A phase II study of high-dose calcitriol combined with mitoxantrone and prednisone for androgen-independent prostate cancer

OBJECTIVE

To evaluate the preliminary efficacy, safety, and impact on quality of life (QoL) of high‐dose calcitriol (DN‐101) combined with mitoxantrone and glucocorticoids in androgen‐independent prostate cancer (AIPC).

PATIENTS AND METHODS

Nineteen patients with metastatic AIPC and no previous chemotherapy received DN‐101 180 µg orally on day 1 and mitoxantrone 12 mg/m² intravenously on day 2 every 21 days with continuous daily prednisone 10 mg orally for a maximum of 12 cycles. A confirmed decline in prostate‐specific antigen (PSA) levels by half was the primary endpoint. QoL was evaluated using the European Organization for Research and Treatment of Cancer QLQ‐C30 questionnaire, and pain and analgesic use were evaluated.

RESULTS

Five of 19 patients (26%; 95% confidence interval, CI, 9–51) achieved a ≥50% decline in PSA level. The median (95% CI) time to PSA progression was 16 (6–26) weeks. The overall median (95% CI) survival was 16 (6–26) months; 47 (21–73)% of patients achieved an analgesic response. Toxicity was similar to that expected with mitoxantrone and prednisone alone. The QoL analysis suggested a decrease in physical functioning and increase in fatigue, insomnia, and diarrhoea.

CONCLUSIONS

DN‐101 given every 3 weeks does not add significant activity to mitoxantrone and prednisone in AIPC, as measured by the PSA decline. The high rate of analgesic response is encouraging. The addition of DN‐101 does not appear to increase the toxicity of mitoxantrone.     

Developmental profile and mechanisms of GABA-induced calcium signaling in hippocampal astrocytes

GABA (gamma-aminobutyric acid) is a transmitter with dual action. Whereas it excites neurons during the first week of postnatal development, it represents the major inhibitory transmitter in the mature brain. GABA also activates astrocytes by binding to ionotropic (GABA(A)) and metabotropic (GABA(B)) receptors. This results in glial calcium transients which can induce the release of gliotransmitters, rendering GABA an important mediator of neuron-glia interaction. Using whole-cell patch-clamp and ratiometric calcium imaging in hippocampal slices from rats at postnatal days 3-34, we have analyzed the developmental profile as well as the cellular mechanisms of calcium signals induced by GABA(A) and GABA(B) receptor activation in astrocytes. We found that GABA-evoked glial calcium transients are mediated by both GABA(A) and GABA(B) receptors. Throughout development, GABA(A)-receptor activation resulted in immediate calcium transients in the vast majority of astrocytes, most likely by influx of calcium through voltage-gated calcium channels. GABA(B) receptor activation, in contrast, resulted in delayed calcium transients, which were blocked following depletion of intracellular calcium stores and during persistent activation of heterotrimeric G-proteins. GABA(B) receptor-mediated calcium signals exhibited a clear developmental profile with less than 10% of astrocytes responding at P3 or P32-34, and about 60% of cells between P11 and P15. Our data thus indicate that GABA(B) receptor-mediated calcium transients are due to calcium release from intracellular stores following G-protein activation. Moreover, GABA(B) receptor-mediated calcium signaling in astrocytes preferentially occurs at a period during postnatal development when hippocampal networks are established.     

纳布啡通过调控 miR-4301/BRD4抑制肝癌细胞增殖、迁移和侵袭的机制研究

目的 探讨纳布啡对肝癌细胞增殖、迁移和侵袭的影响及作用机制.方法 本研究时间为2019年1—7月.肝癌细胞株Huh7购自美国ATCC,将Huh7细胞分为对照组、不同浓度纳布啡(50μmol、100μmol、200μmol)组、微小RNA-4301(miR-4301)组、miR-4301模拟物阴性对照(miR-NC)组、纳布啡+miR-4301抑制表达载体(anti-miR-4301)组、纳布啡+miR-4301抑制表达载体阴性对照(anti-miR-NC)组.四甲基偶氮唑盐比色法(MTT)检测Huh7细胞的增殖抑制率;Transwell检测Huh7细胞的迁移和侵袭数;蛋白质印迹检测细胞周期蛋白依赖性激酶抑制剂1A(p21)、细胞周期蛋白D1(Cyclin D1)、基质金属蛋白酶2(MMP-2)、基质金属蛋白酶9(MMP-9)和溴结构域蛋白4(BRD4)蛋白表达水平;实时荧光定量聚合酶链式反应(RT-qPCR)检测miR-4301和BRD4 mRNA表达水平;荧光素酶报告实验检测miR-4301和BRD4的靶向关系.结果 与对照组相比,不同浓度纳布啡组Huh7细胞抑制率升高[(11.25±1.12)％、(22.63±2.25)％、(37.65±3.74)％比(0.00±0.01)％],迁移数减少[(79.32±7.38)个、(61.36±5.48)个、(45.69±5.37)个比(98.63±9.54)个],侵袭数减少[(67.53±6.65)个、(53.41±5.22)个、(37.64±3.87)个比(81.36±8.13)个],p21表达水平升高,Cyclin D1、MMP-2、MMP-9表达水平降低,miR-4301表达水平升高,BRD4mRNA和蛋白表达水平降低(P<0.05),且呈浓度依赖性.与miR-NC组相比,miR-4301组Huh7细胞抑制率升高[(31.25±3.15)％比(6.32±0.63)％],迁移数减少[(52.33±5.26)个比(96.32±9.54)个],侵袭数减少[(41.69±4.32)个比(83.15±8.33)个],p21表达水平升高,Cyclin D1、MMP-2、MMP-9表达水平降低(均P<0.05).miR-4301靶向调控BRD4的表达,抑制miR-4301表达逆转了纳布啡对Huh7细胞的作用.结论 纳布啡可抑制肝癌Huh7细胞增殖、迁移和侵袭,其机制可能与miR-4301和BRD4有关.     

外周血miRNA-150与CD19+CD24hiCD38hiBreg细胞在儿童免疫性血小板减少症中的变化及意义

目的探讨外周血miRNA-150与CD19⁺CD24^hiCD38^hiBreg细胞在儿童免疫性血小板减少症（ITP）中变化及意义。 方法选取南通大学附属妇幼保健院在2017年3月至2020年9月接受诊治的ITP患儿93例,其中新诊断ITP患儿（病程＜3个月）56例,持续性ITP患儿（病程3~12个月）37例。另选取本院同期健康体检儿童50例作为对照组。采用实时荧光定量PCR反应测定外周血miRNA-150表达,采用流式细胞术测定CD19⁺CD24^hiCD38^hiBreg细胞表达。 结果ITP患儿外周血miRNA-150表达高于对照组,且CD19⁺CD24^hiCD38^hiBreg细胞表达率低于对照组（P＜0.05）。持续性ITP组外周血miRNA-150表达高于新诊断ITP组,而CD19⁺CD24^hiCD38^hiBreg细胞表达率低于新诊断ITP组（P＜0.05）。全反应（CR）组外周血miRNA-150表达低于反应（R）组和无效（NR）组,且R组低于NR组（P＜0.05）。CR组CD19⁺CD24^hiCD38^hiBreg细胞表达率高于R组和NR组,且R组高于NR组（P＜0.05）。外周血miRNA-150对ITP诊断敏感度和特异度分别为78.18%和60.53%;CD19⁺CD24^hiCD38^hiBreg细胞表达对ITP诊断敏感度和特异度分别为83.63%和71.05%。miRNA-150与PLT呈负相关（r=-0.738）,而CD19⁺CD24^hiCD38^hiBreg细胞与PLT呈正相关（r=0.796）。 结论外周血miRNA-150在ITP患儿中高表达,而CD19⁺CD24^hiCD38^hiBreg细胞在ITP患儿中低表达,且外周血miRNA-150和CD19⁺CD24^hiCD38^hiBreg细胞表达与疗效密切相关。     

lncRNA OIP5-AS1调节miR-942-5p/CHEK1轴对脑胶质瘤细胞生物学行为的影响

目的 探讨长链非编码RNA（lncRNA）OPA相互作用蛋白5反义转录本1（OIP5-AS1）对脑胶质瘤细胞增殖、凋亡、迁移和侵袭的影响机制。方法 收集33例胶质瘤患者（低级别胶质瘤14例、高级别胶质瘤19例）和33例颅脑损伤患者的组织标本。实时荧光定量PCR（qPCR）检测组织和细胞中OIP5-AS1、微小RNA-942-5p（miR-942-5p）和检查点激酶1（CHEK1）mRNA表达,分析胶质瘤组织中OIP5-AS1、miR-942-5p和CHEK1 mRNA表达水平的相关性。体外培养人脑胶质瘤细胞系U87、SHG-44、U251、H4和正常人星形胶质细胞NHA,Western blot检测细胞中CHEK1蛋白表达。将U87细胞分为对照（NC）组、siRNA阴性对照（si-NC）组、OIP5-AS1 siRNA（si-OIP5-AS1）组、si-OIP5-AS1+inhibitor阴性对照（si-OIP5-AS1+anti-NC）组、si-OIP5-AS1+miR-942-5p抑制剂（si-OIP5-AS1+anti-miR-942-5p）组,采用Lipofectamine 3000试剂进行转染。转染后,qPCR和Western blot检测细胞中OIP5-AS1、miR-942-5p和CHEK1 mRNA和蛋白表达水平;MTT法测定细胞增殖活性;流式细胞术检测细胞凋亡;Transwell实验检测细胞迁移和侵袭能力。最后通过双荧光素酶和RNA免疫沉淀（RIP）实验验证OIP5-AS1和miR-942-5p以及CHEK1和miR-942-5p的相互作用。结果 OIP5-AS1和CHEK1在脑胶质瘤组织和细胞中过表达,miR-942-5p呈低表达（均P<0.05）;相关分析显示,脑胶质瘤组织中OIP5-AS1与miR-942-5p的表达水平呈负相关,miR-942-5p与CHEK1 mRNA的表达水平呈负相关,CHEK1与OIP5-AS1 mRNA的表达水平呈正相关;且OIP5-AS1、CHEK1 mRNA在高级别胶质瘤组织中的表达明显高于低级别组织,而高级别胶质瘤中的miR-942-5p水平明显低于低级别组织（P<0.01）。沉默OIP5-AS1可显著上调miR-942-5p,抑制CHEK1的mRNA和蛋白表达（均P<0.05）;沉默OIP5-AS1可显著抑制U87细胞增殖、迁移和侵袭,促进U87细胞凋亡（均P<0.05）;下调miR-942-5p可上调CHEK1表达,阻断OIP5-AS1沉默对脑胶质瘤细胞生物学行为的影响（均P<0.05）。双荧光素酶和RIP实验证实miR-942-5p是OIP5-AS1的靶基因,CHEK1是miR-942-5p的下游靶基因。结论 沉默OIP5-AS1可能通过上调miR-942-5p抑制CHEK1表达,抑制脑胶质瘤细胞的侵袭和迁移,并促进细胞凋亡。     

Cutaneous phenotype and MC1R variants as modifying factors for the development of melanoma in CDKN2A G101W mutation carriers from 4 countries

The G101W founder mutation is the most common CDKN2A mutation in Italy, Spain, and France. As the background of modifying genes, environmental exposures, and sun behavior vary across countries, studying G101W carriers from distinct countries offers a unique opportunity to evaluate possible modifying factors in melanoma development. We evaluated 76 G101W cases and 59 carrier controls from France, Italy, Spain, and the United States. Hair color and dysplastic nevi distributions differed significantly in cases and controls across the 4 study groups. Cases also varied significantly for eye color, freckling, and nevi. The distribution of MC1R variants in cases differed significantly across study groups because 12% of Italian melanoma patients had > or =2 MC1R variants vs. >50% for the other case groups. Several MC1R covariates showed significant associations with melanoma risk in all groups combined and in the American, French, and Spanish samples; no significant findings were observed in the Italian sample. In multiple-case families, the number and type of MC1R variants varied significantly between multiple-primary-melanoma and single-primary-melanoma patients from the 4 groups; there was also a significant decrease in median age at melanoma diagnosis as the number or type of MC1R variants increased. The variation in the effects of the cutaneous phenotypic and MC1R factors across the study sample suggests that these factors differentially contribute to development of melanoma even on a common genetic background of a germline CDKN2A mutation. Differences in melanoma risk across geographic regions justify the need for individual studies in each country before counseling should be considered.     

A new pure ω-3 eicosapentaenoic acid ethyl ester (AMR101) for the management of hypertriglyceridemia: the MARINE trial

ω-3 fatty acids reduce triglyceride (TG) levels, but corresponding increases in low-density lipoprotein cholesterol (LDL-C) levels may compromise achievement of lipid goals in patients with elevated cardiovascular risk. AMR101 is an investigational agent containing ≥96% of pure icosapent ethyl (the ethyl ester of eicosapentaenoic acid). The Phase III Multi-Center, Placebo-Controlled, Randomized, Double-Blind, 12-Week Study with an Open-Label Extension (MARINE) investigated the efficacy and safety of AMR101 in 229 patients with very high TG levels (≥500 mg/dl). AMR101 4 g/day significantly reduced median placebo-adjusted TG levels from baseline by 33.1% (p < 0.0001), and AMR101 2 g/day reduced TG levels by 19.7% (p = 0.0051). Changes in LDL-C were minimal and nonsignificant. AMR101 may offer substantial TG lowering without increases in LDL-C levels.     

Serotonin 2A receptor antagonists for treatment of schizophrenia

Introduction: All approved antipsychotic drugs share an affinity for the dopamine 2 (D₂) receptor; however, these drugs only partially ameliorate the symptoms of schizophrenia. It is, therefore, of paramount importance to identify new treatment strategies for schizophrenia.

Areas covered: Preclinical, clinical and post-mortem studies of the serotonin 5-HT_2A system in schizophrenia are reviewed. The implications of a combined D₂ and 5-HT_2A receptor blockade, which is obtained by several current antipsychotic drugs, are discussed, and the rationale for the development of more selective 5-HT_2A receptor antagonists is evaluated. Moreover, the investigational pipeline of major pharmaceutical companies is examined and an Internet search conducted to identify other pharmaceutical companies investigating 5-HT_2A receptor antagonists for the treatment of schizophrenia.

Expert opinion: 5-HT_2A receptor antagonists appear to assume an intermediate position by being marginally superior to placebo but inferior to conventional antipsychotic drugs. Three previous 5-HT_2A receptor antagonists have been discontinued after Phase II or III trials, and available Phase IIa data on the remaining 5-HT_2A receptor antagonist will need substantial additional validation to be approved as a new treatment strategy against schizophrenia.     

miR-4319通过靶向调控USP2抑制乳腺癌细胞侵袭

目的探讨miR-4319与泛素特异性蛋白酶2(USP2)表达的相关性以及miR-4319靶向USP2通过核转录因子κB(NF-κB)信号通路对乳腺癌细胞侵袭的影响。方法实时荧光定量PCR(qRT-PCR)检测miR-4319在正常乳腺癌上皮细胞(MCF10A)、低侵袭性乳腺癌细胞(MCF7)和高侵袭性乳腺癌细胞(MDA-MB-231)中的表达量。将MCF10A、MCF7和MDA-MB-231细胞分为6组进行转染:(1)MDA-MB-231/NC组,瞬时转染插入一段乱码序列(scramble 1),即作为miR-4319过表达对照质粒;(2)MDA-MB-231/miR-4319组,瞬时转染插入目的片段miR-4319质粒过表达miR-4319,即miR-4319过表达;(3)MDA-MB-231/miR-4319+Con组,瞬时转染同时转入miR-4319过表达质粒和USP2过表达对照质粒,即miR-4319过表达以及USP2过表达对照;(4)MDA-MB-231/miR-4319+USP2组,瞬时转染同时转入miR-4319过表达质粒和USP2过表达质粒,即miR-4319过表达和USP2过表达;(5)MDA-MB-231/miR-4319inhibitor组,瞬时转染miR-4319的反义序列抑制miR-4319的表达,即抑制miR-4319的表达;(6)MDA-MB-231/miR-4319inhibitor NC组,瞬时转染插入一段乱码序列(scramble 2),即作为抑制miR-4319组的对照组。qRT-PCR检测miR-4319在MDA-MB-231细胞中的转染效率。荧光素酶实验检测miR-4319与USP2 mRNA是否存在结合位点。qRT-PCR检测miR-4319在乳腺癌细胞中过表达后的USP2 mRNA表达水平。蛋白质印迹法检测过表达miR-4319或抑制miR-4319表达后的USP2蛋白表达水平。Transwell侵袭实验检测过表达miR-4319或USP2后MDA-MB-231细胞侵袭能力。双荧光素酶实验检测miR-4319对NF-κB信号通路活性的影响以及过表达USP2后miR-4319对NF-κB信号通路活性的影响。结果 qRT-PCR结果显示,miR-4319相对表达量在细胞MDA-MB-231(t=14.860,P<0.001)和MCF7(t=12.770,P<0.001)中分别为0.330±0.075和0.570±0.082,均低于细胞MCF10A(1.012±0.051);miR-4319相对表达量MDA-MB-231/miR-4319组(3.980±0.083)高于MDA-MB-231/NC组(1.009±0.058),差异有统计学意义,t=102.90,P<0.001。荧光素酶实验结果显示,pGL3-USP2 3’-UTR-WT报告载体与miR-4319质粒共转染后的荧光素酶活性下降,差异有统计学意义,t=15.740,P<0.001。qRT-PCR结果显示,USP2 mRNA相对表达量MDA-MB-231/NC组(1.013±0.058)和MDA-MB-231/miR-4319组(0.988±0.062)基本没有变化,差异无统计学意义,t=1.201,P=0.296。而蛋白质印迹法结果显示,USP2蛋白相对表达量miR-4319组(0.371±0.083)低于miR-4319/NC组(1.003±0.064),miR-4319/inhibitor组(1.982±0.093)高于inhibitor/NC组(1.104±0.072),USP2蛋白相对表达量的组间比较差异有统计学意义,F=212.4,P<0.001。Transwell侵袭实验结果显示,穿过Matrigel细胞数MDA-MB-231/miR-4319组(58.337±5.972)低于MDA-MB-231/NC组(192.371±5.476),差异有统计学意义,t=29.720,P<0.001;穿过Matrigel细胞数MDA-MB-231/miR-4319+USP2组(167.197±8.292)高于MDA-MB-231/miR-4319+Con组(63.283±10.397),差异有统计学意义,t=14.150,P=0.001。双荧光素酶结果显示,miR-4319/NF-κB-luc组荧光素酶活性(0.324±0.06)低于NC/pRL-TK组(1.024±0.080)、NC/NF-κB-luc组(2.023±0.110)和miR-4319/pRL-TK组(1.109±0.050),组间比较差异有统计学意义,F=237.1,P<0.001;miR-4319+USP2/NF-κB-luc组荧光素酶活性(2.032±0.12)高于miR-4319+USP2/pRL-TK组(1.094±0.100)、miR-4319+Con/pRL-TK组(1.063±0.080)和miR-4319+Con/NF-κB-luc组(0.334±0.050),组间比较差异有统计学意义,F=164.2,P<0.001。结论 miR-4319在乳腺癌细胞中低表达,miR-4319靶向结合USP2,过表达USP2逆转了miR-4319对乳腺癌细胞侵袭能力的抑制作用和NF-κB转录活性的抑制作用。miR-4319通过靶向结合USP2抑制NF-κB信号通路,从而抑制乳腺癌细胞的侵袭。