis as a novel therapeutic strategy to overcome sorafenib resistance of HCC.

The Effect of Estrogen on Intracellular Ca2+ and Na+ Regulation in Heart Failure.

Contradictory findings of estrogen supplementation in cardiac disease highlight the need to investigate the involvement of estrogen in the progression of heart failure in an animal model that lacks traditional comorbidities. Heart failure was induced by aortic constriction (AC) in female guinea pigs. Selected AC animals were ovariectomized (ACOV), and a group of these received 17ß-estradiol supplementation (ACOV+E). One hundred-fifty days post-AC surgery, left-ventricular myocytes were isolated, and their electrophysiology and Ca2+ and Na+ regulation were examined. Long-term absence of ovarian hormones exacerbates the decline in cardiac function during the progression to heart failure. Estrogen supplementation reverses these aggravating effects.

Natural Compounds Modulate Drug Transporter Mediated Oral Cancer Treatment.

Oral cancer (OC) is a serious health problem. Surgery is the best method to treat the disease but might reduce the quality of life of patients. Photodynamic therapy (PDT) may enhance quality of life but with some limitations. Therefore, the development of a new strategy to facilitate PDT effectiveness has become crucial. ATP-binding cassette G2 (ABCG2) is a membrane protein-associated drug resistance and stemness in cancers. Here, we examined whether ABCG2 plays an important role in regulating the treatment efficacy of PDT and whether ABCG2 inhibition by natural compounds can promote the effect of PDT in OC cells. Several head and neck cancer cells were utilized in this study. OECM1 and SAS cells were selected to investigate the relationship between ABCG2 expression and protoporphyrin IX (PpIX) accumulation. Western blot analysis, flow cytometry analysis, and survival probability were performed to determine PDT efficacy and cellular stemness upon treatment of different dietary compounds, including epigallocatechin gallate (EGCG) and curcumin. In this study, we found that ABCG2 expression varied in OC cells. Hypoglycemic culture for SAS cells enhanced ABCG2 expression as higher ABCG2 expression was associated with lower PpIX accumulation and cellular stemness in OC cells. In contrast, suppression of ABCG2 expression by curcumin and tea polyphenol EGCG led to greater PpIX accumulation and enhanced PDT treatment efficiency in OC cells. In conclusion, ABCG2 plays an important role in regulating the effect of PDT. Change in glucose concentration and treatment with natural compounds modulated ABCG2 expression, resulting in altered PDT efficacy for OC cells. These modulations raise a potential new treatment strategy for early-stage OCs.

Effect of ovariectomy on intracellular Ca2+ regulation in guinea pig cardiomyocytes.

This study addressed the hypothesis that long-term deficiency of ovarian hormones after ovariectomy (OVx) alters cellular Ca2+-handling mechanisms in the heart, resulting in the formation of a proarrhythmic substrate. It also tested whether estrogen supplementation to OVx animals reverses any alterations to cardiac Ca2+ handling and rescues proarrhythmic behavior. OVx or sham operations were performed on female guinea pigs using appropriate anesthetic and analgesic regimes. Pellets containing 17ß-estradiol (1 mg, 60-day release) were placed subcutaneously in selected OVx animals (OVx + E). Cardiac myocytes were enzymatically isolated, and electrophysiological measurements were conducted with a switch-clamp system. In fluo-4-loaded cells, Ca2+ transients were 20% larger, and fractional sarcoplasmic reticulum (SR) Ca2+ release was 7% greater in the OVx group compared with the sham group. Peak L-type Ca2+ current was 16% larger in OVx myocytes with channel inactivation shifting to more positive membrane potentials, creating a larger "window" current. SR Ca2+ stores were 22% greater in the OVx group, and these cells showed a higher frequency of Ca2+ sparks and waves and shorter wave-free intervals. OVx myocytes showed higher frequencies of early afterdepolarizations, and a greater percentage of these cells showed delayed afterdepolarizations after exposure to isoprenaline compared with sham myocytes. The altered Ca2+ regulation occurring in the OVx group was not observed in the OVx + E group. These findings suggest that long-term deprivation of ovarian hormones in guinea pigs lead to changes in myocyte Ca2+-handling mechanisms that are considered proarrhythmogenic. 17ß-Estradiol replacement prevented these adverse effects.NEW & NOTEWORTHY Ovariectomized guinea pig cardiomyocytes have higher frequencies of Ca2+ waves, and isoprenaline-challenged cells display more early afterdepolarizations, delayed afterdepolarizations, and extra beats compared with sham myocytes. These alterations to Ca2+ regulation were not observed in myocytes from ovariectomized guinea pigs supplemented with 17ß-estradiol, suggesting that ovarian hormone deficiency modifies cardiac Ca2+ regulation, potentially creating proarrhythmic substrates.

Epigallocatechin-3-gallate diminishes cytokine-stimulated Cyr61 expression in human osteoblastic cells: a therapeutic potential for arthritis.

OBJECTIVE: To assess the effects of epigallocatechin-3-gallate (EGCG) on cytokine-induced Cyr61 synthesis in human osteoblastic cells and the associated signalling pathways. The therapeutic effect of EGCG on CIA in rats was also studied. METHODS: The expression of Cyr61 and NF-κB pathway molecules was examined by western blotting. CCL2 expression was assessed by northern blotting and ELISA. Interaction between NF-κB and Cyr61 promoter was evaluated by electrophoretic mobility shift assay. In rat CIA, osteoblastic expression of Cyr61 was examined by immunohistochemistry and disease progression was assessed by clinical, radiographic and histological examinations. RESULTS: EGCG inhibited Cyr61 expression stimulated by cytokines in primary human osteoblasts and human osteoblastic cell line U2OS. In U2OS, oncostatin M (OSM) induced IκB-α degradation through the mTOR/rictor/Akt pathway, and EGCG attenuated the action. Electrophoretic mobility shift assay revealed that the OSM-enhanced NF-κB/DNA binding was reduced by EGCG, possibly through abrogating nucleus localization of p65 and p50. Cyr61 enhanced OSM-induced expression of CCL2. Moreover, EGCG diminished OSM-stimulated CCL2 expression at least partially via suppressing Cyr61 induction. Co-distribution of CD68(+) macrophages and Cyr61(+) osteoblasts in osteolytic areas was obvious in the CIA model. Clinical, radiographic and immunohistochemical analyses revealed that administration of EGCG markedly diminished the severity of CIA, macrophage infiltration, and the number of Cyr61-synthesizing osteoblasts. CONCLUSION: By modulating the mTOR/rictor/Akt/NF-κB pathway, EGCG attenuated Cyr61 production in osteoblastic cells and in turn diminished macrophage chemotaxis. Our data support the therapeutic potential of EGCG on arthritis.

5-aminolevulinic acid induce apoptosis via NF-κB/JNK pathway in human oral cancer Ca9-22 cells.

BACKGROUND: 5-aminolevulinic acid-based photodynamic therapy (5-ALA-PDT) is being used to treat oral pre-cancerous and cancerous lesions with some encouraging clinical outcomes. However, the exact mechanisms behind the photodynamic treatment are still not fully elucidated. METHOD: Flow cytometry, TdT-mediated dUTP nick end labeling assay and Western blot analysis were used to investigate the effects of 5-ALA-PDT on human oral cancer Ca9-22 cells. RESULTS: We found that 5-ALA-PDT induces apoptosis in Ca9-22 cells. Western blotting showed that 5-ALA-PDT activates both the caspase-8 and caspase-9 pathways, which differed from previous studies conducted in other cell types. Activation of JNK was evident as early as 30 min. The caspases activation was inhibited by JNK inhibitor SP600125. Treatment with NF-κB inhibitor Bay 11-7082 (Bay) completely abrogated ALA-PDT-induced JNK activation. In addition, Bay and SP600125 almost completely abolished ALA-PDT-induced apoptosis. CONCLUSION: These results demonstrate significant involvement of caspase-8 and -9 and their upstream NF-κB-JNK pathways in ALA-PDT-induced apoptosis. Future studies on how NF-κB and JNK activity regulate ALA-PDT response should provide a better strategy for the treatment of oral cancer.

Topical photodynamic therapy is very effective for oral verrucous hyperplasia and oral erythroleukoplakia.

BACKGROUND: Oral verrucous hyperplasia (OVH) and oral erythroleukoplakia (OEL) are two oral precancerous lesions with relatively high malignant transformation potential. One of the best cancer prevention strategies is to use a conservative and effective treatment modality to eliminate oral precancers to stop their further malignant transformation. Our previous studies have shown that the topical 5-aminolevulinic acid-mediated photodynamic therapy (topical ALA-PDT) using the 635-nm light-emitting diode (LED) light is very effective for OVH and OEL lesions. METHODS: Because the laser machine is a more-popular light source than the LED device in PDT clinics, in this study 40 OVH and 40 OEL lesions were treated once a week with the same PDT protocol but using the 635-nm laser light to evaluate whether this laser light-mediated topical ALA-PDT was also effective for OVH and OEL lesions. RESULTS: We found that all the 40 OVH lesions exhibited complete response (CR) after an average of 3.6 PDT treatments. Of the 40 OEL lesions, 38 showed CR after an average of 3.4 PDT treatments and two showed partial response (PR). Better PDT outcomes were significantly associated with OVH and OEL lesions with the smaller size, pink to red color, epithelial dysplasia, or thinner surface keratin layer. CONCLUSION: This study indicates that the laser light-mediated topical ALA-PDT is also very effective for OVH and OEL lesions. Therefore, we suggest that topical ALA-PDT using either the LED or laser light may serve as the first-line treatment of choice for OVH and OEL lesions.

Comparison of clinical outcomes of oral erythroleukoplakia treated with photodynamic therapy using either light-emitting diode or laser light.

BACKGROUND AND OBJECTIVES: Topical 5-aminolevulinic acid-mediated photodynamic therapy (topical ALA-PDT) using a 635-nm light-emitting diode (LED) light is an effective treatment modality for oral verrucous hyperplasia. This study tested whether topical ALA-PDT using either the LED or laser light was also an effective treatment modality for oral erythroleukoplakia (OEL) lesions. STUDY DESIGN/MATERIALS AND METHODS: In this prospective but non-randomized study, 20 OEL lesions were treated with topical ALA-PDT using the 635-nm LED light and 26 OEL lesions were treated with topical ALA-PDT using the 635-nm laser light. The difference in clinical outcomes was compared between the two groups by Fisher exact test. RESULTS: We found that the 20 LED light-treated OEL lesions showed complete response (CR) in 17 and partial response (PR) in 3. The 17 CR OEL lesions required an average of 3.7 (range, 2-7) treatments of ALA-PDT to achieve CR of the lesions. The 26 laser light-treated OEL lesions showed CR in 25 and PR in 1. The 25 CR OEL lesions needed an average of 3.3 (range, 2-6) treatments of ALA-PDT to achieve CR of the lesions. There was no significant difference in PDT outcomes between the 20 LED light-treated and 26 laser light-treated OEL lesions (P = 0.303). When the 42 CR OEL lesions were pooled together, we found that smaller lesions (greatest diameter <1.5 cm) and lesions with thinner surface keratin (keratin layer < or =30 microm) needed significantly fewer mean treatment number of PDT to achieve a CR than the larger lesions (P = 0.000) and lesions with thicker surface keratin (P = 0.000), respectively. CONCLUSIONS: Topical ALA-PDT using either the LED or laser light is an effective treatment modality for OEL lesions. There is no significant difference in clinical outcomes of OEL lesions treated with PDT using either the LED or laser light.

Comparison of clinical effects between warm water spray and sitz bath in post-hemorrhoidectomy period.

BACKGROUND: Warm water sitz bath is advised for a variety of anorectal disorders. However, preparation of the sitz bath is sometimes difficult for patients. As an alternative to the sitz bath, we have adapted a water spray method. A randomized, controlled study was conducted to determine if the water spray method has similar effects to the sitz bath in the post-hemorrhoidectomy period and it is easy to carry out. METHODS: A total of 120 patients were randomly assigned to water spray or sitz bath groups. All patients received analgesics and a fiber-rich diet after hemorrhoidectomy. Clinical parameters including pain, irritation (burning or itching sensations), hygiene, convenience, and overall satisfaction were evaluated by a visual analog scale to assess treatment outcome in both groups. RESULTS: There was no obvious difference in age, gender distribution, body mass index, or duration of disease between groups. There were no significant difference in scores for postoperative pain (p = 0.23), irritation (p = 0.48), or hygiene (p = 0.725) between groups. However, the water spray group reported significantly greater convenience (p < 0.05) and higher overall satisfaction (p < 0.05) compared with the sitz bath group. At the end of the 4-week postoperative follow-up period, 90% of patients in the watery spray group and 93% of patients in the sitz bath group showed complete wound healing. There were no significant differences in postoperative complications between groups. CONCLUSION: Our results demonstrate that the water spray method could provide a safe and reliable alternative to the sitz bath for post-hemorrhoidectomy care. Furthermore, the water spray method could be used instead of the sitz bath as a more convenient and satisfactory form of treatment.