Co-delivery of cisplatin and doxorubicin by covalently conjugating with polyamidoamine dendrimer for enhanced synergistic cancer therapy.

Because of the synergistic effects of drugs and minimal drug dose for cancer therapy, combination chemotherapy is frequently used in the clinic. In this study, hyaluronic acid-modified amine-terminated fourth-generation polyamidoamine dendrimer nanoparticles were synthesized for systemic co-delivery of cisplatin and doxorubicin (HA@PAMAM-Pt-Dox). In vitro data showed that HA@PAMAM-Pt-Dox can enter the cells through the lysosome mediated-pathway in a time-dependent manner. Cell viability studies indicated that HA@PAMAM-Pt-Dox exhibited a higher anticancer activity on MCF-7 and MDA-MB-231 breast cancer cells at a relative low concentration. HA@PAMAM-Pt-Dox not only efficiently inhibited tumor growth but also significantly reduced the toxicity of Dox. Moreover, intravenous administration of HA@PAMAM-Pt-Dox to MDA-MB-231 tumor-bearing BALB/c nude mice resulted in the accumulation of HA@PAMAM-Pt-Dox at the tumor site, thereby significantly inhibiting tumor growth without apparent toxicity. These results suggested that HA@PAMAM-Pt-Dox has great potential to improve the chemotherapeutic efficacy of cisplatin and doxorubicin in breast cancer. STATEMENT OF SIGNIFICANCE: One of the main problems in cancer treatment is the development of drug resistance. To date, it is believed that combination chemotherapy might be an effective strategy for the above problem. However, for two completely different drugs, combination chemotherapy faces huge difficulties including the antagonistic nature of drugs, variations in drugs in terms of solubility, and limited tumor targeting. Recent developments in nanoscience and nanotechnology provide an effective approach for such disadvantages. Considering the advantages of dendrimers such as control of size and molecular weight, bioavailability, and biosafety, we used fourth-generation dendrimers modified by HA as drug vectors by covalently conjugating them with anticancer drugs (cisplatin and doxorubicin) to form a nanodrug delivery system, named HA@PAMAM-Pt-Dox. We observed that the HA@PAMAM-Pt-Dox system can effectively kill breast cancer cells both in vitro and in vivo, which showed a favorable synergistic effect. This strategy can be extended to other drugs, thus providing a highly effective strategy for cancer treatment.

Chronic intermittent hypoxia induces cardiac hypertrophy by impairing autophagy through the adenosine 5'-monophosphate-activated protein kinase pathway.

Autophagy is tightly regulated to maintain cardiac homeostasis. Impaired autophagy is closely associated with pathological cardiac hypertrophy. However, the relationship between autophagy and cardiac hypertrophy induced by chronic intermittent hypoxia (CIH) is not known. In the present study, we measured autophagy-related genes and autophagosomes during 10 weeks of CIH in rats, and 6 days in H9C2 cardiomyocytes, and showed that autophagy was impaired. This conclusion was confirmed by the autophagy flux assay. We detected significant hypertrophic changes in myocardium with impaired autophagy. Rapamycin, an autophagy enhancer, attenuated the cardiac hypertrophy induced by CIH. Moreover, silencing autophagy-related gene 5 (ATG5) exerted the opposite effect. The role of adenosine monophosphate-activated protein kinase (AMPK) in regulating autophagy under CIH was confirmed using AICAR to upregulate this enzyme and restore autophagy flux. Restoring autophagy by AICAR or rapamycin significantly reversed the hypertrophic changes in cardiomyocytes. To investigate the mechanism of autophagy impairment, we compared phospho (p)-AMPK, p-Akt, cathepsin D, and NFAT3 levels, along with calcineurin activity, between sham and CIH groups. CIH activated calcineurin, and inhibited AMPK and AMPK-mediated autophagy in an Akt- and NFAT3-independent manner. Collectively, these data demonstrated that impaired autophagy induced by CIH through the AMPK pathway contributed to cardiac hypertrophy.

P2X7 Receptor Antagonism Attenuates the Intermittent Hypoxia-induced Spatial Deficits in a Murine Model of Sleep Apnea Via Inhibiting Neuroinflammation and Oxidative Stress.

BACKGROUND: The mechanism of the neural injury caused by chronic intermittent hypoxia (CIH) that characterizes obstructive sleep apnea syndrome (OSAS) is not clearly known. The purpose of this study was to investigate whether P2X7 receptor (P2X7R) is responsible for the CIH-induced neural injury and the possible pathway it involves. METHODS: Eight-week-old male C57BL/6 mice were used. For each exposure time point, eight mice divided in room air (RA) and IH group were assigned to the study of P2X7R expression. Whereas in the 21 days-Brilliant Blue G (BBG, a selective P2X7R antagonist) study, 48 mice were randomly divided into CIH group, BBG-treated CIH group, RA group and BBG-treated RA group. The hippocampus P2X7R expression was determined by Western blotting and real-time polymerase chain reaction (PCR). The spatial learning was analyzed by Morris water maze. The nuclear factor kappa B (NFκB) and NADPH oxidase 2 (NOX2) expressions were analyzed by Western blotting. The expressions of tumor necrosis factor α, interleukin 1ß (IL-ß), IL-18, and IL-6 were measured by real-time PCR. The malondialdehyde and superoxide dismutase levels were detected by colorimetric method. Cell damage was evaluated by Hematoxylin and Eosin staining and Terminal Transferase dUTP Nick-end Labeling method. RESULTS: The P2X7R mRNA was elevated and sustained after 3-day IH exposure and the P2X7R protein was elevated and sustained after 7-day IH exposure. In the BBG study, the CIH mice showed severer neuronal cell damage and poorer performance in the behavior test. The increased NFκB and NOX2 expressions along with the inflammation injury and oxidative stress were also observed in the CIH group. BBG alleviated CIH-induced neural injury and consequent functional deficits. CONCLUSIONS: The P2X7R antagonism attenuates the CIH-induced neuroinflammation, oxidative stress, and spatial deficits, demonstrating that the P2X7R is an important therapeutic target in the cognition deficits accompanied OSAS.

Regulatory effects of AT1R-TRAF6-MAPKs signaling on proliferation of intermittent hypoxia-induced human umbilical vein endothelial cells.

Endothelial dysfunction induced by intermittent hypoxia (IH) participates in obstructive sleep apnea syndrome (OSAS)-associated cardiovascular disorders. Myeloid differentiation primary response 88 (MyD88) and tumor necrosis factor receptor-associated factor 6 (TRAF6) regulate numerous downstream adaptors like mitogen-activated protein kinases (MAPKs) and the subsequent oxidative stress and inflammatory responses. This study aimed to characterize the role of MyD88/TRAF6 in IH-treated cell function and its associated signaling. Human umbilical vein endothelial cells (HUVECs) were randomly exposed to IH or normoxia for 0, 2, 4 and 6 h. Western blotting was used to detect the expression pattern of target gene proteins [angiotensin 1 receptor (AT1R), p-ERK1/2, p-p38MAPK, MyD88 and TRAF6], and the relationships among these target genes down-regulated by the corresponding inhibitors were studied. Finally, the influence of these target genes on proliferation of HUVECs was also assessed by EdU analysis. Protein levels of AT1R, TRAF6 and p-ERK1/2 were increased after IH exposure, with a slight rise in MyD88 and a dynamic change in p-p38MAPK. The down-regulation of TRAF6 by siRNA reduced ERK1/2 phosphorylation during IH without any effects on AT1R. Blockade of AT1R with valsartan decreased TRAF6 and p-ERK1/2 protein expression after IH exposure. ERK1/2 inhibition with PD98059 suppressed only AT1R expression. IH promoted HUVECs proliferation, which was significantly suppressed by the inhibition of TRAF6, AT1R and ERK1/2. The findings demonstrate that TRAF6 regulates the proliferation of HUVECs exposed to short-term IH by modulating cell signaling involving ERK1/2 downstream of AT1R. Targeting the AT1R-TRAF6-p-ERK1/2 signaling pathway might be helpful in restoring endothelial function.

Melatonin protects against chronic intermittent hypoxia-induced cardiac hypertrophy by modulating autophagy through the 5' adenosine monophosphate-activated protein kinase pathway.

Obstructive sleep apnea syndrome (OSAS) is usually associated with multiple cardiovascular disorders, including myocardial hypertrophy. Melatonin protects the heart from damaging conditions. However, whether melatonin alleviates heart damage induced by chronic intermittent hypoxia (CIH) is unknown. We investigated the melatonin-induced protective role of AMPK-regulated autophagy in the myocardium by exposing rats to CIH and treating them with melatonin or saline daily for six weeks. In vivo, CIH induced significant myocardial hypertrophy; this trend was strikingly reversed by melatonin. Moreover, AMPK activation and autophagy was enhanced, and the number of autophagosomes increased. CIH induced apoptosis of cardiomyocytes; this was significantly mitigated by melatonin. In vitro, CIH induced hypertrophic changes in cardiomyocytes; this effect was significantly reversed by melatonin. Autophagy decreased after AMPK inhibition, and we found that autophagy was required for the protective function of melatonin. Our results suggest that melatonin ameliorates cardiac hypertrophy caused by CIH by inducing autophagy via the AMPK pathway and by autophagy-regulated apoptosis.

Efficacy of atorvastatin on hippocampal neuronal damage caused by chronic intermittent hypoxia: Involving TLR4 and its downstream signaling pathway.

Hippocampal neuronal damage is critical for the initiation and progression of neurocognitive impairment accompanied obstructive sleep apnea syndrome (OSAS). Toll-like receptor 4 (TLR4) plays an important role in the development of several hippocampus-related neural disorders. Atorvastatin was reported beneficially regulates TLR4. Here, we examined the effects of atorvastatin on hippocampal injury caused by chronic intermittent hypoxia (CIH), the most characteristic pathophysiological change of OSAS. Mice were exposed to intermittent hypoxia with or without atorvastatin for 4 weeks. Cell damage, the expressions of TLR4 and its two downstream factors myeloid differentiation factor 88 (MYD88) and TIR-domain-containing adapter-inducing interferon-ß (TRIF), inflammatory agents (tumor necrosis factor α and interleukin 1ß), and the oxidative stress (superoxide dismutase and malondialdehyde) were determined. Atorvastatin decreased the neural injury and the elevation of TLR4, MyD88, TRIF, pro-inflammatory cytokines and oxidative stress caused by CIH. Our study suggests that atorvastatin may attenuate CIH induced hippocampal neuronal damage partially via TLR4 and its downstream signaling pathway.

Telmisartan attenuates myocardial apoptosis induced by chronic intermittent hypoxia in rats: modulation of nitric oxide metabolism and inflammatory mediators.

PURPOSE: NO and NO synthase (NOS) are known to play key roles in the development of myocardial apoptosis induced by ischemia/hypoxia. Current evidence suggests that angiotensin II type 1 receptor blockers, such as telmisartan, lower blood pressure and produce beneficial regulatory effects on NO and NOS. Here, we examined the protective role of telmisartan in myocardial apoptosis induced by chronic intermittent hypoxia (CIH). METHODS: Adult male Sprague-Dawley rats were subjected to 8 h of intermittent hypoxia/day, with/without telmisartan for 8 weeks. Myocardial apoptosis, NO and NOS activity, and levels of inflammatory mediators and radical oxygen species were determined. RESULTS: Treatment with telmisartan preserved endothelial NOS expression and inhibited inducible NOS and excessive NO generation, while reducing oxidation/nitration stress and inflammatory responses. Administration of telmisartan before CIH significantly ameliorated the CIH-induced myocardial apoptosis. CONCLUSIONS: This study show that pre-CIH telmisartan administration ameliorated myocardial injury following CIH by attenuating CIH-induced myocardial apoptosis via regulation of NOS activity and inhibition of excessive NO generation, oxidation/nitration stress, and inflammatory responses.

Clinical characteristics of hereditary multiple exostoses: a retrospective study of mainland chinese cases in recent 23 years.

Hereditary multiple exostoses (HME) are an autosomal dominant skeletal disease with wide variations in clinical manifestations among different ethnic groups. This study investigated the epidemiology, clinical presentations, pathogenetic features and treatment strategies of HME in mainland China. We searched and reviewed the related cases published since 1990 by searching electronic databases, namely SinoMed database, Wanfang database, CNKI, Web of Science and PubMed as well as Google search engines. A total of 1051 cases of HME (male-to-female ratio 1.5:1) were investigated and the diagnosis was made in 83% before the age of 10 years. Approximately 96% patients had a family history. Long bones, ribs, scapula and pelvis were the frequently affected sites. Most patients were asymptomatic with multiple palpable masses. Common complications included angular deformities, impingement on neighbouring tissues and impaired articular function. Chondrosarcomas transformation occurred in 2% Chinese cases. Among the cases examined, about 18% had mutations in EXT1 and 28% in EXT2. Frameshift, nonsense and missense mutations represented the majority of HME-causing mutations. Diagnosis of HME was made based on the clinical presentations and radiological documentations. Most patients needed no treatment. Surgical treatment was often directed to remove symptomatic exostoses, particularly those of suspected malignancy degeneration, and correction of skeletal deformities. This study shows some variance from current literature regarding other ethnic populations and may provide valuable baseline assessment of the natural history of HME in mainland China.

ERK signaling mediates enhanced angiotensin II-induced rat aortic constriction following chronic intermittent hypoxia.

BACKGROUND: Obstructive sleep apnea (OSA) has been recognized as an independent risk factor for systemic hypertension. The study investigated the functional consequences of chronic intermittent hypoxia (CIH) on aortic constriction induced by angiotensin II (Ang II) and the possible signaling involving ERK1/2 and contractile proteins such as myosin light chain kinase (MLCK), myosin phosphatase targeting subunit (MYPT1) and myosin light chain (MLC). METHODS: Male Wistar rats were randomly divided into CIH group and normoxia group and exposed to either CIH procedure or air-air cycles. Phosphorylation of ERK1/2, MYPT1 and MLC was assessed by Western blotting following constrictor studies in the presence or absence of PD98059 (10 µmol/L). RESULTS: CIH-exposure resulted in more body weight gain and elevated blood pressure, which could be attenuated by pretreatment with PD98059. Endothelium-removed aortic rings from CIH rats exhibited higher constrictor sensitivity to Ang II (Emax: (138.56 ± 5.78)% versus (98.45±5.31)% of KCl; pD2: 7.98 ± 0.14 versus 8.14 ± 0.05, respectively). CIH procedure exerted complex effects on ERK expressions (total ERK1/2 decreased whereas the ratio of phosphorylated to total ERK1/2 increased). CIH aortas had higher MLCK mRNA and basal phosphorylation of MYPT1 and MLC. In parallel to greater increases in phosphorylation of ERK1/2, MYPT1 and MLC, Ang II-induced aortic constriction was significantly enhanced in CIH rats, which was largely reversed by PD98059. However vascular constriction of normoxia rats remained unchanged despite similar but smaller changing tendency of proteins phosphorylation. CONCLUSION: These data suggest that CIH exposure results in aortic hyperresponsiveness to Ang II, presumably owing to more activated ERK1/2 signaling pathway.

Ang II type 1 receptor expression in rat aorta exposed to chronic intermittent hypoxia: effects of p38MAPK and ERK1/2 signaling.

BACKGROUND: Obstructive sleep apnea is a frequent medical condition consisting of repetitive sleep-related episodes of upper air ways obstruction and can lead to hypertension. Ang II type 1 receptor (AT1R) played important roles in hypertension since it binds with Ang II, controlling salt-water and blood pressure homeostasis. This study explores rat aorta AT1R expression during intermittent hypoxia (IH) and the signaling pathways involved. METHODS: A rat model and a cell model used a BioSpherix-OxyCycler A84 system and a ProOx C21 system respectively. The arterial blood pressure was recorded by a Nihon Kohden Polygraph System. Immunohistochemic was used to focus and analyze the expression of AT1R in rat aorta. Real-time PCR and Western blotting were used to explore the signaling pathways that participated in AT1R expression. RESULTS: In this study, we found that chronic intermittent hypoxia (CIH) induced AT1R transcription which increased the blood pressure in rat aorta compared to normoxia and to sustained hypoxia. The AT1R protein expression in the aorta was similar to the real-time PCR results. We explored the signaling mechanisms involved in the AT1R induction in both rat aorta and the aortic endothelial cells by real-time PCR and Western blotting. Compared to normoxia, CIH increased ERK1 mRNA transcription but not ERK2 or p38MAPK in the aorta; whereas sustained hypoxia (SH) upregulated ERK2 but not ERK1 or p38MAPK mRNA. In cells, IH induced AT1R expression with ERK1/2 phosphorylation but reduced p38MAPKs phosphorylation, whereas SH induced only ERK1/2 phosphorylation. The ERK1/2 inhibitor PD98059 attenuated the IHinduced AT1R increase but the p38MAPK inhibitor SB203580 did not. CONCLUSIONS: Our results indicate that CIH induced the elevation of rat blood pressure and aorta AT1R expression. Moreover, AT1R expression in IH and sustained hypoxia might be regulated by different signal transduction pathways, highlighting a novel regulatory function through ERK1/2 signaling in IH.