Supplementary Data from Effects of Plasmid-Based Stat3-Specific Short Hairpin RNA and GRIM-19 on PC-3M Tumor Cell Growth

Melanoma is one of the most lethal skin malignancies in the world. Interferons (IFNs) have been also demonstrated in response to tumor cell and IFNs such as IFN-α have been used for melanoma treatment. The long chain double-stranded RNA (dsRNA) (from a variety of nonviral sources) is a potent activator of the IFN system and an inducer of cell apoptosis. Panaxadiolsaponins (PDS) is a major Panax ginseng-derived active component with known antitumor activity and immune modulation. Here, we investigated a hypothesis that the combination of PDS and total natural dsRNA (as opposed to the synthetic dsRNA) will suppress tumor growth better than the individual agents. We have evaluated the antitumor and immunostimulatory effects of the combination of natural long chain dsRNA (derived from yeast) and PDS on melanoma cell line B16 and mice xenograft model. The underlying mechanisms of growth suppression were investigated by analyzing dsRNA-activated pathways, apoptosis, and cell cycle. Natural dsRNA and PDS exert superior anticancer effects than either agent alone. Natural dsRNA and PDS combination might be a promising strategy for treating malignancies, including melanoma.

Prostate-specific antigen (PSA)-based mass screening remains the most controversial topic in prostate cancer. PSA-based mass screening has not been widely used in China yet. The aim of our study was to evaluate the effect of the PSA-based screening in China. The cohort consisted of 1,012 prostate cancer patients. Data were retrospectively collected and clinical characteristics of the cohorts were investigated. Survival was analyzed for prostatic carcinoma of both PSA screened and clinically diagnosed patients according to clinical characteristics and the National Comprehensive Cancer Network (NCCN) risk classification. Cox Proportional Hazards Model analysis was done for risk predictor identification. The median age was 71 years old. Five-year overall and prostate-cancer-specific survival in prostatic adenocarcinoma patients were 77.52% and 79.65%; 10-year survivals were 62.57% and 68.60%, respectively. Survival was significantly poorer in patients with metastases and non-curative management. T staging and Gleason score by NCCN classification effectively stratified prostatic adenocarcinoma patients into different risk groups. T staging was a significant predictor of survival by COX Proportional Hazard Model. PSA screened patients had a significantly higher percentage diagnosed in early stage. PSA screened prostatic adenocarcinoma patients had a better prognosis in both overall and prostate cancer-specific survivals. This Chinese cohort had a lower overall and prostate cancer survival rate than it is reported in western countries. The incidence of early-stage prostate cancer found in PSA-based mass screening was high and there were significant differences in both overall and prostate cancer-specific survival between the PSA-screened and clinically diagnosed patients.

目的 探究冠心病心脏支架患者的护理对策及效果.方法 随机选取我院收治的74例患者进行分析研究,将全部患者随机均分为观察组和对照组两组各37例.对照组患者术后进行常规护理,观察组患者实施综合护理干预.结果 观察组患者经过综合护理后的心绞痛发作次数、焦虑程度与生活质量以及患者满意度等评分情况均显著优于对照组患者.结论 将综合护理干预措施应用于冠心病患者经皮冠状动脉成形术与支架置入术治疗过程中能够有效改善患者心理状态,提高患者对护理服务的满意度,在临床上具有广泛的推广价值.

1964年,我在第二中心医院实习.当时医院药剂科不设中药房,中药调剂要去西北角万年青药店实习.当我迈进中药房,老师傅一开口就给我讲了一堂课. 万年青药店有个惯例,对新来的学徒进门上第一堂课就是“事故课”,把建店以来重大的事故汇总讲给将要上岗者听.他们不护短,他们说得明白:揭自己的伤疤能不疼吗?为了引以为戒,为了不叫更多的人受伤害,为了救死扶伤,值得!

The present study investigated the effect of signal transducer and activator of transcription 3 (Stat3) interference on RM1 prostate cancer cell viability in vitro, using plasmid‑based Stat3 specific short hairpin RNA (sh‑Stat3) delivered by hydroxyapatite nanoparticles (HAP). HAP carrying sh‑Stat3 plasmids were transfected into tumor cells. MTT assays were used to measure RM1 cell viability 24 and 48 h following transfection, and the apoptosis rate and cell cycle phase distribution were determined by flow cytometry. Stat3 mRNA expression levels were measured by reverse transcription‑quantitative polymerase chain reaction and Stat3, Cyclin D1, B cell lymphoma 2 apoptosis regulator (Bcl‑2), vascular endothelial growth factor (VEGF), Bcl‑2 associated X apoptosis regulator (Bax) and cleaved‑caspase‑3 protein expression levels were detected using western blot analysis. The results demonstrated that HAP‑delivered sh‑Stat3 significantly decreased RM1 cell viability through the promotion of cell cycle arrest and apoptosis. Stat3 mRNA and protein expression levels were significantly downregulated in RM1 cells. Bcl‑2, VEGF and Cyclin D1 were also significantly downregulated, but cleaved‑caspase‑3 and Bax mRNA and protein expression levels were significantly upregulated. HAP‑delivered sh‑Stat3 decreased RM1 cell viability in vitro, and HAP assisted plasmid‑based delivery of shRNA into tumor cells. The present results suggest that HAP may be a useful method for successful shRNA delivery into tumors.

Severe acute pancreatitis (SAP) is a serious necro-inflammatory disease with a high mortality rate due to lack of specific treatments. Previous studies have shown that panaxadiolsaponins (PDS) involved in the regulation of immune function and protection against cell injury. The aim of our study was to investigate the protective effects of PDS on liver and kidney injury in rats with SAP. Sixty-four rats were divided into four groups (n=16): Sham Operation (SO) group, SAP group, Dexamethasone (DEX) group and PDS group. The results showed that PDS significantly decreased the level of serum AMS and GLU of SAP rats (P<0.05), while DEX did not affect the level of serum AMS and GLU. DEX retarded the increasing of serum Hb and HCT of SAP rats (P<0.05), and PDS retarded the increasing of Hb, RBC and HCT (P<0.05). DEX and PDS significantly decreased the level of serum BUN, TNF-alpha and IL-6 (P<0.01). Results of pathological analysis showed that PDS could protect against liver and kidney injury in SAP rats. To the best of our knowledge, this is the first report that PDS could protect against liver and kidney injury in SAP rats and had better therapeutic effects than DEX, indicating a potential drug to be used in treating SAP.

RNA interference (RNAi) has been used for cancer gene therapy in recent years. However, the application of RNAi is hindered in the absence of safe and efficient gene delivery. In this article, a novel vehicle of graphene oxide functionalized with polyethylenimine and polyethylene glycol (GO-PEI-PEG) was successfully synthetized and then used to deliver plasmid-based Stat3 siRNA. The carrier can readily bind plasmid with high transfection efficiency. Moreover, molecular biology studies reveal that Stat3-related gene and protein expressions were significantly inhibited, suggesting that the formation of GO-PEI-PEG complexes could be utilized as a promising gene delivery in cancer therapy.

Aldo-keto reductase family 1 member C3 has recently been regarded as a potential therapeutic target in castrate-resistant prostate cancer. Herein, we investigated whether berberine delayed the progression of castrate-resistant prostate cancer by reducing androgen synthesis through the inhibition of Aldo-keto reductase family 1 member C3. Cell viability and cellular testosterone content were measured in prostate cancer cells. Aldo-keto reductase family 1 member C3 mRNA and protein level were detected by RT-PCR and Western bolt analyses, respectively. Computer analysis with AutoDock Tools explored the molecular interaction of berberine with Aldo-keto reductase family 1 member C3. We found that berberine inhibited 22Rv1 cells proliferation and decreased cellular testosterone formation in a dose-dependent manner. Berberine inhibited Aldo-keto reductase family 1 member C3 enzyme activity, rather than influenced mRNA and protein expressions. Molecular docking study demonstrated that berberine could enter the active center of Aldo-keto reductase family 1 member C3 and form p-p interaction with the amino-acid residue Phe306 and Phe311. In conclusion, the structural interaction of berberine with Aldo-keto reductase family 1 member C3 is attributed to the suppression of Aldo-keto reductase family 1 member C3 enzyme activity and the inhibition of 22Rv1 prostate cancer cell growth by decreasing the intracellular androgen synthesis. Our result provides the experimental basis for the design, research, and development of AKR1C3 inhibitors using berberine as the lead compound.

Gastric cancer remains one of the most prevalent and lethal malignancies in the world. Despite new advances in treatment and diagnosis, patients with advanced gastric cancer are still difficult to cure resulting in a high mortality rate and poor prognosis. Signal transducer and activator of transcription 3 (Stat3) is observed aberrant in multiple tumours, including gastric cancer. Stat3 overexpression was confirmed performing a vital role in tumorigenesis. In the present study, we constructed a pSi-Stat3 plasmid to silence Stat3 and investigated the effect of pSi-Stat3 on cell proliferation, apoptosis and cell cycle progression in gastric cancer cell line SGC-7901 and mice xenograft model. Downstream proteins of Stat3, including Cyclin-D1, Survivin and Bcl-2, were detected as well for the underlying mechanism exploration. It showed that pSi-Stat3 can effectively silence the expression of Stat3 and inhibits the growth of gastric tumour both in vitro and in vivo significantly via cell apoptosis and cell cycle shift induction. The findings suggest that Stat3 signal pathway might be a promising therapeutic target for tumour treatment, including gastric cancer.

目的：尿素通道蛋白B（ urea transporter B， UT－B）是介导尿素快速跨膜转运的膜蛋白，UT－B基因敲除后可导致老龄小鼠发生心肌肥大，本研究拟探讨UT－B基因敲除后对小鼠线粒体功能的影响。方法：应用试剂盒检测线粒体呼吸链复合体的活性， PCR检测mtDNA 基因D－loop区域多态性／突变情况，流式细胞术检测线粒体膜电位和ROS水平。结果：与野生型小鼠相比， UT－B基因敲除小鼠线粒体复合体I和IV活性明显下降，通过复合体Ⅴ产生的ATP量明显降低；线粒体mtDNA发生多处突变，线粒体损伤标志线粒体DNA／核DNA的比率显著下降。 UT－B基因敲除小鼠线粒体膜电位水平较野生型小鼠明显下降，而心肌ROS水平增加约31．42％。结论：UT－B基因敲除后可导致小鼠线粒体功能障碍，这可能是其老龄后发生心肌肥大的机制之一。

Background Acute kidney injury (AKI) is a serious complication of systemic inflammatory response syndrome (SIRS), which has a high mortality rate. Previous studies showed that panaxadiol saponin (PDS) and Dexamethasone have similar anti-inflammatory properties and protect cardiopulmonary function in lipopolysaccharide (LPS)-induced septic shock rats. In the present study, we investigated whether PDS or Dexamethasone has a similar role in improving kidney function in LPS-induced AKI mice. Methods and Results Mice subjected to LPS (10 mg/kg) treatment exhibited AKI demonstrated by markedly increased blood urea nitrogen and creatinine levels compared with controls (P<0.01). However, PDS and Dexamethasone induce similar reverse effects on renal function, such as reduced serum creatinine and blood urea nitrogen levels compared with the LPS group (P<0.05). PDS decreased the production and release of tumor necrosis factor (TNF)-α and interleukin (IL)-6 by inhibiting the NF-κB signaling pathway, down-regulating inducible nitric oxide synthase protein expression levels and inhibiting oxidative stress. In most anti-AKI mechanisms, PDS and dexamethasone were similar, but PDS are better at inhibition of TNF production, promote SOD activity and inhibition of IKB phosphorylation. In addition, nuclear glucocorticoid receptor expression was markedly enhanced in PDS and Dexamethasone treatment groups. Further research is required to determine whether PDS can combine with the glucocorticoid receptor to enter the nucleus. Conclusion This study demonstrated that PDS and dexamethasone have similar reverse amelioration for renal functions, and have potential application prospects in the treatment of sepsis-induced AKI.

BACKGROUNDCastration-resistant progression of prostate cancer after androgen deprivation therapy remains a critical challenge in the clinical management of prostate cancer. Resurgent androgen receptor activity is an established driver of castration-resistant progression, and upregulation of androgen receptor expression has been implicated to contribute to the resurgent androgen receptor activity. We reported previously that methylselenocysteine can decrease the expression and activity of androgen receptor. Here we investigated the ability of methylselenocysteine to inhibit castration-resistant progression of prostate cancer.METHODSThe regrowth of LNCaP prostate cancer xenografts after castration was monitored. The levels of prostate-specific antigen in mouse serum were measured by ELISA. Tumor cell proliferation and apoptosis were analyzed via Ki-67 immunohistochemistry and TUNEL assay, respectively. Intratumoral angiogenesis was assessed by immunohistochemistry staining of vascular endothelial growth factor and CD31.RESULTSWe showed that methylselenocysteine delayed castration-resistant regrowth of LNCaP xenograft tumors after androgen deprivation. This was accompanied by decreased serum levels of prostate-specific antigen, inhibition of prostate cancer cell proliferation and tumor angiogenesis, as well as downregulation of androgen receptor and induction of apoptosis in the relapsed tumors.CONCLUSIONSThe present study represents the first to show the preclinical efficacy of methylselenocysteine in delaying castration-resistant progression of prostate cancer. The findings provide a rationale for evaluating the clinical application of combining methylselenocysteine with androgen deprivation therapy for the treatment of advanced prostate cancer. Prostate 75:1001-1008, 2015. (c) 2015 Wiley Periodicals, Inc.

Aldo-keto reductase family 1 member C3 (AKR1C3) is a key steroidogenic enzyme that is overexpressed in prostate cancer (PCa) and is associated with the development of castration-resistant prostate cancer (CRPC). The aim of this study was to investigate the correlation between the expression level of AKR1C3 and the progression of PCa.

There are so many vehicles in the world and the number of vehicles is increasing rapidly. To alleviate the parking problems caused by that, the smart parking system has been developed. The parking planning is one of the most important parts of it. An effective parking planning strategy makes the better use of parking resources possible. In this paper, we present a feasible method to do parking planning. We transform the parking planning problem into a kind of linear assignment problem. We take vehicles as jobs and parking spaces as agents. We take distances between vehicles and parking spaces as costs for agents doing jobs. Then we design an algorithm for this particular assignment problem and solve the parking planning problem. The method proposed can give timely and efficient guide information to vehicles for a real time smart parking system. Finally, we show the effectiveness of the method with experiments over some data, which can simulate the situation of doing parking planning in the real world.

目的：探讨人参二醇组皂苷（PDS）对内毒素（LPS）诱导的急性肾损伤（AKI）小鼠肾功能的保护作用及机制。方法选择C57BL/6小鼠随机分成3组（n＝8），分别为对照组（control，腹腔注射0.9％氯化钠），LPS组（腹腔注射 LPS 10 mg/kg），PDS＋LPS组（先腹腔注射 PDS 25 mg/kg，1 h后注射LPS 10 mg/kg）。注射LPS 12 h后麻醉下处死小鼠，取肾脏组织冻存或固定，备蛋白表达与形态学观察用，并收集血液用于生化检测。利用免疫组化方法观察形态学变化。利用免疫印迹方法测定肾脏相关蛋白含量及表达情况。结果 LPS组小鼠肾组织凋亡严重，肝肾功能明显异常，而PDS＋LPS组肾脏组织凋亡及肝肾功能损伤较LPS组轻（ P＜0.05）。 LPS组一氧化氮合成酶（ iNOS）蛋白表达水平和NO含量与对照组比较明显增加（P＜0.05）。而PDS＋LPS组iNOS蛋白表达水平和NO含量均显著低于LPS组（P＜0.05）。 LPS组肾脏丙二醛（MDA）含量与对照组比较明显增高（P＜0.05），超氧化物歧化酶（SOD）含量和Mn-SOD的蛋白表达水平都明显低于对照组（P＜0.01，P＜0.05），而PDS＋LPS组 MDA含量较 LPS 组低（P＜0.05），SOD含量和Mn-SOD的蛋白表达水平较LPS组高（P＜0.05）。结论 PDS有逆转LPS诱导的AKI小鼠肾功能的作用，其保护作用机制为抑制肾脏iNOS表达，减少NO产生与释放，抑制MDA的产生和上调SOD的表达而起到抗氧化应激的作用。

Objective To investigate the proapoptotic effects of methylseleninic acid (MSA) on prostatic cancer Lncap cells and explore its mechanism. Methods The treated Lncap cells were divided into four groups such as the control group (mock), MSA low-dose group (1.25μM), middle dose group (2.50μM) and high dose group (5.00μM) respectively. Cell morphology was observed with microscope at the time of 24h, 48h and 72h after treatment. Cell proliferation was detected by SRB assay. Cell cycle and apoptosis was measured by flow cytometry. Stat3 expression was detected by immunofluorescence and immunocytochemical analysis. Results Light microscopy observation and SRB results indicated that the growth of Lncap cells were inhibited obviously with MSA dose and time dependent effect. Flow cytometry analysis showed that MSA promoted LNCap cells apoptosis with dose and time dependent effect and changed the cell cycle distribution. The majority of cells were arrested in G0-G1 phase. Immunofluorescence and immunohistochemistry results showed MSA could inhibit the expression of intracellular stat3 compared with that of the control group. Conclusion MSA might inhibit cell proliferation and induce apoptosis, and its mechanism was associated with decreased stat3 expression.

目的：探讨内毒素休克时大脑皮质损伤状况及人参二醇组皂苷对大鼠脑保护作用机制。方法将28只 Wistar成年大鼠随机分为实验对照（control）组，内毒素休克（LPS）组，地塞米松（LPS＋DEX）组和人参二醇组皂苷（LPS ＋PDS）组。大鼠静脉注射内毒素制作内毒素休克大鼠模型。生物化学法检测脑组织中超氧化物歧化酶（ SOD）活力和丙二醛（ MDA）含量。蛋白印迹检测大鼠核转录因子（ NF）－κB 蛋白 P65亚基表达含量的变化。 ELISA法检测脑组织内白细胞介素（ IL－1β），肿瘤坏死因子（ TNF－α）和 IL－6水平。结果与LPS组比较，LPS＋DEX组和 LPS ＋PDS组大鼠脑组织MDA含量降低，SOD活性上调，NF－κB蛋白表达降低，IL－1β，TNF－α和IL－6水平显著降低（P＜0．05）。结论 PDS能够下调内毒素休克脑组织中NF－κB蛋白表达，改善自由基和炎症因子对脑组织的损伤作用，对中枢神经系统具有保护作用。

In previous research, we showed that 16‐week‐old urea transporter B (UT‐B) null mice have an atrial–ventricular conduction block, and hypothesized myocardial mitochondrial dysfunction. To investigate the mechanism of this block, we examined the proteomic differences in the myocardial mitochondria of UT‐B null and wild‐type mice with nanoscale LC–MS/MS. Of 26 proteins clearly downregulated in the UT‐B null mice, 15 are involved in complexes I, III, IV, and V of the respiratory chain, which would strongly reduce the activity of the electron transport chain. Excess electrons from complexes I and III pass directly to O 2 to generate ROS and deplete ROS‐scavenging enzymes. Myocardial intracellular ROS were significantly higher in UT‐B null mice than in wild‐type mice ( p < 0.01), constituting an important cause of oxidative stress injury in the myocardia of UT‐B null mice. The mitochondrial membrane potential (ΔΨm) was also lower in UT‐B null mice than in wild‐type mice ( p < 0.05), causing oxidative phosphorylation dysfunction of complex V and insufficient ATP in the myocardial cells of UT‐B null mice. HADHA (a trifunctional protein) and HSP60 were also downregulated in the UT‐B null myocardial mitochondria. These results confirm that mitochondrial dysfunction underlies the pathogenesis of the atrial–ventricular conduction block in UT‐B null mice.