MiR-34a targets GAS1 to promote cell proliferation and inhibit apoptosis in papillary thyroid carcinoma via PI3K/Akt/Bad pathway

MicroRNAs (miRNAs) are fundamental regulators of cell proliferation, differentiation, and apoptosis, and are implicated in tumorigenesis of many cancers. MiR-34a is best known as a tumor suppressor through repression of growth factors and oncogenes. Growth arrest specific1 (GAS1) protein is a tumor suppressor that inhibits cancer cell proliferation and induces apoptosis through inhibition of RET receptor tyrosine kinase. Both miR-34a and GAS1 are frequently down-regulated in various tumors. However, it has been reported that while GAS1 is down-regulated in papillary thyroid carcinoma (PTC), miR-34a is up-regulated in this specific type of cancer, although their potential roles in PTC tumorigenesis have not been examined to date. A computational search revealed that miR-34a putatively binds to the 3′-UTR of GAS1 gene. In the present study, we confirmed previous findings that miR-34a is up-regulated and GAS1 down-regulated in PTC tissues. Further studies indicated that GAS1 is directly targeted by miR-34a. Overexpression of miR-34a promoted PTC cell proliferation and colony formation and inhibited apoptosis, whereas knockdown of miR-34a showed the opposite effects. Silencing of GAS1 had similar growth-promoting effects as overexpression of miR-34a. Furthermore, miR-34a overexpression led to activation of PI3K/Akt/Bad signaling pathway in PTC cells, and depletion of Akt reversed the pro-growth, anti-apoptotic effects of miR-34a. Taken together, our results demonstrate that miR-34a regulates GAS1 expression to promote proliferation and suppress apoptosis in PTC cells via PI3K/Akt/Bad pathway. MiR-34a functions as an oncogene in PTC.     

D2/D3 receptor agonist ropinirole protects dopaminergic cell line against rotenone-induced apoptosis through inhibition of caspase- and JNK-dependent pathways

Ropinirole, a D2/D3 receptor agonist has been reported to have neuroprotective effects. We showed that ropinirole can prevent rotenone-induced apoptosis in dopaminergic cell line SH-SY5Y through D3 receptor. We found that ropinirole can block the rotenone-induced phosphorylation of JNK, P38 and p-c-Jun, but promote the phosphorylation of ERK1/2. Furthermore, we demonstrated that ropinirole can reduce the rotenone-induced cleavages of caspase 9, caspase 3 and PARP and elevate the expression of anti-apoptotic proteins of p-Akt and bcl-2. These results provide a basis for neuroprotection by this drug for the treatment of Parkinson disease.     

Genetic mosaicism of a frameshift mutation in the RET gene in a family with Hirschsprung disease

Mutations and polymorphisms in the RET gene are a major cause of Hirschsprung disease (HSCR). Theoretically, all true heterozygous patients with a new manifestation of a genetically determined disease must have parents with a genetic mosaicism of some extent. However, no genetic mosaicism has been described for the RET gene in HSCR yet. Therefore, we analyzed families with mutations in the RET gene for genetic mosaicism in the parents of the patients. Blood samples were taken from patients with HSCR and their families/parents to sequence the RET coding region. Among 125 families with HSCR, 33 families with RET mutations were analyzed. In one family, we detected a frameshift mutation due to a loss of one in a row of four cytosines in codon 117/118 of the RET gene (c.352delC) leading to a frameshift mutation in the protein (p.Leu118Cysfs*105) that affected two siblings. In the blood sample of the asymptomatic father we found a genetic mosaicism of this mutation which was confirmed in two independent samples of saliva and hair roots. Quantification of peak-heights and comparison with different mixtures of normal and mutated plasmid DNA suggested that the mutation occurred in the early morula stadium of the founder, between the 4- and 8-cell stages. We conclude that the presence of a RET mutation leading to loss of one functional allele in 20 to 25% of the cells is not sufficient to cause HSCR. The possibility of a mosaicism has to be kept in mind during genetic counseling for inherited diseases.     

Mesenchymal stem cells stimulate angiogenesis in a murine xenograft model of A549 human adenocarcinoma through an LPA1 receptor-dependent mechanism

Carcinoma-associated fibroblasts play a key role in tumorigenesis and metastasis by providing a tumor-supportive microenvironment. In the present study, we demonstrate that conditioned medium from A549 human lung adenocarcinoma cells induces differentiation of human adipose tissue-derived mesenchymal stem cells (hASCs) to carcinoma-associated fibroblasts expressing α-smooth muscle actin, vascular endothelial growth factor, and stromal cell-derived factor-1. A549 conditioned medium-induced differentiation of hASCs to carcinoma-associated fibroblasts was completely abrogated by treatment of hASCs with Ki16425, a lysophosphatidic acid receptor antagonist, or knockdown of lysophosphatidic acid receptor 1 (LPA₁) expression in hASCs with small interfering RNA or lentiviral short hairpin RNA. Using a murine xenograft transplantation model of A549 cells, we showed that co-transplantation of hASCs with A549 cells stimulated growth of A549 xenograft tumor, angiogenesis, and differentiation of hASCs to carcinoma-associated fibroblasts in vivo. Knockdown of LPA₁ expression in hASCs abrogated hASCs-stimulated growth of A549 xenograft tumor, angiogenesis, and differentiation of hASCs to carcinoma-associated fibroblasts. Moreover, A549 conditioned medium-treated hASCs stimulated tube formation of human umbilical vein endothelial cells by LPA₁-dependent secretion of vascular endothelial growth factor. These results suggest that A549 cells induce in vivo differentiation of hASCs to carcinoma-associated fibroblasts, which play a key role in tumor angiogenesis within tumor microenvironment, through an LPA-LPA₁-mediated paracrine mechanism.     

Thalidomide inhibits epidermal growth factor-induced cell growth in mouse and human monocytic leukemia cells via Ras inactivation

The effect of thalidomide on epidermal growth factor (EGF)-induced cell growth was examined. Thalidomide inhibited EGF-induced cell growth in mouse and human monocytic leukemia cells, RAW 264.7, U937 and THP-1. Thalidomide inhibited EGF-induced phosphorylation of extracellular signal regulated kinase (ERK) 1/2, but not p38 and stress-activated protein kinase (SAPK)/JNK. The phosphorylation of MEK1/2 and Raf at Ser 338 as the upstream molecules of ERK 1/2 was also prevented by thalidomide. Further, it inhibited EGF-induced Ras activation through preventing the transition to GTP-bound active Ras. Thalidomide inhibited the Ras activation induced by lipopolysaccharide (LPS) and vascular endothelial growth factor (VEGF) as well as EGF. There was no significant difference in the expression and function of EGF receptor between thalidomide-treated and non-treated cells. Therefore, thalidomide was suggested to inhibit EGF-induced cell growth via inactivation of Ras.     

Parthenolide generates reactive oxygen species and autophagy in MDA-MB231 cells. A soluble parthenolide analogue inhibits tumour growth and metastasis in a xenograft model of breast cancer

Triple-negative breast cancers (TNBCs) are clinically aggressive forms associated with a poor prognosis. We evaluated the cytotoxic effect exerted on triple-negative MDA-MB231 breast cancer cells both by parthenolide and its soluble analogue dimethylamino parthenolide (DMAPT) and explored the underlying molecular mechanism. The drugs induced a dose- and time-dependent decrement in cell viability, which was not prevented by the caspase inhibitor z-VAD-fmk. In particular in the first hours of treatment (1–3 h), parthenolide and DMAPT strongly stimulated reactive oxygen species (ROS) generation. The drugs induced production of superoxide anion by activating NADPH oxidase. ROS generation caused depletion of thiol groups and glutathione, activation of c-Jun N-terminal kinase (JNK) and downregulation of nuclear factor kB (NF-kB). During this first phase, parthenolide and DMAPT also stimulated autophagic process, as suggested by the enhanced expression of beclin-1, the conversion of microtubule-associated protein light chain 3-I (LC3-I) to LC3-II and the increase in the number of cells positive to monodansylcadaverine. Finally, the drugs increased RIP-1 expression. This effect was accompanied by a decrement of pro-caspase 8, while its cleaved form was not detected and the expression of c-FLIP_S markedly increased. Prolonging the treatment (5–20 h) ROS generation favoured dissipation of mitochondrial membrane potential and the appearance of necrotic events, as suggested by the increased number of cells positive to propidium iodide staining. The administration of DMAPT in nude mice bearing xenografts of MDA-MB231 cells resulted in a significant inhibition of tumour growth, an increment of animal survival and a marked reduction of the lung area invaded by metastasis. Immunohistochemistry data revealed that treatment with DMAPT reduced the levels of NF-kB, metalloproteinase-2 and -9 and vascular endothelial growth factor, while induced upregulation of phosphorylated JNK. Taken together, our data suggest a possible use of parthenolide for the treatment of TNBCs.     

Molecular functions of the iron-regulated metastasis suppressor,NDRG1, and its potential as a molecular target for cancer therapy

N-myc down-regulated gene 1 (NDRG1) is a known metastasis suppressor in multiple cancers, being also involved in embryogenesis and development, cell growth and differentiation, lipid biosynthesis and myelination, stress responses and immunity. In addition to its primary role as a metastasis suppressor, NDRG1 can also influence other stages of carcinogenesis, namely angiogenesis and primary tumour growth. NDRG1 is regulated by multiple effectors in normal and neoplastic cells, including N-myc, histone acetylation, hypoxia, cellular iron levels and intracellular calcium. Further, studies have found that NDRG1 is up-regulated in neoplastic cells after treatment with novel iron chelators, which are a promising therapy for effective cancer management. Although the pathways by which NDRG1 exerts its functions in cancers have been documented, the relationship between the molecular structure of this protein and its functions remains unclear. In fact, recent studies suggest that, in certain cancers, NDRG1 is post-translationally modified, possibly by the activity of endogenous trypsins, leading to a subsequent alteration in its metastasis suppressor activity. This review describes the role of this important metastasis suppressor and discusses interesting unresolved issues regarding this protein.     

The role of metallothionein in oncogenesis and cancer prognosis

The antiapoptotic, antioxidant, proliferative, and angiogenic effects of metallothionein (MT)-I+II has resulted in increased focus on their role in oncogenesis, tumor progression, therapy response, and patient prognosis. Studies have reported increased expression of MT-I+II mRNA and protein in various human cancers; such as breast, kidney, lung, nasopharynx, ovary, prostate, salivary gland, testes, urinary bladder, cervical, endometrial, skin carcinoma, melanoma, acute lymphoblastic leukemia (ALL), and pancreatic cancers, where MT-I+II expression is sometimes correlated to higher tumor grade/stage, chemotherapy/radiation resistance, and poor prognosis. However, MT-I+II are downregulated in other types of tumors (e.g. hepatocellular, gastric, colorectal, central nervous system (CNS), and thyroid cancers) where MT-I+II is either inversely correlated or unrelated to mortality. Large discrepancies exist between different tumor types, and no distinct and reliable association exists between MT-I+II expression in tumor tissues and prognosis and therapy resistance. Furthermore, a parallel has been drawn between MT-I+II expression as a potential marker for prognosis, and MT-I+II's role as oncogenic factors, without any direct evidence supporting such a parallel. This review aims at discussing the role of MT-I+II both as a prognostic marker for survival and therapy response, as well as for the hypothesized role of MT-I+II as causal oncogenes.     

AD-1, a novel ginsenoside derivative,shows anti-lung cancer activity via activation of p38 MAPK pathway and generation of reactive oxygen species

Background

Ginseng is a traditional Chinese herb that has been used for thousands of years. In the present study, effects and mechanisms of AD-1 were evaluated for its development as a novel anti-lung cancer drug.

Methods

The cytotoxic activity was evaluated by MTT assay. Flow cytometry was employed to detect cell cycle, apoptosis and ROS. Western blot and immunohistochemistry were used to analyze signaling pathways. Lung cancer xenograft models were established by subcutaneous implantation of A549 or H292 cells into nude mice.

Results

AD-1 concentration-dependently reduces lung cancer cell viability without affecting normal human lung epithelial cell viability. In A549 and H292 lung cancer cells, AD-1 induces G₀/G₁ cell cycle arrest, apoptosis and ROS production. The apoptosis can be attenuated by a ROS scavenger — N-acetylcysteine (NAC). In addition, AD-1 up-regulates the expression of p38 and ERK phosphorylation. Addition of a p38 inhibitor SB203580, suppresses the AD-1-induced decrease in cell viability. Furthermore, genetic silencing of p38 attenuates the expression of p38 and decreases the AD-1-induced apoptosis. Treatment with NAC reduces AD-1-induced p38 phosphorylation, which indicates that ROS generation is involved in the AD-1-induced p38 activation. In mice, oral administration of AD-1 (10–40 mg/kg) dose-dependently inhibited the growth of xenograft tumors without affecting body weight and decreases the expression of VEGF, MMP-9 and CD34 in tumor tissue. TUNEL staining confirms that the tumors from AD-1 treated mice exhibit a markedly higher apoptotic index.

Conclusions and general significance

These data support development of AD-1 as a potential agent for lung cancer therapy.