Role of DJ-1 siRNA in reverse sensitivity of breast cancer cells to chemotherapy and its possible mechanism

Breast cancer which has a high incidence rate is the 2^nd lethal diseases only followed by lung cancer in women. How to improve the recovery rate is the principal problem should be solved in clinical. Previous studies demonstrated the importance of DJ-1 in the existence of breast cancer for the secreted of protein into serum by breast cancer cells both in vitro and in vivo. So the DJ-1 probably could be selected as the target in breast cancer treatment. Adriamycin resistance breast cancer cells MCF-7 and DJ-1 siRNA plasmid were employed to explore the potential clinical application of DJ-1 in this study. Our results showed that the sensitivity of cancer cells to chemotherapeutics was significantly improved with the transfection of DJ-1 siRNA. Further mechanism studies indicated the role of PI3K/AKT/MTOR pathway in the improvement of apoptosis after treatment with adriamycin in DJ-1 silence group.     

Focal dysplasia of the cerebral cortex and infantile spasms associated with somatic 1q21.1‐q44 duplication including the AKT3 gene

Somatic and germline duplications or activating mutations of AKT3 have been reported in patients with hemimegalencephaly and megalencephaly. We performed array comparative genomic hybridization on brain tissue and blood in 16 consecutive patients with symptomatic epilepsy due to focal or multilobar malformations of cortical development who underwent surgical treatment of epilepsy. One patient with infantile spasms and a dysplastic left frontal lobe harboured a somatic trisomy of the 1q21.1‐q44 chromosomal region, encompassing the AKT3 gene, in the dysplastic brain tissue but not in blood and saliva. Histopathology revealed severe cortical dyslamination, a thin cortex in the premotor area with microgyri and microsulci, immature neurons with disoriented dendrites and areas of cortical heterotopia in the sub‐cortical white matter. These cytoarchitectural changes are close to those defining type Ib focal cortical dysplasia. Immunohistochemistry in brain specimens showed hyperactivation of the PI3K/AKT/mTOR pathway. These findings indicate that AKT3 upregulation may cause focal malformations of cortical development. There appears to be an etiologic continuum between hemimegalencephaly and focal cortical dysplastic lesions. The extent of brain malformations due to AKT3 upregulation may be related to the embryonic stage when the post‐zygotic gene alteration occurs.     

Disruption of Tsc2 in pancreatic beta cells induces beta cell mass expansion and improved glucose tolerance in a TORC1-dependent manner

Regulation of pancreatic beta cell mass and function is a major determinant for the development of diabetes. Growth factors and nutrients are important regulators of beta cell mass and function. The signaling pathways by which these growth signals modulate these processes have not been completely elucidated. Tsc2 is an attractive candidate to modulate these processes, because it is a converging point for growth factor and nutrient signals. In these experiments, we generated mice with conditional deletion of Tsc2 in beta cells (betaTsc2(-/-)). These mice exhibited decreased glucose levels and hyperinsulinemia in the fasting and fed state. Improved glucose tolerance in these mice was observed as early as 4 weeks of age and was still present in 52-week-old mice. Deletion of Tsc2 in beta cells induced expansion of beta cell mass by increased proliferation and cell size. Rapamycin treatment reversed the metabolic changes in betaTsc2(-/-) mice by induction of insulin resistance and reduction of beta cell mass. The reduction of beta cell mass in betaTsc2(-/-) mice by inhibition of the mTOR/Raptor (TORC1) complex with rapamycin treatment suggests that TORC1 mediates proliferative and growth signals induced by deletion of Tsc2 in beta cells. These studies uncover a critical role for the Tsc2/mTOR pathway in regulation of beta cell mass and carbohydrate metabolism in vivo.     

mTOR/Akt/FoxO3信号通路在人参皂苷Rg1抗PC-12细胞OGD损伤的作用

目的:研究人参皂苷Rg1对PC-12细胞缺氧缺糖(oxygen-glucose deprivation,OGD)损伤的保护作用,并初步探讨与mTOR/Akt调控FoxO3核浆穿梭相关的可能的分子机制.方法:建立OGD损伤PC-12细胞模型,MTT 法检测OGD对PC-12细胞存活率.人参皂苷Rg110,20,40 μmol·L-1预处理PC-12细胞24h,加OGD损伤,MTT 检测细胞存活率,比色法检测乳酸脱氢酶(LDH)释放、超氧化物歧化酶(SOD)活力和丙二醛(MDA)含量.Western blot法检测mTOR,p-Aktser473,p-Aktthr308,Akt,p-FoxO3,细胞核、细胞质FoxO3和总FoxO3蛋白表达.结果:OGD作用4～24h显著抑制PC-12细胞增殖,呈时间依赖性.Rg1(20,40 μmol·L-1)预处理24h可显著增加细胞存活率,减少LDH释放,增加SOD活力和降低MDA含量.Rg1可抑制由OGD引起的rmTOR,p-Aktser473表达降低.OGD 6 h可减少FoxO3磷酸化,并促进细胞质FoxO3进入细胞核;预处理Rg1增加FoxO3磷酸化,促进FoxO3进入细胞质.提示Rg1可经由调控mTOR/Akt/FoxO3信号通路抑制PC-12细胞损伤.结论:人参皂苷Rg1可抑制OGD引起PC-12细胞损伤,作用机制可能与激活mTOR/Akt信号通路调控FoxO3核质穿梭密切相关.     

抗纤灵方通过PI3K/AKT/mTOR信号通路干预肾纤维化机制研究

目的:研究抗纤灵方对PI3K/AKT/mTOR信号通路的影响及抗肾纤维化作用机制。方法:将60只C57小鼠,随机分为假手术组10只和手术组50只,手术组行5/6肾切除术。术后2周,手术组随机分为模型组、抗纤灵低、中、高剂量组及雷帕霉素阳性药组,各组10只。假手术组给予0.5 mL生理盐水ig,抗纤灵方低、中、高剂量组分别给予0.5 mL抗纤灵药物ig(0.1,0.2,0.4 mg·kg-1),阳性药组给予0.5 mL雷帕霉素ig(0.016μg·kg-1),ig 12周后处死小鼠,在处死小鼠前1 d收集24 h尿液检测24 h蛋白定量,眼眶采血测血肌酐、尿素氮,取残肾采用HE观察肾脏组织形态改变,PCR法检测肾组织中PI3K/AKT/mTOR mRNA表达。结果:与假手术组比较,模型组24 h尿蛋白定量,血肌酐,尿素氮,PI3K/AKT/mTOR mRNA表达均显著升高(P0.01),肾脏病理形态改变明显;与模型组比较,各治疗组24 h尿蛋白定量,血肌酐,尿素氮,PI3K/AKT/mTOR mRNA表达均下降(P0.05),肾脏组织学形态改善。结论:抗纤灵方能降低小鼠24 h尿蛋白定量,改善肾功能,延缓肾纤维化发生;其机制可能与抑制PI3K/AKT/mTOR信号通路表达相关。     

Role of the Fyn −93A>G polymorphism (rs706895) in acute rejection after liver transplantation

The tyrosine kinase Fyn phosphorylates tyrosine residues on key targets involved in early T-cell signal transduction. T-cell signal transduction is one essential step for acute transplant rejection. The aim of this study was to evaluate the association of Fyn −93A>G single nucleotide polymorphism (SNP) (rs706895) with the susceptibility to acute rejection episodes in liver transplantation. In total, 72 liver transplant recipients with one biopsy proven acute rejection (S-BPAR), 56 with multiple BPAR (M-BPAR), 105 without BPAR (No-BPAR), and 145 healthy controls were enrolled in this case-control study. The SNP was genotyped by polymerase chain reaction–allele specific restriction enzyme analysis (PCR–ASRA) and was analyzed for a recessive and a dominant model. The Fyn −93G allele exhibits in healthy controls a statistically significant lower frequency than in liver recipients (18% vs. 24%; p = 0.046) or in liver recipients with BPAR (18% vs. 27%; p = 0.017). However, the genotype and allele frequencies of the Fyn −93A>G SNP demonstrate no significant differences between recipients with acute rejection episodes (S-BPAR and M-BPAR) and No-BPAR recipients. Thus our results provide no evidence that the Fyn −93A>G SNP contributes to the susceptibility to acute liver transplant rejection in a Caucasian population.     

Sirolimus for progressive neurofibromatosis type 1–associated plexiform neurofibromas: a Neurofibromatosis Clinical Trials Consortium phase II study

BackgroundPlexiform neurofibromas (PNs) are benign peripheral nerve sheath tumors that arise in one-third of individuals with neurofibromatosis type 1 (NF1). They may cause significant disfigurement, compression of vital structures, neurologic dysfunction, and/or pain. Currently, the only effective management strategy is surgical resection. Converging evidence has demonstrated that the NF1 tumor suppressor protein, neurofibromin, negatively regulates activity in the mammalian Target of Rapamycin pathway.MethodsWe employed a 2-strata clinical trial design. Stratum 1 included subjects with inoperable, NF1-associated progressive PN and sought to determine whether sirolimus safely and tolerably increases time to progression (TTP). Volumetric MRI analysis conducted at regular intervals was used to determine TTP relative to baseline imaging.ResultsThe estimated median TTP of subjects receiving sirolimus was 15.4 months (95% CI:14.3–23.7 mo), which was significantly longer than 11.9 months (P < .001), the median TTP of the placebo arm of a previous PN clinical trial with similar eligibility criteria.ConclusionsThis study demonstrated that sirolimus prolongs TTP by almost 4 months in patients with NF1-associated progressive PN. Although the improvement in TTP is modest, given the lack of significant or frequent toxicity and the availability of few other treatment options, the use of sirolimus to slow the growth of progressive PN could be considered in select patients.     

The preclinical evaluation of the dual mTORC1/2 inhibitor INK-128 as a potential anti-colorectal cancer agent

The colorectal cancer is the leading contributor of cancer-related mortality. Mammalian target of rapamycin (mTOR), existing in 2 complexes (mTORC1/2), is frequently dysregulated and constitutively activated in colorectal cancers. It represents an important drug target. Here we found that INK-128, the novel ATP-competitive kinase inhibitor of mTOR, blocked both mTORC1 and mTORC2 activation in colorectal cancer cells (both primary and transformed cells). The immunoprecipitation results showed that the assembly of mTORC1 (mTOR-Raptor association) and mTORC2 (mTOR-Rictor-Sin1 association) was also disrupted by INK-128. INK-128 inhibited colorectal cancer cell growth and survival, and induced both apoptotic and non-apoptotic cancer cell death. Further, INK-128 showed no effect on Erk/MAPK activation, while MEK/Erk inhibition by MEK-162 enhanced INK-128-induced cytotoxicity in colorectal cancer cells. Meanwhile, INK-128 downregulated Fascin1 (FSCN1)/E-Cadherin expressions and inhibited HT-29 cell in vitro migration. In vivo, daily INK-128 oral administration inhibited HT-29 xenograft growth in mice, which was further enhanced by MEK-162 administration. Finally, we found that INK-128 sensitized 5-fluorouracil-(5-FU)-mediated anti-HT-29 activity in vivo and in vitro. Thus, our preclinical studies strongly suggest that INK-128 might be investigated for colorectal cancer treatment in clinical trials.     

Both mTORC1 and mTORC2 are involved in the regulation of cell adhesion

mTOR is a central controller for cell growth/proliferation and survival. Recent studies have shown that mTOR also regulates cell adhesion, yet the underlying mechanism is not known. Here we found that inhibition of mTOR by rapamycin reduced the basal or type I insulin-like growth factor (IGF-1)-stimulated adhesion of cancer cells. Further research revealed that both mTORC1 and mTORC2 were involved in the regulation of cell adhesion, as silencing expression of raptor or rictor inhibited cell adhesion. Also, PP242, an mTORC1/2 kinase inhibitor, inhibited cell adhesion more potently than rapamycin (mTORC1 inhibitor). Of interest, ectopic expression of constitutively active and rapamycin-resistant mutant of p70 kinase 1 (S6K1) or downregulation of eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) conferred resistance to rapamycin inhibition of cell adhesion, whereas expression of constitutively hypophosphorylated 4E-BP1 (4EBP1-5A) or downregulation of S6K1 suppressed cell adhesion. In contrast, neither genetic manipulation of Akt activity nor pharmacological inhibition of Akt affected cell adhesion. The results suggest that both mTORC1 and mTORC2 are involved in the regulation of cell adhesion; and mTORC1 regulates cell adhesion through S6K1 and 4E-BP1 pathways, but mTORC2 regulates cell adhesion via Akt-independent mechanism.