Effect of Glucocorticosteroids in Diamond-Blackfan Anaemia: Maybe Not as Elusive as It Seems

Diamond-Blackfan anaemia (DBA) is a red blood cell aplasia that in the majority of cases is associated with ribosomal protein (RP) aberrations. However, the mechanism by which this disorder leads to such a specific phenotype remains unclear. Even more elusive is the reason why non-specific agents such as glucocorticosteroids (GCs), also known as glucocorticoids, are an effective therapy for DBA. In this review, we (1) explore why GCs are successful in DBA treatment, (2) discuss the effect of GCs on erythropoiesis, and (3) summarise the GC impact on crucial pathways deregulated in DBA. Furthermore, we show that GCs do not regulate DBA erythropoiesis via a single mechanism but more likely via several interdependent pathways.     

The Role of mTOR and eIF Signaling in Benign Endometrial Diseases

Adenomyosis, endometriosis, endometritis, and typical endometrial hyperplasia are common non-cancerous diseases of the endometrium that afflict many women with life-impacting consequences. The mammalian target of the rapamycin (mTOR) pathway interacts with estrogen signaling and is known to be dysregulated in endometrial cancer. Based on this knowledge, we attempt to investigate the role of mTOR signaling in benign endometrial diseases while focusing on how the interplay between mTOR and eukaryotic translation initiation factors (eIFs) affects their development. In fact, mTOR overactivity is apparent in adenomyosis, endometriosis, and typical endometrial hyperplasia, where it promotes endometrial cell proliferation and invasiveness. Recent data show aberrant expression of various components of the mTOR pathway in both eutopic and ectopic endometrium of patients with adenomyosis or endometriosis and in hyperplastic endometrium as well. Moreover, studies on endometritis show that derangement of mTOR signaling is linked to the establishment of endometrial dysfunction caused by chronic inflammation. This review shows that inhibition of the mTOR pathway has a promising therapeutic effect in benign endometrial conditions, concluding that mTOR signaling dysregulation plays a critical part in their pathogenesis.     

双PI3K/mTOR抑制剂NVPBEZ235对宫颈癌Hela细胞的放射增敏作用

目的: 探讨双PI3K/mTOR 抑制剂 NVPBEZ235对宫颈癌Hela细胞的放射增敏作用。方法: 四甲基偶氮唑盐（MTT）及集落克隆法测定各因素对Hela细胞的抑制作用。通过流式细胞仪检测药物联合射线（irradiation,IR）对宫颈癌细胞周期的影响。结果: 与对照组相比,NVPBEZ235在体外能够抑制人宫颈癌细胞的生长。NVPBEZ235、IR联合作用细胞,在体外可以使细胞周期阻滞于G2/M期,二者具有协同作用,联合作用效果显著大于单药作用。结论: 20% IC50 NVPBEZ235在体外能够促进细胞对射线照射的敏感性,抑制细胞的增殖从而实现对宫颈癌细胞的放射增敏作用。     

Spontaneous Cell Detachment and Reattachment in Cancer Cell Lines: An In Vitro Model of Metastasis and Malignancy

There is an unmet need for simplified in vitro models of malignancy and metastasis that facilitate fast, affordable and scalable gene and compound analysis. “Adherent” cancer cell lines frequently release “free-floating” cells into suspension that are viable and can reattach. This, in a simplistic way, mimics the metastatic process. We compared the gene expression profiles of naturally co-existing populations of floating and adherent cells in SW620 (colon), C33a (cervix) and HeLa (cervix) cancer cells. We found that 1227, 1367 and 1333 genes were at least 2-fold differentially expressed in the respective cell lines, of which 122 were shared among the three cell lines. As proof of principle, we focused on the anti-metastatic gene NM23-H1, which was downregulated both at the RNA and protein level in the floating cell populations of all three cell lines. Knockdown of NM23-H1 significantly increased the number of floating (and viable) cells, whereas overexpression of NM23-H1 significantly reduced the proportion of floating cells. Other potential regulators of these cellular states were identified through pathway analysis, including hypoxia, mTOR (mechanistic target of rapamycin), cell adhesion and cell polarity signal transduction pathways. Hypoxia, a condition linked to malignancy and metastasis, reduced NM23-H1 expression and significantly increased the number of free-floating cells. Inhibition of mTOR or Rho-associated protein kinase (ROCK) significantly increased cell death specifically in the floating and not the adherent cell population. In conclusion, our study suggests that dynamic subpopulations of free-floating and adherent cells is a useful model to screen and identify genes, drugs and pathways that regulate the process of cancer metastasis, such as cell detachment and anoikis.     

Role of PI3K-AKT-mTOR Pathway as a Pro-Survival Signaling and Resistance-Mediating Mechanism to Therapy of Prostate Cancer

Androgen deprivation therapy (ADT) and androgen receptor (AR)-targeted therapy are the gold standard options for treating prostate cancer (PCa). These are initially effective, as localized and the early stage of metastatic disease are androgen- and castration-sensitive. The tumor strongly relies on systemic/circulating androgens for activating AR signaling to stimulate growth and progression. However, after a certain point, the tumor will eventually develop a resistant stage, where ADT and AR antagonists are no longer effective. Mechanistically, it seems that the tumor becomes more aggressive through adaptive responses, relies more on alternative activated pathways, and is less dependent on AR signaling. This includes hyperactivation of PI3K-AKT-mTOR pathway, which is a central signal that regulates cell pro-survival/anti-apoptotic pathways, thus, compensating the blockade of AR signaling. The PI3K-AKT-mTOR pathway is well-documented for its crosstalk between genomic and non-genomic AR signaling, as well as other signaling cascades. Such a reciprocal feedback loop makes it more complicated to target individual factor/signaling for treating PCa. Here, we highlight the role of PI3K-AKT-mTOR signaling as a resistance mechanism for PCa therapy and illustrate the transition of prostate tumor from AR signaling-dependent to PI3K-AKT-mTOR pathway-dependent. Moreover, therapeutic strategies with inhibitors targeting the PI3K-AKT-mTOR signal used in clinic and ongoing clinical trials are discussed.     

Novel Dual PI3K/mTOR Inhibitor,Apitolisib (GDC-0980), Inhibits Growth and Induces Apoptosis in Human Glioblastoma Cells

Deregulated PI3K/AKT/mTOR signalling commonly exists in glioblastoma, making this axis an attractive target for therapeutic manipulation. Given that activation of PI3K/AKT/mTOR promotes tumour growth, metastasis, and resistance to anticancer therapies, mTOR inhibitors show promise in the treatment of cancer. The aim of this study was to investigate the underlying mechanism of novel dual PI3K/mTOR inhibitor, Apitolisib (GDC-0980), in A-172 and U-118-MG GBM tumour cell line suppression. It has been demonstrated that GDC-0980 induces time- and dose-dependent cytotoxicity and apoptosis in investigated glioma cell lines. In our study, the strongest induction of apoptosis was exhibited in the A-172 line after 48 h of incubation with 20 µM GDC-0980, where we observed 46.47% of apoptotic cells. In conclusion, we first discovered that dual PI3K/mTOR blockade by GDC-0980 markedly suppressed survival of human GBM cells and induced apoptosis, independent of the ER stress-mediated DR5 activation. We suggest that GDC-0980, by exerting an inhibitory effect on PERK expression, may thus block its inhibitory effect on protein synthesis, leading to intensification of translation, and this may result in an increase in apoptosis. On the other hand, CHOP stimulates protein synthesis and increases apoptosis. These findings suggest that GDC-0980 may be a candidate for further evaluation as a chemotherapeutic agent for anti-GBM therapy.     

X-ray Diffraction Analysis to Explore Molecular Traces of Eccentric Contraction on Rat Skeletal Muscle Parallelly Evaluated with Signal Protein Phosphorylation Levels

We performed X-ray diffraction analyses on rat plantaris muscle to determine if there are strain-specific structural changes at the molecular level after eccentric contraction (ECC). ECC was elicited in situ by supramaximal electrical stimulation through the tibial nerve. One hour after a series of ECC sessions, the structural changes that remained in the sarcomere were evaluated using X-ray diffraction. Proteins involved in cell signaling pathways in the muscle were also examined. ECC elicited by 100, 75, and 50 Hz stimulation respectively developed peak tension of 1.34, 1.12 and 0.79 times the isometric maximal tetanus tension. The series of ECC sessions phosphorylated the forkhead box O proteins (FoxO) in a tension-time integral-dependent manner, as well as phosphorylated the mitogen-activated protein kinases (MAPK) and a protein in the mammalian target of rapamycin (mTOR) pathway in a maximal tension dependent manner. Compared to isometric contractions, ECC was more efficient in phosphorylating the signaling proteins. X-ray diffraction revealed that the myofilament lattice was preserved even after intense ECC stimulation at 100 Hz. Additionally, ECC < 75 Hz preserved the molecular alignment of myoproteins along the myofilaments, while 75-Hz stimulation induced a slight but significant decrease in the intensity of meridional troponin reflection at 1/38 nm⁻¹, and of myosin reflection at 1/14.4 nm⁻¹. These two reflections demonstrated no appreciable decrease with triple repetitions of the standard series of ECC sessions at 50 Hz, suggesting that the intensity decrease depended on the instantaneous maximal tension development rather than the total load of contraction, and was more likely linked with the phosphorylation of MAPK and mTOR signaling proteins.     

EGCG inhibits protein synthesis,lipogenesis, and cell cycle progression through activation of AMPK in p53 positive and negative human hepatoma cells

In the previous studies, (–)‐epigallocatechin‐3‐gallate (EGCG) has been shown to have anticarcinogenic effects via modulation in protein expression of p53. Using p53 positive Hep G2 and p53 negative Hep 3B cells, we found that treatment of EGCG resulted in dose‐dependent inhibition of cellular proliferation, which suggests that the interaction of EGCG with p53 may not fully explain its inhibitory effect on proliferation. Caloric restriction (CR) reduces the incidence and progression of spontaneous and induced tumors in laboratory rodents. EGCG has multiple beneficial activities similar to those associated with CR. One key enzyme thought to be activated during CR is AMP‐activated kinase (AMPK), a sensor of cellular energy levels. Here, we showed that EGCG activated AMPK in both p53 positive and negative human hepatoma cells. The activation of AMPK suppressed downstream substrates, such as mammalian target of rapamycin (mTOR) and eukaryotic initiation factor 4E‐binding protein‐1 (4E‐BP1) and a general decrease in mRNA translation. Moreover, EGCG activated AMPK decreases the activity and/or expression of lipogenic enzymes, such as fatty acid synthase (FASN) and acetyl‐CoA carboxylase (ACC). Interestingly, the decision between apoptosis and growth arrest following AMPK activation is greatly influenced by p53 status. In p53 positive Hep G2 cells, EGCG blocked the progression of cell cycle at G1 phase by inducing p53 expression and further up‐regulating p21 expression. However, EGCG inducted apoptosis in p53 negative Hep 3B cells. Based on these results, we have demonstrated that EGCG has a potential to be a chemoprevention and anti‐lipogenesis agent for human hepatoma cells.